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Am J Physiol Endocrinol Metab 292: E1694-E1701, 2007. First published February 13, 2007; doi:10.1152/ajpendo.00430.2006
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Analysis of compensatory beta-cell response in mice with combined mutations of Insr and Irs2

Jane J. Kim,1,2 Yoshiaki Kido,3 Philipp E. Scherer,5 Morris F. White,4 and Domenico Accili2

1Deparment of Pediatrics, University of California, San Diego, California; 2Department of Medicine, Columbia University, New York, New York; 3Department of Medicine, Kobe University, Kobe, Japan; 4Children's Hospital, Harvard Medical School, Boston, Massachusetts; 5Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York

Submitted 18 August 2006 ; accepted in final form 9 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Type 2 diabetes results from impaired insulin action and beta-cell dysfunction. There are at least two components to beta-cell dysfunction: impaired insulin secretion and decreased beta-cell mass. To analyze how these two variables contribute to the progressive deterioration of metabolic control seen in diabetes, we asked whether mice with impaired beta-cell growth due to Irs2 ablation would be able to mount a compensatory response in the background of insulin resistance caused by Insr haploinsufficiency. As previously reported, ~70% of mice with combined Insr and Irs2 mutations developed diabetes as a consequence of markedly decreased beta-cell mass. In the initial phases of the disease, we observed a robust increase in circulating insulin levels, even as beta-cell mass gradually declined, indicating that replication-defective beta-cells compensate for insulin resistance by increasing insulin secretion. These data provide further evidence for a heterogeneous beta-cell response to insulin resistance, in which compensation can be temporarily achieved by increasing function when mass is limited. The eventual failure of compensatory insulin secretion suggests that a comprehensive treatment of beta-cell dysfunction in type 2 diabetes should positively affect both aspects of beta-cell physiology.

insulin receptor; insulin receptor substrate-2; insulin resistance; beta-cell compensation; genetics; insulin signaling; knockout mice

TYPE 2 DIABETES IS CHARACTERIZED by insulin resistance and beta-cell failure (1, 3). Prospective studies of metabolic control in diabetics indicate that, whereas insulin resistance is relatively constant, beta-cell failure is progressive in nature (24). The decrease in beta-cell function appears to have a complex pathogenesis, with defects of both insulin secretion (3) and beta-cell number (9). It is unclear how these two variables are interrelated.

The insulin receptor (Insr) and its substrates (Irs) play key roles in beta-cell function (1). Ablation of Insr (19) or Igf1 receptor (Igf1r) (20, 41) causes defects of insulin secretion. In contrast, ablation of Irs2, either in the whole body (40) or selectively in beta-cells (10, 25), brings about beta-cell failure due to decreased beta-cell proliferation and increased apoptosis. A similar defect can be caused by double ablation of Insr and Igf1r (38), indicating that both receptors signal through Irs2. The effect of Irs2 ablation can be rescued by haploinsufficient mutations of the forkhead transcription factor FoxO1 (17) or by a Pdx1 transgene (21), indicating that beta-cell failure associated with reduced Pdx1 expression is the key defect in these mice. Recent studies have also demonstrated a role for Irs2 in promoting islet cell survival by Creb induction via incretin hormones such as Glp1 (13, 31).

In this study, we used genetic crosses of mice with Irs2 and Insr mutations to analyze the pathophysiology of beta-cell compensation to insulin resistance. Because Irs2–/– mice have reduced beta-cell mass and impaired beta-cell proliferation (17), we asked whether they would be able to mount a compensatory beta-cell response to peripheral insulin resistance. To cause insulin resistance, we superimposed an Insr haploinsufficient mutation on the Irs2–/– background (5, 14, 16, 22). The human genes INSR and IRS2 are located on chromosomes 19 and 13, respectively. In contrast, the corresponding murine genes are located 3.8 cM apart on the short arm of chromosome 8 (33). By intercrossing mice with heterozygous mutations in both genes, we obtained mice with cis allelic mutations of Insr and Irs2 through meiotic recombination events. This recombinant congenic strain (Insr+/–Irs2+/–) enabled us to generate mice lacking Irs2 and haploinsufficient for Insr. We have preliminarily reported that these Insr+/–Irs2+/– mice develop diabetes (17). In the present study, we characterized their pancreatic islet response to insulin resistance.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal husbandry and genotyping. Generation of mice with null alleles of Insr and Irs2 has been described previously (14). Mice with combined mutations of Insr and Irs2 were obtained by crossing recombinant congenic Insr+/–Irs2+/– (cis) (14) with Irs2+/– mice. The mutations were maintained on a mixed genetic background (C57BL/6J x 129/Sv), and littermates were used as controls. All animals were housed in clear, ventilated Plexiglas cages within a pathogen-free barrier facility that maintained a 12:12-h light-dark cycle and were fed a standard pellet diet. Genotyping was performed as described (14). Only male animals were analyzed.

Phenotypic analysis. Blood was drawn from the retroorbital sinus of anesthetized animals between 9 and 11 AM. Glucose levels were measured using a glucometer (Accucheck; Boehringer Mannheim, Indianapolis, IN). Diabetes was defined as random plasma glucose > mean + 2 SD of wild-type controls on at least two separate occasions. Insulin was measured in plasma samples by RIA using a rat insulin standard (Linco, St. Charles, MO). Whole body composition of 4- to 8-wk-old mice was assessed by dual-energy X-ray absorptiometry (DEXA) measurements on a Lunar PIXIMUS scanner (GE Medical Systems, Waukesha, WI) functioning in the pencil beam mode. Before each series of scans, a tissue calibration scan was performed using the manufacturer's provided phantom. Live mice were anesthetized with intraperitoneal phenobarbital. Each mouse was placed on the scanner dish in a prone position with fore- and hindlegs outspread. Scans provided determinations of fractional body fat content, total body fat mass, and total fat-free mass. All procedures were approved by the Institutional Animal Care and Utilization Committee at Columbia University.

Glucose tolerance tests. Male mice were fasted overnight. They were then anesthetized with 50 mg/kg phenobarbital, and dextrose (1 g/kg) was injected into the peritoneal cavity. Blood samples were drawn from the retroorbital sinus at 0, 15, 30, 60, and 120 min for glucose measurement.

Insulin tolerance tests. Male mice were fed freely and then fasted for 4–6 h. They were subsequently anesthetized with 50 mg/kg intraperitoneal phenobarbital and treated with an intraperitoneal injection of human insulin at a dose of 0.5 U/kg (Humulin R; Eli Lilly, Indianapolis, IN). Blood samples were drawn from the retroorbital sinus at the beginning of the test and after 15, 30, and 60 min.

Leptin and adiponectin measurements. Total adiponectin and leptin levels were determined using ELISA-based colorimetric kits (Linco Research). Separation of high- and low-molecular-weight adiponectin complexes was determined by velocity sedimentation-gel filtration chromatography as previously described (30). Briefly, plasma was diluted 1:5 in 125 mM NaCl-10 mM HEPES, pH 8, and layered onto 5–20% sucrose step gradients and then spun at 55,000 rpm for 4 h at 4°C. Gradient fractions (150 µl) were retrieved sequentially from the top of the gradient and analyzed by quantitative Western blot analysis. Adiponectin complex distribution was independently confirmed by gel filtration chromatography.

Immunoprecipitation, immunoblotting, and PI 3-kinase assay. We carried out experiments in overnight-fasted, 8- to 12-wk-old mice. Animals were anesthetized by intraperitoneal administration of pentobarbital sodium (65 mg/kg), and the upper liver lobe and left soleus muscle were removed. Humulin R (5 U) was then injected through the inferior vena cava, and sections of the liver and right hindlimb muscles (gluteus and soleus) were taken at 1 and 3 min, respectively, after insulin injection. Tissues were homogenized in buffer containing 20 mM Tris, pH 7.6, 10% glycerol, 1% NP-40, 140 mM sodium chloride, 2.5 mM calcium chloride, 1 mM magnesium chloride, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. To evaluate the association between p85 and phosphotyrosine-containing proteins, Western blots were performed on tissue extracts first immunoprecipitated with anti-phosphotyrosine antibodies (Transduction laboratories, Lexington, KY) coupled to protein G-agarose beads. The beads were boiled in SDS sample buffer, separated on 8% SDS-polyacrylamide gel, and transferred to nitrocellulose membranes. The filter was then immunoblotted with anti-p85 antibodies (Upstate Biotechnology, Lake Placid, NY). Bound antibodies were detected with horseradish peroxidase-coupled antibodies to rabbit immunogloblin G by use of the ECL detection system (Amersham, Buckingshire, UK). Bands corresponding to phosphorylated PI 3-kinase were quantitated with NIH Image software (version 1.6; National Institutes of Health, Bethesda, MD). To measure Akt phosphorylation, solubilized extracts containing equal amounts of tissue protein were immunoblotted with rabbit polyclonal antibodies against either Akt or phospho-Ser473 Akt (Cell Signaling Technology, Beverly, MA), followed by second antibody detection and quantitation as previously described (14).

Pancreas histomorphometry. Animals were killed by sodium amytal injection. Pancreata were removed, cleared of fat and spleen, weighed, and fixed overnight in Bouin's solution. Tissues were embedded in paraffin, and consecutive 5-µm-thick sections were mounted on slides. Following rehydration and permeabilization in 0.1% Triton X-100, sections were immunostained for beta-cells with mouse monoclonal anti-glucagon antibodies (Sigma). beta-Cells were immunostained using either guinea pig anti-insulin (Linco) or mouse anti-insulin antibodies (Sigma). For quantitation of beta-cell mass, sections were viewed using a Nikon Eclipse E-400 microscope and video camera magnification of x10. Three sections of each pancreas were covered systematically by acquiring tiled images from at least 48 nonoverlapping fields of 1 mm2. Analyses of beta-cell area were performed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) and defined as the total surveyed area containing cells positive for insulin. The ratio of beta-cell to non-beta-cell areas in each section was then multiplied by pancreatic weight to obtain absolute beta-cell mass (14).

Statistical analyses. All values are expressed as means ± SE unless otherwise noted. Unpaired nonparametric Student's t-test was employed with a threshold for statistical significance of P < 0.05 to make comparisons between genotypes.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Generation of mice with combined mutations of Insr and Irs2. We generated mice of five genotypes by intercrossing Irs2+/– with Insr+/–Irs2+/– (cis) or Irs2+/– mice: wild-type (WT), Irs2+/–, Insr+/–Irs2+/–, Irs2–/–, and Insr+/–Irs2–/–. The mutations were maintained on a mixed genetic background, and littermates were used as controls. Progeny with Insr+/–Irs2–/– and Irs2–/– genotypes were obtained at lower frequency than expected on the basis of a Mendelian distribution of alleles (10% actual vs. 25% expected; n = 588 males), suggesting a significant effect on prenatal development in these genotypes. In crosses between Insr+/–Irs2+/– (cis) and Irs2+/– mice, offspring with Insr+/– or Irs2–/– genotypes were observed 3.8% of the time, in concordance with the expected ratio (4%). Interestingly, we were unable to recover Insr–/–Irs2–/– double-mutant mice.

Double-mutant Insr+/–Irs2–/–and single mutants Irs2–/– mice show postnatal growth retardation. Offspring with the five genotypes analyzed had normal birth weight. By 4 wk, we observed a 25% decrease in body weight of Insr+/–Irs2–/– compared with WT mice (P < 0.0005; Fig. 1A). Irs2–/– mice were also 10% smaller at 4 wk, as reported previously (40). The decline in growth became more pronounced in both genotypes with the onset of diabetes. Diminished growth was also observed in heterozygous Irs2+/– and Insr+/–Irs2+/– mice, but was more modest and did not reach statistical significance.


Figure 1
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  		Fig. 1. Growth curves and body composition of mutant mice.A: mice were weighed at birth and then at 4-wk intervals up to 16 wk of age. Values represent mean body weight of ≥25 mice per genotype ± SE; P < 0.0005 for Insr+/–Irs2–/– vs. wild type (WT), P < 0.001 for Irs2–/– vs. WT. B: epididymal adipose fat pads were isolated from 4- to 6-wk-old mice and their relative mass determined by dividing their weight by total body weight. Each bar represents individual means of 10 mice ± SE; P < 0.05 for Insr+/–Irs2–/– and Irs2–/– vs. WT. C: whole body adipose mass measured by DEXA scan at 4 to 6 wk of age. Data represent means ± SE; n = 7 for each genotype. P < 0.02 for Insr+/–Irs2–/– vs. Irs2–/–.

 
Reduced body fat content in Insr+/–Irs2–/– mice. Although we observed significant growth retardation in both Insr+/–Irs2–/– and Irs2–/– mice, body composition characteristics were divergent between these two genotypes. By 4–6 wk of age, adipose tissue mass was markedly reduced in Insr+/–Irs2–/– mice and significantly increased in Irs2–/– mice. Direct measurement of epididymal fat pads revealed that these depots constituted 1.84 ± 0.43 vs. 4.01 ± 0.26% of total body weight in Insr+/–Irs2–/– vs. Irs2–/– mice, respectively (P <0.05; Fig. 1B). This observation was also borne out from DEXA scan measurements in 6-wk-old mice, showing whole body adipose content of 17.3 ± 0.99% in Insr+/–Irs2–/– mice vs. 19.8 ± 0.89% in Irs2–/– mice (P < 0.05; Fig. 1C). Blood glucose measurements were similar between these two genotypes at this stage, indicating that changes in body composition were not secondary to diabetes. We also measured adipocyte size and number using histomorphometry but failed to identify any difference among the various genotypes (data not shown).

Increased serum leptin and adiponectin concentrations in mice lacking Irs2. Leptin levels were elevated in 6-wk-old Irs2–/– and Insr+/–Irs2–/– mice (P < 0.05; Fig. 2A), consistent with the hypothalamic leptin resistance described previously in Irs2–/– animals (6). This effect was independent of Insr haploinsufficiency. Interestingly, total plasma adiponectin levels were also significantly increased in Irs2–/– and Insr+/–Irs2–/– mice (P < 0.05), primarily due to increased high-molecular weight adiponectin (Fig. 2, B and C). This differs from the distribution of high- vs. low-molecular weight complexes found in obese humans and rodents with type 2 diabetes (30), suggesting that Irs2 may contribute to adiponectin-mediated effects on insulin action downstream of AdipoR1/R2 (37).


Figure 2
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  		Fig. 2. Increased serum leptin and adiponectin concentrations in Irs2 knockout mice. A: circulating leptin levels. B: total adiponectin concentrations. C: percentage of high-molecular-weight adiponectin forms. For each group at 6 wk of age; n = 10. *P < 0.05 vs. WT.

 
Diabetes in Irs2–/– and Insr+/–Irs2–/– mice. We examined the development of diabetes in mice with the different genotypes. As shown in Fig. 3A, glucose values began to rise in 4-wk-old Insr+/–Irs2–/– and Irs2–/– mice. Sixty-seven percent of Insr+/–Irs2–/– and 50% of Irs2–/– mice had hyperglycemia at 8 wk (Fig. 3B), with postprandial glucose levels of 324 ± 50 and 269 ± 61 mg/dl, respectively. Fasting glucose levels were also elevated in both groups to a similar degree (Fig. 3C). In contrast, only 6% of Insr+/–Irs2+/– heterozygotes became hyperglycemic in the same period, whereas WT and Irs2+/– mice remained euglycemic throughout. Kaplan-Meyer survival curves showed that Insr+/–Irs2–/– mice exhibited the highest mortality, with only 44% surviving longer than 12 wk, whereas 77% of Irs2–/– mice and 95% of Insr+/–Irs2+/– mice did so (n ≥ 20 per genotype group; data not shown).


Figure 3
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  		Fig. 3. Effect of combined Insr and Irs2 mutations on glucose levels. A: mean ± SE whole blood glucose levels in random-fed animals at various ages (n = 30 per genotype). **P < 0.005 and *P < 0.05 vs. WT. B: individual random glucose values. C: mean ± SE whole blood glucose levels in fasted animals at various ages; n = 30 per genotype.

 
Compensatory hyperinsulinemia and failure to expand beta-cell mass in Insr+/–Irs2–/– mice. Fasting plasma insulin levels were similar in all mice at 4 wk of age (Fig. 4A). By 8 wk, we detected a twofold increment in Irs2–/– and a 15-fold increment in Insr+/–Irs2–/– mice relative to WT controls (P < 0.05). Fed insulin levels were fivefold higher than fasting levels (data not shown). The increase in insulin levels was temporary, and was followed by a progressive decline, in association with worsening hyperglycemia. Scatter plots of insulin and glucose levels in fed and fasted 8-wk-old mice (Fig. 4, B and C) indicate that Insr+/–Irs2–/– mice have higher insulin levels for any given glucose level compared with Irs2–/– mice (Fig. 4, A–C), resulting in lower glucose-to-insulin ratios (GIR; Fig. 4D). The change is particularly evident during fasting. The fall in GIR at 8 wk was followed by a sharp increase at 12 wk, due to the unremitting progression of hyperglycemia and beta-cell failure. In contrast to Insr+/–Irs2–/– mice, only slight increases in fasting GIR were observed in Irs2–/– mice at the same ages (Fig. 4D).


Figure 4
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  		Fig. 4. Correlation between glucose and insulin values. A: fasting insulin values in mice of different genotypes between 4 and 12 wk of age. B and C: scattergram representation of fasting and fed plasma insulin vs. glucose values in all genotype groups at 8 wk of age. D: fasting glucose-to-insulin ratios and insulin values in all genotypes. Each bar represents the mean of ≥12 mice ± SE. *P < 0.05 and **P < 0.01 for Insr+/–Irs2–/– vs. WT.

 
Changes in islet cell mass in double-mutant Insr+/–Irs2–/– and single-mutant Irs2–/– mice. In view of the differences in circulating insulin levels between Insr+/–Irs2–/– and Irs2–/– groups, we analyzed islet histomorphometry in 4- and 8-wk-old mice (Fig. 5A). In contrast to previous studies in mice with combined heterozygous mutations in Insr and Irs2 genes (14), insulin levels in Insr+/–Irs2–/– mice did not correlate with beta-cell mass. Despite significantly higher fed and fasting insulin levels, Insr+/–Irs2–/– mice displayed a reduction in beta-cell mass at 8 wk compared with controls (0.20 ± 0.06 mg vs. 0.84 ± 0.12 mg, P < 0.005; Fig. 5B). The dissociation of islet cell mass and insulin levels was not seen in other genotypes. In comparison, beta-cell mass in Irs2–/– mice increased with age but was still smaller relative to nondiabetic controls. By 8 wk, beta-cell mass was reduced by 45% in Irs2–/– and 75% in Insr+/–Irs2–/– mice.


Figure 5
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  		Fig. 5. Pancreatic islet cell morphology. A: typical islet cell morphology with insulin immunostaining in 8-wk-old mice of indicated genotypes. B: morphometric evaluation of beta-cell mass in 4- and 8-wk-old mice of indicated genotypes. Data represent means ± SE; n = 8 per genotype. *P < 0.01 and **P < 0.003 vs. WT.

 
Glucose and insulin tolerance tests. Ins+/–Irs2+/–, Irs2–/–, and Insr+/–Irs2–/– animals became glucose intolerant by 6–8 wk of age (Fig. 6A). Despite similar levels of fasting hyperglycemia in Insr+/–Irs2–/– and Irs2–/– mice at test initiation, significantly higher glucose values were seen in Insr+/–Irs2–/– mice at all time points (P < 0.01). This is consistent with the observation that, although fasting insulin levels are higher in Insr+/–Irs2–/– than in Irs2–/– mice, fed levels increase to a lesser extent in the double mutants compared with the single Irs2 mutants. Impaired insulin sensitivity was also manifested in Insr+/–Irs2–/– and Irs2–/– animals following intraperitoneal insulin administration at 8 wk of age, the greatest deficit again being evident in Insr+/–Irs2–/– mice (Fig. 6B; P < 0.005).


Figure 6
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  		Fig. 6. Metabolic tests. A and B: ip glucose tolerance and insulin tolerance tests in 6- to 8-wk-old mice; n = 8 per genotype. Animals were fasted for 12 and 4 h prior to glucose and insulin tolerance tests. P < 0.005 for Insr+/–Irs2–/– vs. WT and P < 0.05 for Irs2–/– vs. WT at all time points after injection.

 
Impaired insulin-stimulated PI 3-kinase and Akt activity in liver and muscle. We next examined insulin action in selected target organs from diabetic and nondiabetic mice with various combinations of mutations. Because of the pivotal roles of PI 3-kinase and Akt in mediating the many biological actions of insulin through Insr and Irs2, we measured the activation of these signaling proteins as an indicator of insulin action. PI 3-kinase and Akt activities were notably diminished in Insr+/–Irs2–/– and Irs2–/– mice. The extent of the reduction paralleled the number of mutant alleles introduced. PI 3-kinase activity in liver and muscle was reduced by >50% in Irs2–/– mice (40), ~60% in Insr+/–Irs2+/– mice, and 80% in Insr+/–Irs2–/– mice (Fig. 7, A and B). Insulin-stimulated Akt activity in liver was similarly reduced by 80 and 90% in Insr+/–Irs2+/– and Insr+/–Irs2–/– mice, respectively. In contrast, insulin-dependent signaling in muscle extracts (Fig. 7, B and C) was relatively spared in Insr+/–Irs2+/– mice, with 40 and 30% reductions in PI 3-kinase and Akt activity, respectively. Insulin-dependent activation of these proteins in muscle was depressed by ~70% in Insr+/–Irs2–/– mice.


Figure 7
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  		Fig. 7. Insulin-stimulated PI 3-kinase and Akt activity in liver and muscle. A: representative blot from liver extracts. B: association between PI 3-kinase p85 subunit and phosphotyrosine-containing proteins in liver and hindlimb muscle from 8- to 12-wk-old mice following insulin stimulation. Solubilized tissue extracts were immunoprecipitated (IP) with anti-phosphotyrosine followed by immunoblotting (IB) with anti-p85 antibody, as described in METHODS. Results are expressed as percentage of p85 levels in WT mice. Data represent means from 5 independent experiments. C: Akt phosphorylation in liver and hindlimb muscle from 8- to 12-wk-old mice following insulin stimulation. Intensity of bands corresponding to phospho-Akt was corrected by total Akt levels to obtain relative measures of Akt phosphorylation between samples. Quantitation of results from 3 independent experiments is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main new finding of this study is that beta-cell compensation to insulin resistance can temporarily occur by increasing insulin secretion when beta-cell mass is limited. However, the increase in insulin secretion appears to strain replication-defective beta-cells and accelerate beta-cell failure.

Earlier studies have suggested that Insr and Irs2 control distinct aspects of beta-cell function, with Insr playing a role in insulin secretion (19) and Irs2 in the regulation of beta-cell mass (18, 39, 40). Analyses of beta-cell mass in the present report indicate that mice with combined Insr+/–Irs2–/– mutations are unable to compensate for peripheral insulin resistance by expanding beta-cell mass. This is consistent with recent reports indicating that blocking insulin and IGF signaling impairs beta-cell replication (8, 28, 38). Of note, mice with combined mutations show greater reductions in islet size compared with mice with Irs2 mutations alone. The increase in serum insulin levels in 8-wk-old Insr+/–Irs2–/– mice despite severely reduced beta-cell mass, suggests that beta-cells can temporarily compensate by augmenting insulin secretion. However, this increase is associated with more rapid beta-cell failure, perhaps by increasing endoplasmic reticulum stress or cellular apoptosis (12, 26, 29). A dissociation between insulin secretion and beta-cell mass has also been observed in mice with targeted mutations of Irs1 and liver-specific Irs2 compared with Irs1 knockouts alone (11). The mechanism by which increased insulin secretion contributes to beta-cell failure remains unclear.

Differential effects of Insr and Irs2 signaling on adipose tissue development. Both Insr-heterozygous (15) and Irs2-null mice (6) have been previously reported to have mild growth retardation. Combined Insr haploinsufficiency and Irs2 nullizygosity result in normal birth weights but reduced postnatal growth. This effect may be attributed to hepatic Irs2 function, perhaps in the production of circulating Igf1, as suggested by observations in liver-specific Irs2 knockouts (11). The moderate degree of growth retardation seen in Insr+/–Irs2–/– mice in this study suggests that their combined effect is additive but not synergistic, and consistent with the hypothesis that growth effects by Insr are mediated primarily via Irs1 (2, 5, 14, 35). Since the growth deficit in Insr and Irs2 mutants is already evident at 4 wk, when mice are still euglycemic, these differences likely reflect intrinsic signaling defects rather than unrestrained catabolism resulting from diabetes.

Although reduced growth was observed in both Irs2–/– and Insr+/–Irs2–/– mice, we detected significant differences in body composition between these two groups. Male Irs2 knockout mice showed increased visceral adiposity (6). On the other hand, Insr+/–Irs2–/– mice had markedly reduced abdominal and whole body adipose tissue stores. Insulin signaling has been shown to play an important role in lipid storage and adipogenesis. Conditional knockout of Insr in mature adipocytes leads to markedly reduced fat mass and triglyceride synthesis and storage (4). In contrast, the functional contribution of Irs2 in adipose tissue signaling is complex. In cultured cells, Irs2 is necessary for white adipocyte differentiation (27), but not for brown adipocyte differentiation (36), and studies of Irs knockout mice indicate that Irs2 does not play a role in adipogenesis in vivo (23). Moreover, although hyperinsulinemic euglycemic clamp studies demonstrate insulin resistance in adipose tissue of Irs2–/– mice (32), caloric restriction and resultant weight reduction lead to improved insulin sensitivity in Irs2–/– mice without affecting hepatic glucose production (34). This suggests that insulin resistance in adipocytes of Irs2-null mice results from obesity secondary to hypothalamic resistance to insulin and leptin. Together, these data imply that adipocyte hypertrophy, rather than impaired Irs2-dependent signaling, accounts for increased glycerol turnover and decreased glucose disposal observed under clamp conditions in Irs2–/– mice. The marked reduction in adipose stores of Insr+/–Irs2–/– mice likely reflects impaired adipogenesis secondary to Insr haploinsufficiency rather than Irs2 signaling defects in vivo.

Peripheral insulin resistance in Insr+/–Irs2–/– mice. In this study, Insr haploinsufficiency augments the severity of diabetes and glucose intolerance with further exacerbations in peripheral insulin resistance in both muscle and liver. Our data support recent findings indicating that both Irs2 and Irs1 mediate hepatic insulin action in vivo (11). Other models of hypothalamic insulin resistance have shown that central Irs2 signaling modulates hepatic metabolism (25). In addition, impaired responses to adiponectin and leptin (6) may contribute to the marked insulin resistance of Insr+/–Irs2–/– mice in this study.

In summary, we have explored the genetic interactions in Insr signaling that integrate peripheral insulin signaling and beta-cell response. Our findings indicate that insulin resistance and beta-cell failure can share a common etiology. The association of increased insulin secretion with deterioration of beta-cell function in Insr+/–Irs2–/– mice should strike a note of caution about treatments that aim to prevent diabetes by increasing beta-cell function (7).


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-58282 and DK-63608 (Columbia Diabetes and Endocrinology Research Center). J. J. Kim was supported by a Mentor-Based Postdoctoral Fellowship Award to D. Accili by the American Diabetes Association.


   ACKNOWLEDGMENTS
 
We thank members of the Accili laboratory for helpful discussions of the data.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Accili, Berrie Research Pavilion, 1150 St. Nicholas Ave., Rm. 238A, New York, NY 10032 (e-mail: da230{at}columbia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Accili D. Lilly lecture 2003. The struggle for mastery in insulin action: from triumvirate to republic. Diabetes 53: 1633–1642, 2004.[Abstract/Free Full Text]
  2. Araki E, Lipes MA, Patti ME, Bruning JC, Haag Br Johnson RS, Kahn CR. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186–190, 1994.[CrossRef][Medline]
  3. Bell GI, Polonsky KS. Diabetes mellitus and genetically programmed defects in beta-cell function. Nature 414: 788–791, 2001.[CrossRef][Medline]
  4. Bluher M, Michael MD, Peroni OD, Ueki K, Carter N, Kahn BB, Kahn CR. Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev Cell 3: 25–38, 2002.[CrossRef][Web of Science][Medline]
  5. Bruning JC, Winnay J, Bonner-Weir S, Taylor SI, Accili D, Kahn CR. Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell 88: 561–572, 1997.[CrossRef][Web of Science][Medline]
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