REGULATION AND FUNCTlON OF GLUCOSE TRANSPORTERS IN RAT MYOBLASTS
Deifina Maria Mazzuca
Department of Biochemistry
Submitted in partial fulnlment of the requirements for the degree
of Master of Science
Faculty of Graduate Studies The University of Western Ontario
London, Ontario Feb '1998
O Delfina M. Mazzuca 1998
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ABSTRACT
The objectives of this thesis are to examine the regulatory and functional roles of
glucose transporters in rat myoblasts. The first part of the thesis examined the properties
of GLUT 3- mutants. Three independentiy isolated GLUT 3- mutants @2. Dg. and D23)
were found to be d e f d v e not only in GLUT 3 expression but also in myogenic
differentiation. Studies with these mutants revealed a lack of direct correlation between
the expression of GLUT 3 and various myogenesis-associated genes. First, the expression
of myogenzn and muscle-specific genes varied wnsiderably amongst the mutants, even
though they contained sirnilar GLUT 3 transcript levels. Second, transfection of D2 and
D23 mutants with the GLUT 3 cDNA did not restore the cells' ability to express myogenin
and muscle-specific proteins and to form myotubes. To further examine the myogenic
defect in mutant D23, this ce11 line was transfected with the myogenzn prornoter, or with
the myogenin coding sequence. These studies suggested that mutant D23 was defective in
a component (Factor M) essential for myogenzn promoter activity, and in a component
(Factor S) required for the transcription of muscle-specific genes. A tentative working
mode1 is proposed to explain Our observations using various GLUT 3- mutants and L6
GLUT 3 transfectants. It is wnceivable that the GLUT 3 transporter may regulate the
fûnctional state, level andor stability of these factors by direct interaction.
The second part of the thesis deals with the construction, expression and isolation
of GST-fusion proteins containing the centrai loop (G4L) or the carboxyl terminus (G4C)
regions of the GLUT 4 transporters. These fusion proteins can be used as powefil tools
in identifjing and isolating proteins that can interact with glucose transporters.
iii
ACKNOWLEDGEMENTS
Special thanks to Dr. Lo for believing in me. Your guidance and enthusiasm made
my time in the lab an enjoyable one. I would also Wte to thank my lab mates over the
years, Michelle, Mei, Fariha, Dale, Patrick and Rob for keeping me grounded and teaching
me it is o.k. to laugh at myself. Thank-you to the Sanwal and Ske janc labs, Anne, Ilona,
S h a r o ~ Al and Helen for always king there when 1 needed technical help or just a good
"belly" laugh. Thanks Ted JaMs, Bah, Maureen and Ruth for all your great advice on
Iife,
I would also like to sincerely thank my fiends Schmitty, Kazala, Ridgeway, K.C.,
Miller and Kevin for al1 your suppon and encouragement.
DEDICATlON
Ricordo i nonni Mazzuca. Ricordo la nonna di essere sempre allegra anche se in
realtà fisicalmente era ammalata. End col suo parlare mi à inspirato di scrivere questo
Libro. Il nonno, vogiio ringraziarlo per la sua pasione di legere i libn che mi à inspirato e
anche lui di studiare scienza e scrivere questo libro. E mianno insegnato a scnvere e
Iegere la lingua italiana. E sono contenta di queîio che mianno insegnato e per queao gli
ricordo sempre.
Ringrazio i miei genitori che mianno mandat0 e pagato la mola. Io gii ricordo
sempre con questo libro. La'quaie possono legere anche loro.
PAGE
CHAPTER 3 . ALTERATIONS IN MYOGENIC REGULATORY COMPONENTS IN MUTANT D23
................................................................................................. 3.1 INTRODUCTION 66
3.2 MATERIALS AND METHODS 3.2.1 Bacteriai Strains and Plasmids ......................................................................... 6 8 3 .2.2 Cell Culture ..................................................................................................... 69
... 3.2.3 Transient Transfdon W1th Constmcts Containing the Myogenin Promoter -69 .................. 3 .2.4 Assay for &Galactosidase and Chlonunphenicol Acety l tderase -69
....................... 3.2.5 Stable Transfdon studies using the PGK-myogenin Constnict 71 3 .2.6 Southern Blot Analysis .................................................................................... 71 3 .2.7 Fusion Index Measurement ............................................................................. 7 1 3.2.8 Northern Blot Analysis ................................................................................... 7 1 3.2.9 Irnrnunofluorescence Microscopy Studies .................................................. 72
3.3 RESULTS ..................... 3.3.1 Myogeenin Promoter Activities in L6 and Mutant D23 Myoblasts 72
3.3.2 Restonng Myogenin Expression in D23 Myoblasts ........................................ 7 5 3 .3.3 Ability of D23/myogenin Transfectants to Differentiate ................................... -84
3.4 DISCUSSION ....................................................................................................... 89
CHAPTER 4 . EXPRESSION OF THE GLUT 4 CENTRAL LOOP AND C-TERMINAL DOMAINS IN BACTERIA
INTRODUCTION ............................................................................................... 92
4.2 MATERIALS AM> METHODS Bacterial Culture Media .................................................................................. 95 Bacterial Strains and Plasrnids ......................................................................... 95 Amplification of the G4L and G4C Regions .............................................. 97 Ligation and Transformation of the p ~ ~ ~ - T @ Vector with G4L and G4C PCR products ....................................................................................................... 98 Ligation of G4L and G4C into pGEX-KG ....................................................... 99 Transformation and Screening of pGEX-GLUT4 Constructs ......................... 100 Expression of GST-G4L Fusion Protein ....................................................... 101 Andysis of Soluble and Insoluble Fractions of the GST-G4L fusion protein .. 10 1 Solubliration of the GST-G4L From Ce11 Pellet ........................................ 102 Coupling and Elution of G4L Fusion Protein from Glutathione Agarose Beads ........................................................................................................... 104 Expressing, Coupling and Eluting Soluble GST and GST-G4C proteins ........ 1 OS
PAGE 4.3 RESULTS
4.3.1 Construction of the GST-G4L and GST-G4C Constmcts ............................... 105 4.3.2 Expression of the GST Fusion Proteins .......................................................... 115
............... 4.3 -3 Coupling o f the GST-fùsion Proteins to Glutathione-agarose Beads 118
..................................................................................................... 4.4 DISCUSSION 118
. ................. APPENDIX 1 Sample Caladations of Transcript Levels From Raw Data 142
APPENDIX 2 . Sample Cdculations of Myogenxn Promoter Activity in L6 and D23 Ceus ................................................................................................ 144
REFERENCES .................. .. ................................................................................... 146
VITAE ......................................................................................................................... 159
LIST OF FIGURES PAGE
CHAPTER I
FIGURE 1.1 Prediaed Secondary Structure of the Glucose Transporter ......... .. ........ 6
CHAPTER 2
FIGURE 2.1 FIGURE 2.2 FIGURE 2.3
FIGURE 2.4
FIGURE 2.5 FIGURE 2.6
FIGURE 2.7
FIGURE 2.8
FIGURES 2.9
FIGURE 2. 10
FIGURE 2.1 1
FIGURE 2.12
FIGURE 2.1 3
FIGURE 2.14
FIGURE 2.15
GL (IT 3 T m r i p t Levels in G L W - Mutants.. ............................... 2 8 Myogenic Ability of GLUn- Mutants .................................................. 30 m 5 , MOGEMN, M C , and IcNTTranscript Levels in L6 and GLUT3- Mutants (D23, D2, Dg) ..................................................... 33 Southem Blot Anaiysis of GLUT 3- Myoblast Transfected with the GL UT 3 cDNA .................................................... . 3 7 Northem BIot Analysis of Dex-Induced Myoblasts. ............................... 39 Morphology of Day 2 Dex-Induced and Uninduced
.............................................................. Cultures of D23 Transfectants. 42 Morphology of Day 6 Dex-Induced and Uninduced Cultures of D23 Transfectants. .............................................................. 44 Imrnunofluorescence Staining of Myogenin in Day 2 Uninduced Cultures.. .................... ,, .................................................... 47 Irnmunofluorescence Staining for Myogenin in Day 2 Dex-Induced Cultures. ......................................................................... 4 9 Irnmunofluorescence Staining for MHC in Day 2 Dex-
.................................................... Uninduced Cultures ........................ 5 1 Immunofluorescence Staining for MHC in Day 2 Dex- Induced Cultures .................................................................................. .53 Immunofluorescence Staining of Myogenin in Day 6 Uninduced Cultures. .............................................................................. 55 Immunofluorescence Staining for Myogenin in Day 6 Dex-induced dtures.. ........................................................................... 5 7 Immunofluorescence Staining for MHC in Day 6 Uninduceci CuItures. ..................................... ... ................................. 5 9 Immunofluorescence staining for MHC in Day 6 Dex-
................................................................................. Induced Cultures. 6 1
FIGURE 3.1 Myogenin Promoter Activities in L6 and D23 Myoblasts .............................................................................................. 73
FIGURE 3.2 Southem Blot Analysis of D23 Transfaants ........................................ 76 FIGURE 3.3 Northem Blot Analysis of D23lmyogenin
Myoblasts ............................................................................................... 78 FIGURE 3.4 Immuno£luorescence Staining of Myogenin in Day 2
................................................................ D23/myogenin Transfectant S. 8 0
FIGURE 3 -5 Immunofluorescenee Staining of Myogenin in Day 6 D23/myogenin Transfectants .................................................................. 8 2
FIGURE 3 -6 Immunofluorescence Staining of MHC in Day 2 D23/myogenin Tdec tan t s ................................................................. 8 5
FIGURE 3.7 Lmmunofluorescence Staining of MHC in Day 6 D23 /myogenin Transfectants ................................................................. 87
CHAPTER 4
FIGURE 4.1 FIGURE 4.2 FIGURE 4.3
FIGURE 4.4
FIGURE 4.5 FIGURE 4.6
FIGURE 4.7
FIGURE 4.9
CHAPTER 5
Design of the pGEX-G4L Constmct ..................................................... 106 Design of the pGEX-G4C Constnict ..................................................... 109 Diagnostic Restriction Digestion of G4L and G4C
..................................... ....................................................... Clones ,... 113 Expression of the GST-G4L and GST-G4C in E-COW BL-2 1 @E3) .......................................................................................... 116 Solubilization of the GST-G4L Fusion Proteins ..................................... 119 Elution of GST-G4L and GST-G4C Proteins From Glutathione- Agarose ............................................................................. 121 Cleavage ofGST/GST-Fusion Proteins From Coupled Glutathione-Agarose Beads ................................................................... 123 Predicted Secondary Structure of the GLUT 4
.................. ...................... Centrai Loop and Carboxyl Terminus ....... 128 HeIical-Wheel Presentation of the Predicted Helical
.................................................................................. Structure of G4L 130
FIGURE 5.1 Tentative Working Mode1 ..................................................................... 136
LIST OF APPENDICES PAGE
APPENDIX 1. Sample Caldations of Transcxipt Levels Frorn Raw Data . . . . . .. . . . . .. . . . . 142
APPENDIX 2. Sample Caiculations of Myogenin Promoter Activity in L6 and D23 Ceils ....... . .... . ... . . .. . . .. . . . ... .. . . ... . .. . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 144
LIST OF ABBREVIATI[ONS
Ab ATP b H L H &MG bp BS A OC CAME' cDNA CIAP COOH cST D2 D23 D9 DEPC dex dGlc dNTP dpm DTT EDTA FBS F5D G4 18 G4C G4L GLUT GSC GST GXC HS HAHT HEPES HBS buEer
hr IPTG kb kDa L6/G3 A L6/G3 S L m
antibody adenosine triphosphate basic helix-bop-helix motif /%2-microgIobuIin gene base pair bovine s e m albumin degrees Celsius cyclic adenosine monophosphate cornplementary DNA calf intestinal alkaiine p hosphatase carboxyl cytoplasmic signal transducer L6 mutant defective in the GLUT 3 isoform L6 mutant defective in the GLUT 3 isoform L6 mutant defective in the GLUT 3 isofonn diethyl pyrocarbonate dexamethasone 2-de0~y-D-glu~ se deoxynucleotide triphosphates disintegrations per minute dithiothreitol ethylenediaminetriacetic acid fetal bovine senun monoclonal anti-myogenin antibody geneticin GLUT 4 C-terminal domain constmct GLUT 4 loop domain wnstnid giucose transporter 1092 bp, full length myogenin prornoter constmct glutathione S-tranferase 133 bp, truncated myogenin promoter constmct horse serum high af£inity hexose transporter N-2-hydroxyethylpiperazine-N-2-ethane-dphoric acid 270 mM NaCl, 9mM KCL, 1mM Na2HPOr2H20, dextrose92 mM HEPES, pH 7.04) hour isopropyl-P-D-thiogalactosidase kilobase kilodalton L6 cells transfected with GLUT 3 in the antisense orientation L6 ceus transf-ed with GLUT 3 in the sense orientation low atfinity hexose transporter
LB LMW MADS MCS MEF 2 MeGlc MF-20 MHC min mL MLC MMTV-Lm MRFs mRNA Wf-4 myf-, Myf-5 mg-5 NaAc OD p l 12 PAX-3 PBS
PGK PMSF PPD PVP pRdCMV SB
SDS SDS-PAGE SOB
SOC sPBS
SSC TAE TB TE TM TnT clr,
Luria-Bertani Medium Low rnolecular weight protein markers MCM 1, agamous, deficiens, semm response factor multiple cioning site myocyte enhancer factor 2 3 -0-methyI-D-glyw se monodonai anti-myosin heavy chah antibody myosin heavy chah rninu te &ter myosin light chah mouse mammary -or virus long terminal repeat promoter muscle regulatory factors messenger RNA myogenic factor 4 (protein) myogenic factor 4 gene myogenic factor 5 @rotein) myogenic factor 5 gene sodium acetate optical density 1 12 kDa phosphoprotein paired-type homeobox gene phosphate buffered saline (1 37 mM NaCl 2.7 mM KCI, 8.1 rnM Na2HPO4 1.5mM K&PO,) p hosp hoglycerate kinase phenylmethyl sulfonyl fluoride p henylendiamine polyvinylpyrrolidone CMV expression vector Sarnple buffer (50 mM Tris-CI (pH 6.8), 1ûûm.M dithiothreitol, 2% SDS, 0.1 % bromophenol blue, 1 0% glycerol) sodium dodecyl sulfate sodium dodecyl sulfate-polyacqlamide gel electrophoresis 20 g/L Bacto-tryptone, 5 g/L Bacto yeast extract, 0.01 M NaCl, 250 mM KCL, 2 M MgC12 SOB with 20 mM glucose and 2 mM MgCl2 stockholm PBS (137 mM NaCl, 2.7 rnM KCl, 4.3 mM Na2HPO4, 1.9mM KH2PO4) standard (saline) citrate (3M NaCl, 0.3M sodium citrate) 0.04 M Tris-acetate, 0.00 1 M EDTA Temfic Broth 1 O mM Tris-Hel, pH 7.5; 1 mM EDTA trammembrane troponin-T microliter
CHAPTER 1
INTRODUCITON
1.1 The Glucose Transporter Super-famiIy
The traffic of moleailes through biological membranes is vital for most cellular
processes. The passage of moa molecules across the membrane involves the mediation of
specific membrane transport proteins. Molecules such as glucose, a major energy source
of m a d i a n cells, are taken up into cells via a famiy of specific glucose transporters.
This protein-rnediated transport process is characterized by a high degree of
stereoselectivity. This saturable, bidirectional transport system acts to equalize
concentrations of glucose in the cytoplasm and extracellular fluid, assuring that a constant
supply of glucose will be available for metabolism (Bell et ai., 1993; Wright, 1993).
The facilitative glucose transporter (GLUT) super-family consists of six
functionally distinct membrane integral proteins. These proteins are referred to as GLUT
1-7, based on the chronological order cf identification and isolation of their cDNAs. This
seven member supergene family shares signifiant sequence similarity and has unique
tissue distribution and biochemical properties
Al1 marnmalian ceiis contain one or more members of the GLUT super-fdy
(Olson et al., 1996). Initially identifieci as the major glucose transport protein in human
erythrocytes (Mueckler et al., 1985), GLUT 1 is expressed at highest levels in the
endotheliai cells of barrier tissues such as blood vessels and the blood brain barrier. The
expression of GLUT 1 in endothehl celis is thought to provide a mechanism by which
glucose can be transported across the blood brah bamier to the central nervous system,
which is dependent on glucose as its prirnary energy source. Because of its hi&
abundance (3 to 5% of total membrane protein) in red blood celi membrane, the ability to
p w GLUT 1 has allowed for the initial biochemical characterkation of this protein and
for the generation of antibodies. Antibodies r a i d against GLUT 1 were used to clone
the GLUT 1 cDNA ffom human HepG2 cells (Mueckler et al., 1985) and rat brah
(Birnbaurn et al., 1986). The cDNAs for GLUT 2-4 have since been isolated and
characterized in tenns of tissue specificity, expression and finctional activity.
GLUT 1 is expressed at high levels in al1 f d tissues and is widely expressed. It is
most abundant in fibroblasts, erythrocytes and endothelial cells. It is expressed in reduced
levels in muscle, liver and adipose tissue (Kayano et al., 1988; Torcino et al., 1994). The
variable molecular mass (45-55 kDa) of GLUT 1 in tissues can be accounted for by
specific difFerences in N-linked glycosylation (Olson et al. 1996).
GLUT 2, a 524 amino acid protein, is predominantly expressed in hepatocytes and
pancreatic P-cells, with lower levels in the kidney and intestine. It has a low affinity for
glucose and a high turnover rate. Its relatively high Km value results in transport activity
in direct proportion to the physiological range of glucose concentration (3.9 to 5.6 mM)
(Thorens et al., 1988). It is believed that GLUT 2 finctionally coordinates with
glucokinase in maintainhg a physiologicai range of intracellular concentration of fke
glucose (Heimberg et al., 1993). During states of glycogen synthesis, glucokinase is
upregulated and increases the formation of glucose-6-phosphate allowing a continuous
influx of glucose by maintaining its concentration low. Reduced glucokhase, such as in
the giuwneogenesis suite, increases the cells' intracellular concentration of fiee glucose,
greater than that present in the plasma, such that there is a net efflux of intracellular
glucose into the circulation. This coordinated regulation is required for appropriate
glucose sensing by the pcells. Upon changes in the plasma glucose concentration, the
highly sensitive fi-ceils regulate the amount of insulin secreted. The high Km glucose
transporter ensures that the transporter is not saturated at physiological levels so that the
flux will be directly proportional to the plasma glucose concentration. GLUT 2 also
serves as a low affinty h a o s e transporter (Colville et al., 1993)
The GLUT 3 transporter was origuially cloned fkom a human fetal skeletal muscle
library, suggesting a possible role for GLUT 3 in muscle development (Kayano et al.,
1988). The GLUT 3 transporter has a low Km value and is believed to transport glucose
at its maximal level in neurons, at normal plasma glucose concentrations. This transporter
is found at highest levels in neuronal tissue of al1 species studied (Kayano et al., 1988). It
is wnsidered the major GLUT responsible for maintainhg glucose supply to neurons by
transporting glucose into the brain and peripherai nerves. Unlike GLUT 1, GLUT 3
expression is primarily localized to neurons and has not been detected in the
microvasatlature of human or rat brains. GLUT 3 expression was detected in neurons at
approximately 10 days after birth, in fetal and neonatal brains of rodents, while the
GLUT 1 transporter was primarily expressed in al1 ce11 types (Nagarntsu et al., 1994).
GLUT 4 is predominantly found in insulin-sensitive (responsive) tissues. It is
expressed in adult skeletal and cardiac muscle, as well as in brown and white adipose
tissue (Bimbaum, 1989). Photolabeiling techniques using skeletal muscle cells and
adipocytes have shown that the insulin-stimulated increase in glucose transport activity is
due to an increase in surface-accessible GLUT 4 protein (Wilson et al., 1994). Insulin
reduces circulating glucose concentrations and promotes muscle glycogen storage and
adipocyte triglyceride synthesis. In vitro, innilin binds to a unique tyrosine kinase receptor
to initiate events that rapidly increase ceIl-surface locaiization of GLUT 4 by redistributing
this transporter fiom low-density microsornes to the plasma membrane. The intraceliular
distribution of GLUT 4 in the basal state appean to be directed by a dileucine motif
located in GLUT 4 COOH-terminus (Covera et al., 1994)
The GLUT 5 transporter is actually a haose transporter. It is located in both the
apical and basolaterai membranes of the intestine. GLUT 5 transcript was upregulated
only by D-hctose (Eilakeman et al., 1995). Transport of h a o s e by GLUT 2 has a 6-
fold lower affinity than that for GLU' 5 (Colville et al., 1993).
GLUT 6 contains multiple translation termination signais. It is a pseudo gene that
does not encode for a protein (Kayano et al., 1990).
The direct identification of the GLUT 7 protein as a fiindional facilitative glucose
transporter is yet to be established and remains under question. First located in the
endoplasmic reticulum, GLUT 7 has been identifieci as a component of the glucose-6-
phosphatase complex in the liver (Waddell et al., 1992).
1.2 Structure and Function of the Glucose Transporter
Tryptic digestion studies (Cairns et ai., 1 987) and hydropathy analysis (MuecWer
et al., 1985) of the GLUT 1 transporter predicted that this protein possessed a twelve
transmembrane-spanning (TM) domain with the amino (12 a.a.) and carboxyl (42 a-a.)
termini and a large loop (64 a.a.) comecting TM6 and TM7 oriented intracellularly
(Fig. 1.1). The 35 residue exofacid loop comecting TM1 and TM2 of the GLUT protein
appears to contain the only N-linked glycosylation site. In the glucose transporter
secondary structure (Fig. 1. l), TM 2, 3, 4, 5 , 7, 8 and 1 1 were reporteci to be
amphiphathic, each containhg more than four polar amino acid residues (Mueckler et al.,
1985; Zeng et al., 1996). The short connecting loops contain 8 to 12 residues. This
suggests very close packing of the helices in a tertiary structure at the imer sufice of the
membrane (Bell et al., 1993). The general transmembrane mode1 was confirmeci by
giycosylation scanning mutagenesis (Hresko et al., 1994). In these studies a N-Linked
glycosylation consensus site was independently inserted into each putative hydrophilic
region of an aglyco-GLUT1 mutant cDNA constmct. Expression in Xenopus laevis
oocytes confirmeci the exofacial and cytoplasmic orientation of each hydrophilic region.
The loop sizes and the ends of helices have not yet been determined with any certainty.
Circular dichroism spectral studies revealed that punfied GLUT 1 protein has 82%
a-helical, 1W P tums, and 8% random coi1 with no detectable P-sheet (Zeng et al.,
1996). Similarly, IR studies confirmed the GLUT 1 protein is highly a-helicd and
suggested the transmembrane domain to be mainly a-helical. Linear dichroism and
Fourier transfomi infiared spectral measurements on oriented films of purified GLUT 1
reconstituted in vesicles indicated that al1 TMs were nearly perpendicular to the plane of
the membrane lipid bilayer (Chin et al., 1986, Alvarez et al., 1987).
Extensive studies of the GLUT's structure have been performed to determine the
region required for glucose transport activity. Members of GLUT isoforms differ in the
lengths andor çequences of the amino and carboxyl termini, the large intracellular
hydrophilic loop region and the exofacial loop connecting TM1 and TM2 (Bell et al.,
1993; Zeng et al., 1996). These domains were thought to be responsible for tissue-
specific regdation of glucose transporter function to each isoform. Even though various
GLUT isoforms have a similar size (492 to 528 residues) and a topology similar to that for
GLUT 1, only 38% of the amino acids are cunserved between GLUT 1-4 isoforms. The
Figure 1.1 Predicted Secondary Structure of the Glucose Transporter
The glucose transporter is an integral membrane protein with twelve
transmembrane dornains s h o w in boxes numbered 1 through 12. The amino (NI&) and
carboxyl (COOH) termini are located on the cytoplasmic side of the protein. The carboxyi
terminus, different for d GLUT isoforms, is a distinct region for an antigenic determinant
site (black solid Iine). The black circle denotes a N-glycoqdation site of the transporter.
The diagram was modified nom Olson and Pessin, 19%.
greatest degree of amino acid sequence identity is found within the TM domains (Bell et
al., 1993, Zeng et al., 1996).
Several reagents have been used to define the substrate recognition (binding) sites
of the GLUT transporter. ATB-BMPA [NU-( 1 -aP-2,2,2-trifluoroethyl)benzoyI-l-3-
bi~mamose4yl-oxy)-2-propylamine J binds to the extemal face and inhibits sugar
idlux. Cytochalasin B and IAPS-forskoh ([1ZI~-iodo-4-azidophenetylamido-7-~-
succinyl-deacetyl-forskolin) bind to the cytoplasrnic site and inhibits sugar efflw. These
studies reveal two rnutually exclusive binding sites on the glucose transporter (Cairns et
al., 1987; Holrnan et al., 1987; Holrnan et al,. 1990). The binding of glucose to either the
outward or inward face of the substrate-binding site, is thought to induce the transporter
to switch between two conformations; thus resulting in movement of the substrate across
the plasma membrane (Walmsley, 1988). In comparing sequence similarities of putative
transmembrane regions within the glucose transporter farnily, several invariant polar
residues were found. Mutation of these residues has revealed structure-hction
relationships for the glucose transporters (Wandel et al., 1994 and 1995; Olson et al.,
1996).
Two rnodels have since been proposed by Jung's group on the tertiaxy structure of
the GLUT 1 transmembrane domains (Zeng et al., 1996). Model 1 suggests TM 3, 4, 7,
8, and 11 rnay form a channel, whereas Model 2 predicts that the channel is fonned by TM
2, 5, 7, 8, and 11. Both models predict that the charme1 is lined by polar residues present
in TM 7, 8, and 11. The channel is large enough in both models to d o w passage of
glucose. It should be noted that the a-helicai transmembrane mode1 has b e n chaiienged
by Fischbarg's group (Fischbarg et al., 1993). Fischbarg (1994) suggested that the
predicted transmembrane domains of the protein were consistently shorter than expected
for transmembrane a-helices. Analysis of the hydrophobicity, arnphiphilicity and tum
propensity, suggested that GLUT 1 had the correzt length and number to fold as porin-
like P-barrels. It should be noted that the P-barre1 model does not agree with data
generated by spectral studies which indicated a lack of enough possible P-sheets to
support a P-barre1 structure (Alvarez et al., 1987; Chin et al., 1987, Zeng et al., 1996).
The P-barre1 model also does not agree with the biochemical and molecular biological
findings on the topology of the GLUT 1 transporter (Hersko et al., 1994, Mueckler et al.,
1994).
1.3 Myogenesis
Early in skeletal muscle development, multipotential precursor cells become
committed to the myoblast limage. These mononucleated proliferating myoblasts will
then undergo biochernical and morphological differentiation (Cossu et al., 1 995). This
biochemical differentiation is characterized by the expression of muscle specific proteins
and enzymes such as myosin heavy chah (MHC), myosin light chah (MLC), troponin-T
(TnT), muscle creatine kinase, and acetylcholine recepton (Sassoon et al., 1988; Endo et
al., 1987; Kauhan et al., 1988; Weintraub et al., 1989; Medford et ai., 1980; Adolph et
ai., 1993; Edmondson et al., 1993; Garnnkel et al., 1982). Morphological differentiation
ocnin when small, spindle-shaped myoblasts align, adhere and fuse to form longer
tubular-like, multinucleated structures (myotubes), which then mature into various classes
of myofibers (YafEe, 1968).
The regdation of skeletai muscle determination and differentiation in vertebrates is
controlled by a network of two families of transcription factors, the basic helix-loophelix
(bHLH) muscle regulatory factors (MRFs) and the rnyocyte enhancer factor 2 (MEFZ)
group of MADS-box (MCMI, agamous, defiàens, serum response factor) regulators
(Yun et al., 1996; Rawls et al., 1997). The W s , each capable of activating the program
for skeletal muscle dserentiation, consist of the four bHLH transcription factors
myogenin, MyoD, Myf-5, and MRF4. This family of nuclear transcription factors shares a
cornmon region of 70 amino acids at the N-terminus with the mycsnwgene family
(Edmondson et al., 1992; Olson et al., 1993). These factors are thought to be part of a
much larger family of bHLH proteins. Similar domains were found in the achaete-scute
proteins (Alonso et ai., 1988), the positive-acting CO-regdators, daughterless (Caudy et
al., l988), and in the ubiquitously expressed E proteins, El2 and E47 (Murre et al., 1989).
Al1 four MRFs heterodimerize in vitro with members of the ubiquitous E2A and E2-2
bHLH family and bind the DNA consensus sites (CANNTG)(E-boxes) found in the
promoters of the muscle specific genes (Ludoph et. al., 1995).
MyoD andor Myf-5 can be detected in cell culture as well as during muscle
development in the embryo (Braun et ai., 1989a). Myogenin can be found in al1 skeletal
muscle types (Montamas et al., 1991) and is required for myogenesis to occur (Florini et
al., 1990, Hasty et al., 1993, Nabeshima et ai., 1993). MRF4 (herculin/Myf O), found only
in a few ceil lines, is believed to fundon in later stages of myogenesis (Miner et ai., 1990).
In siiu hybridization studies on developing mouse embryos have shown that
myogenic regulatory genes are expressed in a temporal order. Myf-5 is first detected in
the developing somite and declines by day 14 (Hannon et al., 1992). M'nin mRNA is
seen on day 8.5 and is expressed throughout the development of the sornite (Sassoon et
al., 1989). MRF4 is present on day 10 and 1 1 and is again expressed on day 16 (Bober et
al., 1991). MyoD is expressed on day 10.5 and then remains expressed during the rest of
mouse development (Sassoon et al., 1989). The temporal order of expression of the MRFs
varies in different species.
Valuable information about the role of each of the MRFs has been deduced fiom
knockout mice. No skeletal muscle celis were detected in the MyoD and Myf-5 double
knockout mice (Rudnicki et al., 1993); however skeletal muscle formation was detected
when either MyoD or Myf-5 was individually knocked out (Braun et al., 1992; Rudnicki et
al., 1992). Myogenin-nuil mice exhibited a variety of abnomalities (Hasty et al., 1993,
Nabeshima et al., 1993). The axial muscles were normal but the fibers were disorganized.
In the limb muscle, myoblasts were found to be arrested, mononucieated and unable to
fuse (Lasser et al., 1994a). Late stages of embryonic and fetal development were found to
be dependent on myogenin, more so than early stages (Venuti et al., 1995). Myogenin
was not required for myotome formation and the appearance of myoblasts during
embryogenesis. The MRF4-nul1 rnice developed normal skeletal muscle, but a high level
of myogenin was thought to compensate for the absence M W 4 (Zhang et al., 1995). It is
believed that MyoD and Myf-5 can compensate for each other in vivo and that myogenin
is not needed for cornmitment of skeletal muscle precursor ceus, but for terminal
differentiation of some muscle ceIl lineages.
Some myoblasts express determination MRFs, MyoD a d o r Myf 5 (rat L6 ceIl
line (YafFe, 1968) expresses only Myf-S), where they have exited the ceIl cycle at a
permanent GdGi arrest and daerentiate to produce mature muscle cells (myocytes). In
L6 ceiis, the expression of myogenin initiates muscle differentiation.
Expression of the differentiation genes has been shown to be enhanced by the
MEF2 family factors for terminal dierentiation (Edrnondson a al., 1992). This activation
of the myogenic network has been well documentai (Molkentin et al., 1996). In
coîransfiection assays, the expression of one of the four, mamrnalian MEF2 genes
(MEFZA-D) increases the efficiency of MRF-initiateci myogenesis (Moikentin et al., 1995;
Kaushal et ai., 1994). One member of the MEF 2 famiIy, MEFZC, is believed to control
skeletal muscle development by controlling myogenin expression in tissue culture cells
(Edmondson et al., 1992). During skeletal muscle development, MyoD or Myf-5 may
initiate a cascade of events that tum on MEFZC expression, which in turn activates
myogenin expression (Edmondson et al., 1992). Myogenin and MEF2C are involved in a
reinforcing positive regdatory loop where enhancement of myogenzn expression by
MEF2C enhances MEFZC expression. This ensures the levels of MEFZC and myogenin
remain high throughout skeletal muscle dserentiation (Lasser et al., 1994b). This
cooperative activation occurs when the MRF-E protein heterodimers (thought to interact
with the MEF2 protein) produce heterotypic MEF2-MRF-E-complexes. These complexes
require the DNA-binding capacity of only one of either the MEF2 or MRF-E facton to
produce a myogenic eEect (Mokentin et al., 1995).
The myogenic bHLH facton have been well doaunented as the earliest marken
specific for the skeletai muscle lineage in vertebrate embryos (Cossu et al., 1996; Dias et
al., 1994; Lasser et al., 1994b; Molkentin et ai., 1996; Yun et al., 1996). Whether their
expression tnggers initiation of this developmental pathway or is preceded by an even
earlier muscle-specific factor has not been determined. Recently it has been demonstratecl
that the paired-type homeobox gene, Pax-3 is a key regulator of skeletal muscle
development. It is found to be necessary and sufficient to activate MyoD expression and
initiate the myogenic program in vivo and in vitro (Tajbakhsh et al., 1997; Maroto et al.,
1997). It may a h fùnction to inhibit myogenic dïerentiation in migrating cells. When
Par-3 expression decreases, rnigrating ceils begin to différentiate. Pax-3, expressed in a
wide range of ceU types, is believed to act through combinatorid mechanisms to control
cornmitment to the myogenic lineage (Epstein et al., 1995; Ludolph et al., 1995).
There is further evidence that supports the myogenic network can be
downregulated or inactivated by a large and diverse group of positive and negative
regdators (Yun et al., 1996; Rawls et ai., 1997). There are regulators that promote Go-
Gi-S phase cell cycle progression together with their associated upstream signal
transduction apparatus (Lasser et al., 1 994a). Regulaton can also dominantly specify
other nonmuscle fates such as fat (Hu et al., 1995). Members of the Notch/Delta cell-ce11
signalling family have been found to regdate myogenesis (Kopan et al., 1994). Inhibitory
helix-loop-helix (HLH-) and bKLH-class regulaton such as Id and Twist can slow down
or abolish muscle differentiation. Id (able to heterodimerize with bHLH E-proteins) Iacks
the basic region and is unable to bind to DNA (Jen et al., 1992). Twist is thought to
interact with components involved in the myogenic network (MRFs), and to compete with
E-protein partners like Id. Twist can heterodimerize with E-proteins and bind to DNA
with an E-box consensus sequence uniike the preferred muscle E-box wre or twist can
interact with MEF2 factors and inhibit MEF2-MRF pergy (Spicer et al., 1996).
1.4 Involvement o f Glucose Transporters in Myogenesis
Characterization of glucose transporter expression during muscle dserentiation
has not been weli shidied. Studies using skeletal muscle (W~enstein et al., 1994;
Santalucia et al., 1992; Etgen et al., 1993) and L6 myoblasts (Xia et al., 1993; Mitsumoto
et al., 1 992; Richardson et al., 1993; Kudo et al., 1 990; Chen et al. 1 993; Klip et al., 1992;
Sleeman et al., 1995; Moyers et al., 19%) indicated GLUT transaipt, transporter levels
and transport activity were aitered during myogenesis. A high level of GLUT I expression
was reported in undifferentiated myoblasts and declined with muscle differentiation
(Mitsumoto et al., 1991; Kiip et al., 1992). GLUT 3 expression was also observed to
decline in myotubes @a et al., 1993). The retinoic acid circulating factor, known to
promote cellular differentiation, was reported to increase giuwse uptake and GLW 4
expression in L6 muscle cells (Sleeman et al., 1995). Although its level was low in
undifferentiated myoblasts, GL UT 4 mRNA level was elevated in myotubes (Mitsumoto et
al., 199 1; Klip et al., 1992; Xia et al., 1993). A myocyte enhancer factor 2 (MEFZ)
binding site (103 base pair fragment) was reported to be essential for myotube specific
expression of GLUT 4 in C2C 12 ceils (Liu et ai., 1994). A proximal skeletal muscle-
specific activation domain was thought to be essentiai for both myotube-specific GLUT 4
expression and thyroid hormone responsiveness (Richardson et al., 1 993).
1.5 Objective
The objective of this thesis is to examine the funaion and regdation of the glucose
transporters in rat myoblast. The two GLUT isoforms studied in this investigation were
GLUT 3 and GLUT 4. By examining other myogenesis-def~ive L6 myoblasts, mutants
with similar GLUT 3 expression patterns can be identified. Attempts were made to
restore myotube formation and expression of various myogenesis-associated genes in
these myoblasts by transfecting these mutants with the GLUT 3 cDNA The efféct of
overexpressing myogenin in the myogenesis defectve mutant, D23 was also examinecl.
These studies showed that components, in addition to the GLUT 3 transporter, are also
involveci in regulating myogenic differentiation. Since the GLUT 4 transporter is the only
fùnctional GLUT transporter present in mutant D23, attempts were made to idente
protellis that wuld bind specincdy to the GLUT 4 transporter. GST-fiision proteins
containing the central loop (G4L) and C-terminal (G4C) regions were wnstruaed,
expresseci and purifieci using ghtathione-agarose beads. These distinct regions of the
GLUT 4 protein can be used as tools to identq cytoplasmic signal transducen (cST) that
may be present in the L6 myoblast but not in the D23 myoblast. Studies using these
GLUT 4 fusion proteins may help to elucidate the fiinction of the GLUT 4 transporter in
regulating metabolic processes in myoblasts.
CHAPTER 2
INVOLVEMENT OF THE GLUT 3 TRANSPORTER IN MYOGENIC
REGULATION
2.1 INTRODUCTION
Myogenesis is a cornplex process characterized by morphological and biochemical
differentiation (Emerson et al., 1993; Olson et al., 1990; Buckingham et al., 1994). The
rat L6 skeletal myoblast line (YafFe, 1968) has been used to shidy the in vitro expression
of myogenic components (Chen et al., 1993). These myoblasts express the myf-5 gene,
but not the myoD gene. There are several advantages in studying the myogenic pathway
using this c d line. Defined growth conditions (fke of hormonal and physiological
changes nonnally present in animals) can be used to study the myogenic events. Growth
of cells in different concentrations of horse or fetal bovine serum can alter rates of
myogenesis (Chen et al. 1991). Myogenesis-defective mutants can be isolateci from L6
myoblasts. These mutants are usefùl in studying the myogenic components and their
temporal order of expression (Chen et al., 1991a; Kudo et ai., 1990). Myogenesis-
defective mutants transf-ed with appropriate cDNAs can aiso be used to determine the
role of specific components in myogenesis. For example, a mutant expressing low levels
of a ce11 surface 1 12 kDa phosphoprotein @112) was Unpaired in myogenesis (Chen et al.,
199 la; 1991 b). Transfection of the myogenzn cDNA into this mutant restored the
endogenous expression of myogenin, M C , MYC and TnT as well as their ability to fonn
myotubes (Chen et ai., 1993).
The rat L6 myoblast cell iine has dso been used to examine the glucose transport
process @'Amore et al., 1986; Xia et al., 1993; Kudo et al., 1990; Broydell, 1994).
Three GLUT isofonns, GLUT 1, 3 and 4, are found in rat L6 rnyoblasts (Xia et al., 1993).
The GLUT 1 transporter was inactive in glucose-grown myoblasts; however its transcript
level and transport activity were elevated in glucose-starved myoblasts (Lu et al., 1995).
The GLUT 3 isoform is the major GLUT isoform present in undifferentiated myoblasts
and the GLUT 4 isoform is the predominant isofom found in rnyotubes.
A close correlation has been observeci between GL UT 3 and 4 expression and the
celi's myogenic ability. GLUT 3 transcript levels were reduced, whereas GLUT 4
transcnpt levels were elevated during myogenesis (Xia et al., 1993; Lu et ai., 1995).
Inhibition of myogenesis by phloretin or 5-bromo-2'-deoxyundine (BrdUrd) was
accornpanied by a reduced decline of GLUT 3 expression (Chen et ai., 1989). More
interestingly, dl rat myoblast GLUT 3- mutants examined were impaireci in myogenesis
(Xia et ai., 1993; Kudo et ai., 1990). Clone D23, a GLUT 3- mutant, was essentially
devoid of myogenin, M C , MW, and TnT transcripts (Broydell et al., 1 997). Since these
mutants possessed normal levels of GLUT 1 and GLUT 4 isoforms, the myogenic defect
was not likely a consequence of reduced intracellular glucose concentration (Mesmer et
ai., 1995).
Over- and under-expression of GLUT 3 in L6 myoblasts revealed that a critical
GLUT 3 level was important for the expression of myogenin and muscle specific genes
(Broydell thesis, 1994; Broydell et al., 1997). In these studies, L6 myoblasts were
transfected with the GLUT 3 sense (L6/G3S) or antisense (L6/G3A) cDNA. Transfectant
L61G3A expressed oniy 39% of the L6 GLUT 3 aanscript level and were not altered in the
rate and extent of fusion. The amount of myogenin present in this transfectant was
wfficient to activate and to maintain temiinai differentiation (Broydell et. ai., 1997).
However, when the GLUT 3 transcsipt level was reduced to 16% (as in the case of mutant
D23), the cells were impaired in myogenic differentiation and in the expression of various
muscle specific-genes (Broydell et al., 1997). Transfectant L6/G3S expressed 3 fold
higher GLUT 3 transaipt level than L6 myoblast. Even though its initial myogenzn
transcript levels were higher than these of L6fG3A transfectants (Broydell et al., 1997),
this transfectant was irnpaired in the expression of muscle-sp&c genes. This
transfectant was thought to be deficient in factors, other than myogenin, required for
myogenic differentiation (Broydell et. al., 1997).
The aim of this study was to determine if GLUT 3 was the key component
involved in regulating the expression of myogenin and other muscle-specific (MHC, M C
and Tn7) genes. If this were the case, then GLUT 3- mutants should be impaired in
myogenesis, and their myogenic defects should be rescued by Limitecl expression of an
exogenous GLUT 3 cDNA. To test the hypothesis, GLUT 3- mutants @23, D2 and Dg)
were transf-ed with a GLUT 3 cDNA placed under the cuntrol of the MMTV-promoter.
These mutants were previously shown to be impaired in myogenesis (Kudo et al., 1990).
Stable transfectants were isolated and cloned. Studies with these stable transfectants
revealed that components, in addition to GLUT 3, are involved in regulating the
expression of myogenin and other rnusde-specific genes.
2.2 MATERIALS AND METHODS
2.2.1 Plasmids and Culture Media
The pMAMneo Mammdan Expression Vector (Clonetech Laboratones) contains
the Rous Sarcoma Virus-Long Tenninal Repeat ('SV-LTR) enhancer linked to the
dexamethasone-inducible Mouse Mammary Tumor Virus-Long Terminal Repeat (MMTV-
LTR) promoter. The latter d o w s dexamethasone-inducible high level expression of the
cloned cDNAs. This vector also contains SV40 splicing and polyadenylation sites, thus
allowing RNA processing in mammalian cells. The neomycin gene, dnven by the SV40
early promoter, enables selection of trmfectants by growth of cells in medium containing
geneticin (G4 1 8) (Gibco).
For transfection studies, the MUW-GLUT 3 construct was subcloned by Fariha
Abidi in our laboratory. The human GLUT 3 cDNA (Repository of Human DNA Probes
and Libraries, ATCC) contains the coding sequence fiom 1 1 5-2742 bp. The CMV-
GLUT3 wnstnict was digested using Hind III, and the GLUT 3 hgment was then
subcloned into the Bluescript (KS3 vector's Hind ïII site. The KSGLUT 3 constmct was
then cleaved with EcoR V and the GLUT 3 fiagrnent was subcloned into EcoR V of the
pG3EX (Bluescript (KS3 vector which contains a t 50 bp his-tag fragment (a gift fiom Dr.
S .P. Yee's laboratory at London Regional Cancer Center, London)). The KSGL CIT 3his
construct was then digested with Xho 1 and Srna 1 and subcloned into the Mie 1 and Xho 1
sites of the MAMneo vector's multipledoning site (MCS).
The human fetal skeletal muscle myl-5 and myf-4 cDNAs were purchased fiom the
Repository of Human and Mouse DNA probes and Libraries, ATCC. The myf-5 cDNA
does not wntain the first 185 nucleotides of the coding sequence (Braun et al., 1989a;
Braun et al., 1989b). The m y f l cDNA contains the wding sequence fkom nucleotide
170- 1420 (Braun et al., 1989). Both cDNAs encode for the bHLH homology motif. The
MLC, MHC and TnT cDNAs were gifts from B. Nadal Ginard (Endo et al., 1987). The
2-mzcrogIobuh (WG) cDNA was a generous gift fiom F. Daniel (F. Daniel et al., 1983).
W G is a housekeeping gene expressed at a constant level during myogenesis. To
quanti@ the amount of RNA loaded ont0 the gel, the W G transcript level in each sample
was determineci.
Plasrnid DNAs were punfied using the Qiagen DNA purification kit (Qiagen).
This kit uses the alkaline lysis method (Sambrook et al., 1989) and a column to enable
elution of the plasmid DNA. Restriction enzymes were purchased 60m Promega. h DNA
BstE Il digest (New England Labs), i kb ladder and 100 bp standard (Gibco hc) were
used to detennine the sizes of the digested DNA fiagments.
Reagents used in bacteriai cultures were purchased from BDH Inc, Toronto, On.
Bacterial cultures were grown in T e d c Broth (TB) medium containing the appropnate
antibiotics (Sambrook et ai., 1989). Cells were grown in SOC medium (SOB + 20mM
glucose) in transformation experiments (Sambrook et al., 1 989).
Labeled cDNA probes were prepared using a-32~ dCTP (Amersham) and the
Prime-a-Gene labeling kit (Promega). 30 ng of the desired DNA was denatured at 100°C
for 5 min and placed on ice for another 5 min. 1ûx labelling buffer containing primers,
dNTP, B S 4 d 2 p dCTP and DNA polymerase was then added to the denatured DNA,
and the mixture was incubated overnight at 21°C. A Sephadex G50 DNA grade Nick
Column (Phmacia Biotech) was pre-equilibrated with 3 mL of Tris-HCV EDTq pH 7.5
buffer (TE). The labelled DNA was purified by heating the mixture at 100°C for 5 min,
eluting from the Mck Column using 400 5 of the TrislEDTA buffer and then with
another 400 pL, to elute the labeled probe. This procedure enables purification of DNA
fiagments larger than 20 bp in length and also removes unincorporatecl radiolaùelled
NTPs.
AU other chemicals were purchased from BDH Inc (Toronto, ON) or Sigma Inc
(Mississauga, ON) and were of the highest available quality.
2.2.2 Ca Cultures
The parental cell Line was the rat L6 skeletd myoblast (YafTe, 1968). Clones D2,
D9 and D23 are mutants isolated fkom L6 myoblasts @'Amore et al., 1986); they contain
around M, 17% and 15% of the L6 GLUT 3 transporter, respectively. These clones
were kept under selection with 0.1 mM of 2-D-deoxy-giucose (dGlc), a glucose analogue.
Alpha Minimal Essential Medium (Gibco), supplemented with 25 m M glucose, 50 pg/mL
of gentamycin sulphate (Gibco) and 1% fetal calf s e m (FCS) (Hyclone Inc) was us& as
the growth medium. Cells were maintained at 3PC in a humidiied atmosphere of 5%
COz on 150 mm tissue culture dishes (Nunc) for regular ce11 culture and on 6-well culture
dishes (Falcon) for fusion nudies. Cells were detached by first washing with citrate saline
b a e r followed by incubation for 5 min at 37°C with 4% trypsin (Gibco) in citrate saline
buffer.
2.2.3 Transfmtion of the MMTV-GLUT3 Constmct into Myoblasts
The MMTV-GLUT3 construct was transfected into L6, D23, D2 and D9
myoblasts using the Cap04 precipitation method (Sambrook et al., 1989). As controls,
the pMAMneo vector was also transfected into L6 (L6/MMTV), D23 @23/MMTV), D2
@2/MMTV) and D9 @9/MMTv> myoblasts. Plasmid DNAs were isolated using the
Qiagen Midi Kit. Cells were plated at a density of 4x10' cells/lOOmm dish and grown for
6 hrs. A 10 mL pipette was used to bubble 500 pL of 2x HEPES Buffer Solution W S )
(270 rnM NaC1, 9 mM KCI, 1 rnM Na2HP04.2H20, 11 m . dextrose, and 42 mM N-2-
HydroxyethyIpiperaPne-N-2-ethane-sulphonic acid (HEPES), pH 7.04 ) as a fiesh mamire
of 0.25 M CaC12/10 pg DNA was added dropwise. The mixture was aiiowed to sit for 30
min at 21°C to d o w formation of the DNA precipitate. The DNA precipitate was added
dropwise to each plate. Cells were incubated overnight at 37°C in a humidifieci
atmosphere of 5% COz. After washing each plate twice with l x phosphate-buffered saline
(PBS) (1 3 7 mM NaCl 2.7 mM KCl, 8.1 m M Na2HP04 and 1.5 rnM Kl&PO4), ceUs were
incubated with fiesh growth medium for 24 hrs. Transfectants were then selected by
growth in growth medium containing 10 pg/mL of G418. Medium was changed every 4
days. Individual colonies were trypsinwd in a giass coliar and transfemed to 6-well dishes
where they were fùrther selected with G4 18. The stable transfectants obtained were
grown in growth medium containing lû?? horse sem (HS), instead of 1% FCS.
2.2.4 Southern Blot Analysis
Genomic DNA was isolated fiom myoblasts using a previously described
procedure (Chen et al., 1993). Cells were seeded at a density of l x 10' ceIldl 50 mm plate
and dowed to grow for 2.5 days. After washing with PBS, cells were incubated at 3PC
with a DNA lysis bufler (10 mM Tris-base (pH 7.9), 10 m M EDTA, 10 mM NaCl, 0.1%
SDS, proteinase K (200 pB/mL )) for 4 hrs. The resulting viscous liquid was transfemed
to a tube, which was then incubated at 55°C ovemight. An equal volume of
phenoVchloroform solution (24 parts saturated phenol (Gibco) with 0.1%
hydroxyquinoline antioxidant (Sigma): 2 5 parts chloroform (BDK) : 1 part isoarnyl alco ho1
(BDH)) was added to each tube and centrifùged. M e r removing the supernatant. two
volumes of absolute ethanol were added to extract the D N 4 which was spooled out of
the solution using a flame sealed Pasteur pipette. The DNA, which remaineci at the end of
the Pasteur pipette, was then washed first in 70% ethanol and then in lW! ethanol. The
DNA was air-dried briefly and then dissolved ovemight in 400 pL of TE buffer (pH 8 .O).
The DNA was quantifieci by determining its optical density at a wavelength of 260 m.
To determine if the exogenous DNA was Uiwrpomted into the genome, 10 pg of
genomic DNA was digesteci with EwR V and Huid III, and separated on a 0.8% agarose
gel. The gel was transferred to ICN Biotrans positive nylon membrane using a VacuGene
XL vacuum blotter (Pharmacia Biotech). Mer being air dried for 30 m h the DNA was
crosslùiked to the nylon membrane by irradiating for 12 sec at 1200 pjoules in a
Stratagene Crosslinker. The blot was pre-hybridized for 2 hrs and then hybridized with a
32~-labelled GLUT 3 cDNA probe overnight at 4Z°C. The blot was washed with high salt
(4x SSC, 0.1% SDS) at 21°C for 1 hr, changing the wash solution every 30 min. It was
then washed for 2 hrs in a low salt (O. lx SSC, O. 1% SDS) wash buffer at 6S°C, changing
the solution every hour (Sarnbrook et al., 1989). The resulting blot was exposed to a
phosphoimaging screen and to a Kodak X-OMAT AR film kept at -80°C.
2.2.5 Fusion Index Measurement
The ability of transfectants to form multinucleated myo tubes was examined. Cells
were seeded at a density of 5 x 105 cells/well in Falcon 6-welI plates. At the appropriate
t he , celis were washed twice with wld PBS, and then treated with 1 mM ZnSOs for 45
sec to swell the nuclei. They were then fixed with 2.5 % glutaldehyde for 3 min, and
followed by a 500h ethanol wash for 2 min. Cells were air-dried briefly before being
washed twice with ice cold PBS. After staining with 6% Giemsa overnight, cells were
washed with de-ionized water severai times to remove excess stain. Six fields per well
were chosen to determine the fusion index. In a field of at least 150 nuclei, a myotube was
scored oniy if it contained three or more nuclei; otherwise it was scored as a myoblast.
The fiision index of each field was calculateci as a ratio of the number of nuclei in
myotubes to the number of nuclei in each field.
2.2.6 Northern Blot Andysis
Poly (A)' RNAs were extracted Eom myoblasts using the Invitrogen Fast TrackW
kit. The kit's protocol was slightly modified. Twelve 120 mm Nunc tissue culture plates
seeded at a density of 1 x 106 celldplate were used for harvesting on &y 2, 4 and 6. Cens
were removed fiom dishes using PBS and a rubber policeman, and pelleteci by centrifiiging
at 2000 x g for 5 min. Cells were suspended in 10 rnL of Stock buffer and 200 pL of
Protein Degrader (provideci by the Fast Track kit), and homogenized with a Dounce
homogenizer (20 strokes) and passed 3 times through a 21 gauge needle. The lysate was
rocked for 1 hr at 45°C. This lysate was then incubateci with 630 pL of 0.5 M NaCl and
5 pg oligo dT cellulose (half a tablet) for 1 hr at 2 1°C. Mer washing three times with the
binding buffer and t h e times with the low sait wash buffer, the oligo dT cel1ulose was
resuspended in 0.63 mL of low salt wash buffer. The suspension was transferred to a spin
column and washed 4 times in the low salt wash buffer. Poly (A)+ RNA was eluted from
the spin column using the elution buf5er (supplied by the kit). The RNA was precipitated
with 100% ethanol and 2 M NaAc for two days at -80°C. One pg of each RNA sample
was run on an 1% formaidehyde gel (Sambrook et al., 1989) in MOPS Running Buffer (10
mM EDTq 0.2 M MOPS, 10 mM NaAc, pH 7.0). After vacuum (Pharmacia) transfer to
an ICN neutrai nylon membrane, the blot was first probed with the W G cDNA to
determine sample loading. The blots were then probed with rnfi5* myf-4* myogenln,
ICMC, MLC, TnT or GL LIT3 cDNAs. The blot was stripped each tirne before hybndizing
with a new probe. The 10x stripping biiffer (50 rnM Tns pH 8.0, 2 mM EDTA 0.5% Na
pyrophosphate, 0.02% bovine serum aibumin (BSA), 0.02% polyvinylpyrrolidone (PVP)
and 0.02% Ficoll) was heated to 65°C and used at a lx concentration to strïp off the old
probe by incubating with the blot for 2 hrs at 65°C. The buffer was changed 2-3 times
(Thomas, 1980). The blot was wrapped in Saran Wrap and exposed tu a phosphoimaging
screen or a Kodak X-ray film. Band intensities were determined in the linear range of the
optical density (Appendix # 1). In caidating the relative t r h p t levels, the tninscript
levels of day 2 L6 cultures were used as lW!. A sarnple calculation is shown in
Appendk #1. Two diffierent poly(A)' RNA preparations were used for each ce11 line.
Sarnples from each preparation were probed at least twice to ascertain the consistency of
Our findings.
2.2.7 Immunofluorescence Studies
Cells were seeded at a density of 5x10~ cells/well in Falcon 6-well plates
containing stenle coverslips precoated with O. 1% gelatin. Cultures were washed twice in
Stockholm PBS (sPBS) (4.3 mM Na2HP04, 1.9 mM Na&P04.2H20, 136.9 mM NaCI,
and 2.7 mM KCl) before fixing.
The monocional anti-myogenin antibody (FSD) was used to label myogenin. This
antibody was a generous gi f t from W. E. Wright (Southwestern Medical Center,
University of Texas. DA.). In this shidy, cells were treated with Lana's Fixative (4%
depolymerized paraformaldehyde, 14% satwated picric acid, 0.5 M sodium phosphate
buffer, pH 7.1) for 30 min, washed several times with sPBS and then incubated with 0.2%
Triton X-100 in sPBS for 10 min. M e r blocking non-specific sites with lP? FCS in
sPBS for 30 min, cells were incubated with the primary antibody, FSD (50 pL of non-
diiuted serum with 1û% FCS) ovemight at 4OC. This was foliowed by washing ceils three
tirnes with sPBS, and inaibathg with the secondary antibody, rabbit anti-mouse CY3
(diluted 150) (Jackson Laboratones: East Acres Biologicals, Southbridge, MA.), in sPBS
for 1 hr. M e r washing t h e times with sPBS, coverslips were mounted on slides with
50 pL of mounthg medium (50% glycerol, p-phenylendiamine (PPD) and sPBS),
containing the Hoechst DNA stain.
The location of myosin heavy chah was determineci by immunofluorescence
studies using a monoclonal anti-myosin heavy chah antibody (MF-20), originally obtained
firom the Developmentai Studies Hybridoma Bank and grown in culture in Dr. 1. S.
Skejanc's laboratory (University of Western Ontario, London, ON.). Cells were h e d in
-20°C methanol for 5 min, air-dried and blocked with FCS/sPBS for 30 min. Cells
were incubated with 50 pL, of the first antibody, MF-20 in sPBS /lm FCS for 1 hr at
21°C. M e r washing three times with sPBS, cells were then incubated with 50 pL of the
secondary antibody, CY3 (diluted 1 :50), in sPBS. M e r washing three times in sPBS,
coverslips were mounted on slides, with mounting medium wntaining Hoechst DNA stain.
Slides were examined under oil emersion using a Zeiss Axiophot Immunofluorescence
microscope (Carl Ziess, Oberkochen, Germany). Pictures were captureci using Northem
Exposure Software (IrnagExperts Inc., Toronto, ON.), opened in Adobe Photoshop
(Adobe Systems, Inc., San Jose, CA) cropped and resized before importing into Corel
Draw (Corel Corporation, Ottawa, ON) for final placement and printing.
2.3 RESULTS
2.3.1 GLUT 3 Trnnscript Leveis in GLUT 3- Mutants
Three independent GLUT 3- mutants fiom L6 myoblasts were used to examine the
role of GLüT 3 in myogenesis. Mutant D2 was selected fiom ethyl methane suifhate-
mutagenized L6 myoblast (Kudo et al., 1990). D9 and D23 cells were two independent
spontaneous mutants afso isolateci from L6 myoblast (Kudo et al., 1990). Poly (A)' RNAs
were prepared from day 2 and 6 cultures of L6, D23, D2 and D9 myoblasts and used to
determine GLUT 3 tranmipt levels.
Northem blot studies revealed that GLUT 3 tnuiscnpt levels in day 2 cultures of
D2. Dg, D23 were 14%?4.8, 25%+5.3, and 15%+ 1.4 of that in L6 myoblasts (Fig. 2.1).
Day 6 cultures of L6, D2, D9 and D23 exhibit 3 1%+0.87, 100?+3.07, 7%+2.21 and
1 % 2 0.86 of the day 2 L6 level (Fig. 2.1). This study showed that D23, D2, and D9 ceUs
harboured reduced levels of the GLUT 3 transcript; thus indicating al1 three ce11 lines were
defective in GLUT 3.
2.3.2 Ability of the GLUT 3- Mutants to fonn Multinucle~ted Myotubes
Previous studies indicated that GLUT 3- mutants were impaired in myogenesis
(Kudo et al., 1990). In this study, the rates of fusion of these mutants were determineci
over a 10 day period (Fig. 2.2). L6 cells had fùsion indices of 81%+0.54 by day 6 and
100% by day 10. D23 cells were unable to fom myotubes. D2 cells had a slightly lower
rate of fusion; day 6 and day 10 cultures had a fusion index of 44%2 1.2 and 79%+0.32,
respectively. The fusion index of D9 cells was l l%e 0.1 7 on day 6 and increased only
slightly to 16%+0.43 on day 10 (Fig. 2.2). These studies showed that the reduction in
GLUT 3 expression was accompanied by the inability of these mutants to forrn myotubes.
It is also important to point out that the rates of fusion did not correspond directiy with
the amount of GLUT 3 transporter present. Thus other myogenic factors might also be
aitered in these GLUT 3-mutants.
Figure 2.1 GLUT 3 transcript levels in GLUT3- Mutants
Poly A' RNA was isolated from day 2 and day 6 C U ~ ~ S of L6 and GLUT 3-
mutants 0 2 3 , D2 and Dg). Northem blot studies were carried out to determine the
GLUT 3 and PZ-rnicrogloblh W G ) transcript levels in these ceIl types. The intensity of
the bands from the phosphoimage was measured using Image Quant Software (Molecular
Dynamcs, Inc., Sumyvale, CA). G L U 3 transcript levels were normalized according to
the level of W G mRNA present in each sample. The G L W 3 transcript levels of day 2
L6 cultures were taken as 100%. The dotted bar and slashed bar denote samples fiom day
2 and day 6 cultures, respectively. The standard deviations were calculated for three
different samples (n=3 ) .
CELL LINES
Figure 2.2 Myogenic Ability of GLUT3- Mutants
Fusion indices were determined for the L6 controls and the GLUT 3- mutants. O,
e, A, and V denote the rates of fusion by L6, D2, D9 and D23 myoblasts, respectively.
The standard error was calculated for each cell line for each day (n=18). Since the values
were less than 2%, they were not apparent on the graph.
DAYS
2.3.3 Expression of MRFs and MuscIcSpecific Genes in GLUT 3- Mutants
As mentioned in Chapter 1, morphological differentiation is preceded by
biochemical ditferentiation. Biochemical merentiation is initiated by myogenin which in
turn activates muscle-specific contractile protein genes such as MHC, MLC and TnT. To
determine the site of alteration in various GLUT 3- mutants, the expression of various
myogenesis-associated genes was examined. Studies were carried out using poly (A)'
RNAs isolateci fiom day 2 and day 6 cultures of L6 and GLUT 3- mutants @23, D2 and
D9) under conditions that promoted myotube formation. These rnRNAs were probed
with m m , myogenin, M C and TOT cDNAs and were compared with the corresponding
L6 day 2 levels (10%) (Fig. 2.3). These studies showed that the L6 myf5i transcript
level decreased from 1 O P ! to 3 1% 2 0.6 on day 6. The initial myf-S transcript levels in
GLUT 3- mutants 0 2 (96%24.8), D9 (lO7%f 7.0) and D23 (84%*4.9)) were similar
to that of day 2 L6 cultures (Fig. 2.3). In day 6 cultures, the myjf-5 transcript levels were
also reduced in these cells (86%+4.1, 68%+2.9 and 5 1%11.4, respectively) (Fig. 2.3).
Thus, there was a slight reduction of myj-5 expression in various GLUT 3- mutants.
Studies with L6 ceils reveaied that their day 6 myogenin (mg- transcript level
was about 2 fold (200% + 9.1 7) higher than that of day 2 (Fig. 2.3). It was important to
note that mutant D23 possessed ody 3%+ 1.5 of the L6 cell's myogenin level on day 2
and there was no subsequent increase in day 6 cultures. Myogenin expression was
reduced in D2 (77%+6.2) and D9 (12%+5.0) myoblasts. Even though their myogenin
levels were increased by day 6 (1 28% + 22.5 and 16 1 % 248.1, respectively). These levels
never reached the levels seen in day 6 L6 celis (Fig. 2.3).
Whiie both MLC and TnT expression in L6 c d s were elevated 2.5 fold fiom day 2
Figure 2.3 Myf-5. Myogenin, MLC, and TnT Transcript Levels in L6 and GLUT3-
Mutants @23, D2, Dg)
Northem blot analysis and quantification of the mRNA levels were perfomed as
outlined in Figure 2.1. The levels of myf-5, myogenin, M C and TnT *As were
determined by probing with their respective cDNAs. The L6 day 2 level was taken as
10Ph for each probe. Panels A, B, C and D indicate m ~ 5 , myogenin, M C and TnT
transcript levels, respectively. The doned bar and the slashed bar denote samples from
day 2 and day 6 cultures, respectively. The standard deviations were cdculated for three
different samples (n=3).
MLC mRNA LEVELS (O / )
TnT mRNA LEVEL (%)
MYF-5 mRNA LEVEL ("w
MYOGENIN mRNA LEVEL (%)
to day 6, theû expression was hardly detectable in D23 myoblasts (Fig. 2.3). D2 cells
expressed initidly low levels of MLC and TnT transcript levels but increases were
obswved nom day 2 to day 6. The level of increase never reached that seen in day 6 L6
cells (Fig. 2.3).
These studies indicate the absence of biochemical differentiation in D23 myoblasts.
Whiie D2 and D9 cells could undergo biochemical differentiation, they preceded at slower
rates than that seen in the parental L6 culture. Even though these mutants were sirnilady
reduced in their GLUT 3 transcript levels (Fig. 2. l), their ability to express myogenesis-
associated genes and to form multinucleated myotubes differ significantly. This suggests
that components, in addition to GLUT 3, must be involved in myogenic regulation. It is
possible that changes in some regdatory components may lead to reduced expression of
GLUT 3 and some myogenesis-associated genes.
2.3.4 Transfection of GLUT 3- Mutants with a MMW-GLUT3 cDNA
Previous studies revealed that a critical level of the GLUT 3 transponer was
required for myogenic differentiation (Broydell et. al., 1997). Myogenic ability was
abolished upon over- or under expression of the GLUT 3 transporter. This suggests that
the myogenic ability of GLUT 3- mutants rnay be rescued by lirnited expression, but not
the over-expression, of an exogenous GLLIT 3 cDNA. In this audy, D23, D2 and D9
mutants were transfected with an inducible MlMTY-GLUT 3 constnrct @23/GLUT 3,
DUGLUT 3 and D9/GLUT 3 myoblasts) or with the pMAMneo expression vector
@23/MMTV, DUMMTV and D9MMTV myoblaas). The MMTV promoter activity
can be turned onfoff by addition/removal of dexamethasone (dex) (10'~ M) to myoblast
cultures (Arnold et al., 1994). This very low dex concentration should not affêct
endogenous metabolic processes, as it alters myoblast metabolism oniy when used at a
concentration above 12 pM (Clarke et al., 1993).
To determine if the GLUT 3 cDNA was incorporated into the genomic DNA of
various transfectants, genomic DNA sarnples were digested with Hind III and EcoX V,
and then probed with 32~-labeled GL UT 3 cDNA. Southem blot analysis revealed the
presence of a band similar in size (1.5 kb) to the GLUT 3 cDNA in different clones of
D23/GLUT 3, D2/GLUT 3 and D9/GLUT 3 transfectants, but not in D23, D2, Dg,
D23/MMTV, D2/MMTV or D9/MMTV myoblasts (Fig. 2.4). These findings suggested
that the exogenous GLUT 3 was incorporated into the genomic DNA of the GLUT 3-
transfectants.
2.3.5 Der-induced expression of exogenous GLUT 3
Several transfectants harbounng either MMTV-GLUT 3 cDNA or the vector
pMAMneo were chosen to examine the dex effect. Northern blot analysis revealed that
GLUT3 expression was induced only upon incubation of D23/GLUT 3 clones 2-4 and 2-6
(Fig. 2.5) and D2IGLUT 3 clones 1 4 and 1-6 (data not shown) myoblasts with IO-' M
dex. This suggested that GLUT 3 expression was restored in these transfectants. It was
important to note that the levels of induced GLUT 3 expression were at least 3-5 times
higher than that of L6 cells. These transfectants will therefore be usehl in studying the
role of GLWT 3 in myogenic differentiation.
2.3.6 Effects of Des-induced GLUTJ Expression on the Morphology of D23 and D2
Transfectan ts
Since GLUT 3 expression in D23lGLUT 3 and D2/GLUT 3 transfectants was
induced by dex, its effect on myotube formation could be monitored. Both dex-induced
Figure 2.4 Southern Blot Analysis of GLUT 3- Myoblsst Trnnsfected with the
GLUT3 cDNA
A. Genomic DNAs were isolated fiom the D23/GLUT 3 transfmants digested
with EcoR V and Hind III and then probed with the GLLIT 3 cDNA A 1.5 kb band was
observeci in transfectants D23/GLUT3 clones 2-1,2-2, 24,2-6,2-7,2-8, 2-9, 2-1 1 and 2-
12 (lanes 3, 4, 6, 8, 9, 10, 1 1, 13 and 14, respectively). No band was detected in the
controls:D23 (lane 1) rnyoblast and D23/MMTV transfectant (lane 2), nor in
D23/GLUT 3 clones 2-3, 2-5 and 2- 10 transfectants (lanes 5, 7 and 12).
B. Ten pg of genomic DNA was isolated from the DZGLUT 3 transfectants,
digested with EwR V and Hind HI and then probed with the GLLIT 3 cDNA- The
GLVT3 insert was detected as a 1.5 kb band (lane 1). A similar size band was observed
in samples frorn DUGLUT 3 clones 1-1, 1-3, 1-5 and 1-6 (lanes 2, 4, 6 and 7,
respectively). No bands were observed in the transfectant DZIGLUT 3 clone 1-2 (lane 5)
and D2/MMTV (lane 8) and control D9 (lane 9).
C. Genornic DNAs were isolated nom D9/GLUT 3 transfectants digested with
EcoR V and Kind III and then probed with the GLUT 3 cDNA. No bands were observed
in the contro1s:-D9 (lane 1) and transfectant D9/MMTV (lane 2) nor in D9/GLUT 3 clone
5 (Iane 7). A 1.5 kb band was observed when genomic DNAs nom transfectants
D9/GLUT 3 clones 1, 2, 3, 4 and 6 (lanes 3-6 and 8, respectively) were probed with the
GL UT 3 cDNA. The GL UT 3 cDNA insert was detected as a 1.5 kb band in lane 9.
Figure 2.5 Northern Blot Analysis of Dex-Ind uced Myo blasts.
Northern blot analysis was carried out as described in the text (Section 2.2.6).
Poly (A)' RNAs were extracted ftom day 2 cultures of L6, D23, D23/MMTV( 1 - 1) and
D23IGLUT3 transfectants clones 2-2, 2-4 and 2-6. Cultures were treated with or without
1 x IO-' M dex. Two pg of RNA was loaded into each lane. GLUT 3, myogenzn, and
W G cDNAs (Panels 4 B and C, respectively) were used as probes in these studies. The
endogenous GLUT 3 transcript level (4.1 kb) was evident in L6 myoblasts, but not in
other ce11 lines (Panel A). A transcript correspondmg to the size of the exogenous
GLUT 3 cDNA (1.5 kb) was detected in dex-induced transfectants 2-4, 2-6, but not in
transfectant 2-2, nor in uninduced cells. The other bands observed in these induced
transfectants were probably transcripts fiom genes that have integrated the exogenous
GLUT3 cDNA (Panel A). The endogenous myogenin transcript level(1.9 kb) was evident
in L6 myoblasts, but not in other ce11 lines (Panel B). RNA Ioading was indicated by
probing the blot with M G cDNA (Panel C).
GLUT 3
Myogenin (I
and uninduced D23 transfectants were fixed and stained with Giemsa stain (Section 2.2.5).
Myotube formation was not detected in uninduced D23lGLUT 3 transfectants (Figs. 2.6
and 2.7). The incubation of D23 and D23/MMTV myoblasts with dex did not alter the
cells' myogenic ability. However, alignrnent of myoblast and formation of multinucleated
myotubes were observed in the dex-induced D23lGLUT 3 cultures (Fig. 2.7). Other
clones of D23/GLUT 3 transfectants were also capable of fonning myotubes upon
induction with dex (data not shown). The fusion index was only 57% of that expected
for L6 myoblast. Induction of DUGLUT 3 transfectants with dex did not increase the
extent of myotube formation (data not shown).
Attempts were also made to determine the optimal and suboptimal conditions for
the induction of G L W 3 in both the D23 and D2 transfectants. The effects of cell density,
induction penod with dex, serum concentrations and dex concentrations on the extent of
fusion were determined. Despite numerous attempts we could not find a condition that
would further increase the myogenic ability of D23/GLUT 3 clones (2-4 and 2-6)
transfectants or the DZIGLUT 3 clones (1-4 and 16) transfectants (data not shown).
It is interesting to note that GLUT 3 transcript levels in the induced clones (2-4)
and (2-6) of D23lGLUT 3 myoblasts were at lest 4 fold higher than that of L6 myoblasts
(Fig. 2.5). This substantially higher GL LIT 3 expression in D23/GLUT 3 myoblasts was
similar to that of L6/G3S transfectants (Broydell et al., 1997), which exhibited only 5-7%
of the L6 fusion index. Attempts to screen for transf-ants expressing very low levels of
the GLUT 3 transcript upon induction with dex had not been successful. The observation
that the myogenic defect in D23 myoblasts can be partially rescued by G L W 3 expression
suggests that GLUT 3 may be involved in regulating the myogenic program.
Figure 2.6 Morphology of Day 2 Dex-Induced and Uninduced Cultures of D23
Transfectan ts.
Cells were seeded at a density of 5 x10' /well. Cultures D23 (Panel A),
D23/MMTV (1-1) (Panel C) and D23/GLUT 3 clones (2-4 and 2-6) (Panels E and G,
respectively) were uninduced controls. The foliowing cultures were induced with
1x1 O-? M dex for 24 hrs: D23 (Panel B), D23lMMTV (1- 1) (Panel D) and D23IGLUT 3
clones (2-4 and 2-6) (Panels F and FI, respectively). Day 2 celis were treated with 1 m .
ZnSO1, fixed in 2.5% glutaidehyde, washed with Sû?? ethanol and stained with 6%
Giemsa. Both uninduced (Panels 4 B, C, E and G) and induced (Panels D, F, and H)
cells appeared as rnononucleat ed myoblasts. The mapification of cells was 40x.
Figure 2.7 Morphology of Diy 6 Dex-Induced and Uninduced Cultures of D23
Transfectants.
D23/MMTV 1-1 and D23/GLUT 3 clones (24 and 2-6) are stable transfectants
harbouring only the pMAMneo vector, and the MMTV-GLUT 3 construct, respectively.
GLZIT 3 expression was induced on day 1 by incubating with lu7 M dex for 24 hn.
Cultures were then fixeci and stained on day 6 as mentioned in Fig. 2.6. Panel A &6),
Panel B @23), Panel C @23/MMTV 1-1) and Panels E and G @23/GLUT 3 clones 2-4
and 2-6, respectively) denote uninduced cells. Panel D @ 2 3 W 1-1) and Panels F
and H @23/GLUT 3 clones (2-4 and 2-6). respectively) denote dex-induced cells.
Alignment of myoblasts and formation of multinucleated myotubes can be seen in dl dex-
induced D23/GLUT 3 transfmants (Panels F and H). The maBnification of cells was 40x.
2.3.7 Biochemid Differentiation of Der-induced D23 Transfectan ts
To W h e r examine the myogenic ability of the induced D23/GLUT 3 transfectants,
the expression of myogenesis-associated genes was examineci. Poly (A3 RNAs were
prepared corn day 2 and day 6 cultures of dex-induced and uninduced transfectants. They
were then probed with 32~-labe~ed myogenin, M C , MHC or TnT cDNAs. These midies
indicated that the induction of G L U 3 did not cause an increase in the expression of any
one of the myogenesis-associated genes (Fig. 2.5 and data not shown).
Immunofluorescence studies were also carried out to detect the presence of
myogenesis-associated proteins. Neither myogenin nor MHC could be detected in day 2
L6, D23, D23hfMTV and D23lGLUT 3 (Figs. 2.8 to 2.11). While myogenin nuclear
staining was observed in day 6 cultures of L6 cells (Figs. 2.12 and 2.13), it wuld not be
detected in dex-induced or uninduced day 6 cultures of D23 or the D23 transfectants.
Similarly, while MHC staining was apparent in day 6 cultures of L6 cells, it could not be
otxerved in day 6 dex-induced or uninduced D23 and D23 transfaants (Figs. 2.14 and
2.15). Thus similar to Northem biot studies (Fig. 2.5 and data not shown), the induction
of GLUT 3 did not activate the expression of myogenin and MHC. It was interesting to
note that a similar reduction of biochemical and morphological dserentiation was al=
observed in L6 transfectants overexpressing the GLUT3 transporier (Broydell et al.,
1997).
2.4 DISCUSSION
Our laboratory has used a number of biochemical, genetic and molecular biological
approaches to examine the properties of the rat L6 myoblast glucose transport system.
Figure 2.8 Irnmunofluorescence Stnining of Myogenin in Day 2 Uninduced
Cultures
D23/GLUT 3 transfectants were st ained wit h an anti-myogenin monoclonal
antibody (F5D) (Section 2.2.7). Day 2 cultures of L6 (Panels 4 B and C), D23 (Panels
D, E and F), D23/MMTV (Panels G, H, and 1) and D23/GLUT 3 clones (2-4 and 2-6)
(Panels J, K, L, and M., N, and 0, respectively) were observed under phase contrast
microscope (Panels 4 D, G, J, and M), stained with Hoechst DNA sain (Panels B. E, H,
K and N) and labelled with anti-myogenin antibody (Panels C, F, 1, L, and 0). Under
phase contrast microscope cells were observed as non-confluent mononucleated myoblasts
(Panels 4 D, G, J, and M). Hoechst nuclear staining of the DNA was observed in al1
cultures (Panels B, E, H, y and N). No nuclear myogenin staining was observed in any of
the cultures (Panels C, F, 1, L, and 0).
Figures 2.9 Immunofluorescence Staining For Myogenin in Day 2 Dex-Induced
Cuitures.
Dex-induced stable transfectants harboring the GLUT 3 cDNA were stained on
day 2 with an anti-myogenin antibody (F5D) (Seciion 2.2.7). Cultures of L6 (Panels A, B
and C), D23 (Panels D, E and F), D23/MMTV induced with dex (Panels G, H, and I), and
D23lGLUT 3 clones (2-4 and 2-6) induced with IO-' M dex, on day 1 for 24 hrs. (Panels
J, K, L, and M, N, and 0, respectively) were observed under phase contrast microscope
(Panels A., D, G, J. and M), for the presence of Hoechst DNA stain (Panels B, E, H, K and
N) and for nuclear myogenin staining (Panels C, F, 1, L, and 0). Under phase contrast
microscope, the cells were observed as non-confluent mononucleated myoblasts (Panels
4 D, G, J, and M). Hoechst nuclear staining of the DNA was observed in dl of the
cultures (Panels B, E, H, K, and N). No nuclear rnyogenin stain was observed in any of
the cultures (Panels C, F, 1, L, and 0).
Figure 2.10 Immunofluorescence Staining For MHC in Day 2 Der-Uninduced
Cultures
Stable transfectants harboring the GLUT 3 cDNA construa were stained with an
anti-MHC antibody (MF-20) (Section 2.2.7). Day 2 cultures of L6 (Panels 4 B and C),
D23 (Panels D, E and F), D23/MMTV clone (1 - 1) (Panels G, H, and 1), and D23/MMTV-
GLUT 3 clones (2-4 and 2-6) (Panels I, K, L, and M, N, and 0, respectively) were
observed under phase contrast microscope (Panels 4 D, G, J, and M), for the presence of
Hoechst DNA stain (Panels B, E, H, K and N) and for cytoplasmic MHC staining (Panels
C, F, 1, L, and 0). Under phase contrast microscope cells were observed as non-contluent
mononucleated myoblasts (Panels A, D, G, J, and M). Hoechst nuclear aaining of the
DNA was observed in al1 of the cultures (Panels B, E, H, K, and N). No cytoplasmic
MHC staining was observed in any of the cultures (Panels C, F, 1, L, and 0).
Figure 2.11 Immunofluorescence Staining For MHC in Day 2 Dex-Induced
Cultures
Stable transfectants harboring the G L W 3 cDNA were stained on day 2 with an
anti-MHC antibody (MF-20) (Section 2.2.7). Cultures of L6 (Panels 4 B and C), D23
(Panels D, E and F), D23fMMTV clone (1-1) induced with lu7 M dex on day 1 for 24
hours (Panels G, FI, and 1), and D23lGLUT 3 clones (2-4 and 2-6) induced with dex
(Panels J, K, L, and M, N, and 0, respectively) were observed under phase contrast
microscope (Panels 4 D, G, J, and M), for the presence of Hoechst DNA stain (Panel B,
E, H, K and N) and for cytoplasmic MHC staining (Panels C, F, 1, L, and 0). Under
phase contrast microscope, cells were observed as non-confluent mononucleated
myoblasts (Panels 4 Dy Gy J, and M). Hoechst nuclear staining of the DNA was observed
in al1 of the cultures (Panels B, E, Y K, and N). No cytoplasmic MHC staining was
observed in any of the cultures (Panels C, F, 1, L, and 0).
Figure 2.12 Immunofluorescence Staining of Myogenin in Day 6 Uninduced
Cultures.
Stable transfectants of mutant D23 harboring the G L W 3 cDNA conaruct were
stained with an anti-myogenin antibody (FSD) (Section 2.2.7). Day 6 cultures of L6
(Panels A, B and C), D23 (Panels D, E and F), D23maîTV (Panels G, Y and I) and
D23IGLUT 3 clones (2-4 and 2-6) (Panels I, K, L, and M, N, and 0, respectively) were
observed under phase contrast microscope (Panels 4 D, G, J, and M), for Hoechst DNA
stain (Panels B, E, FI, K and N) and for nuclear myogenin staining (Panels C, F, 1, L, and
O). Under phase contrast microscope, L6 cells were present as multi-nucleated myotubes
(Panel A), whereas D23 celIs and the D23 transfectants were observed as confluent
mononucleated myoblasts, (Panels D, G, J, and M). Hoechst nuclear staining of the DNA
was observed in al1 of the cultures (Panels B, E, H, K., and N). Myogenin staining was
observed in the L6 culture (Panel C), but not in other cultures (Panels F, 1, L, and 0).
Figures 2.13 Immunofluoreseence Staining For Myogenin in Day 6 Der-Induced
Cultures
Stable transfectants harboring the GLUT 3 cDNA were stained on day 6 with an
anti-myogenin antibody (F5D) (Section 2.2.7). Cultures of L6 (Panels 4 B and C), D23
(Panels D, E and F), D23/MMTV induced with lU7 M dex, on day 1 for 24 hours (Panels
G, H, and 1), and dex-induced D23lGLUT 3 clones (2-4 and 2-6) (Panels J, K, L, and M,
N, and 0, respectively) were observed under phase contrast microscopie (Panels A, D, G,
J, and M), for the presence of Hoechst DNA aain (Panel B, E, H, K and N) and for
nuclear myogenin staining (Panels C, F, 1, L, and 0). Under phase contrast microscope,
L6 cells appeared as multi-nucleated myotubes (Panel A). D23 and D23IMMTV cultures
were observed as confluent mononucleated myoblasts (Panels D and G). D23/GLUT 3
transfectants were observed as possible myotubes (Panels D, G, J, and M). Hoechst
nuclear staining of the DNA was observed in al1 cultures (Panels B, E, H, K., and N).
Myogenin staining was only observed in the nuclei of L6 myotubes (Panel C), but not in
other cultures (Panels F, 1, L, and 0).
Figure 2.14 Immunofluorescence Staining For MHC in Day 6 Uninduced Cultures.
Stable transfectants harboring the GLUT 3 cDNA were stained on day 6 with an
anti-MHC antibody (MF-20) (Section 2.2.7). Cultures of L6 (Panels 4 B and C), D23
(Panels D, E and F), D23/MMTV clone (1-1) (Panels G, H, and I), and D 2 3 M V -
GLUT 3 clones (2-4 and 2-6) (Panels J, K., L, and M., N, and 0, respectively) were
observed under phase contrast microscope (Panels A., D, G, J, and M), for the presence of
Hoechst DNA sain (Panels B, E, Y K and N) and for cytoplasmic MHC staining (Panels
C, F, 1, L, and 0). Under phase contrast microscope, the L6 culture appeared as
multinucleated myotubes (Panel A), D23 cells and D23 transfectants were observed as
confluent mono-nucleated myoblasts (Panels D, G, J, and M). Hoechst nuclear staining of
the DNA was observed in cultures (Panels B, E, H, K, and N). Cytoplasmic MHC
staining was observed in L6 cells ( Panel C), but not in other cultures panels F, 1, L, and
0) -
Figure 2.15 Immunofluorescence Staining For MHC in Day 6 Des-Induced
Cultures
Stable transfectants harboring the GLCIT 3 cDNA were stained on day 6 with an
anti-MHC antibody (MF-20) (Section 2.2.7). Cultures of L6 (Panels A, B and C), D23
(Panels D, E and F), D23/MMTV clone (1-1) induced with 1 0 - ' ~ de- on day 1 for 24
hrs,(Panels G, FI, and 1), and dex-induced D23/GLUT 3 clones (2-4 and 2-6) (Panels J, K,
L, and M, N, and 0, respectively) were observed under phase contrast microscope (Panels
4 D, G, J, and M), for the presence of Hoechst DNA stain (Panels B, E, H, K and N) and
for cytoplasmic MHC staining (Panels C, F, 1, L, and 0). Under phase contrast
microscope, L6 cells were observed as multinucleated myotubes (Panel A). D23 and
D23/MMTV cultures were observed as confluent mononucleated myoblasts (Panels D and
G). Transfectants D23/GLUT 3 clones (2-4 and 2-6) were observed as possible myotubes
(Panels J, and M). Hoechst nuclear staining of the DNA was observed in al1 of the
cultures (Panels B, E, H, K, and N). Cytoplasmic MHC staining was only observed in L6
cultures (Panel C), but not in other cultures (Panels F, 1, L, and 0).
Two rat L6 rnyoblast hexose transport systems, a high affinity (HAHT) and a low (LAHT)
transport system, have been characterized and they are associated with the GLUT 3 and
GLUT 4 genes, respectively (Xia et al., 1993). Several correlations exia between GLUT
expression and myogenic ability. In L6 myoblasts, the GLUT 3 transcript level was
reduced at an uniform rate during growth, whereas the GLUT 1 transcript level was
increased during myogenesis (Xia et al., 1993). Overexpression of GLUT 3 in L6
myoblast was found to have a detrimentai effect on biochemical and morphological
differentiation (Broydell, 1994). Most importantly, several independently isolated
GLUT 3- mutants were impaired in myogenesis.
This chapter examined whether the G L U 3 isoform was the only key component
essential for regulating myogenesis. The properties of several independently isolated
GLUT 3- mutants were examined.
Studies using several GLUT 3- mutants revealed that even though these GLUT 3-
mutants may have sirnilar GL UT 3 transcript levels (Fig . 2.1 ), their myogenic ability varied
considerably (Figs. 2.2 and 2.3). Mutant D23 (which expressed oniy 15% of L6 GLCITJ
transcript level (Fig. 2.1)) was unable to express myogenin, muscle-specific contractile
protein genes (MLC and Tnï) or to form multinucleated myotubes (Fig. 2.2 and 2.3). On
the other hand, mutant D2 (which expressed around 14% of the L6 GLUT 3 transcript
level (Fig. 2. l)), was able to fom multinucleated myotubes, alkit at a slower rate than L6
cells (Fig. 2.2). This mutant also expressed significant levels of the myogenin, M C and
TnT transcript (Fig. 2.3). Similarly, mutant D9 (which contained 25% of the L6 GLLIT 3
transcript level (Fig. 2.1)) dso exhibited a much slower rate of fusion (Fig. 2.2). Day 6
cuitures of this mutant contained significant amount of the myogenin, M C and TnT
transcripts (Fig. 2.3). These studies suggest that factors in addition to the GLUT 3
transporter, mua be altered in these GLUT 3- mutants. It is possible that these mutants
may be defective in regdatory elements involved in regulating the expression of GLUT 3
and components involved in myogenesis.
To fiirther explore the possibility that components other than GLUT 3 are altered
in these mutants, GLUT 3- mutants were transfected with the GL UT 3 cDNA Attempts
were made to see if the myogenic defects in these mutants could be rescued by the
exogenous GLUT 3 cDNA. To be able to regulate the expression of the GLUT 3 cDN4
the latter was placed under the control of a MMTV promoter. Expression of the GLUT 3
cDNA can then be induced by the addition of dexarnethasone. Several GLUT 3
transfectants @23/GLUT 3, DUGLUT 3) were isolated. They possessed not only
significant levels of the GLUT 3 cDNA (Fig. 2.3), but dso much elevated GLUT 3
transcript Ievels upon induction with dexamethasone (Fig. 2.5). Despite a number of
attempts, we were unable to induce GLWT 3 expression in D9/GLUT 3 transfectants by
the addition of dexamethasone.
The possibility of rescuing the myogenic ability of GLUT 3- mutants upon the
induction of GL(IT3 expression was examineci. Dex-induced day 2 D23/GLUT 3 cultures
appeared as mononucleated myoblasts (Fig. 2.6). About 57% fusion was observed in day
6 cultures of dex-induced D23lGLUT 3 myoblasts (Fig. 2.7). However, we were unable
to detect the expression of myogenesis-associated genes (myogenin, and MHC) (Figs.
2.13 and 2.15) in these induced transfectants. Induction of GLUT 3 expression in
DZGLUT 3 myoblasts failed to increase the rate of fusion and the expression of
myogenesis-associated genes (data not shown).
It was important to note that the fusion index observed in the induced
D23/GLUT 3 transfectants was only a fiaction (5-7%) of that in L6 myoblasts (1000/o).
The GLUT 3 transcnpt levels in the induced D23/GLUn myoblasts (clones 2-4 and 2-6)
were at least 4 fold (400%) higher than that in L6 myoblast. Our laboratory has
previously shown that about 5-7% fusion was also observed in day 6 cultures of L6/G3S
transfectants overexpressing the GLUT 3 transporter (Bro ydeil et al., 1 997). Thus similar
to L6/G3S transfectants, the myogenic ability of D23 cannot be rescued by over-
expression of the GLUT 3 transporter. Despite numerous attempts to reduce GLUT 3
expression by altering the ce11 density, dexamethasone induction and removal times, serurn
concentrations and dexarnethasone concentrations, we were unable to increase the extent
of hsion.
Our findings also suggest that these GLUT 3- mutants may also be altered in
regdatory components responsible for the expression of GLUT 3 and the expression of
myogenesis-associated genes. For example, mutant D2 may be altered in a factor that
regulates both GLUT 3 expression and myogenesis. If this factor is mutated at a site
involved in regulating GLUT 3 expression, one can then explain the low levels of GLUT 3
expression. The mutated factor may still be able to regulate expression of myogenesis-
associateci genes. This may explain why mutant D2 can still retain its myogenic ability.
Our tentative working mode1 will be discussed in greater detail in Chapter S.
In summaq, the extensive correlation between GLUT 3 expression and
myogenesis still suggests that GLUT 3 may play a role in regulating myogenesis dong
with other components.
CaAPTER 3
ALTERATIONS IN WOGENIC REGULATORY COMPONENTS IN
MUTANT D23
3.1 INTRODUCTION
A number of distinct sequential events are involved in myogenic differentiation.
As rnentioned in Chapter 1, myogenesis involves biochemical differentiation, total
cornmitment, and morphological differentiation (Buckingham, 1 994; Emerson, 1993).
Biochemical differentiation is initiated in prolifkrating myoblasts when myogenin
expression is upregulated. Myogenin activates muscle-specific contractile protein genes:-
myosin heavy chah (MHC), myosin light chah (MX), and troponin-T (TnT), thus leading
to myotube formation. Transcription of myogenin and muscle-specific contractile protein
genes is dependent on the binding of the myogenin-oligomeric wmplex to the E-box
(CANNTG) and the AT-rich sequence motifs present in these muscle-specific genes (Funk
et al., 1992).
Through the use of a ce11 surface (p 1 123 myoblast mutant, F72, a temporal order
of in vitro expression for L6 myoblasts was suggested (Chen et al., 1993). This mutant
was unable to fom myotubes or express any of the muscle-specific genes. By transfecting
this pl 12- mutant with the myogenin (myJ-4) cDN& the ability to form myotubes was
restored. This study suggested the in vifro temporal order of expression of components in
the myogenic pathway was Myf-5, myogenin, and muscle-specific contractile proteins
(Chen et al., 1993). It is also interesting that the GLUT 3- mutant, D23, does not express
myogenin or any of the muscle-specific contractile proteins and it is also unable to form
myotubes (Figs. 2.3 and 2.7).
Chapter 2 examined the possibility of restoring G L U 3 expression in GLUT 3-
mutants and regaining the myogenic ability. Aithough D23, D2 and D9 myoblasts
expressed sirnilar low levels of the GLUT 3 transcript, they varied considerably in their
myogenic ability. Mutants D2 and D9 were able to form myotubes dbeit at a slower rate
than L6 myoblasts (Fig. 2.2). While transfection of these mutants with an inducible
GL UT 3 wnstruct restored GLLIT 3 expression in D2 and D23 myoblasts (Fig. 2.5), these
tranfectants were ail1 impaired in biochernical and morphological differentiation. These
studies suggeaed component(s) regulating both GLUT 3 expression and myogenesis
might be altered in these mutants.
The objective of this chapter is to examine why mutant D23 is impaired in the
expression of myogenin and other muscle-specific genes. To examine if mutant D23 is
defective in components required for the transcription of myogenin, constnicts containing
the myogenin promoter were transfected into L6 and D23 myoblasts. Data presented in
this chapter show that mutant D23 is def-ive in faaor(s) required for myogenin
prornoter activity. Since myogenin is involved in the transcription of M C , TnT, and
MHC, the inability of mutant D23 to express myogenin may be the primary cause for the
lack of expression of various muscle-specific contractile proteins.
If this was the case, then overexpression of myogenzn should activate expression of
the muscle-specific genes. In this study, myogenin cDNA was transfected into D23
myoblasts. These studies showed that myogenic factors, in addition to myogenin, are also
altered in this mutant.
3.2 MATERIALS AND METHODS
3.2.1 Bacterial Strains and Plasmids
E- coli strain HB I O I (F. hdS2O (r-Bm-B), supE44, ara- 1 4, gaiK2, ZacY 1 , proA2,
rpsl20(aï), xyl-5, ml/-1, recA13) (Sambrwk et al., 1989) was used in transformation
-dies using the myogenin constructs.
The rnouse myogenin promoter wnstructs used in transient transfection studies
were generous gifts fiorn Dr. S. P. Yee. The pGSC conma is comprised of a 1092 bp
myogenin promoter, a chloramphenicol acetyltrwerase (CAO gene (1.6 kb) and a
SV40 t-antigen sequence with poly(A)-signal. This construd was subcloned into the
pBluescript@ II KS+ (3 -0 kb) (Stratagene) (Yee et ai., 1993). A truncated myogenin
promoter construct, pGXC, containing oniy the first 133 bp of the myogenin promoter
was also used in these studies (Yee et al., 1993). To masure the efficiency of
transfection, a pgafactosidare (lac Z) gene placed under the control of the constitutive
CMV promoter (pRcCMV-Bgal) (Stratagene) was also used in transfection studies. The
pRc/CMV vector (containing no lac Z gene) was used as a negative control in the P-
galactosidase assay. The pBluescnpt@ KS+ II vector was used as a negative control in
the CAT assay.
The PGK-myogenirr construct and the PKJl AR vector used in stable transfection
studies were gifts from Dr. 1. S. Skejanc. PGK-myogenin consists of the 1.5 kb
myogenin coding sequence (Wright et al., 1989). Myogenin is placed under the control of
the pho~phoglycerutekinase promoter (PGK) ( Adra, 1 987). For selection of stable
transfectants with G4 1 8, PGK-myogenin was CO-transfected with the pRdCMV-pgal
vector (Stratagene) containing the neomycin gene.
The plasmid DNAs used in the transfmion studies were purifmi using the
Qiagen's DNA purification kit as outlined in Section 2.2.1
Ali media and chemicals used were the sarne as in Section 2.2.1.
3.2.2 CeU Culture
Mutant D23 (GLU?' 3 3 and its parental rat L6 myoblast were used in transfection
studies. Al1 cell culturing conditions were the same as in Section 2.2.2.
3.2.3 Transient Trnnsfcetion With Constructs Containing the Myogenin Promoter
pGSC and pGXC constmas were CO-transfected with pRdCMV-Bgal into L6 and
D23 myoblasts using the Cap04 precipitation method (Section 2.2.3). Vector pRc/CMV
was transfected and vectors pRdCMV-Bgal with pBluescript KS+ were cotransfected into
L6 (L6/CMV, L6/KS+/Pgal) and D23 @23/CMV, D23/KS+/~gal) myoblasts. Cells were
plated at a density of 4 x 10' cellJlOOrnm dish in the moming and allowed to grow for 6
hrs in growth media (Section 2.2.2). Transf-ion studies were carried out as described in
Section 2.2.3. M e r washing twice with 1 x PBS, cells were incubated for 48 hrs with two
changes of fiesh growth medium. M e r several washes in l x PBS, cells were harvested
with 1 mL of PBS and scraped with a mbber policeman into a 1 mL Ependorf tube. Ce11
lysate was &en at -80°C. They were lys& by thawing and passage through an 18-gauge
needle and then through a 25-gauge needle three times.
3.2.4 Assay for fbGalactosidase and Chloramphenicol Acetyltransferase
P-galactosidase activity was meanired according to the procedure as described by
Sambrook (1989). In this assay, 60 pL, of ce11 extract was incubated with 3 pL of lOûx
Mg solution (0.1 M MgCl2, 4.5 M P-rnercaptoethanol), 201 of 0.1 M sodium
phosphate (pH 7 3 , 66 pL. of O-Ntrophenyl-P-D-gdactopyranoside (ONPG) (Sigma)
(4 m g / d ONPG in 0.1 M sodium phosphate) at 3PC for 30 rnin. The reaction was
terrninated by the addition of 500 pL of 1M Na2C03. The optical density of the solution
was determined at a wavelength of 420 nm. The readings were within the linear range
(0.2-0.8) of the assay. The positive control included 30 pL of extract fiom mock-
transfected cells and 1 pL, of a 50 unitslrnL P-galactosidase (Sigma) stock. The negative
control was compnsed of 1 pL of ddH20.
Chloramphenicol acetyltransferase (CAT) assays were carrieci out by first heating
the sample at 6S°C for 10 min to inactivate deacetylases. M e r which samples were
centrifùged at 12000 x g, at 4OC for 10 min. The supernatant was then used in the CAT
assay (Kingston, et al., Current Protocols in Molecular Biology Vol. 1, 1990). An 87.5
pL ceIl extract mixture was incubated with 27 pL of 1 M Tris pH 7.8, 64 fi of 5 rnM
chioramphenicol in Hz0 (Sigma) and 1.5 pL of ' ~ - a c e t ~ l coenzyme A (ICN) (conc. 1.06 x
1 o4 mmoles/pL, specific activity of 1 -7 16 x 1 o6 dpdmmole) for 2 hrs at 37C. The
reaction was terminated by transfemng the wunple to ice. Each sarnple mixture was
extracted twice with 200 pL ice-cold ethyl acetate (BDH), mixeci thoroughly by vortexing
for 1 rnin. Samples were centrifùged for 5 min at 12000 x g. Only 160-1 80 of the
upper (organic) phase (the acetylated, radiolabelled forms of chloramphenicol) was
transferred to scintillation vials. The '~-acet~i coenzyme A and non-acetylated
chloramphenicol remainecl in the bottom (aqueous) phase. The amount of radioactivity
(3~-acetylated chloramphenicol) in the extractecl sample was determined using Non-
Aqueous Scintanalyzed Scintilene (Fisher).
3.2.5 Stable Transfection studies using the G m y o g e n i n Coastmct
The PGK-myogenin construct was co-aansfécted with pRdCMV-pgal into rat L6
myoblast and its GLLJT 3- mutant 0 2 3 ) . Stable transfectants were selected by growth in
the presence of G418 and cloned (Section 2.2.3). The PKTlAR vector was also CO-
transfected with pRc/CMV-pgal as a control.
3.2.6 Southern Blot Analysis
Genomic DNA were isolated fiorn the L6, and D23 transfectants using procedures
as outlined in Section 2.2.4. To determine the presence of the cDNA insert, 10 pg of
DNA was digested with BamH 1 and Xho 1 and analyzed on a 0.8% agarose gel. The gel
was then transferred to an ICN Biotrans positive nylon membrane, crosslinked, probed
with an ~-'*P-~cTP labelled mouse myogenin cDN& and exposed to Kodak X-OMAT
AR film (Section 2.2.4). The labelled bands on the autoradiogram were compared with
those generated by digesting the plasrnid DNA with the sarne restriction enzymes.
3.2.7 Fusion Index Measurement
The ability of stable transfectants to fom myotubes was determineci as described in
Section 2.2.5.
3.2.8 Northern Blot Analysis
Poly (A)' RNAs were extracted from L6, D23, D23/PKJl& and D23PGK-
myogenin ce11 lines (Section 2.2.6). One pg of each mRNA sample was run on an 1%
formaldehyde gel, transferred to an ICN neutral nylon membrane, crosslinked and
hybridized with various cDNA probes. The blot was uütially probed with W G cDNA to
determine sarnple loading. Gels were then further probed with myogenzn, M C , M C , or
T'T cDNAs (Section 2.2.6). The blot was wrapped in Saran Wrap and exposed to a
phosphorirnaging screen or a Kodak X-ray film.
3.2.9 Immunofluorescence Microscopy Studies
Immunofluorescence studies were conducteci using mouse monoclonal anti-
myogenin (FSD) and anti-MHC (MF-20) antibodies (Section 2.2.7). Sarnples were
examined under oil emersion using a Zeiss Axiophot Immunofluorescence microscope.
Pictures were captured using Northern Exposure Software, cropped and resized by Adobe
Photoshop Software and importeci into Corel Draw for final placement before printing.
3.3 RESULTS
3.3.1 Myogenin Promoter Activities in L6 and Mutant D U Myoblasts
To determine why mutant D23 expressed very low myogenin transcnpt level,
myogenïn prornoter adivities in L6 and D23 myoblasts were examined. L6 and D23
myoblans were transiently CO-transfected with pGSC/pGXC (Section 3 -2.1) and a
pRclCMV-ka1 conamct (Section 3 -2.4). To determine the efficiency of transfection, the
P-galactosidase activity was assessed. Since the CAT gene was placed under the control
of the myogenin promoter, one can determine the myogenin promoter activity by
meamring CAT activity (Section 3.2.4). Very high myogenin promoter activity was
observed in L6 cells transfected with pGSC which contained the full length (1.1 kb)
myogenin promoter (Fig. 3.1). Ody 35% of this activity was observed with cells
transfected with the tmncated (133 bp) rnyogenin promoter @GXC). More importantly,
less than 10% of myogenin promoter activity was obsewed in D23 myoblasts transfmed
with niII length or truncated conaructs (Fig. 3.1). This suggests that mutant D23 is
defective in component(s) required for the myogenin promoter activity.
Figure 3.1 Myagenin Promoter Activities in L6 and D23 Myoblasts
CNorarnphenicol acetyltransferase (CAT) and P-galactosidase (P-gai) activities
were measured in L6 and D23 transient transfectants harbouring a full length (GSC) or
truncated (GXC) myogenin promoter construct dong with a pRdCMV-pgai vector
(Section 3.2.3). The expression vector pRc/CMV was also transiently transfeaed into
5 x 1 0 ~ L6 and D23 cells, as negative controls in assessing P-gaiactosidase activity. The
Bluescript KS* vector served as a negative control for measuring CAT activity. To
normalize for transfection efficiencies, the myogenin promoter activity was measured as a
ratio of the CAT and the P-gal activities (Appendix #2). The promoter adivities were
compared to the L6/GSC sample which was expressed as 10W. The error bars denote
standard deviations for a sample size of 4.
MYOGENIN PROMOTER
- L6/GSC L6/GXC D23/GSC D23lGXC
CELL LINES
3.3.2 Restoring Myogenin Expression in D23 M y O blasts
In this study, the PGK-myogeenin cunstrud and the P U I d R vector were
t d e c t e d into D23 myoblasts. Genomic DNAs were isolated fiom stable D23
transfectants (Section 2.2.4). Samples were digested with Xho 1 and B d 1 and
compared to a digested PGK-myogenin plasmid DNA sample by Southern blot analysis.
B lots were probed with 32~-labelled myogenin cDNA. Southern blot analysis revealed the
presence of a band similar in size (1.5 kb) to the myogenin cDNA in D23lmyogenin
transfectants, but not in D23 or D23RKJlAR myoblasts (Fig. 3.2). These findings
indicated that a number of D23/myogenin transfectants (clones 1-2, 1-6, 1- 10, 2-3, 2-5
and 2-7) harboured significant levels of the exogenous myogenin cDNA.
To determine ifmyogenin was expressed in the D23 transfectants, poly (A)+ RNAs
prepared from day 4.5 cultures were isolated from L6, D23, D231PKJlA.R and
D23lmyogenin cells and probed with "P-labelled myogenin cDNA (Section 2.2.6). A
band, sirnilar in size (1 -9 kb) to that of the endogenous myogenin transcnpt was seen the
L6, D23lmyogenin myoblasts but not in D23 or D23IPKJlAR myoblasts (Fig. 3.3). This
indicates that the myogenin transcript was expressed in significant levels in the
D23lmyogenin transfectants.
Irnmunofluorescence staining using a anti-myogenin antibody (F5D) was
performed to determine if myogenin was expressed in D23lmyogenin transfectants. Day 2
and day 6 cultures of L6, D23 and D23/PKJlAR and D23lmyogenin celIs were prepared
as outlined in Section 2.2.7 and stained for the presence of myogenin. No myogenin was
present in day 2 cultures (Fig. 3.4). In day 6 cultures, myogenin was detectable in L6 and
D23lmyogenin cells, but not in D23 or D23/PKJlbR cells (Fig. 3.5). Thus these studies
Figure 3.2 Southern Blot Anaiysis o f D23 Trnnsfectants
Genomic DNAs were isolated from the D23hyogenin transfectants and tested for
the presence of the myogenzn cDNA. Panels A and B denote genomic DNAs from D23,
D23IPKTldR (1-6) and D23/myogenin tels digested with Xho 1 and BarnH 1. Panel A:-
lane 1 indicates myogenin cDNA digested with Xho 1 and BamH 1; Lanes 2 to 9 indicate
genomic DNAs of D23, D23/PKJ 1 AR clone 1-6. and D231myogenin transfectants clones
1-2, 1-4, 1-6, 1-8, 1-10, 1-12, respectively. Panel B:- lanes 1 to 10 indicate D23,
D23/PKT 1AR clone 1-6, and D23/myogenin transfectants clones 2-1, 2-2, 2-3, 2-4, 2-5. 2-
6, 2-7, and 2-8, respectively.
Figure 3.3 Northetm Blot Analysis of D23/myogenin Myoblasts
Northern blot analysis of various transfectants was camed out as described in
Section 2.2.6. mRNAs were extracted on day 4.5 cultures of L6, D23, D23/PKJlbR 1-6
and D23hyogenin clones 1-4, 1-6,2-3 and 2-5. Two pg of sarnpie was loaded ont0 each
Iane. The blots were probed with 32~-labeled MLC (Panel A) and myogenin (Panel B)
cDNAs. RNA loading was indicated by probing the blot with 32~-labeled PZ-
microglobulin (BMG) cDNA (Panel C).
A MLC
C BMG -1.0 kb
D23/ n D231 3 myogenin C1 PJKIAR
Figure 3.4 Immunofluorescence Staining of Myogenin in Day 2 DU/myogenin
transfectants.
Stable transfectants of mutant D23 harbouring the myogenin cDNA construct
were stained with an anti-myogenin antibody (FSD) (Section 2.2.7). Day 2 cultures of L6
(Panels A-C), D23 (Panels D-F), D23/PKJlAR (Panels G-1), and D23/myogenin clones 1 -
6 and 2-3 (Panels J-L and M-O, respectively) were observai under phase contrast
microscope (Panels A, D, G, J, and M), stained with Hoechst DNA stain (Panels B, E, H,
K and N), and stained with the anti-myogenin Ab (Panels C, F, 1, L and 0).
Figure 3.5 Immunofluorescence Staining of Myogenin in Day 6 D23fmyogenin
transfectants.
Stable transfectants of mutant D23 hahouring the myogenin cDNA construct
were stained with an anti-rnyogenin antibody (F5D) (Section 2.2.7). Day 6 cultures of L6
(Panels A-C), D23 (Panels D-F), D23PKJlAR (Panels G-I), and D23/myogenin clones 1 -
6 and 2-3 (Panels J-L and M-O, respectively) were observed under phase contrast
microscope (Panels 4 D, G, J, and M), stained with Hoechst DNA stain (Panels B. E, FI.,
K and N), and stained with the anti-myogenin Ab (Panels C, F, 1, L and 0).
showed that myogenin was expresseci in the D23lmyogenin clones 1-6 and 2.3.
3.3.3 AbiIity of DUlmyogenin Transfeetants to Differentiate
To determine if' the D23/myogenin transfêctants can regain their myogenic ability,
their abilities to fom myotubes and to express muscle-specific contractile proteins were
exarnined. Day 2 cultures of L6, D23 and D23 transfectants appeared as mononucleated
myoblasts (Fig. 3.4). Multinucleated myotubes were observed in only day 6 L6 cultures.
but not in day 6 D23 or D23/PKllAR d t u r e s (Fig. 3.5). More importantly, the
D23lmyogenin clones 1-6 and 2-3 cells contained only mononucleated myoblasts even
though they possessed signifiant levels of myogenin (Fig. 3 S).
The expression of various muscle-specific protein genes was also exarnined.
Northem blot studies were c-ed out using mRNAs £?om L6, D23, D23/PKJIAR and
D23lmyogenin myoblasts. As expected, the MLC, MHC and TnT transcripts were
detected in day 6 L6 culture but not in D23 nor D23lPKJlA.R cultures (Fig. 3.3 and data
not shown). More importantly these muscle-specific transcripts could not be detected in
D23lmyogenin transfectants (Fig. 3 -3). In other words, these transfectants were impaired
in their ability to transcribe MLC, M C and TnT, even though they possessed a significant
amount of the myogenin protein.
The presence of MHC in various ce11 lines was also examined by imrnunofluorescence
studies (Figs. 3.6 and 3.7). MHC could not be detected in day 2 cultures mg. 3.6). As
shown in Fig 3.7, the anti-MHC (h4F-20) antibody was able to recognize MHC in day 6
L6 cultures but not in D23 nor D23/PKJlAR cultures. As expected from Northern blot
studies MHC protein could not be detected in DZ3/myogenin transfaants 1-6 and 2-3
(Fig. 3.7).
Figure 3.6 Irnmunofluorescence Stsining of MHC in Day 2 D23lmyogenin
transfectants.
Stable transfectants of mutant D23 harbouring the myogenin cDNA constmct
were stained with an anti-MHC antibody @IF-20) (Section 2.2.7). Day 2 cultures of L6
(Panels A-C), D23 (Panels D-F), D23/PKJlAR (E%nels G-I), and D23/myogenin clones 1-
6 and 2-3 (Panels J-L and M-O, respectively) were observed under phase contrast
microscope panels 4 D, G, J, and M), stained with Hoechst DNA gain (Panets B, E, H,
K and N), and aained with the anti-MHC Ab (Panels C, F, I, L and 0).
Figure 3.7 Immunofluorescence Staining of MHC in Day 6 DUlmyogenin
transfectants.
Stable transfectants of mutant D23 hahouring the myogenin cDNA conamct
were stained with an anti-MHC antibody (MF-20) (Section 2.2.7). Day 6 cultures of L6
(Panels A-C), D23 (Panels D-F), D23IPKJlAR (Panels G-I), and D23/myogenin clones 1 -
6 and 2-3 (Fhels J-L and M-O, respeaively) were observed under phase contrast (Panels
4 D, G, J, and M), stained with Hoechst DNA stain (Panels B, E, H, K and N), and
stained with the anti-MHC Ab (Panels C, F, 1, L and 0).
The above studies indicated that both clones of D23/myogenin transfectants were
still unable to express the muscle-specific genes, and to form myotubes. Since these
transfectants possessed significant levels of myogenh, it may be ~ r m i s e d that they are
d e f ~ i v e in components required for transcription of muscle-specinc genes. These
components may act in conjunction with myogenin to activate muscle-specific gene
expression.
3.4 DISCUSSION
The biochemical and morphological events involved in myogenesis are complex
processes. An examination of the properties of several myogenesis-defective mutants
from rat L6 myoblasts revealed that the temporal order of the in vitro expression of
myogenic components was Myf-5, myogenin and muscle-specific contractile proteins
(Chen et al., 1993). Studies with L6 myoblasts over- and under-expressing the GLUT 3
transporter and with GLUT 3- mutants indicated that myogenic differentiation could
proceed only within a critical level of the GLUT 3 transporter (Eiroydell et al., 1997).
In chapter 2, we examined the properties of several independently isolated
GLUT 3- mutants. Even though they possessed much reduced levels of the GLUT 3
transporter, their myogenic abilities were affecteci differently. Mutant D23 did not fuse
and expressed essentially no myogenin and no muscle-specific contractile proteins (Section
2.3.2 and 2.3.3). Even though a similar GLUT 3 transcript level was present in mutant
D2, this mutant was able to fuse and express myogenin and the muscle-specific contractile
proteins, ulbeit at a reduced level when compareci with the parental L6 myoblast.
Furthemore, overexpression of an exogenous GLUT 3 cDNA in these mutants could not
increase their myogenic ability (Section 2.3 -6 and 2.3.7). This suggested that components,
in addition to GLUT 3, could be involved in regulating myogenesis.
To gain more insight into the regulation of the myogenic pathway, we examineci
why mutant D23 was impair4 in the expression of myogenin. Studies were carried out
using constructs containhg fidl length or truncated myogenin promoter. The
chiotamphenicoi acetyitransferase (CAO gene was used as the reporter gene for
determining the activity of the myogenin promoter. As indicated in Fig. 3.1, myogenin
promoter activity was readily deteaed when this construct was transfected into rat L6
myoblasts. However, such activity could not be detected in mutant D23. These findings
indicat e mutant D23 is defective in component (s) required for the myogenin promoter
activity .
If the inability of D23 myoblasts to express muscle-specific transcripts is only due
to insufficient myogenin, then over-expression of myogenin should restore their ability to
express muscle-specific genes. D23 myoblasts were therefore transfmed with the
myogenin cDNA. This vector contained the myogenin coding sequence and has been
show by Dr. Ske janc to code for an intact myogenin protein (personal communication).
We have shown in this study that D23/myogenin transfectants harboured the exogenous
myogenin cDNA (Kg. 3 2). These transfectant s could express the myogenin transcnpt
(Fig. 3.3) and myogenin protein (Fig. 3.5).
We have also shown in this study that these D23/myogenin transfectants failed to
fom myotubes or to express M C , MHC and TnTtranscrïpts (Figs. 3.3, 3.4, 3.5 and data
not shown). This suggests that mutant D23 was defective in component(s) required not
only for myogeniin expression but also for activating transcription of muscle-speci fic
contractile protein genes. It is also interesting to note that the inability of L6/G3S
t rans fmts to fom myotubes was due to the absence of muscle-specific transcripts, and
not due to indc ien t myogenin (Broydell et al., 1997).
As will be discussed in Chapter 5, Our studies suggest that mutant D23 is aitered in
component(s) acting on at least two different sites of the myogenic pathway in rat L6
myoblasts (Fig. 5.1). The first site is associated with the activation of myogenin
transcription, whereas the second site is related to the transcription of muscle-specific
proteins. Factors acting on the second site are required even in the presence of myogenin.
Studies with L6lG3S transfectants suggea that the level or funaional state of these
factors are very sensitive to elevated levels of the GLUT 3 transporter (ESroydell et al.,
1997).
CaAPTER 4
EXPRESSION OF THE GLUT 4 CENTRAL LOOP AND C-TE-AL
DOMAINS IN BACTERIA
4.1 INTRODUCTION
Glucose transporters are thought to be compriseci of 12 trammembrane (TM) a-
helical regions with the amino and carboxyl termini and a large central loop oriented
intracellularly (Olson et al., 1996; Zeng et ai., 1996; Cairns et al., 1987; Hresko et al.,
1994; Alvarez et al., 1987). Despite their overall similar structural organization, there is a
considerable divergence in the amino acid sequences of difFerent GLUT isoforms. The
most divergent regions are in the large intracelldar loop, and in the amino and carboxyl
termini. These regions possess isoform-specific amino acid sequences and are most likely
involved in regulating the function and properties of specific GLUT transporters (Zeng et
al., 1996).
The GLUT 4 transporter is the predominant giucose transporter isoform found in
insulin sensitive tissues such as skeletal muscle, cardiac muscle and adipocytes (Fukumoto
et al., 1989). Considerable efforts have been made in studying the regulation, stiucture
and hnction of GLUT 4 (Olson et al., 1996). The GLUT 4 transporier is responsible for
the major increase in glucose uptake activity following insuiin stimulation (Holman et al.,
1990; Slot et al., 1991). Glucose uptake is noted to increase 20-30 fold upon addition of
insulin to rat adipocytes (Czech et al., 1992). Insulin stimulates not only the translocation
of an intracellular pool of GLUT 4 to the plasma membrane, but also the intrinsic activity
of this transporter. The observed insulin-meâiated stimulation of GLUT 4 translocation
and activation are likely brought about by direct andlor Uiduect interaction of insulin-
modulated components with specific regions of the GLUT 4 transporter.
A number of regions of the GLUT 4 transporter are known to harbour the
information necessary for intracellular sorting, processing, and targeting to the plasma
membrane (McGowan et al., 1995). The N-terminal, middle intracellular loop and C-
terminal regions of GLUT 4 have independent intraceiiular targeting signas (Ishii et al.,
1995). The N-terminal haif' of the glucose transporter has been shown to be important in
intracellular trafncking and in maintaining the stability and intrinsic activity of the
transporter (Asano et al., 199 1). The carboxyl terminal half is involved in detemiining
substrate specificity and transport afnnity (ArbucWe et al., 1996). The carboxyl terminus
of GLUT 4 contains a serine-leucine-leucine sequence (SLL). This dileucine motif is
thought to operate as a rapid endocyîosis and retention signai Ui GLUT 4, localizing it to
intracellular compartments in the absence of insulin (Garippa et al., 1996). During insulin
stimulation, unmasking of the GLUT 4 at this site was found to increase the glucose
transpoxt activity by increasing the number of GLUT 4 protein in the sarcolernma and
tubule of skeletal muscle and adipocytes (Wang et ai., 1996).
Not much is known about the mechanisms by which these functional regions can
operate. Aside from maintainhg the proper conformation, they may funaion as sites of
interaction for proteins involved in regulating the intrinsic activity, intracdlular trafficking
a d o r stability of the transporters (McGowan et ai., 1995).
An inverse relationship between GLUT 4 and GLUT 1 has been observed in celis
undergoing physiological changes, and in ceils treated with CAMP, or tumor necrosis
factor (Gemts et al., 1993; Block et ai., 1991; Santalucia et al., 1992; Stephen et al.,
1991). It is conceivable that this relationship may be regulated by the interaction of
specific wmponents with the GLUT 4 transporter.
Several factors have been shown to interact with glucose transporters and affect
their transport activity. At least two cytosolic proteins (28 and 70 kDa) were found to
bind to the GLUT 1 and 4 carboxyl termini and change the i n t ~ s i c activity of these
proteins (Shi et al., 1995). A 70 kDa cytosolic protein was found to bind to the GLUT 4
centrai loop in an ATP-sensitive manner (Liu et al., 1995). These studies show that
interaction of the GLUT transporters with cytoplasmic proteins is likely to play an
important role in rnodulating the activity, and intracellular location of GLUT transporters.
GLUT 1 and GLUT 4 isoforms are also regulated during rat muscle development
(Santalucia et al., 1992). GLUT 1 is expressed during rat fetal muscle development.
Following birth, GLUT 1 expression declines. Declme in GLUT 1 levels is accompanied
by an increased GLLIT 4 level. This GLUT 4 isoform expression corresponds to changes
in the myogenic factor expression in rat muscle. In L6 myoblast and the mouse C2C12
ceIl line, GLUT 4 transcripts are initially low and their levels are elevated during
myogenesis (Xia et al., 1993; Mitsumoto et al., 1991; Klip et al., 1992; Richardson et al.,
1993). It is important to note that E boxes (which are binding sites for myogenic
transcription factors) are present in the GLUT 4 promoter (Richardson et al., 1993). It is
possible that a correlation may exist between GLUT 4 expression and processes involved
in myogenesis. The rat myoblast mutant, D23, discussed in Chapters 2 and 3, is known to
harbour the GLUT 4 and GLUT 1 transporters (Xia et al., 1993; Kudo et al., 1990).
However, when it is grown in the presence of 25 mM D-glucose, only the GLUT 4
transporter is fundonal in this mutant. This mutant can therefore be used to study the
intrinsic activity of the GLUT 4 transporter.
The objective of this chapter was to generate construas of the central loop (G4L)
(residues 223-287), and carboxyl terminus (G4C) (residues 467-509) of the GLUT 4
transporter fiised to the glutathione-S-tramferase (GST) protein. These two regions are
the largest GLUT 4 intracellular domains (Olson et al., 1996). They are highly conserveci
in GLUT 4 isoforms fkom different animals, and are distinct from the other GLUT
isoforms. In this study, we detennined the optimal growth condition for expression of the
GST-fusion proteins containing either one of these two regions. These GST-fusion
proteins are essentiai in identifjing and isolating cytoplasmic proteins that can interact
with specific GLUT 4 regions.
4.2 MATERIALS AND METHODS
4.2.1 Bacterial Culture Media
Bacterial cu i~res were grown in a Luna-Bertani (LB), Temfic Broth (TB) or
2xYT medium (Sambrook et al., 1989). Transformation experiments were c-ed out with
SOC Medium (Sambrook et ai., 1989).
4.2.2 Bacterial Strains and Plasmids
E.coli strain HI3101 was used for large scale production of plasmids. E.coli strain
BL-21(DE3) (hdS gai (hcI1s857 indl Sam7 nin5 lacW5-T7 gene I ) (Pharmacia
Biotech) was used for high level expression of genes cloned into expression vectors
containing bacteriophage T7 promoter. Strain RRl (supE44 h~&2O(r-~ me) mu44
proA2 IacYl gaAC2 rpsL2O xyl-5 mil-1) (Phannacia Biotech) was a r e c ~ + derivative of
HB 101, it cm be transformai with high efficiency. Strain DHSa (supE44 AhcY l69($8O
IacZAM15) hsW 1 7 recAl endAl gyrA96 thi- l relAl ) (Pharmacia Biotech) was used for
plasmid growth. The above strains were made into wmpetent c d s by growth in 2xYT
medium (Sambrook et al., 1989).
The pRc/CMV-GLUT4 vector was constmcted in Our laboratory by Patricia Kudo
(Kudo thesis, 1993). This constmct contains the codig region of the rat GLUT4 cDNA
and was used as a template in the PCR amplification of the cytoplasrnic loop (G4L) and C-
terminal domain (G4C) of the GLUT 4 transporter. To irnprove the efficiency of ligation
of the PCR product into the plasmid, the amplified cDNA inserts were first cloned into the
p ~ ~ M @ - ~ Vector System (Promega), which contains 3'-T overhangs at the insertion site
( M d et al., 1994).
The pGEX vectors are prokaryotic gene fusion vectors (around 4950 bp) used for
expression, purification and detection of GST fusion proteins (Pharmacia). These vectors
code for a 26 kDa Schistosomrr jquninrm glutathione S-transferase (GST) which can be
induced by isopropyl P-D-thiogalactoside (IPTG) for high-level expression. The pGEX
vectors contain a rnultiplecloning site (MCS) downstream from the GST wding region.
This contains three restriction sites for unidirectional clonlng of cDNA inserts. A
thrombin cleavage site is also present downstream £?om MCS, this allows cleavage of the
fùsed protein fiom the GST-protein.
The pGEX-KG vector was constructeci by Guan and Dkon, (1991). A glycine-
rich linker region (56 bp) was added to pGEX-2T's EcoR 1 site. This providecl the GST
fusion protein a glycine-rich "kinker" immediately f i e r the thrombin cleavage site and
allowed for more efficient thrombin cleavage of fusion proteins.
4.2.3 Amplification of the G4L and G4C Regions
Primers for the PCR studies were synthesized by the Protein Anaiytical Centre at
the Department of Biochemistry, University of Western Ontario, London, ON. The G4L
5' primer (RG4LPS') 5'-CGGGATCCATG-AGAGTGCCTGAAACCAG 3 ' (28 bp)
was designeci to contain a BamH I site (CG-GGATCC) and a start codon (ATG)
upstream of position 772 on the GLUT4 gene. The G4L 3' primer (RG4LP3')
S'CGGAATTCTTA-AGGCTGCCGGTGGGT 3' (26 bp) includes a stop codon (TïA)
and a EcoR 1 site, CG-GAA'ITC, downstrearn of position 951 on the GLLIT 4 gene.
Pnmers RG4LP5' and RG4LP3' were used in the amplification of the G4L region in a
GTC-2 Genetic Thermal Cycler (Frecision Scientific Inc. Chicago, IL). The reaction was
hot-staried at 94°C for 3 min, brought down to 84°C for one min before the addition of
DNA Taq Polyrnerase (0.5 units) (Gibco). The subsequent cycle, consisting of 94°C for 1
min (denaturing), 65°C for 2 min (annealing), 72OC for 2 min (elongation) was repeated 20
times. Afier which the temperature was kept at 72OC for 10 min, and then at 4°C.
Sirnilar procedures were used to ampli& the G4C region, except that primers G4C
5' (RG4CTDS') and G4C 3' (RG4CTD3') were used. The RG4CTDS' primer (5'
CGGAATTCTTA-AGAGTGCCTGAAACCAG 3') (29 bp) includes a B a d 1 site,
CG-GGATCC and a star& codon (ATG) upstream of position 1504 on the G L W 4 gene.
The RG4CTD3' primer (5' CGGAATTCTTA-GTCATTCTCATCTGGCCC 3') (29 bp)
contains a stop codon (=A) and EcoR 1 site CG-GAAïTC downstrearn of position
163 1 on the GLLIT4 gene. The optimai annealing temperature for the amplification of the
G4C region was 65OC.
Ail samples were chloroform extracteci. Five pL of the PCR product was mixed
with 1 pL of a 6x gel loading buffer (0.25% bromphenol blue, 0.25% xylene cyan01 FF
and 30% glycerol), and this was nui on a 1û% DNA acrylamide gel (29% acrylarnide/l%
bisacrylamide mixture, 5x TBE (0.45M Tris-borate, 0.01M EDTA), 10?4 ammonium
persulfate and TEMED). The appropriate band was excised nom the gel and eluted with
DNA elution b d e r (3.33 mL of 7.5 M ammonium acetate, 500 pL of 1 M MgAc, 100 pL
of 500 mM EDTA, pH 8.0 and 500 pL of 100/o SDS, in a 50 mL total volume) (Sambrook
et al., 1989). The sample was precipitated using an equal volume of 100% isopropanol,
and kept at -20°C for 1 hr. A DNA spot test using varying concentrations of DNA and
ethidium bromide stain was carrieci out to determine DNA concentration of the sample.
4.2.4 Ligation and Transformation of the p ~ ~ ~ - T e Vector with G4L and G4C
PCR products.
The ligation reactions included: T S N A Ligase, pGEM-T vector (50 ng) and
G4L or G4C in a ligation buffer. The rnass ratios for the vector (pGEM-T (3000 bp)) to
insert size was calculated to be 14: 1 and 2 1 : 1 for G4L (216 bp) and G4C (143 bp),
respectively. The molar ratios (1 : 1, 3 : 1, 1 :3) for each ligation were calculated according
to equations described in Fishers Protocols and Applications (Fisher Scientific, 1995).
Each reaction was incubated for 3 hrs at lS°C. Positive and negative controls included
ligation of pGEM-T vith control insert DNA and ligation in the absence of insert DNA.
The GLUT 4 ligation products were transfonned into E.coli strain DH5a
(Sambrook et al. 1989). Transfomecl celis were plated ont0 LB plates containhg
carbenicillin (Sigma), 25pgIpL of X-gal and grown overnight at 37°C. White wlonies
contained the insert of interest, whereas blue colonies contained an intact Iuc Z gene.
Selected white colonies were grown overnight in 6 m . of T e d c Broth wntaining 120
pg/mL of carbeniciliin. m e r extraction by the alkaline iysis rnini-prep method (Sambrook
et al., 1989), the presence of the insert was confirmed by digestion with B a d 1 and EcoR
1. Individuai clones were then grom on a larger scde (100 mL) in TB medium. Their
plasmid DNAs were isolatecl using the Qiagen Midi Kit and were sequenced using
Pharmacia's T7 Sequencing Kit (Phannacia Biotech).
4.2.5 Ligation of G4L and G4C into pGEX-KG
The pGEX vector was cleaved with BamH 1 and EcoR 1 to create aicky ends; this
treatment also removed the glycine rich "kinkef' region of the pGEX-KG vector. The
linearized vector was dephosphorylated using 5 units of calf intestinal alkaline phosphatase
(CIAP) in 10x CIAP dephosphorylation buffer (10 rnM ZnCh, 10 rnM MgCI2, 100 mM
Tris-Cl (pH8.3)) at 37°C for 30 min. This ~ e ~ e d to reduce the reannealing of Iinear,
single nit pieces. The dephosphorylated vector was then run on an 1% low melting grade
agarose gel (BIO-RAD) and purified using Gene lea an@ Kit (BIOKAN Scientific, Vista,
CA). The pGEMT-GLUT4 constructs were digested with BamH 1 and EcoR 1 and the
inserts were purified fiom an 1% agarose gel using the ~ ~ ~ r n a i d @ K . i t @IO/CAN
ScientSc). Both the vector DNA and the insert DNAs were nui on a 5% DNA
acrylamide gel dong with a 1 kb DNA ladder and the ethidium bromide stained band
intensities were cornpared. The amount of insert and vector used in each ligaton reaction
was calculated according to standard procedure (Sarnbrook et al., 1989). Ligation
reactions were carriecl out at 1S0C for 16 hrs.
4.2.6 Transformation and Screening of pGEX-GLUT 4 Coostnicts
HBlOl cumpetent cells were t rdonned with either the pGEX-G4L or the
pGEX-G4C constnicts and selected by growth on LB/carbenicillin plates. Colony las
were then performed (Sarnbrook et ai., 1989). Colonies were transferred to a sterile
nitrocellulose membrane (ICN Inc), which was then placed on a Whatman filter paper
saturated with denaturation buffer for 5 min and then placed in a neutralirlng solution for
5 min. The membrane was then placed onto a 2x SSC saturated Whatman paper for 5
min. After drying on a dry Whatman filter, the membrane was exposed to W light in a
W Strataiinker 1800 Q Crosslinker (Stratagene). The nitrocellulose membrane was
prehybridized for 2 hrs, probed with 32~-labelled G4L or G4C cDNA, and exposed to
Kodak X-OMAT AR film. Positive colonies were then picked, and the presence of
specific pGEX-G4L or pGEX-G4C vectors in these cells were confirmeci by diagnostic
restriction digestion. Plasmid DNAs fiom these clones were içolated by Qiagen's Midi
Kit, denatured and sequenced with Phannacia's T7 Sequencing Kit with primers 781 and
El @fis £?om Dr. Eric B a h Iab and Dr. B. Sanwal's lab, respectively, University of
Western Ontario, London, ON). These clones were also sent for analysis by the
Sequencing Analysis Facility in the John Robart's Research Institute at the University of
Western Ontario, London, ON.
Clones were fiirther transformed into E. cofi strains RRl , BIS 1 and DH5a. Mini-
preps were performed and diagnostic digestion on the plasmid DNA was performed to
confirm the transformation. The plasmid DNA was fiirther checked by sequencing with
primers El and 78 1 to detennine whether mutations had occurred during transformation.
4.2.7 Expression of GST-G4L Fusion Protein
Ovemight cultures of 4 strains (BL21, DH5a, HB 1 O 1 and RRl) containing
construa pGEX-G4L or the pGEX-KG vector were grown in 2xYT medium at 3PC.
The cultures were then grown in three dserent media (TB, LB and 2xYT) wntaining
carbenicillin. The d tures were grown to an ODm of 0.6-0.65 and induced with 0.1 mM
IPTG before being fiirther divided into 5 mL aliquots. Cells were grown in a shaking
innibator at 2 l0C, 30°C and 37°C for either 3, 6, or 12 hrs. Three mL of each culture
were harvested and stored as pellets at -80°C. Pellets were resuspended in 800 pL of cold
TE containing 2 rnM DTT and protease inhibitors: pepstatin A, (1 pg/mL), aprotinin (1
pg/mL), leupeptin (1 pg/mL), and phenylmethyl nilfunyl fluoride (PMSF) (0.1 rnM)
(Sigma). Cells were kept on ice and lysed by sonkation using the Sonifier Ce11 Disruptor
350 for 20 sec. Protein concentrations were detemiined by the Lowry Protein
Deterrnination Method (Sambrook et al., 1989). Ten pg of protein from each cell Iysate
were loaded on to a 10% SDS denaturing acrylamide gel consisting of a 3.5% stacking gel
and a 10% separating gel (Sambrook et al., 1989). The gels were then stained with 0.5%
Page Blue 83 (Sigma) stain containing 1% glacial acetic acid and 5% ethanol for 2 hrs and
destained in 10% glacial acetic acid with 1% glycerol ovemight.
4.2.8 Analysis of Soluble and Insoluble Fractions of the GST-G4L fusion protein
DHSa, BL-21 and RRI strains transformed with pGEX-G4L were grown in TB
medium, induced with 0.1 mM IPTG and grown for 3 hrs at 30°C or 37°C. M e r
sonication, 250 pL of bacterial lysates were centrifbged at 5000 x g to separate soluble
and particulate fractions. The pellet and soluble fiactions were resuspended in 500 JL of
lx sample bufKer and 250 pL of a 2x sample buffer, respectively (both sample buffers
contained no P-mercaptoethanol and no bromphenol blue dye so that protein
concentration could be determined). 10?4 SDS d e n a n i ~ g acrylamide gels were loaded
with either 20 pg or 10 pg of protein. The 20 pg protein gel was stained with Page Blue
83 stain. The 10 J . L ~ pprtein gel was transferred to Nylon ImmobilonTY-P Transfer
Membrane (Millipore) and probed (Bio-Rad's Protein Blotting 2nd Ed., (1996)) with anti-
GST monoclonal mouse antibody diluted 15000 (WITS, University of Western Ontario,
London, ON). The blot was rocked in IxTTBS (20 mM Tris-HCI, 500 mM NaCl, pH 7.5
and 0.2% Tween-20 @IO-RAD)) that contained 2.5% skim milk for 1 hr at 21°C before
washing three times with 1 xTTBS every 10 rnins. The secondary antibody, a rabbit IgG
horse radish peroxidase-linked antibody ( Amersham) was diluted 1 : 5000 and incubated for
1 hr in IxTTBS. The blot again was rinsed with lxTTBS and washed 5 times with 10 mL
of IxTTBS every 10 min. Cherniluminescence was performed on the blot with Gibco's
LumiGLO substrates, which react with the conjugated secondary antibody.
4.2.9 Solublization of the GST-G4L From CeH Pellet
Celi pellets were thawed and resuspended in 15 mL of TridEDTA with 2 pg/mL
of protease inhibitors: pepstatin A, leupeptin, aprotinin and 0.2 mM PMSF. Suspensions
were sonicated for 2 x 45 sec and kept on ice. The particdate fraction was collected by
centrifugation at 4000 x g for 15 min. Proteiw associated with the particulate fiactions
were first removed by washing the particulate fraction with 10 mL of 0.1 M Tris HCI
containing 1 M urea and protein inhibitors. The pellet was resuspended in 10 mL of 0.1M
Tris-CI pH 8.5, 1 M urea and protease inhibiton. Mer another round of sonication, the
suspension was rocked at 21°C for 15 min and then centrifigeci to collect insoluble
proteins. The suspensions were then centrfiged at 9000 x g for 15 min. Insoluble
proteins were soiubilized by incubating with 10 rnL of 8 M urea, 0.1 M Tris-Cl (pH 8.5)
and protease inhibitors. The samples were incubated on a rocker at 21°C for 1 hr.
Solubilized proteins were recovered by centrifiiging at 4000 x g for 10 min. The
solubilized proteins were diluted slowly with renaturation buEer (50 mM Tris-HC1, 1 mM
DTT, 20% glycerol and 0.1 rnM EDTA) and protease inhibitors to 6 M urea. Samples
were diaiysed (using membrane with a rnolecular weight cut off of 3000 Da (Spectrapor,
Los Angles, CA.)) slowly for 2 hrs in 1 L of renaturation buffer containing 4 M urea and
protease inhibitors, and then for another 2 hrs in renaturation buffer containing 2 M urea
with protease inhibitors and DTT. Didysis tubing was changed f i e r 2 hrs and new
protease inhibitors and 2 rnM DTT were added. Finally samples were dialyzed in 2 L
renaturation buffer wntaining protease inhibitors and DTT, but no urea, for 4 hrs with one
change of buffer. M e r dialysis, samples were centrifbged at 5000 x g for 15 min at 4°C
and both pellet and supernatant were fiozen separately at -80°C. The protein pellets
obtained were brought up in an appropriate volume of lx sample buffer (SB). 25 pg of
each sample was analyzed by SDS-PAGE. Gels were stained with Page Blue 83 for 2 hrs
and destained in 10% glacial acetic acid overnight.
4.2.10 Coupling and Elution of the G4L Fusion Protein fmm Glutathione Agarose
Beads
Glutathione agarose CL-4B beads (Pharmacia) were washed several times with 50
rnL of lx TBS containhg 2 mM DTT and 0.05% Triton X-100. Ten mL of the
solubilized GST-G4L protein containing 0.15 M NaCl and 0.1% Triton X-100 was
incubated with 1 mL of the washed glutathione agarose CL-4B beads overnight on a
rotator at 4 ' ~ .
After coupling the GST-G4L protein to the glutathione beads each 0.5 mL of
agarose beads was washed twice with 1 m . of pre-glutathione elution buffer (50 mM Tris
pH 7.5, 200 mM NaCI, 0.1 mM EDTq 10 % glycerol, 0.05 % Triton X-100, 2 mM
DTT) or thrombin elution buffer ( l x TBS, 2.5 rnM CaC12, 2 mM DTT, 0.05% Triton X-
100). For glutathione elution of the GST-proteins, 0.5 mL of glutathione elution buffer
containing 20 mM glutathione was added to each 0.5 mL of conjugated beads and
incubated for IS min at 4°C with agitation. The supernatant was recovered d e r a brief
centriftgation in a microcentrifuge. This was repeated and supematants were wmbined
and stored at -80°C.
The G4L peptide can also be cleaved from the GST-G4L fusion protein by
digestion with thrombin. In this study, 0.5 mL of Ix TES and 10 pL of thrombin (0.2
pg/rnL) were incubated overnight at 21°C with gentle agitation. The eluted proteins were
recovered by centrifugation. This process was repeated twice. Supematants were
combined and stored at -80°C. A control experiment was c-ed out by innibathg the
coupled beads with the elution b d e r containing no thrombin overnight at 2 1°C. This was
intended to detemine if the long incubation penod could cause protein degradation,
independent of the presence of thrombin.
4.2.1 1 Expressing, Coupling and Eluting SoIuble GST and GST-G4C proteins
Bacterial cultures transformeci with either the pGEX-KG vector, the pGE X-G4C
or a mutated pGEXOG4C constmct were grown ovemight at 37" C in 5 mL
TB/carbenicUin. Clone 19 contains a mutated pGEX-G4C (G4Ctmt) constmct. In this
mutation, amino acid ~ e r ~ ~ ~ of the GLUT 4 C-terminai domain was replaced with amino
acid ~ro"*. Ovemight cultures were used to inoculate into 150 mL of the same medium,
grown at 3 7 C to an ODm of 0.65 and induced with 0.1 mM IPTG. The induced cultures
were grown at 30°C for 3 hrs. CeUs were harveaed and sonicated. The supernatant was
coupled to glutathione agarose CL-4B beads as mentioned above. The presence of
coupled proteins were confirmed by elution with glutathione or by cleavage with
thrombin. The eluted proteins were then subjected to SDS-PAGE.
4.3 RESULTS
4.3.1 Construction of the GST-G4L and GST-G4C Constlvcts
The cDNA coding for the central loop (G4L) (residues 223-287) or the carboxyl
terminus (G4C) (residues 466-509) of the GLUT 4 transporter was arnplified by PCR
using appropriate primers (Section 4.2.3) and the rat GLUT 4 cDNA was used as a
templats (Figs. 4.1A and 4.2A). Primers were designed to contain a BamH 1 restriction
site at the 5' end and a EcoR 1 restriction site at the 3' end of the DNA fragment. These
sites were added to facilitate the eventuai incorporation of G4UG4C cDNAs into the
Figure 4.1 Design of the pGEX-G4L Consmict
The cDNA coding for the central loop of the GLUT 4 transporter (G4L) was
amplified by PCR using appropriate primers and the rat GLLIT 4 cDNA as a template
(Section 4.2.3). Panel A shows the presence of a single band (216 bp) when resolved on
an 8% DNA acrylamide gel. Lanes 1, 2, 3, 5 and 6 denote samples from reactions using
varying concentration (O ng, 0.5 ng, 1 ng, 5 ng and 10 ng) of the template DNA Lane 4
indicates the 1 kb DNA molecular weight marker.
Panel B shows a schematic diagrarn of the amplified G4L PCR product subcloned
into the linear p ~ ~ M ? - ~ vector, using T4 DNA ligase.
Panel C is a schematic diagram of the insertion of G4L cDNA into pGEX-KG
vector. The G4L insert DNA was digested with BamH I and EcoR I fiom the PGEP-T
vector. The pGEX-KG vector was linearized with B a d 1 and EcoR 1 and
dephosphorylated with 5 units of calf intestinal alkaline phosphatase (CIAP). The GIL
insert was then Iigated to the dephosphorylated 1ineanZed pGEX-KG vector using T4
DNA ligase.
pGEM-T vector G4L PCR product (3.0 kb) \ / (216 bp)
T4 DNA Ligase 3 brs. 1 S F
GJL W r t (216 bp)
BamH 1 and
EcoR 1 BamH I and EcoR 1
37"C,30 min
Figure 4.2 Design of the pGEX-G4C Construct
The cDNA coding for the C-terminal domain of the GLWT 4 transporter (G4C)
was arnplified by PCR using appropriate pnmen and the rat GLUT 4 cDNA as a template
(Section 4.2.3). Panel A shows the presence of a single band (156 bp in size) upon
resolving the PCR product on an 8% DNA acrylarnide gel. Lanes 1, 2, 4, 5, and 6, denote
samples from reactions using varying concentrations (O ng, 0.5 ng, 1 ng, 5 ng and 10 ng)
of the template DNA. Lane 3 is the 1 kb DNA molecular weight marker.
Panel B shows a schematic diagram of the arnplified G4C PCR product subcloned
into the linear pGEM-T vector, using T4 DNA ligase.
Panel C is a schematic diagram showing the insertion of the G K cDNA into the
PGEX-KG vector. The G4C insert DNA was digested with B a d 1 and EcoR 1 fiom the
p ~ ~ M ? - ~ vector. pGEX-KG veaor was linearized with BamH I and EcoR 1 and
dephosphorylated with caIf intestinal alkaline phosphatase (CIAP). The G-IC insert was
Iigated to the dephosphorylated linearized pGEX-KG vector using T.t DNA ligase.
pGEM-T vector G4C PCR product (3.0 kb) I J (156 bp)
T4 DNA Ligase 3 bn. IS'C
BamH 1 and EcoR 1
BamH 1 and EcoR 1
P
i P
c w , 1 3 f C . 30 min
a bio , c#loor 4
G4c (5.16 kb)
multiplecloning site (MCS) of the expression vector pGEX-KG. A start codon (ATG)
was added to both G4L and G4C 5' primers, whereas a stop codon (TTA) was included in
both G4L and G4C 3' end primers. PCR reactions were carried out as describecl in the
Methods Section 4.2.3. The amplifieci products were resolved on an 8% DNA-acrylamide
gel (Figs. 4.1 A and 4.2A).
The amplied G4UG4C PCR products were first ligated to the p ~ ~ M 8 - ~ vector
using Td DNA ligase (Figs. 4.1B and 4.2B). The ligation products were then tmsformed
into the E coli strain DH5a. Colonies were screened for the absence of an intact
galac~osihe gene (Section 4.2.4). Plasmid DNAs nom four G4L Clones (L 1 , L3, L4
and L5) and four G4C Clones (C 1, C2, Cl0 and C12) were extracted using the Plasmid
Midi Kit, digested with the appropnate restriction enzymes (Fig. 4.3A) and sequenced
using the T7 Sequencing Kit. Clones L3 and Cl0 were used in subsequent studies.
Purifid DNA of the expression vector pGEX-KG was digested with EcoR 1 and
BamH 1 (Fig. 4.1 C and 4.2C). The linearized vector was dephosphorylated using calf
intestinal alkaline phosphatase (CIAP) and purified as mentioned in Section 4.2.5. The
G W G K insert DNA was isolated by digesting Clones L3 and Cl0 cDNAs with BamH 1
and EcoR 1, and punfied frorn an 1% agarose gel using the ~ ~ ~ r n a i d ~ Kit (Section 4.2.5)
(Figs. 4.1 C and 4.2C). The G4UG4C inserts were ligated to the linearized pGEX-KG
vector for 16 hrs at 15°C (Section 4.2.5). The ligated products were used to transform the
E coli strain HI3 1 O 1 . Positive colonies were selected by the colony lifis met hod (Section
4.2.6). Plasmid DNAs fiom pGEX-G4L Clones 39, 41, and 42 and pGEXOG4C Clones 7
and 12 were extracted by midiprep method. Restriction digestion of these pDNAs
reveaied an insert band of the expected 2 16 bp for pGEX-G4L and 156 bp for pGEX-G4C
Figure 4.3 Diagnostic Restriction Digestion of G4L and G4C Clones.
Diagnostic restriction digestion was carried out using EcoR 1 and BamH 1. Panel
A indicate digestion of the pGEMT-G4C and pGEMT-G4L clones. M e r digestion, the
DNA fiagments were resolved on an 1.2 % agarose gel. Lanes 1-4 denote sarnples @om
pGEMT-G4C clones C 1, C2, C 10 and C 12, respectively. The presence of the 0.1 56 kb
fiagment is apparent in al] pGEMT-G4C clones. Lanes 7-10 denote sarnples from
pGEMT-G4L clones LI, L3, L4 and L5, respectively. Al1 clones contain the 0.21 6 kb
G4L insert. Lanes 5 and 6 contain the 100 bp DNA standard and the BstE II DNA
molecular weight markers, respectively.
Panel B denotes the diagnostic restriction digestion of the pGL.Ix-G4L and pGEY-
G4C cDNAs. The DNA fragments were resolved on an 8% DNA acrylamide gel. Lanes
2, 5, and 6 denote the digested pGEXG4L clones 39, 41, and 42, respectively. The
presence of the 0.216 kb fiagment is apparent in al1 pGEX44L clones. Lanes 3 and 4
denote sarnples tiom the pGEX-G4C clones 7 and 12, respectively. Both pGEX-G4C
clones contain the 0.156 kb insert. Lane 1 contains the 1 kb DNA standard.
(Fig. 4.3B). The plasmid DNAs were also verified by sequencing studies. Sequencing
studies of some pGEX-G4C clones revealed a mutation (senne to proline change) at
residue 488 in the GLüT 4 protein. in other words, the SLL motif has b e n replaced with
PLL. G4L Clones 41 and 42, G4C Clones 7 and 12 and the mutated G4C (G4Cmut)
Clone 17 and 19 were used in subsequent expression studies.
4.3.2 Expression of the GST Fusioa Pmteins
To determine the optimal conditions for the expression of GST fusion proteins,
plasmid DNAs from pGEX-G4L Clones 41 and 42, pGEX-G4C Clones 7 and 12 and
pGEX-G4Cmut Clones 1 7 and 1 9 were used to transform t hree different strains of E- coli
@H5a, BL-21 and RRI). Afier venfjmg their plasmid DNAs by diagnostic restriction
digestion (Fig. 4.3) and sequencing studies, the transformed cells were grown under
different conditions. The growth media (2xYT, TB, and LB), induction temperature
(22'C, 30°C and 37"C), induction time (3 hrs, 6 hrs, and 12 hrs) and IPTG concentrations
(0.1 mM, 0.5 mM and 1 mM) were varied and the expression of the GST-G4L, GST-G4C
and GST-G4Cmut proteins were monitored by SDS-PAGE stained with Page Blue 83.
The optimal conditions were:- E-col1 BL-21 transformed with pGEX-G4L Clone 42,
pGEX-G4C Clone 12 or pGEX-G4mut Clone 19, grown in 400 rnL of TB medium and
induced with 0.1 mM IPTG for 3 hrs at 3 0°C (Fig. 4.4).
Under these optimal conditions, the 33 kDa GST-G4L fusion protein existed
predominantly in the particulate fiaction, whereas the 26 kDa GST protein and the 3 1 kDa
GST-G4C and GST-G4Cmut fusion proteins were found mainly in the soluble fonn (Fig.
4.4).
Attempts were made to solublilize the GST-G4L fusion protein from the
Figure 4.4 Expression of the GST-G4L and GST-G4C in Ecoli BL2l@E3)
Bacterial cultures were grown at 30°C in TB medium supplemented with
carbenicillin and induced with 0.1 mM IPTG. Samples were removed 3 hrs after
induction, and the ce11 extract was separated into particulate (P) and soluble (S) fraaions,
and resolved on a 10% SDS-polyacrylamide gel stained with Page Blue 83. Panel A
indicates samples from pGEX-KG Clone 1 (GST), pGEX-G4C Clone 7 (G4C-7) and
pGEX-G4Cmut Clone 10 (G4Cmut- 10). Samples from uninduced cultures were also
included in this study. The 26 kDa GST-protein, the 31 kDa GST-G4C and GST-
G4Cmut fusion proteins were apparent in this study. Panel B depicts pGEX-G4L Clone
42 (G4L-42) soluble (S) and particulate (P) fiactions and GST sarnple, resolved on a 10%
SDS-PAGE. A 33 kDa GST-G4L fusion protein and a 26 kDa GST protein was detected
by immunoblotting studies using an anti-GST antibody.
dit-7 NI S P NI S P N I S P
GST G4C-7 G4Cmut-10 G4L-42 GST
particulate hction (Fig. 4.5). This protein was associateci with the particulate fraction
even &er treatment with 1 M or 2 M urea for 1 h at 21°C. The rnajority of this protein
was solubilized upon treatment with 5 M urea, and complete solubiiization was achieved
using 8 M urea (Fig. 4.5). Subsequent isolation procedure for the GST-G4L protein
included removal of non-specific proteins f'rom the particulate hction by washing twice
with 2 M urea. M e r which, the GST-G4L protein was solubilized by incubating with 8 M
urea at 21°C for 1 h. To remove the urea and to dow refolding of proteins, the
solubilized proteins were dialyzed at 4OC in varying concentrations of urea (Section 4.2.9)
43.3 Coupling of the GST-fusion Proteins to Glutathiooe-agarose Beads.
Al1 three types of GST proteins were purified by coupling to glutathione-agarose.
Non-specific proteins were removed by washing the coupled beads three times with TBS
buffer containing 2 mM DTT and 0.05% NP-40. They were then stored at 4°C in TBS
buffer containing 100 rnM NaCl. The GST fusion proteins and the GST protein were
coupled to glutathione beads (Sections 4.12 and 4.13). Binding of the GST/GST-fusion
proteins to the agarose was confirrned by releasing the bound proteins upon treatment
with 20 rnM glutathione (Fig. 4.6) overnight at 21°C. The cleavage of the GLüT 4 hsed
proteins fiom the GST was confinnecl by treatment of the coupled beads with 0.2 m g / d
thrombin (Fig. 4.7) overnight at 21°C. The beads and eluates were subjected to SDS-
PAGE.
4.4 DISCUSSION
The activity of the glucose transporters has been extensively studied and is
believed to be regulated in many ditferent ways (Olson et al., 1996; Thorens et al., 1996).
Figure 4.5 Solubüizatioo of the GST-G4L Fusion Proteins
Cell pellet from PX-induced E.coli BL-21(DE3) transfomed with pGEX-G4L
Clone 42 was harvested and sonicated as described in Section 4.2.9. The particulate
m i o n was solubilized using 10 rnL of 1, 2, 5 and 8 M urea at 2 1°C for 1 hr. The
solubilized proteins (S) were separated from the particulate fiaction (P) by centrifugation
and resolved on a 10% SDS-polyacrylamide gel stained with Page Blue 83. The 33 kDa
GST-G4L protein was apparent in this study. The outside lanes contain the LMW
standards in kDa.
P S P S P S P S u u u u
Figure 4.6 Elution of GST-G4L and GST-G4C Proteins From GIutathioncAgarose
Glutathione-agarose beads were coupled with protehs from transformed bactena
harbouring the GST, G4C, G4Cmut, and G4L proteins. The beads were treated with or
without 20 mM glutathione oveniight at 21°C. The eluted proteins were recovered by
centrifugation. Panels 4 B, and C depict Page blue-83 aained SDS-polyacrylamide gels
loaded with beads and eluates from various samples of coupled beads. The outside lanes
indicate the LMW protein standards in ma.
control treated contml treated u GST u
G4C
controt treated control treated
u GST u
G4Cmut
control treated conîrol treated u GST u
G4L
Figure 4.7 Cieavage of GST/GST-Fusion Proteins From Coupled Glutathione-
agarose Beads
Glutathione-agarose beads were first coupled with GST, G4C, GKrnut, and G4L
proteins. These beads were treated with or without 0.2 mg/mL thrombin oveniight at
21°C. The eluted proteins were recovered by centrifugation. Proteins were resolved on a
10% SDS-polyacrylamide gel and stained with Blue PAGE 83. A 33 kDa G4L protein,
the 3 1 kDa G4C and G4Cmut proteins and a 26 kDa GST protein were apparent in the
untreated (control) samples. A 23 kDa GST protein fiagrnent was apparent in the
thrombin-treated samples. The outside lanes indicate the LMW protein standards in kDa.
u u u u GST G4L G4C G4Cmut
Processes that regulate their gene expression, intrinsic activity, intracellular trafncking,
and stability, may involve the interaction of specific proteins with the GLUT transporters.
Merent regions in the GLUT 1 and GLUT 4 transporten are knom to function in
regulating changes in these processes (Ishii et al., 1995; Sleeman et ai., 1995; Olson et al.,
1996; Trocino et al., 1994; Wilson et al., 1994; Verhey et al., 1994). A number of
observations indicate the importance of the N- and C- termini and a large central loop
domain as possible molecular determinants for regdation of transporter targeting to the
plasma membrane (Liu et al, 1995). These regions may serve as binding sites for specific
cytosolic regdatory proteins of the GLUT transporters which in tum elicit changes in
these processes. To study two of these functional regions, this Chapter describes the
construction, expression and purification of glutathione-S-transferase (GST)-fusion
proteins containing either the central loop domain or carboxyl terminus region of the
GLUT 4 transporter.
The central loop (G4L) and C-teminus (G4C) regions of the GLUT 4 were
amplifieci and ligated into the pGEX-KG expression vector (Figs. 4.1 and 4.2). Several
clones were içolated and confirmed by restriction digestion and sequencing studies so they
contain the correct DNAs (Fig. 4.3). Severai bacterial strains were transformed with the
plasmid DNAs of the GST or GST-fusion proteins, and their optimal growth conditions
were detennined (Fig. 4.4). The optimal conditions were:- E-coli strain BL-21(DE3)
transformed with plasmid DNAs of the GST and GST-fusion proteins, grown in TB
media, induced with O. 1 mM IPTG at 30°C for 3 hn (Fig. 4.4).
Several mutants were identified during our screening of the GST-G4C clones.
GSTG4Cmut Clone 19 contains a mutation at position 488 of the GLUT 4 protein.
Sequencing studies of this mutant revealed a Ser a8 change to a Pro "'. This serine is
adjacent to the dileucine motif identified in the carboxyl tenninus of the GLUT 4
transporter. In other words the S U motif has been replaced by a PLL. The S U motif
has been postulated to play a role in GLUT 4 intracellular trafficking; as this region is
thought to be unmasked by insulin stimulation (Petrush et al., 1996; Verhey et al., 1994;
Wang et ai., 1996). The regdatory proteins involved in this unmasking have yet to be
identified. It is also not clear whether the phosporylation of the serine and/or the presence
of the two adjacent leucines in this region are responsible for the insulin-mediated effect.
This mutant (GST-G4mut) can be used as a tool to determine the significance of the serine
site.
Unlike the GST, the GST-G4C and the GST-G4Cmut proteins, the GST-G4L
fusion protein was associated with the particulate £?action after sonication (Fig. 4.4). It
could be solubilized using 8M urea (Fig. 4.5). The insoluble nature of the G4L fùsion
protein is suprising in view of the fact that G4L is thought to be the large cytoplasmic
region of the GLUT 4 transporter and there are no apparent stretches of hydrophobic
amino acids in this region. To understand the insoluble nature of this peptide, we
examined the secondary structure of G4L. Ten different methods (ExPasy program (http:
expasy.hcuge.ch/tools.htd)) (Roa et al., 1994; Garnier et al., 1996; Geou jon et al.,
1995) were used to predict the secondary structure of the G4L. These studies revealed
that the G4L region was likely comprised of two helical stmctures, separated by a loop
region (Fig. 4.8). Helical wheel presentation (Tempe1 et ai., 1995) predicted that both
helices adop ted an a-helical amp hiphilic structure: -non- polar arnino acids were distributed
on one side of the helix whereas polar amino acids were on the other side of the helix (Fig.
4.9). The presence of these two arnphiphilic helices may explain why G4L exists in an
insoluble form. Similar amphiphilic helices are also observed in the central loop of the
GLUT 3 transporter (manuscript submitted for publication). Analysis of the secondary
structure of the G4C region did not reveal any a helical structure (Fig. 4.8).
The GST and GST-fusion proteins were p d e d by wupling to glutathione
agarose. Their elution from the agarose beads was performed by the addition of 20 mM
glutathione (Fig. 4.6). The proteins wuld also be cleaved fiom the GST by digestion with
thrombin (Fig. 4.7).
The GST-fusion proteins generated fiom this study can therefore be us& to
identifL and to isolate proteins that can interact with loop or carboxyl terminal regions of
the GLUT 4 transporter.
Figure 4.8 Predicted secondary structure of the GLUT 4 central loop and carboql
terminus.
This indicates the secondary structure of these regions as predicted by the PHD
program. H, L, and E denote helical, Ioop and sheet structures, respectively.
1. STRUCïURE OF THE GLUT 4 CENTRAL LOOP (C4L)
HELIX #1
(3 PARKS LKRL
H HHHHH HHHH
Reliability Index of Prediction
LOOP #l HELlX # l LOOP #2 HELlX #2 LOOP #3 r------- 4 rœœ--œ-am-mœœœ-
1 N i -
m 4 - C 1 O L--A--=: 1 1 s" 60
m&r.l FLLI RGTAD VTILEF
CLUT3 FLLl WGTQD VTVLEF (variable)
LOOP #2
T QWAD
. LLL.
1 7653
G.l.uu YLYl TOWAD LSLLQLL
- - - -
99986 36626 8 4 2 s
II. STRUCTURE OF THE CLUT 4 CARUOXYL TERMINUS (C4C)
HELIX 12
V SDALA RLKDB KRKLB R
H HHHHH HHHHH HHHHH H
6 99999 99967 99999 7
- -- B 99999 9997
1 SECTION #1
LWP # J
ERPL S L W L WSRT HRQP
.LL. . . . . . . . . LL LLLL
1551 1 2 3 3 4 13467 7099
OLUT 4 CARBOXYL TERMINAL SEQUENCE
PHD Prediction
Reliability Index of Prediction
SECTION # 2
DQJSA TFRRT RVPQT RaRTF
LLLLL LLL..
99999 95533
SIICI'ION # 1 SISGI'ION #2 SECWI'ION H3
N C l O 20 30 4 0
G . J d u DEISAGFRQG GASQSDK
GLuu EDITRAFEGQ (Vnrinble ) - Identical scquencc
GLUU DQISATFRR?' PSIAI XQ Isoform-speci fic scqucnce
SECTION #3
PSLL8 QEVKP STELE YWPD PHD
. . . . . . . . .L LLL.. . . . LL LL.EE B.LLL LLL
Figure 4.9 Helical-wheel presentation of the predicted helical structure of G4L.
This is a helical wheel presentation of Heik #1 and Helix #2 of the centrai Ioop
region of the GLUT 4 transporter.
CHAPTER 5 - SUMlMARY
The sequences and structural aspects of the glucose triinsporten (GLUTs) have
been extensively examined for severai years. These studies have identified seven different
GLUT isoforms (GLUT 1-7) with a high degree of amino acid sequence similarity in the
TM domains (Bell et al., 1993; Zeng et al., 1996). The various GLUT isoforms difKer in
the length and sequences of the amino and carboxyl temini, the large intraceliular
hydrophüic loop region and the exofacial loop comecting TM1 and TM2 (Fig. 1.1).
These divergent (isoform-specific) domains are thought to play a role in tissue-specific
regulation of glucose transporters.
Al1 mamrnalian ceIls contain at lest one GLUT isoforrn. However, Iittle is known
about the fundion and regulation of these isoform(s) within each tissue type. Recent
studies indicate that the expression of GLUT isoforms is uniquely regulated dunng the
process of myogenesis (Xia et al., 1993; Mitsumoto et ai., 1991; KIip et al., 1992).
Isoforrn-specific arnino acid sequences are thought to be important in determining the
subcellular location and tùnctional state of GLUT transporters. These sites may function
as sites of interaction for proteins involved in regulating the intrinsic activity, intracellular
trafficking and/or stability of the transporters (McGowan et ai., 1995).
The objectives of this thesis were two fold. The first part of the thesis exarnined
the regulation and function of the glucose transporier isofonn 3 (GLUT 3) and the
properties of GLUT 3- mutants. This was studied by monitoring the ability of GLUT 3-
mutant ce11 lines to form myohibes and to express myogenesis-associated components in
the presence of exogenously expressed GLUT 3 or myogenin.
GLUT 3- mutants expressing similar low GLUT 3 transcript levels (Fig. 2.1) were
found to Vary in their myogenic ability (Figs. 2.1 and 2.3). These studies suggested that
components, in addition to the GLUT 3 isofom, might be dtered in these myogenesis-
defdive mutants. To examine the direct role of GLUT 3 in myogenesis, attempts were
made to see if increased GLUT 3 expression couid rescue the myogenic ability of these
GLUT 3- mutants. To control the arnount of GLUT 3 expressed in these mutants, the
GLUT 3 cDNA was placed under the wntrol of a dexamethasone-inducible promoter.
This was then transfected into GLUT 3' mutants, D2 and D23. Dexamethasone-induced
D23/GLUT 3 transfectants were found to possess at least 40PA more GLUT 3
transporter than nomal L6 ceUs. Although they were able to form multinucleated
myotubes, the nision index observed in these ceUs was ody 5-7% of the day 6 L6 levels.
The extent of fusion was so low that the expression of myogenesis-associated genes wuld
not be detected (Figs. 2.5, 2.13 and 2.1 5). This observation was sirnikir to that observed
with L6 transfectants overexpressing the GLUT 3 transporter (L6/G3S) (Broydell et al.,
1997). Thus in agreement with previous studies, Our present study using transfectants
shows that a critical level of the GLUT 3 transporter may be required for myogenesis.
Further work is required to determine the level of GLUT 3 expression essential for
activating myogenesis.
To fùrther explore the mechanisms leading to the myogenic defects observed in
mutant D23, this mutant was transfected with constructs wntaining the myogenin
promoter or the myogenin coding sequence. While the parental L6 myoblast exhibited
very active myogenin promoter activity, this activity could hardly be detected in mutant
D23 (Fig. 3.1). This indicated that mutant D23 was defective in factor(s) required for the
myogenin promoter activity. This rnight explain why myogenin expression could not be
detected in mutant D23.
If the observeci lack of expression of muscle-specific proteins in mutant D23 was
solely due to the absence of myogenin, then one would expect increased level of myogenin
should restore the mutant's ability to express MLC, MHC, and TnT, and to fom
myotubes. To explore this possibility, mutant D23 was transfected with a construct
wntaining the myugenin coding sequence. This cDNA was placed under the controi of a
pgk prornoter. Both Northem blot and irnrnunofluorescence studies revealed that the
D23/myogenin transfectants possessed significant levels of myogenin and its transcript
(Figs. 3.3 and 3 S). Despite their very high myogenin levels, these transfectants were still
impaired in their ability to express muscle-specinc genes (Figs. 3.3 and 3.7) and to fonn
myotubes (Fig. 3.7). This study clearly indicated that mutant D23 was defective in
wmponents, in addition to myogenin, required for the transcription of muscle-specific
contractile protein genes. In agreement with this observation, we have previously shown
that the myogenic defects of the L61G3S transfectants were due to their inability to
express muscle-specific genes, and not due to insufficient myogenin (Broydell et al.,
1997).
The above studies suggest that mutant D23 is dtered in components acting on at
least two different sites of the myogenic pathway in rat L6 myoblasts. The fira site is
associated with the activation of myogenin transcription, whereas the second site is related
to the transcription of muscle-specific protein genes. Factors acting on the second site are
required even in the presence of myogenin. These results dong with previous shidies
using L6 transfectants overexpressing GLUT 3 suggest that the level or fwictional state of
these factors are very sensitive to the elevated level of the GLUT 3 transporter.
A tentative working model (Fig. 5.1) can be postulateci to explain the regdation of
the myogenic pathway and the potential regulatory role of the GLUT 3 transporter.
Factor R is postulated to be involvesi in regulating the expression of GLUT 3 and the
expression of two other factors, M and S. Factor M is essential for the rnyogenin
promoter activity, whereas Factor S is required for the expression of muscle-specific
proteins such as MLC, MHC and TnT. It may act in conjunction with myogenin to allow
transcription of these muscle-specific genes.
As indicated in Chapter 2, even though mutant D2 has very low GLUT 3 level
(Fig. 2.1), it still retains significant levels of myogenin and muscle-specific proteins, and
the ability to form myotubes (Figs. 2.2 and 2.3). According to Our working model, this
mutant is tikely mutated in Factor R, such that GLUT3 expression is suppressed, whereas
the expression of Factor M and Factor S is still permissible. This may explain why this
mutant still possesses substantial arnounts of the myogenin, M C , and TnT transcripts
(Figs. 2.3) and the ability to form myotubes. The reduced rates of myogenesis are
probably due to reduced GLUT3 expression (Fig. 2.2).
According to Our working model, mutant D23 is Wtely defective in Factor such
that the expression of GLUT 3, Factor M and Factor S is abolished. This may explain
why this mutant is defective in the myogenin promoter activity (Fig. 3.1), and in the
transcription of muscle-specific genes, even in the presence of increased level of myogenin
(Fig. 3.3).
This model can also explain the properties of L6/G3A and L6/G3S transfectants
(Broydell et al., 1997). As mentioned in Section 2.1, transfectant L6/G3A contains only
39% of the L6 GLUT 3 level. Even though it possesses a reduced myogenin transcript
Figure 5.1 Tentative WorkÏng Mode1
A schematic diagram of the tentative working mode1 suggesting an explmation for
the defects in GLUT 3 mutants. The D2, D23, L6/G3A and L6/G3S mutants are shown
to have defects denoted by an X. & M, and S represent factors present in regulating the
expression of GLUT 3, myogenin, andor M C , MLC, and TnT.
level, it contains normal levels of the MLC, MHC, and TnT transcripts, and exhibits
sunilar rates of fusion as L6 cells (Broydell et al., 1997). It is conceivable that this
transfectant contains a reduced level of Factor M, but a normal level of Factor S;
consequently, its rate of fùsion is not iifF'ected. Unlike tmfectant L6/G3q transfêctant
L6/G3 S possessed not ody reduced trawcript levels of myogenin, but also much reduced
levels of muscle-specific transcripts and rates of fusion. Accordingly, Our working model
predicts that this transfectant contains reduced ievels of both Factor M and Factor S. This
can then explain the myogenic defects of this transfectant. More importantly, these
observations suggest that increased GLUT 3 level may affiect the stability, level, andlor
funaional States of Factor M and Factor S. Needless to Say, fùrther work has to be
carried out to identify and to isolate these three factors before one can veriQ this working
model.
Proteins involved in regulating the subcellular location, stability a d o r intrinsic
activity have been poshilated by various workers (Czech et ai., 1992; Hamison et al.,
1991). Different regions in the GLUT 4 transporter are known to fundion in regulating
changes in its gene expression, intnnsic activity, intracellular trficking, and in
maintainhg its stability (Xshii et ai., 1995; Sleeman et al., 1995; Olson et al., 1996; Trocino
et al., 1994; Wilson et al., 1994; Verhey et ai., 1994).For exarnple, the activity of the
GLUT 4 transporter is altered by its interaction with Ca* -controlled protein hases,
phosphatases (Remch et al., 1993), CAMP (Piper et ai., 1993), and the Rad protein
(Moyers et al., 1996). Further, changes in the intrinsic activity of the GLUT 1 and GLUT
4 transporten were brought about by the binding of cytoplasmic factors (28 and 70 kDa)
to the carboxyI termini of these transporters (Dauterive et al., 1996; Shi et al., 1995). A
70 kDa cytosolic protein was found to bind to the GLUT 4 central loop in an ATP-
sensitive marner (Liu et al., 1995). These studies indicate that interaction of GLUT
transporters with cytoplasrnic proteins is likely to play an important role in mociulating the
activity of the GLUT transporter.
The second part of the thesis was to c o m a GST-hsion proteins containhg the
central loop or the carboxyl terminal regions of the GLUT 4 transporter. The purpose of
this work is to generate tools that can be used to identify and to isolate components that
wi interact with the GLUT transporters. GST-fusion proteins of these regions were
constmcted and expressed in E.coli BL-21@E3). Purifieci fragments of the GLUT 4
central loop and carboxyl terminai regions were successfully obtained (Chapter 4).
During the preparation of this thesis, binding studies using the central loop and
carboxyl terminai regions of the GLUT 4 were perfomed by Pat Teimer in Our laboratory.
Seven rat myoblast proteins were found to bind consistently and specifically to the GST-
G4L fusion protein, but not the GST-G4C fusion protein, nor to the GST protein alone
(Maauca et al., manuscript subrnitted for publication). These studies also revealed that
the binding of some of these proteins to the G4L region was enhanced upon chronic
insulin treatment of L6 cells. These findings codirm the use of the GST-tiision proteins as
a tool for studying the interaction of cytoplasrnic components with the G L U 4
transporter.
The interaction of specific proteins to the central loop and carboxyl terminus of the
GLUT 3 transporter was also recently examined (Abidi et al., manuscript submitted for
publication). Six different rat myoblast proteins were able to bind specificdy to the
central loop domain but not to the carboxyl domain of the GLUT 3 transporter. The
identification and characterization of these various proteins d help to understand the
role of GLUT transporters in regulating various metabolic processes in the L6 myoblasts.
In sumrnary, work presented in this thesis revealed several interesthg aspects of
the relationship between the GLUT 3 transporter and the myogenic pathway. While more
work is required to test our working hypothesis, Our studies do reveal the possible role of
the GLUT 3 transporter in modulating components required for the myogeniin promoter
aaivity, and for the transcription of muscle-spdc genes. We have also construaed
GST-fusion proteins that cm be used for identifjhg and isolating proteins which wi
interact with the GLUT transporters.
Appendix 1. Sample Caiculations of Transcript Leveis From Raw Data
Each band was scanneci using the Molecular Dynamics Phosphorimager System
and measured using ImageQuant software (Molecular Dynamics). To assess the
background levels for each sample, the volume reading of another area was also taken.
The Sample Volume represents the volume (integrated pixel intensity of a band) less the
measured background volume (most m u e n t pixel intensity of a band). To control for
over or under loading of the mRNA ont0 the gel, the transcript levels in each sample
(Sample Volume) was normaiized acwrding to the amount of P-microglobulin (BMG)
present (Corrected Volume). To determine the relative transcnpt levels, day 2 L6
transcript level was taken as 10P! (% Volume). The standard deviation, standard error
and the % average are calculateci eom three different northern blots (see enclosed charts).
Probe: Myogenin 2 L6 D23 D2 D9
2 6 2 6 2 4 6 2 4 6 Sample Volume 282439 66 1029 1624 346 451488 180580 586014 52708 228043 544545
%BMG 86% 142% 103% 65% 113% Correcteci Volumes 282439 586930 381 7 285 224326 197381 433566 49160 335760 523308
%Volume 100% 208% 1% 0% 79% 70% 154% 17% 119.h 185%.
Sample Volume %BMG
Corrccted Volumes %Volume
Probe: Myogenin 3
'standard ~eviatio Standard Emr % Avera~e
0.00% 0.00% 100%
9.17% 5.29% 200%
L6 2
109 19 100%
1 0 19 19 100%
1.53% 0.88%
3%
6 24886 113%
22096 202%
D23 2
162 43%, 380 3%
6 77
122% 63 1%
DZ
0.58% 0.33%
0%
2 15418 201% 7660 70%
D9
6.24% 3.60%
77%
2 1257 107% 1172
1%
13.00%' 7.51%
5%
4 4440 91% 4853 44%
5.03% 2.90%
12%
22.85% 13.19%
128%
6 17066 135% 12626 116%
4 3937 68% 5797 53%
6 11993 104% 11525 106%
55.83% 32.23%
60%
48.09% 27.76%
161%
Appendh 2. Sample Cdculations of Myogeniin Promoter Activity in L6 and D U
Cells
To determine promoter activity in L6 and D23 cells, 8-gai and CAT activities were
performed on ceils transfected with various plasmids (Section 3.2.1). The protein
concentration for each sarnple was determineci. P-gaiactosidase activities were detemiined
by reading the optical densities for each sample at 420 nrn over a period of tirne. The
slope of the values was calculated as OD/rnin/mg of protein (A). The CAT aaivity of
each sarnple was determined as dpdmg of protein (B). The rnyogenzn promoter activity
was calculated as a ratio of CAT activity (£3) : (A) P-gal activity (C). The control sample
(L6KS) ratio represents background and was subtracted fiom each sample ratio. To
determine the relative level of activity (% activity), the L61GSC ratio was taken as 100%
(E). The standard deviation, standard error and the % average of four dEerent transient
transfection studies are show as four charts.
A B C D E
Phte 1
OD/minhilg protein dpmimsprdein
ratio B:A less backpund
% activity
A B C D E
UIKS 0.04662 1 90 17 193403
Plate 3
O D ~ m g p r o t e i n dpmhngprotein
ratio B:A
Staudard Dewiation Standard Etror Average
L6MS 0.022208 16663 750306
less background % activitv
WGSC 0.026329 58088
,. 2206248 20 12845
1W/o
582 1907 1000?%
1.W! 0.58% 1 . W ?
O.W! O.Wh
lOû.W?
WGXC 0.026892 US65 876294 68289 1
34%
W G S C 0.015323 100708 65722 13
DUIGSC 0.034815 53284 15304%
2427287 42%
5.Wh 2.89%
38.Wh
DU/KS 0.030162 10446 346340
DWGXC 0.029872 32308 108 1536
L6/GXC 0.011494 36522 3 177593
2.W! 1.15% 7.Wh
D23/KS 0.012582 13240 1052290
478206 8%
D23/GSC 0.065568 28731 438 183 9 1 843
5%
29246 1%
D231GXC 0.04020 1 14263 354782
8442 O?!
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