Upregulation of insulin receptor substrate-2 in pancreatic β cells prevents diabetes (original) (raw)
Upregulation of Irs2 in β cells. To determine whether β cell dysfunction is a pivotal element in the development of diabetes in Irs2–/– mice, we generated transgenic C57BL/6 mice expressing a low (rip9_→_Irs2) or a high (rip13_→_Irs2) level of Irs2 in pancreatic β cells under the control of rip. Irs2 was measured by specific immunoblotting of islet extracts containing equal amounts of total protein (Figure 1a). Irs2 was detected in WT islet extracts but was undetectable in Irs2–/– islets. Concurrent immunoblots revealed twofold and 12-fold increased Irs2 in rip9_→_Irs2 and rip13_→_Irs2 extracts (Figure 1a), which was similar to that obtained physiologically by activation of the endogenous Irs2 promoter with functional cAMP response element binding protein (21). Moreover, the relative increase of transgenic Irs2 in islets was comparable to the upregulation of endogenous Irs2 in Min6 β cells treated for 10 hours with exendin-4 or dibutyl-cAMP (Figure 1b).
Irs2 protein expression and β cell function. (a) Irs2 protein levels measured by specific immunoblotting in Irs2 immunoprecipitates of 200 μg total protein from islet extracts of 4-week-old WT mice, Irs2–/– mice, or two lines of transgenic mice (rip9_→_Irs2 and rip13_→_Irs2). (b) Irs2 protein levels measured by specific immunoblotting in Irs2 immunoprecipitates of 200 μg total protein from Min6 cells treated for 8 hours with the indicated concentrations of exendin-4 or dibutyl-cAMP (diB-cAMP) (c) Representative WT, rip9_→_Irs2, or rip13_→_Irs2 pancreas sections immunostained with anti-insulin antibodies (left panel) or anti-FLAG antibodies (right panel) that were detected by cyanine (Irs2) or fluorescein (Insulin) conjugated secondary antibodies, respectively. Original magnification, ×100. (d) Pancreatic insulin content per mg total pancreas was measured in acid/ethanol extracts of total pancreas from 6-month-old WT, rip9_→_Irs2, and rip13_→_Irs2 mice. Data are the mean values ± SEM of five mice per genotype. *P < 0.05, **P < 0.01. (e) Glucose-stimulated insulin release measured in 6-week-old WT and rip13→Irs2 mice. D-glucose (3 g/kg body wt) was injected intraperitoneally into 16-hour-fasted mice and blood samples were collected at the indicated timepoints. Results are expressed as mean values ± SEM of six WT and six rip_→_Irs2 mice (*P < 0.05). (f) Irs2 protein levels measured by specific immunoblotting in Irs2 immunoprecipitates from hypothalamic extracts of 4-week-old WT, rip9_→_Irs2, rip13_→_Irs2, Irs2–/–, or Irs2–/–:rip13_→_Irs2 mice. rip9, rip9_→_Irs2; rip13, rip13_→_Irs2.
Our transgenic production strategy incorporated a FLAG tag at the C terminus of Irs2 to facilitate immunostaining. Pancreas sections immunostained with anti-FLAG antibodies revealed that recombinant Irs2 protein was restricted to the insulin-positive β cells in rip9_→_Irs2 and rip13_→_Irs2 islets (Figure 1c). Moreover, the pancreatic insulin content increased 1.3-fold in rip9_→_Irs2 mice and 2.7-fold in rip13_→_Irs2 mice, revealing a dose effect of Irs2 expression on β cell insulin content (Figure 1d). During an intraperitoneal glucose challenge, insulin secretion during the first 30-minute interval was threefold higher in rip13_→_Irs2 mice than in WT mice (AUC WT: 1,880 ± 50 nmol × min/l; AUC rip13_→_Irs2: 5,440 ± 50 nmol × min/l) (Figure 1e). However, glucose homeostasis was normal in rip13_→_Irs2 mice from birth until the experiment was terminated after 24 weeks, as fasting glucose never fell below 79 ± 3 mg/dl and fed glucose never rose above 159 ± 6 mg/dl. Both transgenic mouse lines displayed normal fertility, growth, and adiposity, and had normal life spans (data not shown). Irs2 protein was never detected in hypothalamus (Figure 1f), nor was it detected in liver, muscle, or adipose tissues (data not shown).
Irs2 in β cells prevents diabetes in Irs2–/– mice. Male C57BL/6 Irs2–/– mice developed hyperglycemia between 4 and 6 weeks of age, which progressed to overt diabetes during the next 5–6 weeks until they died (Figure 2a). To determine whether the Irs2 transgene prevented diabetes in the Irs2–/– mice, we crossed rip9_→_Irs2 or rip13_→_Irs2 mice with Irs2–/– mice. As expected, transgenic Irs2 was expressed at a low level in Irs2–/–:rip9_→_Irs2 islets and at a high level in Irs2–/–:rip13_→_Irs2 islets (data not shown). Irs2–/–:rip9_→_Irs2 mice survived for 24 weeks because hyperglycemia progressed slowly toward diabetes between 10 and 24 weeks of age (Figure 2a). By contrast, glucose levels of Irs2–/–:rip13_→_Irs2 mice were normal during the 24-week experiment, revealing a dose effect for β cell Irs2 expression on glucose homeostasis (Figure 2a).
Metabolic effects of Irs2 overexpression in β cells. (a) Blood glucose concentrations were measured from tail bleeds of fed male mice. Values are mean ± SEM obtained from WT, rip13_→_Irs2, Irs2–/–, and rip_→_Irs2 mice crossed into a C57BL/6 Irs2–/– background (Irs2–/–:rip9_→_Irs2 and Irs2–/–:rip13_→_Irs2). Data are the mean values ± SEM of four (Irs2–/–:rip9_→_Irs2) to ten (WT, Irs2–/–, rip13_→_Irs2, and Irs2–/–:rip13_→_Irs2) mice of the indicated ages. (b) Glucose-tolerance tests were performed on 8-week-old fasted mice following intraperitoneal loading with 2 g D-glucose per kg body wt. Blood samples were taken at the timepoints indicated and glucose was determined as described. Results are mean ± SEM of four to ten mice (***P < 0.001, Irs2–/– vs. Irs2–/–:rip13_→_Irs2). (c) Random-fed serum insulin levels (mean values ± SEM, ***P < 0.001) measured by ELISA.
While Irs2–/– mice at 8 weeks of age displayed fasting hyperglycemia (Irs2–/–: 189 ± 10 mg/dl; WT: 109 ± 6 mg/dl), Irs2–/– mice expressing the transgenic Irs2 were nearly normal (Irs2–/–:rip9→Irs2: 124 ± 9 mg/dl; Irs2–/–:rip13_→_Irs2: 121 ± 8 mg/dl). As previously shown, the Irs2–/– mice were severely hyperglycemic during an intraperitoneal glucose challenge (Figure 2b). However, at 8 weeks of age, glucose intolerance was slightly improved in the Irs2–/–:rip9_→_Irs2 mice and completely normalized in the Irs2–/–:rip13_→_Irs2 mice (Figure 2b). Moreover, the Irs2–/–:rip13_→_Irs2 mice survived more than a year after the Irs2–/– mice died, displaying low random fed blood glucose levels at 15 months (WT: 121 ± 7 mg/dl; Irs2–/–:rip13_→_Irs2: 94 ± 6 mg/dl).
Irs2–/– mice are insulin resistant as a result of reduced insulin action in liver, fat, and muscle (9, 22). Before diabetes developed at 6 weeks, serum insulin levels were equally elevated in Irs2–/–, Irs2–/–:rip9_→_Irs2, and Irs2–/–:rip13_→_Irs2 mice to compensate for insulin resistance (Figure 2c). Insulin levels declined dramatically in Irs2–/– mice at 8 weeks, coinciding exactly with the onset of severe diabetes (Figure 2c). Compensatory insulin secretion in Irs2–/–:rip9_→_Irs2 mice gradually declined between 12 and 25 weeks until they died with severe diabetes (Figure 2c). Irs2–/–:rip13_→_Irs2 mice never developed diabetes due to persistent compensatory hyperinsulinemia, revealing the graded physiological response to Irs2 expression in transgenic β cells functioning in a genetically insulin resistant mouse (Figure 2c and data not shown).
Irs2 promotes β cell development and growth. Our previous work suggests that Irs2 signaling regulates survival of pancreatic β cells (18, 19). Compared with WT mice at 8 weeks of age, islet area in Irs2–/– pancreas sections was reduced about threefold, owing to a slightly reduced density of small islets containing 50% fewer β cells (Figure 3a and Table 1). By contrast, islet area in the rip13_→_Irs2 sections increased twofold, mainly due to increased density of normal-sized islets (Table 1). Islet density and the β cell content increased in Irs2–/–:rip13_→_Irs2 mice, a change that was also revealed by the increased ratio of β cells to α cells (Table 1).
Islet morphometry. (a) Insulin immunostaining of representative pancreas sections from 8-week-old C57BL/6 mice and from rip13_→_Irs2, Irs2–/–/rip13_→_Irs2, and Irs2–/– mice (original magnification, ×100). (b) Western blot of Pdx1 protein expression in isolated islets from 4-week-old male mice of the indicated genotypes. Each lane was loaded with 100 μg of total islet protein from one animal and is representative of at least three independent experiments.
To determine whether β cells in Irs2–/–:rip13_→_Irs2 islets were dividing at a higher rate than those in WT or rip13_→_Irs2 islets, we injected 8-week-old mice with the thymidine analogue BrdU to measure mitogenesis. BrdU incorporation into β cells during a 6-hour interval increased three- to fourfold in Irs2–/–:rip13_→_Irs2 mice compared with WT or rip13_→_Irs2 mice; no BrdU-positive cells were detected in Irs2–/– mice (Table 1). The average β cell size never changed upon expression of the Irs2 transgene. Thus, compensatory islet expansion during insulin resistance required Irs2 signaling to increase the number of normal-sized β cells.
The effect of Irs2 upregulation on islet gene expression. A possible mechanism by which Irs2 promotes expansion of β cells is through upregulation of the homeodomain transcription factor Pdx1 (also called Idx1 and Ipf1). Pdx1 is critical for development of the pancreas in mice and humans, and its complete disruption blocks pancreas development; in adult β cells, Pdx1 promotes normal glucose sensing and insulin secretion, and suppresses apoptosis (18, 23–25). Previous results show that Pdx1 expression is nearly lost in Irs2–/– islets, but can be restored by downregulation of Foxo1 (10). Pdx1 was detected equally by immunoblotting in WT and rip9_→_Irs2 islets (data not shown), whereas it was barely detected in our Irs2–/– islet extracts of equal protein concentration (Figure 3b). Consistent with a specific role for Irs2 in Pdx1 expression, Pdx1 protein was strongly upregulated rip13→Irs2 in WT and Irs2–/– mice containing the rip13→Irs2 transgene. (Figure 3b). Since upregulation of Pdx1 increases mitogenesis in Irs2–/– islets, upregulation of Pdx1 might contribute to the increased number of β cells in Irs2–/–:rip13_→_Irs2 islets (18, 25).
Pdx1 is reported to upregulate the expression of many genes that promote glucose-stimulated insulin secretion, including Glut2 (26). We used Affymetrix MG-U74v2 arrays to estimate the change in Glut2 mRNA between WT and rip13→Irs2 islets. The specificity of the Glut2 probe set was validated against the Glut1, Glut2, Glut3, and Glut4 probes using samples from rip13_→_Irs2 islets, brain, fat, liver, and muscle (Figure 4a). As expected, Glut2 was detected in liver and in rip13→Irs2 islets, but absent in the other test tissues; Glut1 and Glut3 were restricted to brain; and Glut4 was expressed exclusively in adipose and muscle tissue (Figure 4a). Compared with WT islets, Glut2 mRNA increased threefold (P < 0.005) in rip13→Irs2 islets (Figure 4b). Immunostaining revealed Glut2 in the plasma membrane of rip13_→_Irs2 and Irs2–/–:rip13_→_Irs2 β cells, whereas it was barely detected in the plasma membrane of WT islets (Figure 4c). The mRNA for other glycolytic enzymes also increased in rip13_→_Irs2 islets, including glucokinase (1.5-fold, P = 0.04), aldolase-1 (1.5-fold, P = 0.03), GAPDH (threefold, P < 0.005), and Pgk-1 (1.9-fold, P = 0.01); the β subunit of pyruvate dehydrogenase was also increased threefold (P < 0.005) (Figure 4b). Identical results were found for Irs2–/–:rip13_→_Irs2 islets. Since the secretion of insulin is tightly coupled to the rate of glucose metabolism, increased activity of glycolytic enzymes is consistent with improved β cell function (27).
Gene expression in WT tissues and rip13_→_Irs2 islets. (a) Affymetrix MG-U74v2 arrays (A, B, and C arrays) were used to estimate the relative mRNA levels for Glut1 (93738_at), Glut2 (103357_at), Glut3 (93804_at), and Glut4 (102314_at) in 6-week-old WT brain (B), adipose (F), rip13_→_Irs2 islets (I), liver (L), and muscle (M); the Affymetrix probe set is indicated in parentheses. Genes called present (Pr) or absent (A) by GeneChip (version 5) are indicated on the figure. The bars show averages of normalized expression measurements obtained from samples of two mice. (b) The fold change for mRNA in rip13_→_Irs2 islets normalized against WT islets is shown for Glut2, enzymes of the glycolytic pathway (glucokinase, Gck; phosphofructokinase-1, Pfk1; aldolase-1, Aldo1; GAPDH, Gapd; phosphoglycerokinase, Pgk1; phosphoglyceromutase-1, Pgam1; enolase, Eno1; pyruvate kinase, Pk3; pyruvate), and the pyruvate dehydrogenase α subunit (Pdha1) and β subunit (Pdhb). The expression of each gene in rip13→Irs2 islets is reported relative to the normalized level in WT islets; the P value for this comparison is indicated. The accession number for each enzyme is indicated above the P value. (c) Glut2 immunostaining of pancreas sections from 8-week-old WT, rip13_→_Irs2, and Irs2–/–:rip13_→_Irs2 mice (original magnification, ×400).
Irs2 in β cells prevents diabetes in obese and old mice. Peripheral insulin resistance develops during obesity and aging in mice and people, and progresses to diabetes when β cells fail to compensate with increased insulin secretion (28). To determine whether expression of Irs2 in β cells promotes compensation for obesity-induced diabetes, WT C57BL/6 mice or rip13Irs2 mice were weaned and maintained for 60 days on a low- or a high-fat diet. Mice fed the high-fat diet were obese at 12 weeks of age (C57BL/6: 36.5 ± 2.7 g, n = 6; rip13_→_Irs2: 37.3 ± 2.5 g, n = 6) compared with those eating a low-fat diet (C57BL/6: 24.6 ± 2.8 g, n = 6; rip13_→_Irs2: 25.4 ± 2.6 g, n = 6). Obese mice displayed fasting hyperglycemia (obese C57BL/6: 206 ± 16 mg/dl; lean C57BL/6: 101 ± 18 mg/dl) and glucose intolerance (Figure 4a). By contrast, obese rip13Irs2 mice had statistically normal fasting glucose levels (129 ± 15 mg/dl) and normal glucose tolerance (Figure 5a). Consistent with these results, fasting hyperinsulinemia was greater in obese rip13Irs2 mice than in obese C57BL/6 WT mice, and during the intraperitoneal glucose challenge insulin levels increased significantly in obese rip13_→_Irs2 mice but not in obese C57BL/6 WT mice (Figure 5b).
Metabolic characterization of WT and rip13_→_Irs2 mice maintained on a low-fat diet (LFD) or high-fat diet (HFD). (a) An intraperitoneal glucose tolerance test (2 g D-glucose per kg body wt) was performed on fasted C57BL/6 WT or rip13_→_Irs2 mice maintained on a high-fat or low-fat diet for 60 days after weaning. Blood samples were taken at the timepoints indicated and glucose levels were determined as described. Results are expressed as mean ± SEM of six WT and six rip13_→_Irs2 male mice (**P < 0.01, ***P < 0.001). (b) Serum insulin levels for the indicated timepoints during a glucose tolerance test. Data are the mean values ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Glucose tolerance test in 6-month-old C57BL/6 WT and rip13_→_Irs2 mice. Data are the mean values ± SEM of eight mice per genotype (*P < 0.05).
Irs2 expression falls as mice age (10). At 6 months, C57BL/6 mice on a normal diet developed mild glucose intolerance, whereas the rip13_→_Irs2 mice were normal (Figure 5c). At 40 weeks, islet area was 3.1% ± 0.6% in WT mice, 6% ± 1% in rip9_→_Irs2 mice, and 10% ± 2% in rip13_→_Irs2 mice, revealing a graded response to a low and high level of Irs2 expression. Thus, transgenic _Irs2_–mediated β cell expansion compensates for insulin resistance that develops during aging.
Irs2 promotes β cell survival signaling. Acute or chronic stress and autoimmune responses upregulate proinflammatory cytokines (including TNF-α, IL-1β, and IFN-γ) that promote destruction of β cells at least in part by contributing to apoptosis (29–32). By contrast, Irs protein signaling promotes cell growth and inhibits apoptosis in various cellular backgrounds (19). In isolated WT murine islets, IGF1 stimulated phosphorylation of Erk1/2, Akt, and the Akt target Foxo1 (Figure 6a). By contrast, in Irs2–/– islets the phosphorylation of these targets was reduced, and cleaved/activated caspase-3 accumulated and was insensitive to IGF1 stimulation, which is consistent with decreased growth and survival of Irs2–/– β cells (Figure 6a). To determine whether upregulation of Irs2 restores these signals, islets were isolated from WT mice, or Irs2–/–:rip13_→_Irs2 and rip13_→_Irs2 mice, cultured for 12 hours, and stimulated with IGF1. IGF1 stimulated phosphorylation of Erk1/2, Akt, and Foxo1 in WT islets; however, the basal and IGF1-stimulated phosphorylation of these proteins was significantly increased in Irs2–/–:rip13_→_Irs2 and rip13_→_Irs2 islets (Figure 6b). Moreover, upregulation of Irs2 eliminated the accumulation of cleaved cascapse-3, even before IGF1 stimulation, suggesting that Irs2 signaling plays a critical role in β cell survival (Figure 6b).
IGF1 and insulin signaling in isolated islets. (a) Islets were isolated from WT or Irs2–/– C57BL/6 mice and incubated overnight before stimulation with insulin or IGF1 for 20 minutes as described in Methods. Each lane was loaded with 100 μg of total islet protein. The results are representative of at least three independent experiments. (b) Islets were isolated from WT, Irs2–/–:rip13_→_Irs2, and rip13_→_Irs2 mice and incubated overnight before stimulation with IGF1 for 20 minutes as described in Methods. The same amount of total protein (300 μg) was loaded onto the gel and transferred to a nitrocellulose membrane. Membranes were analyzed for the presence of Erk1/2 and phosphorylated Erk (pErk1/2); Akt1/2 and phosphorylated Akt (pAkt1/2); Foxo1 and phosphorylated Foxo1 (pFoxo1); and cleaved caspase-3 (Casp3).
Apoptosis and function of rip13→Irs2 islets transplanted in mice. Since recombinant Irs2 dramatically reduced the level of cleaved caspase-3, we reasoned that β cell apoptosis might be reduced in rip13_→_Irs2 islets. We induced β cell apoptosis in situ by injecting a low dose of streptozotocin for 5 consecutive days into 8-week-old WT or rip13_→_Irs2 mice with comparable body weight (21.7 ± 3 g) and comparable levels of random-fed serum glucose (158 ± 11 mg/dl) and plasma insulin (255 ± 60 pmol/l). Blood glucose and serum insulin levels were measured before streptozotocin injection and 6, 12, and 15 days after the first streptozotocin injection. At 15 days, T cell infiltration was comparable in C57BL/6 WT and rip13_→_Irs2 mice (Figure 7a). However, low-dose streptozotocin caused insulinopenic diabetes in C57BL/6 WT mice, whereas the rip13_→_Irs2 mice were normal (Figure 7, b and c). Apoptotic β cells were readily detected in WT mice, but apoptosis was reduced more than 50% in rip13_→_Irs2 mice (Figure 7d). Thus, transgenic Irs2 prevented the onset of diabetes 15 days after streptozotocin injection, at least in part by suppressing β cell apoptosis.
Survival and function of rip13_→_Irs2 mice during streptozotocin-induced diabetes. (a) Representative H&E-stained pancreas sections from untreated WT or rip13Irs2 islets compared with pancreas sections obtained 15 days after one injection per day for 5 days of low-dose (40 mg/kg) streptozotocin (strep; original magnification, ×200). (b) Blood glucose levels in WT or rip13_→_Irs2 mice were measured before and after five (daily) injections of low-dose (40 mg/kg) streptozotocin. Results are expressed as mean ± SEM of eight WT and eight rip13_→_Irs2 mice (**P < 0.01); (c) Glucose levels (mg/dl) divided by serum insulin levels (ng/dl) are shown for experimental days 0, 6, and 15 (***P < 0.001). (d) Apoptotic cells were detected in deparaffinized sections using a rhodamine DNA fragmentation detection assay. The number of apoptotic nuclei per β cell is shown for day 15. Values are expressed as mean ± SEM of eight WT and eight transgenic mice (***P < 0.001). (e) Transplantation of WT islets into streptozotocin-diabetic mice. C57BL/6 mice were treated with 100 mg/kg streptozotocin for 3 consecutive days. Blood glucose levels were measured in samples obtained through tail bleeds of fed mice before transplantation (shaded area) and at the indicated ages after transplantation of 300 (filled circles) or 150 (open circles) WT islets. (f) Transplantation of rip13_→_Irs2 islets into streptozotocin-diabetic mice was conducted in identical fashion with 250 (filled circles), 120 (open circles), or 50 (inverted triangles) islets. Values are mean ± SEM of at least three mice per experiment.
To test whether rip13_→_Irs2 islets are resistant to the effects of stress induced by transplantation, WT or rip13_→_Irs2 islets were transplanted under the kidney capsule of streptozotocin-diabetic mice. In this experiment, 8-week-old C57BL/6 mice were treated with 100 mg/kg streptozotocin for 3 consecutive days to cause severe diabetes (Figure 7, e and f). Without islet transplantation, the diabetic mice died about 10 days after the first injection. By contrast, transplantation of 300 WT islets (>10,000 islets/kg) normalized random-fed glucose levels by 50% within 3–5 days, and glucose levels were nearly normal 60 days later when the experiment was terminated (Figure 7e). By contrast, transplantation of 150 WT islets prevented death but failed to normalize glucose levels (Figure 7e), whereas 80 islets were completely ineffective as the diabetic mice died (not shown). Importantly, 50 rip13_→_Irs2 islets (β cell mass equivalent to 80 WT islets) normalized random-fed glucose levels by 50% after 20 days, and quicker treatment was obtained with 120 and 250 rip13_→_Irs2 islets (Figure 7f). BrdU incorporation measured in the islet grafts 60 days after transplantation revealed that DNA synthesis was twofold greater in rip13_→_Irs2 islets (2.12% ± 0.32% BrdU positive β cells, n = 3) than in WT islets (1.2% ± 0.2% BrdU positive β cells, n = 3). Thus, upregulation of Irs2 in transplanted β cells significantly reduced the number of islets needed to cure diabetes in mice, at least in part by promoting proliferation.