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Endocrinology, doi:10.1210/en.2005-1498
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Endocrinology Vol. 147, No. 6 2764-2772
Copyright © 2006 by The Endocrine Society

Expression of Human Fructose-1,6-Bisphosphatase in the Liver of Transgenic Mice Results in Increased Glycerol Gluconeogenesis

Benjamin J. Lamont, Sherley Visinoni, Barbara C. Fam, Melkam Kebede, Blaise Weinrich, Stavroula Papapostolou, Helene Massinet, Joseph Proietto, Jenny Favaloro and Sofianos Andrikopoulos

Department of Medicine (Austin Health and Northern Health), University of Melbourne, Heidelberg Repatriation Hospital, Heidelberg Heights, Victoria 3081, Australia

Address all correspondence and requests for reprints to: Sofianos Andrikopoulos, Ph.D., Department of Medicine (Austin Health and Northern Health), University of Melbourne, Heidelberg Repatriation Hospital, 300 Waterdale Road, Heidelberg Heights, Victoria 3081, Australia. E-mail: sof{at}unimelb.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In type 2 diabetes, increased endogenous glucose production (EGP) as a result of elevated gluconeogenesis contributes to hyperglycemia. An increase in glycerol gluconeogenesis has led to the suggestion that, in obese human subjects with type 2 diabetes, there may be an increase in the activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase). The aim of this study was to generate transgenic mice that overexpress human liver FBPase in the liver and assess the consequences to whole-body and hepatic glucose metabolism. FBPase transgenic mice had significantly higher levels of transgene expression in the liver and, as a result, had increased FBPase protein and enzyme activity levels in the liver. This resulted in an increase in the rate of glycerol conversion to glucose but not in EGP. The increased expression of FBPase in the liver did not result in any significant differences compared with littermate control mice in insulin or glucose tolerance. Therefore, it appears that, on its own, an increase in FBPase does not lead to impaired regulation of EGP and hence does not affect whole-body glucose metabolism. This suggests that, for EGP to be increased, other factors associated with obesity are also required.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN TYPE 2 DIABETES, endogenous glucose production (EGP) is inappropriately elevated after an overnight fast (1, 2, 3, 4, 5, 6, 7) and is less suppressed after a meal (8, 9, 10, 11, 12). The liver is the major tissue responsible for EGP (13), with a smaller contribution from the kidney (14, 15, 16). EGP is the product of two processes: glycogenolysis and gluconeogenesis. The level of EGP is increased in type 2 diabetes because the rate of gluconeogenesis is inappropriately elevated despite the presence of hyperinsulinemia (1, 3, 17). The rate of gluconeogenesis is governed by the activity of three regulatory and irreversible enzymes, phosphoenolpyruvate carboxy kinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase).

Obesity is strongly associated with an increased risk for the development of type 2 diabetes (18) by contributing to the increase in EGP. As such, increased free fatty acids have been shown to increase the rate of gluconeogenesis (19, 20, 21, 22, 23, 24, 25) by increasing the activity and protein levels of FBPase (26, 27, 28) and the expression of G6Pase and PEPCK (29, 30). Furthermore, increases in these key gluconeogenic enzymes have been seen in the livers of obese, insulin-resistant, and glucose-intolerant animal models (27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39).

Transgenic mouse models that have increased PEPCK expression in the liver display impaired glucose tolerance and increased EGP (40, 41). Our group has also shown that a PEPCK transgenic rat model displays impairments in glucose tolerance and increased EGP during hyperinsulinemic conditions (42, 43). In addition, G6Pase has been overexpressed in the liver of rats using adenovirus and results in hyperinsulinemia and impaired glucose tolerance (33).

An increase in liver FBPase has been suggested as a potential mechanism for increased EGP after it was shown that obese patients with type 2 diabetes have an increase in gluconeogenesis from glycerol (44, 45), a substrate that enters the gluconeogenic pathway immediately before FBPase. Furthermore, it was shown that, for the same plasma glycerol levels, patients with type 2 diabetes convert more to glucose, suggesting an enhanced intracellular conversion (45). This fact led these investigators to hypothesize an increase in FBPase activity as a cause of increased gluconeogenesis. Increased liver FBPase activity has in fact been observed in animal models of obesity and insulin resistance such as New Zealand Obese (NZO) mice (27), db/db mice (34), ob/ob mice (35, 36), fa/fa rats (37), and high-fat fed rats (28). However, the relative importance of increased FBPase to whole-body glucose metabolism and EGP has not been directly assessed by overexpressing the enzyme in the liver in vivo. In this study, we generated and studied transgenic mice with a specific overexpression of human liver FBPase gene (FBP-1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of human liver FBPase transgenic mice
Total human liver RNA, a gift from the Dahl Laboratory (Murdoch Children’s Research Institute, Melbourne, Victoria, Australia), was reverse transcribed using the Thermoscript Reverse Transcriptase kit (Invitrogen, Carlsbad, CA). A PCR with the proofreading Pfu polymerase (Promega, Madison, WI) was used to amplify the liver FBPase coding sequence from human liver cDNA generated by RT. The PCR-generated FBPase fragment was gel purified. To make the FBPase fragment compatible for ligation into the pGem-T-Easy vector, it was A-tailed in a reaction using Taq polymerase (Promega). The A-tailed DNA fragment was ligated into pGem-T-Easy using the reagents supplied with the vector kit (Promega) and transformed into Max Efficiency DH5{alpha} competent cells (Invitrogen) for amplification. To remove the FBPase fragment from the pGem-T-Easy plasmid, a digestion was performed with EcoRI (Promega). The 1.1 kb FBPase fragment was gel purified and ligated into the transthyretin (TTR) vector (pTTR1 ExV3) (46) obtained from Dr. Robert Costa (University of Illinois, Chicago, IL). The sequence of the pTTR-FBPase plasmid construct, shown in Fig. 1AGo, was confirmed by cycle sequencing, using fluorescent-labeled terminator reactions (BigDye Terminator v2; Applied Biosystems, Foster City, CA).


Figure 1
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  		FIG. 1. A, Schematic diagram of the construct containing the human liver FBPase cDNA driven by the TTR promoter, which was used to generate the transgenic mice. Sv40, Simian virus 40. B, Transgenic FBPase mRNA in liver as determined by real-time PCR. The level of transgenic FBPase in line 1 transgenic mice (Tg) was significantly higher than lines 2 and 3 (*, P < 0.05). C, Endogenous mouse FBPase mRNA in liver as determined by real-time PCR. D, Transgenic FBPase mRNA in extrahepatic tissues from FBPase transgenic mice (n = 4) from line 1, as determined by real-time PCR. The transgenic FBPase mRNA levels in brain and kidney were significantly different (*, P < 0.05) from all other tissues. BAT, Brown adipose tissue; WAT, white adipose tissue.

 
Purified, supercoiled plasmid (pTTR-FBPase) DNA was sent to a commercial facility for the generation of the transgenic mice (Ozgene, Murdoch, Western Australia, Australia). The pTTR-FBP construct was linearized by digestion with HindIII (Promega). The purified DNA fragment was microinjected into the pronuclei of fertilized eggs from C57BL/6 mice, and the zygotes that survived the DNA injection and became two-cell-stage embryos were implanted into the oviducts of pseudopregnant females, which were mice (of the C57BL/6 strain) that had been mated previously to vasectomized males. Embryos that developed to term were tail biopsied after weaning to test for the presence of the transgene using Southern blotting. Three transgenic founder mice (designated 1, 2, and 3) were identified and bred with C57BL/6 control mice to determine germ-line transmission of the transgene and to evaluate expression levels. Of the three transgenic founder lines, only one (line 1) showed increased mRNA and protein expression of FBPase in the liver and was thus the subject of the investigation for this study.

Genotyping of liver FBPase transgenic mice
To detect the presence of the TTR-FBPase transgene in total mouse genomic DNA, a PCR using specific primers was performed. The forward primer corresponded to the sequence within the TTR intron-1 region of the transgene: 5'-CCC ATT TCA CTG ACA TTT CTC TT-3'. The reverse primer corresponded to the sequence within the human FBPase cDNA region of the transgene: 5'-ACA CGT GGC AAA GGA TGA CTT T-3'. The PCR amplification from genomic DNA samples that contained the transgene yielded a 393-bp fragment. In addition to primers for the transgene, each PCR mixture also contained primers for the mouse glyceraldehyde-3-phosphate dehydrogenase gene. The forward primer sequence was 5'-CTC CGC CCC TTC TGC CGA T-3'. The reverse primer sequence was 5'-GTC CAC CAC CCT GTT GCT GT-3'. This led to a coamplification of glyceraldehyde-3-phosphate dehydrogenase, which yielded a 614-bp fragment in all samples and served as an internal PCR control.

Housing
Transgenic mice were housed in the Department of Medicine, Royal Melbourne Hospital animal facility. Mice had free access to water and were fed a standard laboratory chow comprising, on a weight basis, 74% carbohydrate, 20% protein, and 6% fat (Barastoc, Pakenham, Victoria, Australia). Temperature was maintained at 22 C with a 12-h light, 12-h dark cycle.

Determination of FBPase mRNA levels
FBPase mRNA was quantitated using the real-time PCR technique. Primers were designed for the SYBR-green real-time PCR using Primer-Express software (Applied Biosystems). The primers for ß-actin, which matched both the rat (GenBank accession no. NM_031144) and mouse (GenBank accession no. NM_007393) sequences were as follows: forward primer, 5'-CGT GAA AAG ATG ACC CAG ATC A-3'; and reverse primer, 5'-CAC AGC CTG GAT GGC TAC GT-3'. The primers for mouse (endogenous) FBPase, which match the mouse sequence of liver FBPase (GenBank accession no. NM_019395) but do not match the human sequence (GenBank accession no. NM_000507), were as follows: forward primer, 5'-GCT CTG CAC CGC GAT CA-3'; and reverse primer, 5'-ACA TTG GTT GAG CCA GCG ATA-3'. The primers that match the expected transgenic TTR-FBPase mRNA sequence, but not the mouse FBPase sequence, were as follows: forward primer, 5'-ACA GAT CCA CAA GCT CCT GAC A-3'; and reverse primer, 5'-CCA TGC TTG AAC CGG GTA GA-3'. All of these primer sets were designed so that they result in amplification of a product that spans an exon-intron boundary.

Enzyme assays and Western blotting
The activities of PEPCK and FBPase were determined as described previously (47). FBPase protein levels were determined by Western blotting as described previously (48).

Intraperitoneal glucose tolerance tests
Intraperitoneal glucose tolerance tests (IPGTTs) were performed as described previously (49). Briefly, mice were fasted overnight (~16 h) with free access to water. Before the experiment, they were weighed and anesthetized with an ip injection of sodium pentobarbitone (100 mg/kg Nembutal; Rhone Merieux, Pinkenba, Queensland, Australia). After a recovery period of 20 min, a blood sample was taken and the mice were injected ip with 2 g glucose per kilogram of body weight. Blood samples (100 µl) were taken from the retroorbital sinus at 15, 30, 60, and 120 min. Each blood sample was centrifuged, and the plasma was removed and stored at –20 C until required for analysis of glucose and insulin concentration.

Intraperitoneal insulin tolerance tests
Intraperitoneal insulin tolerance tests (IPITTs) were performed as described previously (50). Briefly, mice were fasted overnight (~16 h) with free access to water. Before the experiment, they were weighed and anesthetized with an ip injection of sodium pentobarbitone (100 mg/kg). After a recovery period of 20 min, a blood sample was taken and the mice were injected ip with 0.75 U human insulin (Actrapid; Novo Nordisk, Baulkham Hills, New South Wales, Australia) per kilogram of body weight. Blood samples (50 µl) were taken from the retroorbital sinus at 15, 30, 45, and 60 min. The glucose concentration of the blood samples was measured using a glucometer (Precision Q.I.D.; MediSense, Bedford, MA).

Glucose turnover and glycerol gluconeogenesis
Mice were fasted overnight (~16 h) and anesthetized on the morning of the experiment 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. Body temperature was maintained at 37 C using a heat lamp and monitored with a rectal probe.

A primed (2 min, 3 µCi/min) continuous infusion (0.15 µCi/min) of [6-3H]glucose was administered during basal and euglycemic-hyperinsulinemic-clamp experiments to measure whole-body glucose turnover, as described previously (51, 52). During the hyperinsulinemic-clamp experiments, after an initial priming dose, insulin was infused at a constant rate at 5 mU/kg·min. Blood glucose concentration was maintained at basal levels by the infusion of a 5% glucose solution. Blood samples were collected at 90, 100, and 110 min.

Gluconeogenesis from glycerol was measured as described previously (27). Briefly, a bolus of [U-14C]glycerol (3.2 µCi) was infused (during the basal or hyperinsulinemic clamp) via the jugular vein for 2 min and subsequently followed by a constant infusion at 0.1 µCi/min for 110 min. Blood (400 µl) was collected at 90, 100, 110 min, and 100 µl, deproteinized immediately with 500 µl 0.3 mol/liter Ba(OH)2 and 500 µl 0.3 mol/liter ZnSO4, and centrifuged, and the clear supernatant was collected and stored for additional analysis. The rest of the blood was centrifuged, and the plasma was stored at –20 C for measurement of glucose and insulin. At the end of the experiment, a laparotomy was rapidly performed, and the liver was quickly taken into liquid nitrogen and stored at –70 C.

Analytical procedures
To determine the rate of gluconeogenesis from glycerol, 400 µl of the deproteinized sample was passed down three columns. The first column contained 1.7 ml Dowex AG-50W-X8 (H+ form, 100–200 mesh) resin binding amino acids (alanine and glutamine), which were released by eluting with 4 ml of 2 mol/liter NH4OH. The second column containing 1.7 ml AG-1-X8 (Cl– form, 100–200 mesh) resin bound lactate and pyruvate, and they were eluted by 4 ml of 0.1 mol/liter HCl and were not analyzed in this experiment. The third column contained 1.7 ml AG-1-X8 (borate form, 100–200 mesh) resin and bound glycerol and glucose. Glycerol was eluted with 5 ml of 20 mmol/liter sodium tetraborate, and glucose was eluted with 4 ml of 0.5 mol/liter acetic acid. All eluants were dried in a fan-forced oven at 60 C overnight, and each was resuspended in 300 µl distilled H2O, 200 µl of which was combined with 3.5 ml scintillant (ReadyValue; Beckman Instruments, Palo Alto, CA). Radioactivity was determined in a Beckman Instruments (Oakleigh, Victoria, Australia) LS 3081 scintillation counter using a dual-label program. The other 100 µl was used to assay total glucose and alanine concentrations as described below.

Determination of glycerol and D-glucose
Both glycerol and glucose were measured using standard spectrophotometric methods with a Beckman Instruments DU-64 spectrophotometer at a wavelength of 340 nm. The method to determine plasma glycerol was based on Eggstein and Kuhlmann (53), after the conversion of oxidized nicotinamide-adenine dinucleotide to reduced nicotinamide-adenine dinucleotide (27). Glucose was determined by the method of Kunst et al. (54), in which NADP+ was converted to nicotinamide adenine dinucleotide phosphate (27).

Hepatic glycogen assay
Glycogen levels were determined by measuring glucose derived from glycogen in a liver homogenate. A sample of liver (10 mg) and 200 µl ice-cold perchloric acid (0.6 mol/liter) were homogenized using a Polytron homogenizer. A sample of this homogenate (40 µl) was added to 20 µl KHCO3 (1 mol/liter) plus 400 µl glycoamylase/acetate buffer [20 mg amyloglucosidase in 20 ml acetate buffer, 0.2 mol/liter (pH 4.8)] and incubated at 40 C for 2 h. Perchloric acid (0.6 mol/liter, 200 µl) was then added, and the sample was centrifuged at 3000 rpm for 10 min. The remaining tissue preparation was also centrifuged. Samples of these supernatants were analyzed for glucose using a fluorometric method (33). Glycogen levels were determined by subtracting free glucose from total glucose concentrations.

Plasma assays
Plasma insulin was determined by RIA (Linco Research, St. Charles, MO) using a double-antibody technique to separate free from bound insulin. Plasma glucose was determined using a GM7Analox glucose analyzer (Helena Laboratories, Mount Waverley, Victoria, Australia).

Determination of fat pad weight, food intake, and physical activity
Mice were anesthetized as described above, gonadal (epididymal or periovarian), inguinal subcutaneous, and retroperitoneal adipose tissues were removed, and the wet mass was weighed. Having the same person perform the procedure on all mice standardized the dissection of the adipose tissue depots. For the food intake measurement, mice were housed individually, and the food was weighed daily for 5 d. The bedding used in the cage was cellulose bedding (Fibercycle, Mudgeeraba, Queensland, Australia) to enable identification of food particles. Food intake was corrected for spillage. Physical activity levels were determined in a separate group of mice and were measured by continuous monitoring the use of the running wheels using a computerized meter as described previously (55).

Calculations
Specific activity of [6-3H]glucose and [U-14C]glycerol were at steady state after 90 min of infusion in both basal and hyperinsulinemic-clamp experiments. Rate of glucose appearance (Ra), which represents EGP (mainly hepatic) in the basal state, was calculated by dividing the infusion rate of the [6-3H]glucose tracer by its specific activity in plasma. In hyperinsulinemic-clamp experiments, the rate of EGP was determined by the difference between the calculated Ra and the measured rate of infused glucose. Rate of conversion of glycerol to glucose was calculated by multiplying the [14C]glucose-specific activity by the rate of glucose appearance and dividing by [14C]glycerol-specific activity (27).

Statistical analysis
All results are expressed as mean ± SEM. When there were two independent groups, differences were analyzed using Student’s t test. One-way ANOVA followed by post hoc analysis with Fisher’s least significant difference test was used when there were more than two independent groups. A P value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of human liver FBPase transgenic mice
Of the three transgenic founder mice identified, progeny of only one (line 1) was shown to have a significant expression of the human liver FBPase transgene (Fig. 1BGo), whereas expression of endogenous FBPase was not affected (Fig. 1CGo). The levels of transgenic FBPase mRNA in a range of tissues from line 1 FBPase transgenic mice were compared with those in the liver. The levels of transgenic FBPase mRNA in kidney and brain were between 0.15 and 0.20% of those measured in liver (Fig. 1DGo). In other tissues, which included intestine, pancreas, gonads, spleen, heart, skeletal muscle (quadriceps), white adipose tissue, and brown adipose tissue, the levels detected were less than 0.05% of those in the liver (Fig. 1DGo). Transgene inheritance followed a Mendelian pattern, with approximately equal numbers of transgenic and nontransgenic mice born in each line.

Western blotting detected total FBPase protein levels in liver from control and transgenic mice (Fig. 2AGo). The level of liver FBPase in transgenic mice from line 1 was 2-fold higher than in control mice (Fig. 2AGo). In lines 2 and 3, the levels of liver FBPase protein were not different compared with their respective nontransgenic control mice (data not shown). The FBPase enzyme activity was 1.5-fold higher in liver homogenates from line 1 transgenic mice compared with controls (Fig. 2BGo). There was no difference in the FBPase enzyme activity measured in kidney homogenates from transgenic and control mice of line 1 (Fig. 2CGo).


Figure 2
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  		FIG. 2. A, Liver FBPase protein levels in the liver of control mice (C; black bar) and transgenic mice (Tg; white bar) from line 1, as determined by Western blot. Representative blot of liver homogenates (2 µg total protein loaded per lane). A single band corresponding to the reported size of mouse and human FBPase (36 kDa) was detected in each lane with an antirat liver FBPase antibody. Data are presented as mean ± SEM; *, P < 0.005. FBPase enzyme activity in liver (B) and kidney (C) homogenates from control (black bars; n = 5) and FBPase transgenic (white bars; n = 5) mice from line 1, as determined by a spectrophotometric assay. Data are presented as mean ± SEM; *, P < 0.001.

 
The mean body weight of male and female transgenic mice was lower compared with the control mice used in glucose turnover experiments (Table 1Go). Whereas gonadal (Table 1Go), retroperitoneal, and subcutaneous fat pad weights (data not shown) were comparable between the two groups of mice, food intake was significantly lower in both transgenic male and female mice compared with their negative littermates (Table 1Go). Finally, physical activity was similar in female transgenic and control mice (13,018 ± 1781 vs. 13,733 ± 1266 cycles/d). Thus, it seems that the lower body weight in the transgenic FBPase is associated with decreased food intake.


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  		TABLE 1. Body weight, gonadal fat weight, and food intake of control and FBPase transgenic mice at 24 wk of age

 
The concentration of glucose and insulin in plasma was determined in samples collected from random-fed and overnight-fasted mice. Analysis of data combined from male and female mice revealed that, although fasting caused a decrease plasma glucose levels in both groups of mice, there was no difference in these parameters between transgenic and control littermates (Table 2Go).


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  		TABLE 2. Plasma glucose and insulin levels (mean ± SEM) of control and FBPase transgenic mice during random feeding (n = 5) and fasting conditions (n = 13)

 
The levels of glycogen in liver from FBPase transgenic and control mice were not different (Fig. 3AGo). Liver PEPCK activity was also not different between FBPase transgenic and control mice (Fig. 3BGo).


Figure 3
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  		FIG. 3. A, Liver glycogen content in control mice (black bar; n = 10) and FBPase transgenic mice (white bar; n = 12). B, Liver PEPCK activity levels in control mice (black bar; n = 5) and FBPase transgenic mice (white bar; n = 5). Data are presented as mean ± SEM.

 
Glucose turnover
After an overnight fast, there was no difference between FBPase transgenic and control mice in the rate of glucose appearance (Ra) (Fig. 4AGo), indicating that the basal rate of EGP in FBPase transgenic mice was normal. However, the rate of glycerol gluconeogenesis was significantly increased in the transgenic vs. control mice under basal conditions (Fig. 4BGo). Hyperinsulinemic-euglycemic-clamp experiments were also performed and showed no difference in the rate of EGP (Fig. 4CGo) and rate of glucose disappearance (data not shown) between FBPase transgenic and control mice. Again, the rate of glycerol gluconeogenesis under clamp conditions was significantly higher in the FBPase transgenic compared with control mice (Fig. 4DGo). Furthermore, when the data were analyzed such that plasma glycerol concentrations were matched, glycerol gluconeogenesis was almost 2-fold higher in the FBPase transgenic compared with control mice, in line with the level of protein overexpression (Fig. 2AGo).


Figure 4
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  		FIG. 4. Ra (A) and glycerol gluconeogenesis (B) in control (black bar; n = 14) and FBPase transgenic (white bar; n = 14) mice measured under basal conditions. Rate of EGP (C) and glycerol gluconeogenesis (D) in control (black bars; n = 10) and FBPase transgenic (white bars; n = 10) mice measured under hyperinsulinemic-euglycemic-clamp conditions. E, The level of glycerol gluconeogenesis when plasma glycerol levels are matched between control and FBPase transgenic mice. Data are presented as mean ± SEM; *, P < 0.05 vs. control.

 
Glucose and insulin tolerance
Glucose tolerance was assessed in FBPase transgenic and control mice by the IPGTT. The elevation in plasma glucose (Fig. 5AGo) during the IPGTT was the same in both transgenic and control mice. Furthermore, mice were fed a 60% (w/w) fat diet as described previously (55) for 10 wk to assess whether this would result in glucose intolerance in the FBPase transgenic mice. After the high-fat diet, fasting plasma glucose concentrations were similar (7.3 ± 1.1 vs. 7.5 ± 0.6 mM), as was glucose tolerance [area under the curve (glucose), 1560 ± 43 vs. 1561 ± 153 mM for 120 min] between transgenic and control mice.


Figure 5
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  		FIG. 5. A, Comparison of glucose tolerance in control (black squares; n = 13) and FBPase transgenic (white circles; n = 13) mice as assessed by the IPGTT. B, Comparison of the response to an injection of insulin in control (black squares; n = 13) and FBPase transgenic (white circles; n = 14) mice during the IPITT. Data are presented as mean ± SEM.

 
The glucose response to an injection of insulin was assessed in transgenic and control mice at 6 months of age by the IPITT. After injection of insulin, the plasma glucose level fell to the same extent in both FBPase transgenic and control mice (Fig. 5BGo), indicating that increased FBPase in the liver of transgenic mice does not result in an impaired effect of insulin during an IPITT.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gluconeogenesis is a major component of EGP and is inappropriately increased in patients with type 2 diabetes (1, 3, 56). We have shown previously that, in the NZO mouse, a model of obesity and insulin resistance, EGP was elevated as a result of increased gluconeogenesis from alanine and glycerol (27, 57). Furthermore, we showed that this augmentation in glycerol gluconeogenesis was associated with an increase in the gluconeogenic enzyme FBPase (47, 48). In support, it has been shown that glycerol gluconeogenesis is increased and that, for a given concentration of glycerol, the rate of glycerol gluconeogenesis was higher in patients with type 2 diabetes than in control subjects (44, 45). This suggested that an intrahepatic mechanism may be contributing to the elevated gluconeogenesis in obese patients with type 2 diabetes. In fact, Nurjhan et al. (45) implicated an increase in FBPase as a plausible reason for the increased glycerol gluconeogenesis in patients with diabetes. These studies are complicated by the presence of obesity, which can provide substrate as well as directly up-regulate the enzymes and would lead to an increase in gluconeogenesis (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Finally, a specific inhibitor of FBPase has been generated and has been shown to decrease glucose production rates in an obese, diabetic animal model, giving rise to a potential treatment for type 2 diabetes (58, 59). All of this information provided us with the incentive to generate liver FBPase transgenic mice to determine whether a specific increase in this enzyme can, in its own right, lead to increased EGP and possibly impaired glucose tolerance.

Previous studies have shown that overexpression of a single enzyme involved in gluconeogenesis can alter whole-body glucose metabolism. Transgenic mice overexpressing PEPCK in the liver displayed hepatic insulin resistance and impaired glucose tolerance (40, 41), although we have shown that rats that overexpressed PEPCK in the kidney displayed impaired glucose tolerance and hepatic insulin resistance, as well as muscle and fat insulin resistance (42, 43). Increased G6Pase expression in the liver of rats also induced hyperinsulinemia and glucose intolerance (33).

In the present study, we generated transgenic mice that overexpress the human liver form of FBPase and found that glucose and insulin tolerance were normal, as was EGP. However, the rate of glycerol gluconeogenesis was increased as a result of FBPase overexpression. In fact, when we analyzed the data such that plasma glycerol levels were matched, the FBPase transgenic mice displayed an almost 2-fold increase in glycerol gluconeogenesis. This is similar to the situation observed in patients with type 2 diabetes (45) and what we observed previously in the NZO mouse (27). It is important to note that the 2-fold increase in liver FBPase that we observed in our study is comparable with the level of up-regulation that we found in the NZO mouse (47) and in the high-fat fed rats (28) and is similar to that found in other gluconeogenic transgenic models [e.g. G6Pase and PEPCK (33, 41)], which displayed increased EGP and glucose intolerance. We therefore believe that the increase in liver FBPase in our transgenic mice is physiological and sufficient to enhance glycerol gluconeogenesis and would thus be expected to affect glucose production.

So why was EGP unaltered despite an increase in liver FBPase in our transgenic mice? There are several possible reasons for this. It is possible that gluconeogenesis from other substrates (lactate, amino acids) was decreased to compensate for the increase in glycerol gluconeogenesis. When increased levels of liver FBPase were associated with increased rates of EGP, the animals were either obese or placed on a high-fat diet (27, 28). The FBPase transgenic mice are not obese; in fact, the FBPase mice used in the glucose turnover experiments weighed less than their littermate controls. Increased fat can have multiple effects on the liver, including changes in insulin action, changes in the levels of transcription factors [e.g. SREBP-1 (sterol regulatory element-binding protein-1) and PPAR (peroxisome proliferator-activated receptor)], and changes in the levels of the other gluconeogenic enzymes (60). Perhaps the level of FBPase can determine the overall rate of gluconeogenesis and EGP only under conditions of obesity, as appears to be the case in NZO mice (27) and high-fat fed rats (28). Combining the increased liver FBPase in the FBPase transgenic mice with other alterations associated with obesity may provide an opportunity to further investigate this possibility. Conversely, our data also suggest that, under normal conditions, FBPase may not be rate determining in EGP and that other enzymes, particularly PEPCK, are more influential on the rate of glucose production. As discussed above, we (42, 61) and others (40, 41) have shown that hepatic overexpression of PEPCK both in vitro and in vivo can result in increased glucose production. However, the mechanism might not be as simple as increased levels of PEPCK causing a direct increase in the rate of gluconeogenesis. In PEPCK transgenic mice, both liver PEPCK and G6Pase mRNA levels were up-regulated, whereas the levels of IRS-2 (insulin receptor substrate-2) protein and phosphorylation were down-regulated, which resulted in a resistance to the effects of insulin to reduce the expression of gluconeogenic enzymes (41). Conversely, mice in which the PEPCK gene was deleted also displayed alterations in the expression pattern of genes with fatty acid oxidizing genes being up-regulated, in the absence of changes in circulating hormones (62). It is important to note that PEPCK knockout mice had hepatic steatosis and remained normoglycemic, even after an overnight fast, leading the authors to suggest that the newly identified effects of PEPCK on gene expression or the regulation of hepatic lipid metabolism might be more important than a direct role in the regulation of gluconeogenesis (62). The reported association of PEPCK overexpression and hepatic insulin resistance therefore may be related to the effects of PEPCK on the expression of other genes involved in insulin signaling, which would secondarily regulate gluconeogenesis. Another possibility is that an alteration in hepatic lipid metabolism caused by changes in the expression of PEPCK further enhanced glucose production under these conditions, whereas an increase in FBPase expression may not result in obesity because it is purely a gluconeogenic enzyme.

Of interest was the decrease in body weight that was associated with a reduction in food intake in the FBPase transgenic mice. In light of the very low expression of the transgene in the brain compared with the liver, this result may suggest crosstalk between the liver and the brain to regulate body weight by reducing food intake under these circumstances.

In summary, a 2-fold increase of FBPase in the liver resulted in increased glycerol gluconeogenesis but was not sufficient to cause changes in whole-body or liver glucose metabolism. Under normal physiological conditions, the level of FBPase in the liver may not determine the rate of EGP. A complete understanding of the role of the key gluconeogenic enzymes in the regulation of hepatic glucose production in normal and obese states could have significant implications for the implementation of strategies to combat fasting hyperglycemia in type 2 diabetes.


   Acknowledgments
 
We thank Jenny Davis and Christian Rantzau for excellent technical assistance.


   Footnotes
 
This work was supported by National Health and Medical Research Council of Australia Project Grant 145769. S.A. was supported by an R. D. Wright Biomedical Career Development Award from the National Health and Medical Research Council of Australia.

The authors of this manuscript have nothing to declare.

First Published Online February 23, 2006

Abbreviations: EPG, Endogenous glucose production; FBPase, fructose-1,6-bisphosphatase; G6Pase, glucose-6-phosphatase; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test; NZO mice, New Zealand Obese mice; PEPCK, phosphoenolpyruvate carboxy kinase; Ra, rate of glucose appearance; TTR, transthyretin.

Received November 28, 2005.

Accepted for publication February 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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