Perspectives in...

Perspectives in Diabetes Mitochondria and Diabetes Genetic, Biochemical, and Clinical Implications of the Cellular Energy Circuit Klaus-Dieter Gerbitz, Klaus Gempel, and Dieter Brdiczka

Physiologically, a postprandial glucose rise induces metabolic s&d sequences that use several steps in common in both the pancreas and peripheral tissues but result in Merent events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the p-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase I1 in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site- specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose- phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism a t the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three Merent points: first, through NADH transfer to the respiratory chain complex I via the malatehispartate shuttle; second, by providing FADH, to complex I1 through the glycerol-phosphatefdihydroxy-acetone-phos-

phate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolie and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in p-cells. An interplay between the plasma membrane- bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally a t the KAw channels, coordinated to the activity of mitochondrial CrP production. Closure of the K, channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in p-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cyto- solidmitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nuele- oside diphosphate m e . Pathophysiologically, there are at least two genetically Merent forms of diabetes linked to energy metabolism: the Arst example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM. Furthermore, point mutations in the muscle

From the Institutes of Clinical Chemistry and Diabetes Research (K.-D.G., K.G.), hex~kinase 11 have recently been described in some Academic Hospital Schwabing, Miinchen, and the Faculty of Biology (D.B.), NIDDM families, and decreased glycogen synthesis due to University of Konstanz, Germany.

Address correspondence and reprint requests to Dr. Klaus-Dieter Gerbitz, low G-6-P levels has been found as the Arst hint of

Institute fiir Klin Chemie und Diabetesforschung, Krankenhaus Schwabing 80804 peripheral insulin resistance in Miinchen, Kolnerplatz 1, Germany. spring of diabetic parents. Thus, we postulate that ge-

Received for publication l May 1995 and accepted in revised form 5 October 1995. netic defects at quite different sites of energy metabolism CK, creatine kinases; CPEO, chronic progressive external ophthalmoplegia; CrP,

phosphocr~:atine; G-&P, glucose-&phosphate; KSS, Kearns-Sayre syndrome; MDM, Can lead to diabetes and that, in some forms NIDDM, a mitochondrial diabetes mellitus: MELAS, mitochondrial mvowathv, ence~halowa- COlmnOn defect in the cytosolic-mitochondria1 interplay thy, lactic acidosis, stroke-like episodes; MERRF, myoclonic epileby and raked of energy production c& result in both impaired ins-& red fiber disease; Mi-CK, mitochondrial creatine b a s e ; MnSOD, manganese secretion in the ~ - ~ ~ l l and peripheral resistance against superoxide dismutase; MODY, maturity-onset diabetes of the young; mtDNA, mitochondrial DNA; NMR, nuclear magnetic resonance; OXPHOS, oxidative phos the in and tissue. Diabetes phorylation; PCR, polymerase chain reaction. 45113-126,1996

DIABETES, VOL. 45, FEBRUARY 1996 113

T wo forms of the heterogeneous disorder termed NIDDM, which have been genetically defined recently, are caused by defects in energy metabolism. Mutations in the glucokinase gene are

responsible for one form of maturity-onset diabetes of the young (MODY2) (I), and the other type, mitochondrial diabetes mellitus (MDM), is associated with mutations of the rnitochondrial DNA (mtDNA) affecting subunits of respiratory chain complexes (2). These two examples demonstrate that derangements at quite different sites of the cellular energy metabolism can be involved in the pathogenesis of diabetes. It is thus pertinent to revise the current knowledge on the interplay between cytosolic and mitochondrial energy metabolism with respect to possible pathogenic key positions and with special regard to mtDNA defects.

PHYSIOLOGICAL ASPECTS OF MITOCHONDRIAL ENERGY METABOLISM IN PANCREATIC P-CELLS AND IN PERIPHERAL TISSUES 0-cell Mechanisms that w e c t insulin secretion in response to blood glucose increase. According to a generally accepted scheme, insulin secretion in response to postprandial glucose elevation depends on a sequence of metabolic events: 1 ) the uptake of glucose through the GLUT2 transporter, 2 ) phosphorylation of glucose by glucokinase, 3 ) production of NADH and pyruvate by glycolysis, and 4) stimulation of mitochondrial oxidative phosphorylation (OXPHOS). As a consequence of the fourth event, the ATP level rises, leading to closure of ATP-dependent K+ channels. The resulting depolarization of the membrane potential is followed by the opening of ca2+ channels. The increased intracellular Ca2+ triggers insulin secretion (Fig. 1). Uptake and phosphorglation of glucose. GLUT2 is the major, if not the only, glucose transporter in the p-cells (3). The K, of GLUT2 (17 mmoY1) (4) is significantly higher than that of the ubiquitous GLUT1 or the fat and muscle tissue- specific glucose transporter GLUT4. However, experimental data demonstrate that glucose uptake exceeds glucose con-

sumption 100-fold. Because GLUT2 is not rate-limiting, glucokinase (hexokinase N), which has a high K,,, for glucose (>5 mmoH) (6), seems to be the sensor of blood glucose fluctuations in the sequence of metabolic events. Patients suffering from MODY2 cany mutations of the glucokinase gene and are heterozygous in any case. In these patients, a 50% reduction of glucokinase activity in the p-cells causes this subform of NIDDM (1). These and other data suggest that activation of glucokinase by glucose >5 mmoH plays an important role in insulin secretion (5,6). This is confirmed by expression in murine p-cells of yeast hexokinase. Transgenic mice demonstrate an increased insulin secretion and a 50-80% decrease of blood glucose level compared with control animals (7). Stimulation of OXPHOS. Besides appropriate glucokinase - - - function, several lines of evidence indicate that normal mitochondrial oxidative activity is a further important pre- requisite of insulin secretion. First, as discussed below, diabetes is often associated with mitochondrial diseases characterized by defects of the mitochondria1 genome (8). Second, in isolated mouse islets, high glucose stimulates the oxidation rate, and inhibition of OXPHOS inhibits insulin secretion (9,lO). The mechanism by which OXPHOS contrib- utes to insulin secretion is still not fully explained. Blocking of a Kf channel by ATP is assumed to be responsible. The closure of the Kf channel results in cell membrane depolarization followed by subsequent influx of ca2+ and stirnula- tion of insulin exocytosis (11). However, the problem with this hypothesis is that changes of the ATPIADP quotient are not consistently found to correlate with insulin liberation. For instance, Ghosh et al. (12) observed no fluctuation of the ATP-to-ADP ratio in the perfused rat pancreas upon provok- ing a 10-fold increase in insulin release by 8 mmoH glucose. In contrast, Henquin et al. (13) described a good correlation in mouse islets between ATPIADP quotients and insulin release when stimulated by 30 mmoH Kf. It is, therefore, still open whether ATP is the direct signal coupling mitochondrial function and insulin secretion. In fact, nuclear magnetic

FIG. 1. How mitochondrial metabolism may be involved in insulin secretion. Glucoae enters the cell through a specific transporter (GLUT2) and stimulates binding of GK to the mitochondrial pore protein. Because this binding activates GK, increased amounts of G-6-P and ADP are formed. The ADP is transferred across the two boundary membranes through the outer membrane pore (P) and the adenylate translocator (AT) in the inner membrane into the mitochondrial matrix, enhancing OXPHOS. Increased phosphorylation of glucose provides substrate for the glycolytic pathway, resulting in elevated production of substrate (NADH) for the mitochondrial metabolism and phosphoenol pyruvate (PEP). Reducing equivalents from external NADH are transported into mitochondria through the glycerol phosphate shuttle (S l ) o r malate-aspartate shuttle (52). Pyruvate kinase (PK) forms a diazyme complex with CK, and phosphate is directly transferred from PEP to creatine (Cr) yielding CrP. The increase in CrP may change the local concentration of ATF' a t the K+,, channel (K-Ch) through CK activity bound t o the cell membrane. Alternatively, CrP may be produced by activity of Mi-CK from mitochondrial ATP. The preferential export of mitochondrial ATP and uptake of ADP by the AT depends on the membrane potential. Mutation of mtDNA results in defects of the mitochondrial electron transport chain and a reduced activity of oxidative phosphorylation. The local ATF' increase by CK activity closes the K', channel. This leads to depolarization of the membrane potential and opening of Ca2+ channels (Ca-Ch). The increase in intracellular Ca2+ is followed by insulin liberation.

114 DIABETES, VOL. 46, FEBRUARY 1996

resonance (NMR) measurements in excitable tissues (brain, heart muscle) showed only small fluctuations of the ATP and ADP levels due to stimulation (14,15). As many systems in the cytosol depend on the high free energy of ATP, hydroly- sis regulatory mechanisms should exist avoiding large changes of the ATP-to-ADP ratio, even during increased ATP turnover. Coordinated regulation of glucokinase and mitochondrial oxidative metabolism. Although the exact mechanism of insulin secretion is not known, it can be stated that both activation of glucokinase and stimulation of OXPHOS are the two fundamental metabolic prerequisites. Both mechanisms are functions of the recently observed complexes that assemble at the mitochondrial surface between hexokinases and the mitochondrial pore protein porin, resulting in the activation of the enzymes. This leads to the question of whether glucokinase also binds to the mitochondrial surface in p-cells. Binding of glucokinase to the mitochondrial surface. Four hexokinase isozymes are present in different mammalian tissues (16). Comparison of the isolated cDNAs and of amino acid sequences of hexokinases 1-111 revealed that the hexokinases are essentially dimers of glucokinase (17). The hexokinase isozymes 1-111 have a M, of 100 kDa and a K, for glucose in the range of 0.1 mmoVl and are inhibited by physiological concentrations of glucose-6-phosphate (G-6-P). Glucokir~ase, present in liver and p-cells, differs from the other hexokinases by molecular mass of 50 kDa and a K, for glucose of >5 mmoM; it is not inhibited by G-6-P (18). In contrast to that in liver, glucokinase in p-cells is bound to mitochondria (19,20). This tissue-specific difference in glucokinase behavior may be the result of differential splicing of mRNA coding for the NH,-terminus (21). The amino terminal part of hexokinase I was found to be responsible for the binding to the mitochondrial surface (22). However, it appears that the properties of the NH,-terminal sequence of p-cell gl.ucokinase are more comparable to hexokinase isozyme 11. Given that glucokinase in p-cells binds to the mitocho~~drial pore protein, as was observed for hexokinases 1-111 (23,24), this may serve to regulate glucokinase activity. Peripheral tissues Activation of hexokinases by binding to the outer mitochondrial membrane pore. Hexokinase I1 is the predominant isozyme in insulin-sensitive tissues (skeletal muscle, adipose tissue) (16,18). Insulin increases the activity of hexokinase I1 in the mitochondrial fraction in fat pad (25), skeletal muscles (26), and cardiac muscle (27). Also in these tissues, glucose in the absence of insulin stimulates the binding of this isozyme to mitochondria (25,26). Thus, the insulin effect is possibly mediated by an increase in intracellular glucose (26). The association of hexokinase I1 to the surface of muscle mitochondria leads to an activation of the enzyme (26). Comparable results yielding a 5- to 10-fold activation are obtained in binding studies of isolated hexokinase isozymes I and I1 to isolated liver mitochondria (28). In isolated hepatocytes, glucose induces a twofold increase of total hexokinase activity linked to membrane association (29). Induction of hexokinase 11 by insulin. Besides its indi- rect effect through glucose-induced mitochondrial binding, insulin also exerts a direct effect on hexokinase activity. As observed by Katzen (30), insulin induces hexokinase I1 in insulin-sensitive tissues such as muscle and adipose tissue.

In agreement with this, diminished HK I1 mRNA is found in adipose tissue from diabetic rats but is restored to control levels by insulin treatment. Insulin also induces hexokinase I1 mRNA in two adipose cell lines and two skeletal muscle cell lines (31). In view of these findings, insulin resistance in NIDDM was explained by such reduced hexokinase expression, which might develop in parallel to impaired liberation of insulin in NIDDM (see below) (32). Formation of specific hexokinase-porin complexes at the mitochondria1 surface. The location of hexokinase I at the mitochondrial surface was studied by electron micros- copy using immunogold labeling methods. In liver (28,33) and brain (34) mitochondria, the enzyme is concentrated at sites where inner and outer membranes are attached. In contrast, mitochondrial porin as the specific binding site for hexokinases 1-111 is randomly distributed in the outer membrane. This disagreement can be explained by a higher hexokinase affinity to the isolated contact sites (33,35). The specificity of hexokinase binding at the contact points between the inner and outer membranes was recently analyzed in more detail. In these studies, hexokinase I binding to intact liver mitochondria varying in frequency of contact sites and to isolated outer membranes are compared (36). It was observed that, in contrast to binding at the outer membrane, binding at the contact points and activation of the enzyme is a cooperative process. The observed cooper- ativity suggests formation of oligomeric hexokinase complexes at the mitochondrial surface as was described by Xie and Wilson (37). By using cross-linking, these authors showed tetramer formation of the enzyme upon binding to the mitochondrial membrane. In agreement, it is possible to generate tetrameric hexokinase-porin complexes in vitro (38). This in vitro interaction with porin results in a similar activation of the enzyme as that observed by binding to contact sites in intact mitochondria, and the interaction led to complexes of a molecular mass of 400 kDa (38). In view of these findings, a complex between a specific structure of the pore and the adenine translocator in the contact sites is postulated that has a higher affinity for hexokinase (39). The structural expression of these complexes may be the contact sites that have been described and analyzed in freeze- fractured mitochondria (40,41). Stimulation of OXPHOS by peripheral kinases such a s hexokinase. Bessman and colleagues (42) provided evidence that the mitochondrially bound hexokinase preferen- tially uses intramitochondrially generated ATP. On the basis of these results, the authors developed the hexokinase- mitochondrial binding theory of insulin action (43). Recent investigations in this field led to the conclusion that the - mitochondrial binding of hexokinase appears to be more important in supplying ADP to the OXPHOS than to provide ATP for glucose phosphorylation (36,44,45).

THE CELLULAR ENERGY CIRCUIT When glucose is substrate, two energy-providing mechanisms, fermentation or endoxidation to CO, and H20, are alternatives that need coordinated regulation. Besides a number of regulatory metabolites, such as NADH, phosphate, and ca2+, ADP as energy acceptor is certainly one of the important substances. However, as already stated above, the level of free ADP is kept very low during various functional states to avoid dysfunction of energy-consuming

DIABETES, VOL. 45, FEBRUARY 1996

Brain CreaUne induces

Repolanzation phosphory(aHon and uptake of glucose

A

Glucose @ O ~ ~

ATP

Muscle Glucose induces production of UTP and glyoogmn

PGell

Glucose induces local production of

GPD c r phosphosreaune and ATP

QGmw

ATP

FIG. 2. Schematic representation of different ways by which oxidative phosphorylation fits into the cellular energy network. The intramitochondrial ADP is indirectly increased by the activity of peripheral kinases. A ) In case of rapid ATP turnover upon stimulation of muscle or nerve cell activity, the level of Cr increases and stimulates OXPHOS through activity of Mi-CK. This leads to parallel increase of glucose phosphorylation and uptake by activation of hexokinase (HK). B ) In case of p-cells, intramitochondrial ADP rises via bound GK. This stimulates oxidative phosphorylation and leads to higher output of CrP. CrP may lead to high ATP levels close to the K+, channel through cell membrane-associated CK. C ) In case of an increased level of glucose in skeletal muscle, mitochondrial binding and activation of hexokinase I1 is induced. Through the activity of hexokinase 11, intramitochondrial ADP increases and stimulates oxidative phosphorylation. The latter may lead to increased UTP production via mitochondrial nucleoside diphosphate kinase (NuDiKi). Both G-6-P and UTP would stimulate glycogen synthesis. In the contact sites, hexokinase and mitochondrial CK (which is a dimer in the inactive state) form tetramers and octamem respectively. P, porin; AT, adenylate translocator; IM, inner membrane; OM, outer membrane.

systems (39,46). OXPHOS stimulation may thus be per- high ATP turnover during excitation increases ADP levels formed indirectly through substrates of specific kinases that only locally but increases the creatine level (instead of ADP) are organized at the mitochondrial surface and directly globally. The latter would then serve as a signal to stimulate communicate with the inner mitochondrial compartment mitochondrial metabolism (51). To overcome diffusion limi- (47-49). This mitochondrial coupling of kinases, such as tation, the creatine1CrP pool (25-30 mmoyl) is advantageous hexokinase and creatine kinase (CK), may accomplish an because of its approximately five times higher concentration important function in permanently working muscles like the compared with the adenine nucleotide pool (5-8 mmoyl). heart or in nerve cells that depend more or less on blood- The CrP shuttle. Cytosolic and mitochondrial CK (Mi-CK) b o n e substrates. In these tissues, the coupling mechanism are found in excitable tissues such as brain and muscle. The may regulate the uptake of energy-providing substrates (glu- cytosolic isoforms occur as dimers and are BB-CK (found in cose) according to the mitochondrial activity (Fig. 2A). On cells of the neuroendocriniurn), MM-CK (muscle specific), the other hand, the same organization of hexokinase may be and a dimer of both of these, MB-CK (mainly present in important in p-cells to stimulate mitochondrial metabolism cardiac muscle). The equilibrium constant of the reaction according to extramitochondrial substrate (glucose) concen- catalyzed by CK favors ATP synthesis. An interplay between tration (Fig. 2B) (50,51). the cytosolic CK isozymes bound to the energy-consuming Role of phosphocreatine (CrP) in energy homeostasis. ion pumps, and the Mi-CK is postulated to provide an Membrane depolarization and repolarization during excita- energy-shuttling mechanism. In this shuttle, the latter kinase tion, muscle contraction, and relaxation as well as ca2+ is linked to the adenylate translocase in the inner mitochon- release and sequestration are processes with a time scale of drial membrane and plays the part of energy production. milliseconds, while the synthesis of glycogen and proteins Coupling of cytosolic CK t o energy-consuming and proceeds in seconds. In the cases of high energy consump- energy-providing processes. In a variety of tissues, CK tion, ATP may be limiting at the site of its utilization. To isozymes are subcellularly organized. In skeletal muscle, CK avoid large ATPIADP fluctuations during the former fast and is associated with ATP-requiring processes such as Ca2+- energy-consuming processes, creatine1CrP is used as an ATPase (52,53), Naf/K+-ATPase (54), and actomyosin ATPase. energy buffer and energy-transferring system. By this way, In the latter case, MM-CK is specifically located in the M-line

116 DIABETES, VOL. 45, FEBRUARY 1996

Mia Dlrner

Octamer

Br Ins Ht

MM Mi-CK Ins [kDal -

_start MM Mib Dlrner

Octarnel

FIG. 3. Characterization of isozymes of CK in tissue extracts from rat insulinoma cells compared with rat brain and heart tissues. A: the isozymes were separated on cellulose acetate strips according to the method of Marcillat e t al. (152) using a buffer of 50 mmoyl sodium barbital arid 2.7 mmoyl EDTA (pH 8.8). The cellulose acetate strips were soaked in the buffer for 20 min and were then blotted free of excess buffer with Alter paper. The samples were applied in the middle of the strips and were run for 45 min at 3 mA in a Boskamp chamber. After electrophoresis, the isozymes were visualized by incubation of the strips in a medium containing 0.6 mmoVl glycyl-glycine (pH 6.0), 0.04 mmoyl Mg-acetate, 12 mmoyl creatine phosphate, 3 mmoyl ADP, 1 mmoyl NADP, 10 mmoVl AMP, 10 mmoyl Di-adenosine-pentaphosphate, 0.5 mmoyl nitroblue tetrazolium, 0.2 mmoVl phenazine methosulfate, 20 mmoyl glucose, 5 IU hexokinase, and 3 IU G-6-P dehydrogenase. Lane Br, total brain extract; Lane Ins, extract of a mitochondrial enriched fraction from cultured RIN insulinoma cells; Lane Ht, total extract from heart muscle. The cytosolic isozymes are seen: ubiquitous brain type BB and muscle type MM as well as the heterologeous dimer of both MB. The two mitochondrial isozymes, ubiquitous Mia and muscle-specific Mib, are separated into dimem and octamem. B: an extract of a mitochondrial enriched fraction from cultured RIN insulinoma cells (Ins) and isolated Mia-CK were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptides from the gel were blotted to nitrocellulose and there on immunodecorated by specific antibodies against Mia- and Mib-CK. The antibodies bind to the monomer of isolated Mi-CK (molecular mass of 43 kDa) and to a polypeptide in the insulinoma extract of the same M,, suggesting that these cells contain Mi-CK.

(55). Besides this specific location at energy-consuming sites, MIM-CK is also found to be functionally coupled to g1ycolysi.s and glycogenolysis. This is reflected by high MM-CK activity in the I-band, especially in glycolytic fast- twitch fibers. In the I-band, glycolytic enzymes are loosely associated with the thin filaments (56). CK found in the I-band may be linked to this glycolytic complex through pyruvate kinase. A loose association (diazyme) between the two enzymes has been described, and the kinetic advantage of this linkage is demonstrated in vitro (57). In these 3 1 ~

NMR experiments, saturation transfer shows that the phosphate is exchanged between phosphoenol-pyruvate and CrP without change in intermediate ATP. CK activity in pancreatic islet cells. BB-CK (58) is found in pancreatic islet cells. Rat insulinoma cells (INS1, RINm5F') contain high activity of brain type BB-CK and also moderate activity of Mi-CK, as demonstrated in Fig. 3 by isozyme electrophoresis of extracts from cultured RINm5F cells and by immunodecoration with specific antibodies against both forms of Mi-CK on Western blots. Because BB-CK is elevated

in a number of progressive metastatic tumors (for review see 60), its presence in insulinoma cells may not reflect the situation in normal human p-cells. The energy circuit in p-cells. As depicted in Fig. 1, the Naf /K+-ATPase consumes ATP at the cell membrane, which is also important to close the K+ATP channel. The average cytosolic ATP level is between 5-8 mmoYI. It would therefore seem unlikely that the K+,, channel (K, 20-200 krnoM) would ever open. However, as already stated by Ashcroft (ll) , ATP may be compartmentalized in the cell in such a way that its local concentration close to the channel-binding site is very low. Such compartmentation could be performed in p-cells by linking the K+,p channels to glycolysis and OXPHOS through CrP and CK. The involvement of the mitochondrial metabolism in regulation of the Kf ,, channel is depicted in Fig. 2B. Assuming that Mi-CK is present in human p-cells, glucose stimulates OXPHOS via bound glucokinase to produce CrP. The free ATP is increased at the KfATp channels locally by CrP through a cell membrane- bound CK. Respiratory chain and OXPHOS. Electron flux along the respiratory chain involves membrane-integrated enzyme complexes. Respiratory chain complexes I, 111, and IV repre- sent redox-driven proton pumps that translocate protons from the matrix to the outer side of the inner membrane, thus creating an electrochemical gradient. Complex I oxidiz- ing NADH (NADH:ubiquinone oxidoreductase) consists of at least 40 polypeptides. Seven (ND1,2,3,4,4L,5,6) (Fig. 4) are encoded by the mtDNA, the remaining by chromosomal genes. Complex I1 (succinate:ubiquinone dehydrogenase), which contains no mitochondrial encoded subunits, oxidizes FADH, and transfers electrons to ubiquinone (coenzyme Q, CoQ). Complex I11 (ubiquino1:ferricytochrome-c oxidoreductase) contains one subunit (cytochrome b), and complex lV (cytochrome-c oxidase) contains three peptides that are mitochondrially encoded. Complex IV catalyzes the final reaction of two electrons with oxygen to yield water. The energy of the redox reactions during the electron transport is stored as ApH+, consisting of a membrane potential and a proton gradient across the inner membrane. The function of AFH+ is severalfold: 1 ) it drives ATP synthesis in complex V (ATP synthetase), 2 ) it supports electrogenic transport processes such as ATP export as well as glutamatem+ symport versus aspartate- exchange in the glutarnate/aspartate anti- porter, and 3 ) it changes the pore structure in the contact sites, leading to higher affinity for hexokinase (39,50). To sum up, both the ADP supply to OXPHOS and the formation of G-6-P is regulated by an intact membrane potential.

MlTOCHONDRIAL OXIDATIVE METABOLISM AND DIABETES Genetics of mtDNA. Depending on their energy demand, cells contain hundreds to thousands of mitochondria, each carrying 2-10 mtDNA molecules. The mitochondrial energy- producing machinery, i.e., the respiratory chain coupled to OXPHOS, is encoded by both nuclear and mitochondrial genomes. The mtDNA is a closed circular double-stranded molecule that is exclusively maternally inherited. The 16,569 bp-long mt genome codes for 2 ribosomal RNAs (12S, 16S), 22 tRNAs, and 13 polypeptides of the respiratory chain complexes (61) (Fig. 4). Because of its lack of histone protection, its insufficient repair mechanisms, and its highly


MITOCHONDRIA AND DIABETES

point mutations wildtype human length mutations

mt DNA

deletion dirner

partial duplication

with deletion

FIG. 4. mtDNA point and length mutations associated with diabetes. In the central part of the figure, the organization of wild-type mtDNA is shown. The left part summarizes point mutations described so far as being associated with diabetes in sporadic cases as well as in families with maternally inherited diabetes and other neurological symptoms. The right part gives an example (modified from 106) of rearranged mtDNA molecules resulting from a large deletion in a KSS patient with diabetes. Several other deletions of different size are described in the literature.

compact structure, which consists almost entirely of coding regions, the mt genome is vulnerable. It is the preferential target of alkylating reagents as well as of oxygen radicals formed by the electron flux along the respiratory chain. Even under normal conditions, this oxidative damage is extensive, resulting in a 10- to 20-fold higher mutation rate in the mtDNA than in the nuclear DNA (62). This means that besides a variety of neutral and silent mutations, highly deleterious mutations probably arise frequently in the mitochondrial genome. Despite its high mutation rate, mtDNA encodes highly conserved proteins, since in a given cell with several hundred mitochondria, each containing up to 10 mtDNA copies, wild-type mtDNA can usually compensate for the defective function of single mutated mtDNA molecules (63). The amount of heteroplasmic somatic mtDNA mutations accumulates with age, probably as a result of oxygen radical damage. However, the mutation rate varies from tissue to tissue and is highest in postmitotic cells such as neuronal cells, heart, skeletal muscle, etc. As a result of the high mutation rate during evolution, the mtDNA is consider- ably polymorphic. Because most of these polymorphisms are homoplasmic, they have occurred long ago and have long since segregated to homoplasmy (64). Disease-related mutations, in general, are evolutionary new heteroplasmic germ- line mutations. The degree of heteroplasmy, when changing during life and passing a tissue-specific threshold, might be responsible for the onset of a mitochondrial disorder.

MDM mtDNA point mutations and diabetes. The mitochondrial ~RNAL~u("UR) gene is an etiologic hot spot for mtDNA muta-

tions, as at least 10 disease-related mutations have been described so far in this gene (65). Four of them have been associated with diabetes and various other symptoms (Fig. 4). A patient with mitochondrial encephalomyopathy, pig- mentary retinopathy, dementia, and hypothyroidism carried a A3252G mutation (66); an A3260G transition was found in single patients from a large pedigree with maternally inherited myopathy and cardiomyopathy (67); and a C3256T exchange was shown in a patient with a myoclonic epilepsy and ragged red fiber disease (MERRF)-like syndrome (68).

In 1992, two independent publications appeared demonstrating an A/G exchange at np 3243 in the ~ R N A ~ ~ ~ ( ~ ~ ~ ) gene in large Dutch (69) and British families (70) with diabetes and deafness. These first reports were confirmed by several other groups (71-88). We have recently summarized the appropriate publications from different countries (8,89). The ~ R N A ~ ' " ( " ~ ~ ) mutation at np 3243 is found in -0.5-1.5% of unselected diabetic patients, independently of whether they are classified as having type I or type I1 diabetes. In diabetic patients with familial history, the percentage increases up to 10%. The prevalence seems not to be very different between various countries and races. Diabetes is rarely also found in association with the so-called MERRF mutation (90) at np 8344 in the ~ R N A ~ ~ " gene (91). Recently, a T14709C transition

IIIABETES, VOL. 45, FEBRUARY 1996

FIG. 5. Electron micrograph of skeletal muscle biopsy from a patient with KSS. Strong goldlabeling by anti-Mi-CK antibodies of intramitochondrial crystals (type I1 crystal) (122). Lipofuscine particle in between the crystal-bearing mitochondria1 (left upper, right below). Amplification is 35,000-fold.

in the ~ R - Y A ~ ' ~ gene was demonstrated independently by two groups in a syndrome with myopathy and diabetes (92,93). mtDNA length mutations and diabetes. Distinct length abnormalities of the mtDNA were first described in 1988 in patients suffering from mitochondrial myopathies (muscle weakness, chronic progressive external ophthalmoplegia [CPEO]) (94) or the complete Kearns-Sayre syndrome (KSS) (95-99). Endocrine dysfunction, for example hypogonadism, hypothyroidism, hypoparathyroidism, and diabetes, was found in a high degree in KSS and CPEO (100-102). While partial deletions were the first mtDNA defects described in KSS, Poulton et al. (103) 1 year later reported partial direct tandem tluplications. Further studies reinforced a characteristic association between partial duplication and diabetes, as shown schematically in Fig. 4 (104-106). This seems also to be true for other syndromes involving the kidney and the hemopoietic system (107-109) in combination with diabetes. In 1992, Ballinger et al. (110) described a large pedigree with a maternally inherited syndrome of diabetes and deafness carrying a 10.4-kb mtDNA deletion. Revision of this family revealed, besides wild-type mtDNA, a mixture of interrelated and rearranged mtDNA molecules, namely duplications and deletion dimers, but few deletion monomers (111). The proportion of each rearranged mtDNA molecule varies between different tissues, and there is growing evidence that the balance of mtDNA rearrangements may be central to the pathogenesis of this form of MDM (104,106).

A small tandem duplication in the D-loop region of the human mtDNA, 260 bp in length, mapping to nucleotide positions (nps) 308 and 567, was recently described by Brockington et al. (112) in 18 of 58 patients with mitochondrial myopathies and mtDNA deletions but not in 62 control subjects. This duplication seemed to be related to a poly- meric C-insertion at np 567. Torroni et al. (1 13) identified this mtDNA variant as a Caucasian-specific haplotype prone to recurrent somatic duplications. Using polymerase chain reaction 1:PCR)-single-strand conformation polymorphism (SSCP) and subsequent sequencing (114), we have investi- gated 106 randomly selected type I and I1 diabetic patients, 2 KSS patients carrying the so-called common deletion, and 68 control subjects for this haplotype. We obtained the C- insertion at np 567 as well as the 260-bp duplication in 6 of

106 diabetic patients and in both KSS patients but also in 3% of healthy control subjects. However, in none of the subjects carrying this variant were we able to demonstrate the presence of deletions or duplications in blood specimens, either by Southern blotting or by long-distance PCR. Thus, further investigations are necessary to clarify the question of whether the 260-bp D-loop duplication can predispose indi- viduals to mtDNA rearrangements. Mitochondria1 genotype versus phenotype. All the ~ R N A ~ " ' ~ ~ ) point mutations described so far in association with diabetes are heteroplasmic in any case (69-88). The same is true for the family with diabetes and the MERRF mutation at np 8344 (91). Of the 199 affected members from 45 families with diabetes carrying the ~ R N A ~ ~ ~ @ ~ ~ ) mutation at np 3243 described so far, 48% had diabetes and deafness, 13% suffered from diabetes and deafness combined with further neurological symptoms-including those of the mitochondrial myopathy, encephalopathy, lactic acidosis, stroke- like episodes (MELAS) syndrome-21% had only diabetes, and 15% had deafness with or without other neurological symptoms (8,89). Also, the different mtDNA length mutations in association with diabetes described so far are heteroplas- rnic in any case. In the family with diabetes and deafness reported by Ballinger et al. (110) harboring wild-type, duplicated, and deleted mtDNA molecules, the level of these three forms vary between and within family members (111). In EBV-transformed lymphoblastoid cell lines from three p r e bands of this family, the distribution of the three forms changed drastically. While the deleted molecules were lost during culturing, the percentage of duplicated molecules increased up to 95% (111). Because duplications were found in all familial cases, it was suggested that duplicated rather than deleted mtDNA molecules may be transmitted in the germ line (104,106). This was explained by the different proliferative potential of deleted and duplicated mtDNA molecules.

The degree of heteroplasmy of mtDNA point and length mutations varies in different tissues and organs (98,99,115). Because the tissue demand of energy is also different, in decreasing order of the central nervous system, heart, skeletal muscle, kidney, pancreatic p-cells, and liver, organ- specific thresholds, probably declining with age, might be


responsible for the remarkable phenotypic diversity of the clinical symptoms, even between members of the same family (63).

Another possible explanation of the phenotypic variability of mtDNA disorders is the assumption that nuclear genes or additional mitochondrial gene defects might act synergisti- cally (116). An autosomal locus predisposing to deletions of mtDNA has recently been published in the case of autosomal dominant progressive external ophthalmoplegia (1 17).

The frequent combination of MDM with deafness (nearly 70%) is striking and represents a diagnostic criterion of this type of diabetes (8,89). This subform of MDM was therefore described as maternally inherited diabetes and deafness (MIDD) (69,82).

MORPHOLOGICAL IN SITU STUDIES There are only a few morphological and histological studies on pancreatic tissue slices from diabetic patients with mtDNA point or length mutations. Suzuki et al. (118) reported a markedly reduced p-cell number in the pancreas of a diabetic patient canying the A3243G mutation. Poulton et al. (119) compared pancreas tissue from a diabetic KSS patient with tissue from an IDDM patient. In the KSS patient, they found a complete loss of insulin-producing p-cells but regularly shaped islets, while the islets in the IDDM patient were larger and more irregular in shape. Provided that these first observations will be confirmed by further studies, it seems likely that mtDNA point and length mutations can selectively destroy pancreatic p-cells by a thus far unknown mechanism, as already proposed by Oka et al. (120) for the so-called slowly progressive IDDM.

In skeletal muscle, three types of mtDNA mutations, namely the A3243G (MELAS), the A8344G (MERRF), and several deletion/duplication mutations found in KSS, are typically associated with the so-called ragged red fibers. These are accumulations of muscle mitochondria stained by the trichrome Gomori reaction. Ragged red fibers are the result of increased mitochondrial proliferation, presumably to compensate for the insufficient ATP production. Paracrys- talline inclusions were often found inside the mitochondria in ragged red fibers. These inclusions were recently identified as deposits of Mi-CK (122) (Fig. 5). Thus, attempts of the organism to overcome the impaired energy production caused by a given mtDNA mutation results in ragged red fibers at the microscopic level and in Mi-CK deposits at the molecular level inside the mitochondria. This crystalline precipitation of Mi-CK strongly indicates that the CrP cycle is physiologically involved in energy transfer between mitochondria and cytosol. Adult rat cardiomyocytes cultured either in creatine-deficient medium or with the creatine analog 3-guanidino-propionic acid display large mitochondria with paracrystalline inclusion containing Mi-CK. It has been observed that the huge concentrations of crystallized Mi-CK are functionally inactive (E. O'Gorman, unpublished observations). Addition of creatine to the medium of cultured cardiomyocytes caused the disappearance of the giant mitochondria and the paracrystalline inclusions (123), suggesting full reversibility of the adaptations. Thus, treatment with creatine of patients with mitochondrial cytopathies might be beneficial, as described below. Functional consequences at the protein level of mtDNA mutations (in vitro experiments). Along the maternal

lineage, three of the members of the family with diabetes and deafness described by Ballinger et al. (110, 111) were inves- tigated with respect to respiratory chain activities in skeletal muscle biopsies. Two had complex IV defects, and one had a combined complex 111 and IV deficiency. In mitochondrial diabetic patients carrying the A3243G mutation, severe complex I as well as complex I + I11 deficiencies in skeletal muscle were described by several groups (69,71,72,87). Thus, the mtDNA mutations associated with diabetes have a functional correlate at the protein level, demonstrating impaired flux through the respiratory chain and resulting in decreased OXPHOS capacity, i.e., decreased ATP production. Functional in vivo tests. Using oral glucose tolerance tests and various euglycemic clamp techniques, several groups looked for the hormonal response in patients with the t ~ ~ ~ ~ ' " ( " ~ ~ ) - ~ 3 2 4 3 ~ mutation. Although extensive studies are lacking, first results indicate that the A3243G mutation is associated with a defect in the secretory capacity of the pancreatic p-cells rather than a peripheral insulin resistance. Delayed and insufficient insulin and C-peptide responses to a glucose load, together with reduced urinary excretion of C-peptide (76,87), were found in the affected patients (71,77,81,87), while clamp studies revealed no or only moderate peripheral insulin resistance (75,76,100; for review see 8). Cell culture studies. Cell lines can be depleted of mtDNA by long-term exposure to ethidium bromide and can than be repopulated by exogenous mitochondria (124). Such cybrids when transfected with disease-related, i.e., mutated, mtDNA, demonstrate a decline in OXPHOS capacity dependent on the amount of mutated mtDNA (125). Clonal cell lines constructed by fusion of an osteosarcoma cell line depleted of mtDNA with enucleated fibroblasts from diabetic patients canying the t ~ ~ ~ ~ ~ " ( ~ ~ ) - ~ 3 2 4 3 ~ mutation with >80% mu- tant mtDNA show enlarged and swollen mitochondria, a disturbance of the transmembrane potential, and a strongly reduced glucose uptake/phosphorylation capacity when compared with cybrids with wild-type mtDNA (126). To our knowledge, similar studies have not been done so far with human p-cells or insulinoma cell lines.

The influence of cytosolic and mitochondrial calcium fluctuations on insulin secretion (127) has recently been confirmed in elegant studies by Rutter et al. (128). Using an aequorin-transfected INS-1 cell line challenged by ATP or depolarized by high Kf, the authors demonstrated that the transient cytoplasmic Ca2+ increase is accompanied by elevation of mitochondrial ca2+ concentration, more than one order of magnitude above the cytoplasmic Ca2+ levels. This should be sufficient to activate Ca2+-sensitive intramitochondrial dehydrogenases, and this increase in driving force of the respiratory chain should further promote ATP synthesis (128).

As discussed above, insulin secretion in response to glucose stimulation is delayed and insufficient in diabetic patients carrying the t ~ ~ A ~ " ( ~ ~ ~ ) - ~ 3 2 4 3 ~ mutation. There is no information on intracellular Ca2+ fluctuations in p-cells of such patients. A recent publication (129), however, deals with ca2+ homeostasis and mitochondrial polarization in fibroblasts obtained from patients with different mtDNA point mutations (tRNALeU(UUR)-~3243G, T3271C, and t ~ ~ A ~ ~ " - ~ 8 3 4 4 ~ ) . Because these heteroplasmic mutations are usually found in all tissues, it should be acceptable to discuss the results found in the patients'

DIABETES, VOL 45, FEBRUARY 1996

1 0 r rP UI

3 m z 5 TABLE 1 -

Possible pathogenic key positions in the cytoplasnuc/mitochondrial interplay of energy n~etabolism in p-cells B Label in Fig. 1 System Location Function

GLUT2 Glucose transporter, Km for Plasma membrane Facilitated diffusion of glucose glucose 17 mmoM

GK Glucokinase, Km for glucose Cytosol, mitochondrial contact Glucose sensor, stimulates >5 mmoM sites oxidative phosphorylation by

ADP supply S 1 Glycerophosphate shuttle Cytosol and mitochondrial Hydrogen shuttle between

inner membrane cytosol and mitochondria S2 Malate-aspartate shuttle Cytosol and mitochondrial Hydrogen shuttle between

inner membrane, matrix cytosol and mitochondria K-Ch Potassium channel, blocked by Plasma membrane Depolarization of cell

ATP membrane potential

Ca-Ch Voltage dependent Ca2+ channel

P Voltage-dependent pore

AT Adenylate translocator

Plasma membrane Mows extracellular ca2+ influx. This step is essential for insulin secretion

Outer membrane of Exchange of metabolites across mitochondria the outer membrane

Inner mitochondrial membrane Exchanges ATP versus ADP

CK BB-CK Cytosol, plasma membrane? Production of ATP from CrP

Mi-CK Mia-CK Mitochondrial contact sites Production of CrP from mitochondrial ATP

CI-CV Electron transport chain, Mitochondrial inner membrane Generates membrane potential, mitochondrial ATP oxidative phosphorylation, synthetase ATP production

Acyl-CoA, Malonyl-CoA Cytosol, mitochondrial inner Activation of PKC through DAG synthesis compartment production

Regulatory significance Reference no.

Not rate limiting

Activation by glucose and binding to mitochondria

Substrate supply of mitochondrial metabolism

Substrate supply of mitochondrial metabolism

Activates Ca2+ influx, insulin secretion, and mitochondrial metabolism

Opens upon depolarization of cell membrane potential

Changes from anion to cation selectivity at >30 mV

Dependence of electrogenic ATP export on membrane potential

Regulation of potassium channel

Regulation of potassium channel through BB-CK

Regulates porin structure, provides phosphocreatine

Sensitivity, regulation of cellular ca2+ uptake

fibroblasts in analogy to possible effects in p-cells. Fibro- blasts from patients with mtDNA point mutations have significantly elevated intracellular baseline Ca" levels compared with control fibroblasts. Furthermore, when depolarized by high extracellular Ki, the ca2+ levels in control fibroblasts increase transiently and then return to baseline, while identical treatment leads to sustained elevation of intracellular Ca2+ in the mutated fibroblasts (129). Because a decreased electrochemical gradient is found across the patient's fibroblast cell membranes, it can be assumed that the mtDNA-point mutations cause a decreased phosphorylation potential in the cytosol and a reduced capacity to sequester ca2+ into endoplasmic reticulum. Furthermore, it may indicate a reduced mitochondrial membrane potential necessary for voltage-dependent processes such as uptake of ca2+, affinity changes of porin for hexokinase, ATP export, and substrate exchange. Programmed P-cell death in MDM? As mentioned above, first investigations revealed a selective reduction and a complete loss of p-cells in single patients with the A3243G mutation and length mutations, respectively (1 18,119). Some years ago, Karasik et al. (130) suggested that diabetes in Wolfram or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness) syndrome, which is supposed to be a mitochondria-mediated disorder (131) with several mtDNA variants (132), results from genetically programmed selective P-cell death. Because most of the mtDNA deletions as well as the tRNA mutations affect mainly ND gene products, Cortopassi and Wang (133) developed a hypothet- ical explanation of how complex I deficiency could ulti- mately result in cell death. Briefly, the postulated sequence of events is as follows: mtDNA gene defect complex I deficiency + overutilization of the FAD-dependent complex I1 pathway (succinate: ubiquinone dehydrogenase) + enhanced superoxide formation, which cannot suffi- ciently be scavenged by manganese superoxide dismutase (MnSOD) + increased production of the highly toxic hydroxyl radical via the Fenton or the Haber-Weiss reaction + DNA damage, lipid peroxidation, protein modification. Such damage by the hydroxyl radical may be the signal for programmed cell death, possibly involving the mitochondrially located Bcl2AAX system (134). This hypothesis, developed to explain the mitochondrial contribution to normal aging, could also be true for the damage of p-cells in MDM, especially as the activity of the scavenger enzyme MnSOD is very low in p-cells (135).

POSSIBILITIES OF THERAPEUTIC INTERVENTION IN MDM Attempts to treat respiratory chain complex deficiencies in a more causal way by use of carnitine, vitamin C, thiamine, or coenzyme Q,, have been performed but mostly with no or only limited success. Most recently, Hagenfeld et al. (136) tried to use creatine treatment in a 25-year-old man suffering from MELAS and canying the heteroplasmic tRNALeUCUUR)- A3243G mutation. The rational background for such an attempt is discussed above. Furthermore, it is known that athletes can increase high-energy phosphate bonds in their skeletal muscle in the form of creatine phosphate by high- dose oral administration of creatine. In the patient reported by Hagenfeld et al. (136), the creatine treatment (5 g twice daily for 2 weeks and 2 g twice daily thereafter) resulted in reduced headache, less weakness, better appetite, and an

improved general well-being during treatment. In a graded exercise test, the patient was unable to perform 20 W for longer than 15 s before creatine treatment but exercised for 4 min at 30 W after 3 months of treatment. To our knowledge, similar therapeutic attempts in patients with mitochondrial diabetes have not been performed so far.

CONCLUSIONS AND PERSPECTIVES We have revised some aspects of the interplay between cytoplasmic metabolic pathways and mitochondrial OXPHOS. Regulation by binding of hexo(g1uco)kinases to the mitochondrial contact sites, intact membrane potentials due to functionally sufficient respiratory chain activities coupled to OXPHOS, and compartmentation of energy-rich metabolites (ATP, CrP) at sites of cell-specific function are some of the fundamental aspects of glucose metabolism. Other important sites of cytoplasmic-mitochondria1 interactions are listed in the table. As the basic steps of the energy circuit are expected to occur in most energy-providing and -consuming cells, germ-line and somatic mutations of the genes coding for key positions in this cooperative mechanism should lead to cell-specific functional impairment. Thus, the genetically and biochemically quite different subtypes of diabetes, MODY2 and MDM, chosen as examples in this article behave clinically similar as both forms are characterized by a delayed and insufficient insulin response to a glucose load, while peripheral insulin resistance, if ever, is of minor importance. There is evidence that in both forms a reduced mitochondrial ATP production in the p-cells, possibly transmitted via the CrP shuttle, is the common mechanism for impaired insulin secretion. The same point and length mutations of the mtDNA leading to impaired ATP formation and consequently to impaired insulin secretion in MDM can cause, however, a variety of specific symptoms in other cells and tissues, such as muscle weakness, heart failure, enceph- alopathies, etc. Screening of subtraction libraries from skeletal muscles of healthy and diabetic patients revealed an overexpression of several mitochondrial, but not nuclear, genes, indicating the relevance of the mitochondrial genome in diabetes (137). At the nuclear gene level, several rare missense mutations of the hexokinase I1 gene have recently been observed (138-140). Although the relevance of these observations remains to be established by respective family studies, other results point in the same direction. Recent in vivo studies using 13C and 31P NMR spectroscopy under hyperglycemic-hyperinsulinemic clamp conditions demonstrate that reduced insulin-stimulated muscle glycogen synthesis is the first major aspect of insulin resistance not only in NIDDM but also in apparently healthy, normoglycemic, and lean offspring of parents with NIDDM (141). Diminished formation of G-6-P but not impaired glycogen synthetase activity seems to be the limiting step in these cases. As glycogen synthetase activity increases linearly with increas- ing G-6-P concentrations, a decreased G-6-P formation would directly lead to impaired glycogen synthesis.

We suppose that a differentiated expression of gene defects in the translocation/binding mechanism of the hexokinases, in the binding site of mitochondrial porin (142), in the hexose phosphorylating enzyme itself, in the mitochondrial FAD-linked glycerophosphate dehydrogenase (143,144), in mitochondridy encoded subunits, or at other points of the cellular energy circuit (table) could account for different


effects in specialized cells and tissues of diabetic patients, 21. Koran~i u, Tanizawa Y, Welling CM, Rabin DU, Permutt MA: Human

leading, fi3r instance, to peripheral insulin resistance in one islet glucokinase gene: isolation and sequence analysis of full-length cDNA. Diabetes 41:807-811, 1992

case andlor to impaired insulin secretion in the other. 22. Polakis PG, Wilson JE: An intact hvdro~hobic N-terminal seauence is We, therefore, proceed on the assumption that a new critical for binding of rat brain hexoidnase to mitochondha. Arch

Biochem Biophys 234:341-352, 1984 classificat'ion be in the future regarding the 23, fiek CH, Benz R, Roes N, Brdiczka D: Evidence for identity between the obvious genetic and biochemical heterogeneity of diabetes. hexokinasebindine orotein and the mitochondrial ori in in the outer -

ACKNOWLEDGMENTS

membrane of rat Evvkr mitochondria. Biochim ~ i o i h ~ s Acta 688:429- 440, 1982

24. LindCn M. Gellerfors P. Nelson BD: Pore p rote in and the hexokinase- Research work leading to this article was supported in part binding p;otein from the outer membrane bf rat liver mitochondria are

identical. FEBS Lett 141:189-192, 1982 the Deutsche Forschungsgemeinschaft (Ge 21317-1 and 25, Borrebaek B, Spydevold 0: The effect of insulin and glucose on Br 77313-4). We thank Dr. C. Wollheim, Genova, for allowing mitochondrial bound hexokinase activity of rat epididymal adipose us to stutly the INS-1 cell line, Dr. T. Wallimann, Zurich, for tissue. Diabetologia 5:42-43, 1969

antibodies against mitochondrial creatine and Dr. 26. Goncharova NY, Zelenia EV: Influence of insulin on the catalytic activity of hexokinase isozyme I1 of rat skeletal muscles. Biokhimiya 5691%

A.M. Stadhouders, Nirnwegen, for ~roviding us with Fig. 5. 922, 1991

This work is dedicated-to Otto-Heinrich Wieland on the occasion of his 75th birthday.

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