Insulin Receptor-deficient Mice Research Articles

452-P: Liraglutide Decreases Neointimal Growth in Control and Western Diet–Fed Mice, and Its Effect Is Insulin Dependent

Restenosis following percutaneous transluminal angioplasty and stenting is increased in subjects with insulin resistance and diabetes. Our preclinical models of restenosis show that insulin treatment is vasculoprotective in insulin sensitive conditions but insulin's effect is diminished or lost in Western diet induced insulin resistant conditions. Accordingly, early insulin treatment has not proven to provide cardiovascular disease (CVD) protection in subjects with dysglycemia, while the effect of intensive insulin treatment of type 2 diabetes (T2D) is debated. Glucagon-like peptide-1 receptor agonists (GLP-1RA) are a new class of antidiabetic drugs that reduce the CVD risk of T2D. Several animal studies have shown that GLP-1RA decrease neointimal growth in models of restenosis; however, the mechanisms of the CVD actions of these drugs remain elusive. GLP-1RA are insulin secretagogues, therefore their effect may be partly mediated by the insulin receptor (IR) . The objective of this study was to determine if insulin receptor (IR) deficiency in endothelial cells (EC) attenuates the effect of GLP-1RA to decrease neointimal growth after arterial injury. Endothelial deficient IR mice were generated by crossing IR floxed mice with mice expressing tamoxifen-inducible Cre recombinase under the control of Tie2 promoter. Mice were fed a low-fat (LFD) or a high-fat-high-sucrose (HFSD) Western diet and implanted with an osmotic pump delivering liraglutide, a GLP-1RA prototype (1mol/kg/day) , or vehicle prior to femoral artery wire injury. In LFD- as well as in HFSD-fed IR-floxed controls, liraglutide decreased neointimal area (NA) and intima-to-media ratio (I/M) . In LFD-fed endothelial IR deficient mice liraglutide had no effect. These data suggest that IR in ECs is important for the anti-restenotic effect of GLP1-RA, which is preserved in Western diet induced insulin resistant conditions unlike the effect of insulin. *First and second authors contributed equally Disclosure M.Gonzalez: None. Z.Liu: None. A.Giacca: None.

101-OR: Cell-Specific Role of Insulin in a Model of Restenosis in Insulin-Sensitive and Insulin-Resistant Conditions

Angioplasty and stenting are widely used in the treatment of atherosclerosis. However, these procedures may fail due to post-angioplasty restenosis, which remains a problem for patients with diabetes. Currently, the vascular role of insulin is unclear. Its role on endothelial cells (EC) is vasculoprotective, whereas its effects on vascular smooth muscle cells (SMC) are mainly mitogenic according to in vitro data. We have previously shown a suppressive effect of insulin treatment on neointimal growth in rodent models of restenosis in insulin sensitive conditions that was abolished in insulin resistant conditions. The objective of this study was to determine the EC-and SMC-specific effects of insulin treatment on neointimal growth in a murine model of restenosis in insulin sensitive and insulin resistant conditions. Mice were generated by crossing insulin receptor (IR) floxed mice with tamoxifen inducible Cre recombinant mice under the control of Cdh5 (vascular endothelial cadherin) or SMMHC (smooth muscle heavy chain) promoters. Mice were fed with a low-fat (LFD) or a high-fat-high-sucrose (HFSD) diet and implanted with insulin pellet (0.U/day) or vehicle (control) prior to femoral artery wire injury. We found that compared to IR-floxed controls, tamoxifen-induced IRf/f-Cdh5-Cre+ or IRf/f-SMMHC-Cre+ mice showed a 94% and 80% decrease in IR expression in ECs or SMCs, respectively. In LFD-fed insulin sensitive conditions, insulin decreased neointimal area (NA) and intima-to-media ratio (I/M) in controls (Cdh5 and-SMMHC-floxed and wildtype-Cre+ mice) but not in endothelial and SMC IR deficient mice. In HFSD-fed insulin resistant conditions, insulin had no effect in either controls or endothelial and SMC IR deficient mice. These data demonstrate that insulin action in both ECs and SMCs is required for the anti-restenotic effect of insulin in insulin sensitive conditions, which is abolished but not reversed in insulin resistance even when endothelial IR is deficient. Disclosure J.Z. Wang: None. C.X. Zhang: None. A. Giacca: None.

Bi-directional regulation of brown fat adipogenesis by the insulin receptor.

Insulin is a potent inducer of adipogenesis, and differentiation of adipocytes requires many components of the insulin signaling pathway, including the insulin receptor substrate IRS-1 and phosphatidylinositol 3-kinase (PI3K). Brown pre-adipocytes in culture exhibit low levels of insulin receptor (IR), and during differentiation there is both an increase in total IR levels and a shift in the alternatively spliced forms of IR from the A isoform (–exon 11) to the B isoform (+exon 11). Brown pre-adipocyte cell lines from insulin receptor-deficient mice exhibit dramatically impaired differentiation and an inability to regulate alternative splicing of the insulin receptor. Surprisingly, re-expression of either splice isoform of IR in the IR-deficient cells fails to rescue differentiation in these cells. Likewise, overexpression of IR in control IRlox cells also results in inhibition of differentiation and a failure to accumulate expression of the adipogenic markers peroxisome proliferator-activated receptor gamma, Glut4, and fatty acid synthase, although cells overexpressing IR retain the ability to activate PI3K and down-regulate mitogen-activated protein kinase (MAPK) phosphorylation. Thus, differentiation of brown adipocytes requires a timed and regulated expression of IR, and either the absence or overabundance of insulin receptors in these cells dramatically inhibits differentiation.

Transcriptional Activation of Apolipoprotein CIII Expression by Glucose May Contribute to Diabetic Dyslipidemia

Hypertriglyceridemia and fatty liver are common in patients with type 2 diabetes, but the factors connecting alterations in glucose metabolism with plasma and liver lipid metabolism remain unclear. Apolipoprotein CIII (apoCIII), a regulator of hepatic and plasma triglyceride metabolism, is elevated in type 2 diabetes. In this study, we analyzed whether apoCIII is affected by altered glucose metabolism. Liver-specific insulin receptor-deficient mice display lower hepatic apoCIII mRNA levels than controls, suggesting that factors other than insulin regulate apoCIII in vivo. Glucose induces apoCIII transcription in primary rat hepatocytes and immortalized human hepatocytes via a mechanism involving the transcription factors carbohydrate response element-binding protein and hepatocyte nuclear factor-4α. ApoCIII induction by glucose is blunted by treatment with agonists of farnesoid X receptor and peroxisome proliferator-activated receptor-α but not liver X receptor, ie, nuclear receptors controlling triglyceride metabolism. Moreover, in obese humans, plasma apoCIII protein correlates more closely with plasma fasting glucose and glucose excursion after oral glucose load than with insulin. Glucose induces apoCIII transcription, which may represent a mechanism linking hyperglycemia, hypertriglyceridemia, and cardiovascular disease in type 2 diabetes.

Bi-directional Regulation of Brown Fat Adipogenesis by the Insulin Receptor

Insulin is a potent inducer of adipogenesis, and differentiation of adipocytes requires many components of the insulin signaling pathway, including the insulin receptor substrate IRS-1 and phosphatidylinositol 3-kinase (PI3K). Brown pre-adipocytes in culture exhibit low levels of insulin receptor (IR), and during differentiation there is both an increase in total IR levels and a shift in the alternatively spliced forms of IR from the A isoform (-exon 11) to the B isoform (+exon 11). Brown pre-adipocyte cell lines from insulin receptor-deficient mice exhibit dramatically impaired differentiation and an inability to regulate alternative splicing of the insulin receptor. Surprisingly, re-expression of either splice isoform of IR in the IR-deficient cells fails to rescue differentiation in these cells. Likewise, overexpression of IR in control IRlox cells also results in inhibition of differentiation and a failure to accumulate expression of the adipogenic markers peroxisome proliferator-activated receptor gamma, Glut4, and fatty acid synthase, although cells overexpressing IR retain the ability to activate PI3K and down-regulate mitogen-activated protein kinase (MAPK) phosphorylation. Thus, differentiation of brown adipocytes requires a timed and regulated expression of IR, and either the absence or overabundance of insulin receptors in these cells dramatically inhibits differentiation.

The Forkhead Transcription Factor Foxo1 Regulates Adipocyte Differentiation

An outstanding question in adipocyte biology is how hormonal cues are relayed to the nucleus to activate the transcriptional program that promotes adipogenesis. The forkhead transcription factor Foxo1 is regulated by insulin via Akt-dependent phosphorylation and nuclear exclusion. We show that Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion phase. Constitutively active Foxo1 prevents the differentiation of preadipocytes, while dominant-negative Foxo1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. Further, Foxo1 haploinsufficiency protects from diet-induced diabetes in mice. We propose that Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes adipocyte differentiation.

Partial rescue of insulin receptor-deficient mice by transgenic complementation with an activated insulin receptor in the liver

Insulin receptor (IR)-deficient mice develop severe diabetes mellitus, diabetic ketoacidosis (DKA) and liver steatosis and die within 1 week after birth. We examined in this work whether the metabolic phenotype of IR(−/−) mutants could be improved by transgenic complementation with IR selectively in the liver. We first generated transgenic mice expressing a human DNA complementary to RNA encoding a truncated constitutively activated form of IR (IRδ) under the control of liver-specific phenylalanine hydroxylase (PAH) gene promoter. These mice presented more pronounced fasting hypoglycemia and showed slightly improved glucose tolerance as compared to controls. The transgenic mice were crossed with IR(+/−) mutants to generate IR(−/−) mice carrying the PAH-IRΔ transgene. Although such mutants developed glycosuria, DKA was delayed by more than 1 week and survival was prolonged to 8–20 days in ∼10% of mice. In these partially rescued pups, serum glucose and triglyceride levels were lowered, hepatic glycogen stores were reconstituted and liver steatosis was absent as compared with pups which developed strong DKA and died earlier. Thus, lack of insulin action in the liver is responsible in large part for the metabolic disorders seen in IR(+/−) mice. This study should stimulate interest in therapeutic strategies aimed at improving hepatic function in diabetes.

Improved metabolic disorders of insulin receptor-deficient mice by transgenic overexpression of glucokinase in the liver.

Insulin receptor null mutant mice develop severe diabetes, ketoacidosis and liver steatosis and die within 1 week after birth. Since the liver plays an essential role in the control of glucose homeostasis, we examined in this work whether the metabolic disorders of insulin receptor-deficient mice could be improved upon restoration of hepatic glucose metabolism by transgenic constitutive overexpression of glucokinase selectively in the liver. We first generated transgenic mice overexpressing rat glucokinase cDNA under control of the liver-specific phenylalanine hydroxylase gene promoter. These transgenic mice were crossed with heterozygous insulin-receptor-null mutants to produce homozygous insulin-receptor-null mice overexpressing glucokinase in the liver. The transgenic mice overexpressing glucokinase in the liver showed improved glucose tolerance and were mildly hypoglycaemic and hyperlipidaemic under starved conditions. The introduction of the glucokinase transgene in insulin receptor null mice did not prevent the development of glycosuria. However, ketoacidosis was delayed by more than 1 week and survival was prolonged to 10 to 16 days in 16% of the pups. In these longer surviving pups, serum glucose and triglyceride concentrations were lowered, hepatic glycogen stores were reconstituted and liver steatosis was absent as compared with the pups which had developed strong ketoacidosis and died earlier. These results show that overexpression of hepatic glucokinase can compensate, in part, for the metabolic disorders developed by insulin receptor-deficient mice. This shows the importance of improving hepatic function in diabetes and must revive interest in enhancement of glucokinase activity as a therapeutic strategy for the treatment of diabetes.

Differential signaling of insulin and IGF-1 receptors to glycogen synthesis in murine hepatocytes.

We have used SV40-transformed hepatocytes from insulin receptor-deficient mice (-/-) and normal mice (WT) to investigate the different abilities of insulin and IGF-1 receptors to stimulate glycogen synthesis. We report that insulin receptors are more potent than IGF-1 receptors in stimulating glycogen synthesis. Both receptors stimulate glycogen synthesis in a PI 3-kinase-dependent manner, but only the effect of insulin receptors is partially rapamycin-dependent. Insulin and IGF-1 receptors activate Akt to a similar extent, whereas GSK-3 inactivation in response to IGF-1 is considerably lower in both -/- and WT cells, compared to the effect of insulin in WT cells. The findings indicate that (i) the potency of insulin and IGF-1 receptors in stimulating glycogen synthesis correlates with their ability to inactivate GSK-3, (ii) the extent of GSK-3 inactivation does not correlate with the extent of Akt activation mediated by insulin or IGF-1 receptors, indicating that the effect of insulin on GSK-3 requires additional kinases, and (iii) the pathways required for insulin stimulation of glycogen synthesis in mouse hepatocytes are PI 3-kinase-dependent and rapamycin-sensitive.

Identification of sirm, a Novel Insulin-regulated SH3 Binding Protein That Associates with Grb-2 and FYN

We have previously developed a mouse model of insulin-resistant diabetes by targeted inactivation of the insulin receptor gene. During studies of gene expression in livers of insulin receptor-deficient mice, we identified a novel cDNA, which we have termed sirm (Son of Insulin Receptor Mutant mice). sirm is largely, albeit not exclusively, expressed in insulin-responsive tissues. Insulin is a potent modulator of sirm expression, and sirm mRNA levels correlate with tissue sensitivity to insulin. The product of the sirm gene is a serine/threonine-rich protein with several proline-rich motifs and an NPNY motif, conforming to the consensus sequence recognized by the phosphotyrosine binding domains of insulin receptor substrate and Shc proteins. However, Sirm bears no extended homologies with other known proteins. Based on the sequences of the proline-rich domains, we sought to determine whether Sirm binds to the SH3 domains of FYN and Grb-2. We demonstrate here that Sirm binds to FYN and Grb-2 in 3T3-L1 adipocytes and that insulin treatment results in the dissociation of the Sirm.FYN and Sirm.Grb-2 complexes. We also show that Sirm is a substrate for the kinase activity of FYN in vitro. Based on the patterns of expression of sirm, its regulation by insulin, and the interactions with molecules in the insulin signaling pathway, we surmise that Sirm plays a role in modulating tissue sensitivity to insulin.