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INCREASED GLUCOSE PRODUCTION IN MICE OVEREXPRESSING HUMAN

FRUCTOSE-1,6-BISPHOSPHATASE IN THE LIVER

Sherley Visinoni1, Barbara C. Fam1, Amy Blair1, Christian Rantzau1, Benjamin J. Lamont1, Russell

Bouwman1, Matthew J. Watt2, Joseph Proietto1, Jenny M. Favaloro1*, Sofianos Andrikopoulos1*

1Department of Medicine (AH/NH), University of Melbourne, Heidelberg Heights, Victoria, 3081,

Australia

2Department of Physiology, Monash University, Clayton, Victoria, 3800, Australia

* These authors contributed equally to this study

Running Title: Liver FBPase Transgenic Mice KEYWORDS: Fructose 1,6-bisphosphatase, Endogenous Glucose Production, Glucose Intolerance ABBREVIATIONS: Area under the curve (AUC), Brown Adipose Tissue (BAT), Endogenous Glucose Production (EGP), Fructose 1,6-bisphosphatase (FBPase), General Linear Model Analysis of Variance (GLM ANOVA), High Fat (HF), High Fat Fed (HFF), Hyper-Sensitive-4 (HS4), Intraperitoneal (IP), Intraperitoneal Glucose Tolerance Tests (IPGTTs), Polymerase Chain Reaction (PCR), Standard Error of the Mean (SEM), Transthyretin (TTR), Walter and Eliza Hall Institute of Medical Research (WEHI), White Adipose Tissue (WAT) Address correspondence to: Sofianos Andrikopoulos, PhD University of Melbourne Department of Medicine (AH/NH) Heidelberg Repatriation Hospital 300 Waterdale Road Heidelberg Heights Victoria 3081 Australia Tel: +61 3 9496 2403 Fax: +61 3 9497 4554 Email: sof@unimelb.edu.au

Articles in PresS. Am J Physiol Endocrinol Metab (September 9, 2008). doi:10.1152/ajpendo.90552.2008

Copyright © 2008 by the American Physiological Society.

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ABSTRACT

Increased endogenous glucose production (EGP) predominantly from the liver is a characteristic

feature of Type 2 diabetes, which positively correlates with fasting hyperglycemia.

Gluconeogenesis is the biochemical pathway shown to significantly contribute to increased EGP in

diabetes. Fructose-1,6-bisphosphatase (FBPase) is a regulated enzyme in gluconeogenesis that is

increased in animal models of obesity and insulin resistance. However whether a specific increase

in liver FBPase can result in increased EGP has not been shown. The objective of this study was to

determine the role of upregulated liver FBPase in glucose homeostasis. To achieve this goal, we

generated human liver FBPase transgenic mice under the control of the transthyretin promoter,

using insulator sequences to flank the transgene and protect it from site-of-integration effects. This

resulted in a liver-specific model as transgene expression was not detected in other tissues. Mice

were studied under the following conditions - 1) at two ages (24 weeks and 1 year old); 2) following

a 60% high-fat (HF) diet; and 3) when bred to homozygosity. Hemizygous transgenic mice had an

approximately 3–fold increase in total liver FBPase mRNA with concomitant increases in FBPase

protein and enzyme activity levels. Following HF feeding, hemizygous transgenics were glucose

intolerant compared to negative littermates (p<0.02). Furthermore, when bred to homozygosity,

chow-fed transgenic mice showed a 5.5-fold increase in liver FBPase levels and were glucose

intolerant compared to negative littermates, with a significantly higher rate of EGP (p<0.006). This

is the first study to show that FBPase regulates EGP and whole-body glucose homeostasis in a

liver-specific transgenic model. Our homozygous transgenic model may be useful for testing

human FBPase inhibitor compounds with the potential to treat patients with type 2 diabetes.

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INTRODUCTION

Type 2 diabetes is a chronic disorder characterized by hyperglycemia. One of the main defects that

contributes to the fasting hyperglycemia observed in patients with type 2 diabetes is elevated

endogenous glucose production (EGP) (5, 12). After a meal, EGP is also less suppressed,

contributing to impairments in glucose tolerance (15, 20, 34). The liver is the primary organ

responsible for EGP (29) with the kidney playing a smaller role in total glucose output (36).

Glucose is produced in the liver via a combination of two biochemical processes: gluconeogenesis

and glycogenolysis. The abnormal level of EGP in patients with type 2 diabetes is predominately

due to an increase in the rate of gluconeogenesis compared to non-diabetic subjects, as

glycogenolysis is largely unchanged (8, 19). Increased gluconeogenesis from various substrates

such as lactate, alanine, pyruvate and glycerol has been observed in patients with type 2 diabetes

(22, 27, 37, 41).

Glycerol enters the gluconeogenic pathway immediately prior to the step catalyzed by fructose-1,6-

bisphosphatase (FBPase), a regulated enzyme in gluconeogenesis. As mentioned above, the rate of

glycerol gluconeogenesis is increased ~2-fold in obese patients with type 2 diabetes (22, 27).

Furthermore, when matched for plasma glycerol concentrations, patients with type 2 diabetes

retained a higher rate of glycerol gluconeogenesis compared with non-diabetic subjects, which

suggested an increase in the intra-hepatic conversion of glycerol to glucose in diabetes. The study

by Nurjhan and colleagues suggested that this may be due to an increase in FBPase activity (22).

Our laboratory tested the role of FBPase in glucose production by generating a hemizygous

transgenic mouse model (Line 1) with a 2-fold overexpression of human FBPase in the liver. This

resulted in an approximately 3-fold increase in glycerol gluconeogenesis both in the basal state and

following a euglycemic/hyperinsulinemic clamp, compared to littermate controls (18).

Interestingly, these transgenic mice had normal EGP and glucose tolerance (18). This was a

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surprising result, considering the human studies mentioned previously and results from our

laboratory and others. These studies showed liver FBPase expression and/or activity to be double

that of controls in several models of insulin resistance and obesity including New Zealand Obese

(NZO) mice (2), db/db mice (6), ob/ob mice (32, 33), fa/fa rats (39), high-fat fed mice and rats (2,

35) and in liver biopsies from patients with type 2 diabetes (9).

Other groups have previously generated animal models overexpressing the other regulated

gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase

(G6Pase) in the liver (38, 42, 43). When G6Pase was overexpressed in the liver of rats using

adenoviral delivery, they became hyperinsulinemic and glucose intolerant (42). Mice that

overexpressed PEPCK in the liver were glucose intolerant and had elevated levels of EGP (38, 43).

Similarly, our group generated transgenic rats with renal PEPCK overexpression. These transgenic

rats have impaired insulin suppression of EGP, causing peripheral (muscle and fat) insulin

resistance (17, 31).

Considering these studies, the failure of a 2-fold overexpression of FBPase in Line 1 transgenics to

produce a phenotype was unexpected (18). However as reported, this line of transgenic mice also

overexpressed FBPase in the brain (18). Given the known effects of hypothalamic metabolism on

EGP (23, 25), it was possible that the effects of liver overexpression may have been counteracted by

the overexpression of FBPase in the brain. We suspected that site-of-integration effects may be

responsible for this extra-hepatic expression of our transgene. Previous studies have shown that it is

possible to protect against site-of-integration effects with the use of “insulator sequences.” (13),

(26). These sequences are proposed to act by insulating the flanked transgenic sequence from

nearby repressor/enhancer elements thereby resulting in tissue specific expression (7). We took

advantage of this technology and produced a new mouse line aiming for liver-specific transgene

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expression, using the same transgenic cassette as before (18), but flanking it with insulator

sequences from chicken beta-globin, named Hyper-Sensitive-4 (HS4) (26).

The current studies were performed to validate the results observed in the previous transgenic

model (Line 1) and to determine if higher levels of overexpression were necessary to observe a

glucose intolerant phenotype. Additional aims were to further characterize the glucose tolerance

phenotype in Line 1 and the new insulated transgenic mice at two ages (24 weeks and 1 year old),

following a 60% high-fat (HF) diet and when bred to homozygosity, to investigate whether FBPase

has a specific role in EGP and glucose intolerance under these conditions.

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EXPERIMENTAL PROCEDURES

Animals

Transgenic mice overexpressing the human liver FBPase gene (FBP-1) were generated using the

transthyretin (TTR) promoter to direct expression of the transgene to the liver (47). The new

FBPase gene construct used for microinjection contained the same 1.1 kb FBPase cDNA as Line 1

(Accession NM_000507, bases 160-1266) (18) and the same 3 kb segment of the TTR promoter,

using vector pTTR1 Ex V3 (47). However, in the new construct two insulator sequences from

chicken beta-globin, named Hyper-Sensitive-4 (HS4) (26) flanked the transgene to create a barrier

protecting it from position of integration effects (7). All other features were similar to the original

construct (see Fig 1A and B) (18). The 10.5 kb transgenic cassette (Fig 1B) was microinjected into

C57BL/6 fertilized eggs by the Walter and Eliza Hall Institute of Medical Research (WEHI,

Parkville, VIC, Australia) Microinjection Facility and the FBPase transgenic mice were produced

using standard procedures (18).

Founder mice were bred with C57BL/6 wild type mice purchased from WEHI to produce

hemizygous transgenic mice (F1 progeny), and the colony was maintained by breeding male

hemizygous transgenics with female C57BL/6 mice. Homozygous transgenic mice were produced

by breeding together hemizygous mice from the new colony.

Genotyping of transgenic mice

To detect the presence of the TTR-FBPase transgene, a PCR using specific primers spanning the

TTR promoter – FBPase junction was performed. This procedure and primer sequences were

described in detail previously (18).

Homozygous transgenic mice were identified by a 2-step procedure. In the first step, the usual

genotyping PCR was used to show the presence of the transgene. The second step utilized Real-

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Time PCR to differentiate between the hemizygous and homozygous transgenic mice. Primers

were designed for SYBR-green Real-Time PCR using Primer Express Software (Applied

Biosystems, Scoresby, VIC, Australia). The primer sequences for the 18S endogenous control

were: forward 5’-CGG ACA CGG ACA GGA TTG ACA-3’; and reverse 5’-ACA AAT CGC TCC

ACC AAC TAA GA-3’. The primer sequences for detecting the genomic transgenic FBPase were:

forward 5’-TGA CCC ATT TCA CTG ACA TTT CTC-3’; and reverse 5’-CAG CCA TGC TTG

AAC CGG -3’. The primers were positioned within introns in order to amplify genomic DNA and

exclude potential contaminating RNA. Each SYBR-Green Real-Time PCR reaction used 0.19 nM

of 18S, 56 nM of genomic transgenic FBPase primers and 2.5 ng of DNA. A sample from each of

the transgenic mice was analysed by Real-Time PCR to differentiate homozygous from hemizygous

transgenic mice. Samples were analysed by the ABI Sequence Detection Software and absolute

quantification comparative Ct method (Applied Biosystems software). Transgenic samples were

accepted as homozygous when approximately double the absolute quantitation of the hemizygous

samples.

Housing and maintenance of mice

The mice were housed at the University of Melbourne, Department of Medicine Animal Research

Facility at the Heidelberg Repatriation Hospital, at room temperature (~20C) under a 12 hour

light/dark cycle. All mice were supplied with tap water and fed ad libitum a standard laboratory

chow (77% carbohydrate, 20% protein, and 3% fat), except for those used in the High Fat Fed

(HFF) studies, which were fed a 60% fat diet (22% carbohydrate, 18% protein, and 60% fat) for

twelve weeks from 12 weeks of age. The mice were maintained in accordance with guidelines of

the Austin Hospital Animal Ethics Committee (approval number: A2007.2752).

Physiological studies were performed on second generation hemizygous mice (TG +/-) and first

generation homozygous mice (TG +/+) at 24 weeks of age, except for the aging study mice (1 year

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old). Control mice for all experiments were age-matched transgene-negative littermates from each

line (NEG).

Determination of FBPase, PEPCK and G6Pase mRNA levels

RNA was extracted from tissues using the Trizol method (Invitrogen, Mount Waverley, VIC,

Australia). RNA was treated with DNaseI (Ambion, Scoresby, VIC, Australia) and cDNA

synthesized using 1 µg of DNase-treated RNA and random primers with the Promega Reverse

Transcription kit (Annandale, NSW, Australia).

Total FBPase mRNA levels (mouse and human) were quantitated using SYBR-green Real-Time

PCR. Primers for the -actin endogenous control were described previously (18). Primers for total

FBPase which matched the mouse sequence (GenBank: NM_019395) and human sequence of liver

FBPase (GenBank: NM_000507) were: forward 5’-AGC CTT CTG AGA AGG ATG CTC-3’; and

reverse 5’-GTC CAG CAT GAA GCA GTT GAC-3’. Transgenic FBPase mRNA levels were

measured in a range of tissues by Real-Time PCR method as described previously (18).

TaqMan gene expression assay kits (Applied Biosystems, Scoresby, VIC, Australia) were used to

measure PEPCK (Mm00440636_m1) and G6Pase (Mm00839363_m1) mRNA levels using the ABI

PRISM 7900 HT system (Applied Biosystems, Scoresby, VIC, Australia). Reactions containing 10

ng cDNA were analysed by the ABI Sequence Detection Software and relative quantification

comparative Ct method (Applied Biosystems software).

Western blotting and enzyme assays for FBPase

Total FBPase protein levels were determined by Western blotting as described previously (18). The

anti-rat FBPase primary antibody was a kind gift from Dr Hideo Mizunuma (Akita University,

Akita, Japan). Liver homogenates of 1 µg of total protein were loaded and FBPase (37 kDa) was

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detected with the anti-rat liver FBPase antibody. GLUT2 protein was used as a loading control.

Anti-GLUT2 (H-67) primary antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA, USA).

FBPase enzyme activity was determined using a spectrophotometric assay as described previously

(18).

Aging study

Mice were aged to one year old and underwent intraperitoneal glucose tolerance tests (IPGTTs).

High fat feeding study

Mice were fed a 60% HF diet (Specialty Feeds, Glen Forrest, WA, Australia) for twelve weeks

following one week of acclimatisation to the diet. The fat in the diet comprised of ~80% saturated

and ~20% unsaturated fat. The mice were monitored weekly whilst on the diet to ensure they were

gaining weight. Following the twelve weeks on the HF diet IPGTTs were performed.

Intraperitoneal Glucose Tolerance Tests

IPGTTs were performed as previously described using a 2 g/kg glucose bolus, with blood samples

taken for plasma glucose and insulin determination (17). These experiments were performed on

hemizygous transgenic mice at 24 weeks and one year of age, the high fat fed (HFF) mice and the

homozygous transgenic mice. Following completion of the test, mice were sacrificed by cervical

dislocation and the livers and other tissue collected, quickly snap frozen in liquid nitrogen and

stored at -70C for further studies.

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Intraperitoneal Pyruvate Tolerance Tests

Overnight fasted (~16 h) mice were anesthetized with an IP injection of sodium-pentobarbitone

(100 mg/kg). After a 5 l basal blood sample was taken from the tail vein, the mice were injected

IP with 2 g/kg pyruvate (Sigma Aldrich, Castle Hill, NSW, Australia). Subsequent blood samples

were taken at 15, 30, 60 and 120 minutes and blood glucose determined using a Precision Q.I.D.

glucometer (MediSense, Doncaster, VIC, Australia).

Plasma assays

Plasma glucose was determined using an Analox GM7 Micro-stat glucose analyser and reagent

(Helena Laboratories, Mount Waverley, VIC, Australia).

Plasma insulin was measured by a double antibody radioimmunoassay using a rat-specific insulin

antibody (raised in guinea pigs) and highly purified rat insulin standard (Linco Research, Missouri,

USA).

Glucose turnover

Overnight (~16 h) fasted mice were anesthetized with an IP injection of sodium pentobarbitone

(100 mg/kg). Two catheters were inserted, one in the right jugular vein for tracer infusion and the

other in the left carotid artery for blood sampling. A tracheostomy was also performed to prevent

upper respiratory tract obstruction. A 2-min priming bolus (3.0 Ci) followed by a continuous

(0.15 Ci.min-1) infusion of [6-3H]-glucose was given for 120 min to measure basal whole body

glucose turnover. Blood samples were collected at 90, 100 and 110 minutes. The blood was

centrifuged and the plasma collected and stored at –20 C for measurement of glucose and

radioactivity levels as previously described (3, 14, 18, 21).

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Liver triglyceride levels

Triacylglycerol content was analysed as previously described (11). Lipid was extracted from ~20

mg liver by a Folch extraction, the triacylglycerol was saponified in an ethanol/KOH solution at

60oC, and glycerol content was determined by enzymatic spectrophotometric analysis (Free

Glycerol Reagent, Sigma, Castle Hill, NSW, Australia).

Statistical analysis

All results are expressed as mean ± standard error of the mean (SEM). Student’s t-test was used to

analyse differences between two independent groups. Area under the curve (AUC) was calculated

using the trapezoidal rule. A general linear model analysis of variance (GLM ANOVA) with post-

hoc Tukey’s testing was used to determine significances in glucose and insulin levels between the

groups (age, diet and genotypes) in response to IPGTT repeated measures. A p ≤ 0.05 was

considered significant.

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RESULTS

We previously reported (18) that the 2-fold overexpression of FBPase in the liver of hemizygous

transgenic mice generated with the original construct (Line 1, Fig. 1A) did not result in glucose

intolerance or increased EGP, which had been anticipated in a transgenic model overexpressing a

regulatory gluconeogenic enzyme (38, 42, 43). Line 1 mice were also found to express transgenic

FBPase in the brain, which may have affected the phenotype (18). Therefore, we generated a new

liver FBPase construct with HS4 insulator sequences flanking the transgenic cassette (Fig. 1B). The

new construct increased the likelihood of obtaining liver-specific transgene expression and possibly

a line with higher expression levels.

Initial characterization of new FBPase transgenic mice

Three founder mice were identified: one line did not overexpress the transgene (Line 4) and two

lines (Lines 5 and 6) overexpressed the human liver FBPase transgene in the liver to similar levels

as determined by Western blot analysis (data not shown). One of these lines, Line 5, was used for

the studies described in this article.

The endogenous TTR promoter predominately directs expression to the liver and low levels in the

choroid plexus (47). However, when used as a transgenic promoter it has also been documented to

express at low levels in other tissues (1, 24, 40, 46). Therefore, the level of transgenic FBPase

mRNA expression was assessed in a range of tissues from Line 5 transgenic mice. Expression of

the transgene was undetectable by Real Time PCR analysis in kidney, quadriceps, pancreas,

intestine, stomach, heart, spleen, gonads, white adipose tissue (WAT) and brown adipose tissue

(BAT) (data not shown). In Line 1 transgenic mice there was an 8-fold overexpression of total liver

FBPase levels observed in the hypothalamus compared to negative littermates, while hypothalamic

mRNA levels of FBPase in Line 5 hemizygous transgenic mice were comparable to the negative

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littermates (Fig. 1C). Therefore, the current transgenic model had liver-specific overexpression of

FBPase.

Line 5 hemizygous transgenic mice showed a ~3-fold overexpression of total liver FBPase mRNA

(Fig. 1D) with concomitant increases in liver FBPase protein (Fig. 1E) and enzyme activity levels

(Fig. 1F).

Effect of higher level of overexpression on transgenic phenotype

Following an overnight fast, plasma glucose and insulin levels were not different in hemizygous

transgenic mice compared to negative littermates (Table 1). Body weight and food intake was also

reduced in both FBPase transgenic lines compared to negative littermates (Table 1). To determine

if a higher level of overexpression is required to observe a glucose intolerant phenotype, 24 week

old transgenic mice from Line 1 (2-fold overexpression) and Line 5 (3-fold overexpression) were

challenged with a 2 g/kg bolus of glucose. There was no difference in glucose tolerance compared

to the negative littermates even with the higher level of overexpression of FBPase in the liver (Fig.

2B and 2D). Furthermore, the plasma insulin levels during the IPGTT were similar in the

transgenic lines and their negative littermates (Fig. 2C and 2E).

Effect of aging on transgenic phenotype

In order to investigate whether glucose intolerance would develop with age, the mice were studied

at one year of age. Similar findings of normal glucose tolerance were found in Line 1 and Line 5

transgenic mice and age-matched negative littermates (Fig. 2B and 2D), despite higher total liver

FBPase in the aged Line 5 transgenics compared to the aged negative littermates (Fig. 2A). As

aging is associated with insulin resistance (4, 28), all the one year old mice (transgenic and negative

littermates), were glucose intolerant (Fig. 2B and 2D) and had higher insulin levels during the

IPGTT compared to the 24 week old mice of the same genotypes (Fig. 2C and 2E). GLM ANOVA

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with post-hoc analysis found that the age of the mice had a significant effect on glucose levels

during the IPGTT (p<0.001, 24 week old mice vs 1 year old mice). There was no significant

interaction between age x genotype with no effect on plasma glucose levels. At one year of age

there were no differences in body weight between either of the transgenic lines and their negative

littermates (data not shown).

Effect of high fat diet on transgenic phenotype

Since elevated FBPase in spontaneous and diet induced animal models has always been associated

with excess weight gain or obesity, we assessed whether exposure to long-term HF feeding was

necessary for a glucose intolerant phenotype to be observed in our transgenic mice. The HF diet

elevated liver FBPase mRNA (Fig. 3A) and enzyme activity levels (Fig. 3B) in transgenic mice

compared to the negative chow and negative HFF mice. Liver FBPase mRNA levels were even

higher in the HFF Line 5 transgenics (~10-fold of negative) compared to the aged Line 5

transgenics (~4.5-fold of negative). In addition, the HFF diet resulted in ~2.5-fold elevated levels in

both liver PEPCK and G6Pase mRNA in the HFF negative and transgenic mice compared to those

on a chow diet, with no differences in both these genes when comparing the transgenics and

negative littermates on the normal chow diet (Fig. 3G and 3H respectively). Liver triglyceride

levels were not different under chow conditions between the negative and transgenic mice from

Line 5 (data not shown). In response to a high fat diet liver triglyceride levels were ~2-fold higher

in the HFF transgenics compared with the negative littermate mice on the same diet (61.9 ± 10.2 vs

28 ± 7.6 mol/g, p<0.02). Interestingly, both Line 1 and Line 5 transgenic mice were clearly

glucose intolerant following HFF when compared to the negative littermates on the same diet (Fig.

3C and 3E). Analysis with GLM ANOVA and post-hoc testing also revealed that diet and genotype

of the mice had a significant effect on plasma glucose levels during the IPGTT (p<0.001, NEG HFF

vs Line 1 and Line 5 TG HFF). There were no significant differences comparing the two transgenic

lines against each other when both placed on a HF diet. Although there was no effect of the HF diet

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on glucose tolerance in the negative mice (Fig. 3C and 3E), it was sufficient to cause insulin

resistance with significantly higher insulin levels during the IPGTT compared to the negative chow

fed mice (Fig. 3D and 3F).

Effect of homozygosity on transgenic phenotype

To determine whether increasing gene dose would result in glucose intolerance without the

confounding effects of a HF diet, Line 5 mice were made homozygous (+/+). Breeding Line 5

hemizygous mice together generated offspring at the expected 1:2:1 (negative: hemizygous:

homozygous, 26%:49%:25%) ratio. The liver FBPase mRNA levels in Line 5 homozygous

transgenic mice were significantly higher than the negative littermates (~5.5-fold of negative) and

the Line 5 hemizygote transgenics (~3-fold of negative) (Fig. 4A). FBPase enzyme activity levels

were measured in the liver and remained significantly higher in the homozygous mice than the

negative mice, and trended to be higher than the hemizygous littermates (Fig. 4B).

Plasma glucose levels of the homozygous transgenic mice during the IPGTT were mildly, yet

significantly elevated compared to negative and hemizygous transgenic littermates (Fig. 4C and

4E). GLM ANOVA with post-hoc analysis found that the genotypes of the mice had a significant

effect on plasma glucose levels during the IPGTT (p<0.001, NEG and Line 5 TG +/- vs Line 5 TG

+/+). Plasma insulin levels were not different during the IPGTT between the homozygous

transgenics and negative and hemizygous transgenic littermates (Fig. 4D and 4F).

A pyruvate tolerance test was performed to provide an indication of the rate of gluconeogenesis in

the homozygous transgenic mice. The blood glucose levels were higher (Fig. 5A), suggestive of a

higher basal rate of gluconeogenesis in the homozygous transgenic mice than the negative and

hemizygous transgenic littermates. There was no difference in blood glucose levels observed

between hemizygous transgenic mice and negative littermates.

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Glucose turnover was measured in the fasted state to determine if there was an increase in EGP in

the homozygous transgenic mice. There was no difference in the rate of EGP observed between

hemizygous transgenic mice and negative littermates. However, the data in Figure 5B confirmed

the glucose and pyruvate intolerance data and showed that the rate of EGP was significantly higher

in the homozygous transgenic compared to the negative and hemizygous transgenic littermates.

This data supports FBPase having a role in EGP and whole-body glucose intolerance.

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DISCUSSION

FBPase is postulated to be a key regulatory enzyme in the gluconeogenic pathway. It was shown in

the 1970s that the level of FBPase enzyme activity was double in patients with type 2 diabetes

compared to non-diabetics (9), and this increase in FBPase has been confirmed in several animal

models of obesity and insulin resistance (2, 6, 32, 33, 35, 39). Moreover, studies in patients with

type 2 diabetes showed increased glycerol gluconeogenesis (27), which was proposed to be caused

by an increase in FBPase activity (9). Our laboratory previously found that FBPase protein

expression and enzyme activity were elevated in genetic and dietary obesity (2, 35).

In light of this data, FBPase has recently received interest from pharmaceutical companies

producing compounds to inhibit FBPase as a target for type 2 diabetes therapy (10, 16, 44, 45).

These inhibitors decrease EGP in Zucker diabetic fatty rats, an obese diabetic animal model (10,

44), demonstrating a potential therapeutic benefit of FBPase inhibitor compounds for type 2

diabetes. Given the studies showing an increase in FBPase in models of obesity and insulin

resistance, conditions which affect many other genes and biochemical pathways, it was important to

determine whether liver-specific FBPase overexpression elevates EGP.

We previously generated transgenic mice (Line 1) overexpressing human FBPase in the liver under

the control of the TTR promoter (18). The mRNA, protein and enzyme activity levels all showed a

2-fold overexpression of FBPase in the liver of these transgenic mice, and an almost 3-fold increase

in glycerol gluconeogenesis (18), consistent with the data from patients with type 2 diabetes (9) and

our own work in the NZO mouse (2, 3). However the transgenic mice had normal EGP and glucose

tolerance, which was unexpected considering the impairments in glucose tolerance and insulin

resistance in other transgenic models overexpressing gluconeogenic enzymes such as PEPCK (38,

43) and G6Pase (42) in the liver. Additionally, our laboratory has produced rats overexpressing

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PEPCK in the kidney and found that they displayed impaired insulin suppression of EGP, and

peripheral insulin resistance (30, 31).

In addition to liver overexpression, our first transgenic model also expressed transgenic FBPase in

the brain (18). We showed that these Line 1 transgenic mice had lower body weights, which may

have negated the effect of the transgene to cause glucose intolerance. Therefore in this current

study we wanted to produce mice overexpressing FBPase specifically in the liver. To accomplish

this, the new transgenic mouse line (Line 5) was generated with a construct that incorporated

insulator sequences to confer specific expression to the liver by protecting the transgenic cassette

from position of integration effects when randomly inserted into the host genome (7). We achieved

the desired liver transgene specificity without an increased expression in the hypothalamus and

proposed that the use of insulator sequences may be a reasonable means by which a transgene can

be protected from site-of-integration effects. Interestingly, Line 5 transgenic mice still displayed

reduced body weight and food intake compared with their negative littermates, which raises the

possibility of a role for the liver in body weight and food intake regulation.

The effect of a chronic HF diet was tested since, as mentioned earlier, increased levels of FBPase

have been associated with glucose intolerance and increased rates of EGP when the animals were

either obese or placed on a HF diet. By introducing high amounts of fat into their diet, the

transgenic mice had much higher FBPase levels at 24 weeks than the aged transgenic mice and

became glucose intolerant compared to the negative littermate mice on the same diet. This

suggested that lipid oversupply may be necessary for a glucose intolerant phenotype to be observed

in these hemizygous FBPase transgenic mice. Despite the fact that a HF diet upregulated FBPase,

many other genes are also upregulated with a HF diet including PEPCK and G6Pase, as well as

increased lipid substrates such as triglycerides. Thus, we believe that additional effects of the HF

diet contributed to the impairment in glucose tolerance observed in the transgenic mice. The

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triglyceride levels in the livers of transgenic mice on a HF diet were higher compared to the

negative mice on a HF diet which may have contributed to the observed glucose intolerant

phenotype.

Interestingly we did observe a gene-dose effect on glucose metabolism, with homozygous Line 5

transgenic mice developing mild, but significant glucose intolerance and a substantially elevated

EGP. This provides evidence that FBPase in the homozygous state can result in increased EGP,

without the concomitant effects of HF feeding. While upregulated FBPase in the homozygous state

appears to have a role, this is only evident when this enzyme is significantly elevated.. When it is

modestly elevated (2-3 fold as in these transgenic mice) it is clear that FBPase is not rate-

determining and therefore EGP is little affected. This implies that control of gluconeogenesis and

EGP may reside elsewhere. It is possible that other gluconeogenic enzymes such as PEPCK may be

more influential on the flux rate of gluconeogenesis and EGP. This is evident as mice

overexpressing PEPCK in the liver were reported to have increased EGP in the hemizygous state

and fasting glucose and insulin levels were double compared to controls with a significant

impairment in glucose tolerance (38, 43). Furthermore, rats overexpressing G6Pase in the liver also

showed glucose intolerance and hyperinsulinaemia without the need for further stress on the system,

such as a HF diet (42). We therefore suggest that under normal conditions FBPase may not be rate-

determining in EGP, and that there may be a hierarchy of importance for these gluconeogenic

enzymes, with FBPase being less important compared with PEPCK and G6Pase.

In summary, our study provides the first in-vivo evidence in a transgenic mouse model that

confirms a specific upregulation of FBPase in the liver causes glucose intolerance and a significant

elevation in EGP. Therefore if increased levels of FBPase in human type 2 diabetes are high

enough to contribute to the observed increase in EGP, FBPase inhibitors would be a useful therapy.

E-90552-2008-R1

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ACKNOWLEDGEMENTS

We would like to thank Blaise Weinrich, Lisa Billington, Cassie Bush and Zheng Ruan for

excellent technical assistance. This work is supported by a Biomedical Postgraduate Scholarship

from the National Health and Medical Research Council of Australia (S. Visinoni). S.

Andrikopoulos was supported by a RD Wright Biomedical Career Development Award from the

National Health and Medical Research Council of Australia. M. Watt is supported by a RD Wright

and Monash fellowship.

E-90552-2008-R1

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FIGURE LEGENDS

Figure 1. Generation of transgenic construct and FBPase levels in transgenic mice.

(A) Schematic diagram of the original construct used to generate the Line 1 mice. (B) Schematic

diagram of the new construct containing the human liver FBPase cDNA driven by the TTR

promoter, which was used to generate the Line 5 transgenic mice (C) Total hypothalamic FBPase

mRNA levels as determined using Real-Time PCR (n = 3, * p < 0.05 vs NEG and Line 5 TG). (D)

Total liver FBPase mRNA levels (n = 5, * p < 0.05 vs NEG and # p < 0.05 vs Line 1 TG). (E)

Liver FBPase protein levels of transgenic mice and negative littermates as determined by Western

blot (n = 4, * p < 0.05 vs NEG and # p < 0.05 vs Line 1 TG). (F) FBPase enzyme activity as

determined by a spectrophotometric assay (n = 4-8, * p < 0.05 vs NEG and # p < 0.03 vs Line 1

TG) correlated with both mRNA and protein levels from each transgenic line.

Figure 2. Liver FBPase levels and glucose tolerance of hemizygous transgenic mice at 24

weeks and 1 year of age.

(A) Total liver FBPase mRNA levels in 24 week old and 1 year old AGED mice, n = 4 for each

group * p < 0.05 vs NEG and NEG AGED and # p < 0.05 vs LINE 5 TG. (B) Plasma glucose

levels and (C) plasma insulin levels in hemizygous transgenic mice and negative littermates during

IPGTT. The solid lines represent the 24 week old mice; n = 45 for NEG (white circle), n = 21 for

Line 1 TG (gray circle) and n = 17 for Line 5 TG (black circle). The dashed lines represent the 1

year old aged mice; n = 15 for NEG (white triangle), n = 7 for Line 1 TG (gray triangle) and n = 7

for Line 5 TG (black triangle). (D) Plasma glucose data during IPGTT expressed as AUC, * p <

0.05 vs 24 week old mice. (E) Plasma insulin levels expressed as AUC were higher in all aged

mice compared to 24 week old mice, * p < 0.02 vs 24 week old mice.

E-90552-2008-R1

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Figure 3. Liver FBPase, G6Pase and PEPCK levels and glucose tolerance of hemizygous HFF

TG mice.

(A) Total liver FBPase mRNA levels in mice from HFFstudy, n = 4 for each group, * p < 0.05 vs

NEG and NEG HFF. (B) FBPase enzyme activity in the livers of hemizygous HFF TG mice from

Line 1 and 5, HFF and chow fed negative littermates (n = 4-8, * p < 0.02 vs NEG and # p < 0.05 vs

NEG HFF). (C) Plasma glucose levels and (D) plasma insulin levels during IPGTT. The solid lines

represent the chow fed mice; n = 45 for NEG (white circle), n = 21 for Line 1 TG (gray circle) and

n = 17 for Line 5 TG (black circle). The dashed lines represent the HFF mice; n = 18 for NEG

(white triangle), n = 9 for Line 1 TG (gray triangle) and n = 6 for Line 5 TG (black triangle). (E)

Plasma glucose data during IPGTT expressed as AUC, * p < 0.05 vs all other groups. (F) Plasma

insulin levels expressed as AUC, * p < 0.03 vs NEG chow fed mice. (G) Liver PEPCK mRNA

levels in chow and HFF mice, * p < 0.05 vs NEG and LINE 5 TG. (H) Liver G6Pase mRNA levels

in chow and HFF mice, * p < 0.05 vs NEG and LINE 5 TG.

Figure 4. Liver FBPase levels and glucose tolerance of homozygous TG mice.

(A) Total liver FBPase mRNA levels in homozygous and hemizygous Line 5 transgenic mice vs

negative littermates, n = 4 in each group, * p < 0.05 vs NEG and # vs NEG and Line 5 TG +/- mice.

(B) FBPase enzyme activity in the livers of homozygous and hemizygous Line 5 transgenic mice

and negative littermates (n = 4-8, * p < 0.02 vs NEG). (C) Plasma glucose levels and (D) plasma

insulin levels in hemizygous and homozygous transgenic mice and negative littermates during

IPGTT, n = 11 for NEG (white circle), n = 11 for Line 5 TG +/- (gray circle) and n = 13 for Line 5

TG +/+ (black circle). (E) Plasma glucose data during IPGTT expressed as AUC, * p < 0.02 vs

NEG mice and Line 5 TG +/- mice. (F) Plasma insulin levels expressed as AUC.

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Figure 5. Pyruvate tolerance and EGP levels in homozygous TG mice.

(A) Blood glucose levels during the pyruvate tolerance test expressed as AUC, n = 3, * p < 0.05 vs

NEG and TG +/- mice. (B) Rate of endogenous glucose production was significantly elevated in

Line 5 TG +/+ mice compared to negative littermates and hemizygous transgenic mice, n = 5, * p <

0.04 vs NEG and TG +/- mice.

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TABLE 1: Plasma glucose and insulin levels and body weights of male LINE 5 TG +/- mice and

negative littermates under fasted conditions at 24 weeks of age (n=16-44).

* p < 0.05 compared with negative littermates.

NEG LINE 1 TG LINE 5 TG

Plasma Glucose

(mmol/L)

6.8 0.2

7.0 0.3

6.1 0.2

Plasma Insulin

(ng/ml)

0.2 0.03

0.3 0.2

0.3 0.1

Body Weight

(grams)

30.8 ± 1.0

*26.6 ± 1.1

*25.9 ± 0.6

Food Intake

(grams/day)

4.02 ± 0.1

*3.38 ± 0.1

*3.42 ± 0.1

0

2

4

6

8

10

12

14

NEG LINE 1

TG

LINE 5

TG

FBPase E

nzym

e A

ctivity

(um

ol/

min

/mg p

rote

in)

* #

F

* #

* #

*

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

NEG LINE 1 TG LINE 5 TG

Liv

er

FBPase P

rote

in L

evels

(Fold

of negative)

GLUT2 Loading Control

NEG LINE 1 TG LINE 5 TG

E

0

1

2

3

4

5

6

7

8

9

NEG LINE 1

TG

LINE 5

TG

Rela

tive t

ota

l hypoth

ala

mic

FB

Pase m

RN

A levels

*C

0

1

2

3

4

NEG LINE 1

TG

LINE 5

TG

Rela

tive t

ota

l liver

FB

Pase m

RN

A levels

*

D

*

TTR Promoter/Enhancer

TTR Exon 1

TTR Intron 1 TTR Exon 2 TTR Exon 2

SV40 PolyA FBPase cDNA

A

HS4 TTR Promoter/Enhancer

TTR Exon 1

TTR Intron 1 TTR Exon 2

TTR Exon 2

SV40 PolyA

HS4

HS4 FBPase cDNA HS4

B

0

5

10

15

20

25

30

35

0 20 40 60 80 100 120

Time (min)

PlasmaGlucose(mmol/L)

B

0

2

4

6

8

10

12

14

0 20 40 60 80 100 120

Time (min)

PlasmaInsulin(ng/ml)

D

E

C

0

500

1000

1500

2000

2500

3000

NEG LINE 1

TG

LINE 5

TG

NEG

Aged

LINE 1 TG

Aged

LINE 5 TG

Aged

AUCPlasmaGlucose

(mmol/L*120min) *

**

0

100

200

300

400

500

600

700

800

NEG LINE 1

TG

LINE 5

TG

NEG

Aged

LINE 1 TG

Aged

LINE 5 TG

Aged

AUCPlasmaInsulin

(ng/ml*120min)

*

**

(FoldofNegative)

TotalLiverFBPasemRNALevels

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

NEG LINE 5 TG NEG AGED LINE 5 TGAGED

*

* #A

0

1

2

3

4

5

6

7

8

9

10

11

NEG NEG HFF LINE 5

TG HFF

Tota

l Liv

er F

BP

ase m

RN

A L

evels

(Fold

of

Negative)

*

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

NEG LINE

5 TG

NEG

HFF

LINE

5 TG

HFF

Liv

er

PEPC

K m

RN

A L

evels

(Fold

of N

egative)

**

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

NEG LINE 5TG

NEGHFF

LINE 5TG HFF

Liv

er

G6Pase m

RN

A L

evels

(F

old

of

Negati

ve)

**

0

1

2

3

4

5

6

7

8

9

10

NEG NEG

HFF

LINE

1 TG

HFF

LINE

5 TG

HFF

FB

Pase E

nzym

e A

cti

vit

y

(µm

ol/

min

/mg p

rote

in)

*

* #* #

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120

Time (min)

Pla

sm

a G

lucose (

mm

ol/

L)

C

A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 20 40 60 80 100 120Time (min)

Pla

sm

a I

nsulin (

ng/m

L)

D

B

0

500

1000

1500

2000

2500

3000

3500

NEG L1 TG

L5TG

NEGHFF

L1TGHFF

L5TGHFF

AU

C P

lasm

a G

lucose

(mm

ol/

L*120m

in)

*

*

E

G

0

50

100

150

200

250

300

AU

C P

lasm

a I

nsulin

(ng/m

l*120 m

in) *

NEG L1 TG

L5TG

NEGHFF

L1TGHFF

L5TGHFF

F

H

C

A B

E F

0

50

100

150

200

250

NEG LINE 5TG +/-

LINE 5TG +/+

AUCPlasmaInsulins

(ng/mL*120min)

0

500

1000

1500

2000

2500

3000

NEG LINE 5TG +/-

LINE 5TG +/+

AUCPlasmaGlucose

(mmol/L*120min) *

0

5

10

15

20

25

30

35

0 20 40 60 80 100 120PlasmaGlucose(mmol/L)

Time (min)

0

2

4

6

8

10

12

14

16

18

NEG LINE 5TG +/-

LINE 5TG +/+

FBPaseEnzymeActivity

(µmol/min/mgprotein)

*

*

0

1

2

3

4

5

6

NEG LINE 5TG +/-

LINE 5TG +/+

TotalLiverFBPasemRNALevels

(FoldofNegative)

*

* #

D

0.00.51.01.52.02.53.03.54.04.5

0 20 40 60 80 100 120

PlasmaInsulin(ng/mL)

Time (min)

A

B

0

5

10

15

20

25

30

35

40

45

NEG LINE 5 TG +/- LINE 5 TG +/+

*

EndogenousGlucoseProduction

(µmol/min/kg)

0

20

40

60

80

100

120

140

160

NEG LINE 5 TG +/- LINE 5 TG +/+

AUCBloodGlucose(mmol/L*120min) *