Endothelial Cell Insulin Signaling Regulates CXCR4 (C-X-C Motif Chemokine Receptor 4) and Limits Leukocyte Adhesion to Endothelium.

BACKGROUND: An activated, proinflammatory endothelium is a key feature in the development of complications of obesity and type 2 diabetes and can be caused by insulin resistance in endothelial cells. METHODS: We analyzed primary human endothelial cells by RNA sequencing to discover novel insulin-regulated genes and used endothelial cell culture and animal models to characterize signaling through CXCR4 (C-X-C motif chemokine receptor 4) in endothelial cells. RESULTS: CXCR4 was one of the genes most potently regulated by insulin, and this was mediated by PI3K (phosphatidylinositol 3-kinase), likely through FoxO1, which bound to the CXCR4 promoter. CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared with lean controls and 37% higher in db/db mice than db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. SDF-1 (stromal cell-derived factor-1)-the ligand for CXCR4-increased leukocyte adhesion to cultured endothelial cells. This effect was lost after deletion of CXCR4 by gene editing while 80% of the increase was prevented by treatment of endothelial cells with insulin. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling after intravenous injection of SDF-1, but most of this response was prevented in transgenic mice with endothelial overexpression of IRS-1 (insulin receptor substrate-1). CONCLUSIONS: Endothelial cell insulin signaling limits leukocyte/endothelial cell interaction induced by SDF-1 through downregulation of CXCR4. Improving insulin signaling in endothelial cells or inhibiting endothelial CXCR4 may reduce immune cell recruitment to the vascular wall or tissue parenchyma in insulin resistance and thereby help prevent several vascular complications.

Elucidating the Biological Roles of Insulin and Its Receptor in Murine Intestinal Growth and Function.

The role of the intestinal insulin receptor (IR) is not well understood. We therefore explored the effect of insulin (300 nmol/kg per day for 12 days) on the intestine in sex-matched C57Bl/6J mice. The intestinal and metabolic profiles were also characterized in male and female intestinal-epithelial IR knockout (IE-irKO) mice compared with all genetic controls on a chow diet or Western diet (WD) for 4 to 12 weeks. Insulin treatment did not affect intestinal size, intestinal resistance, or metabolic genes, but it reduced proximal-colon crypt depth and acutely increased colonic serine/threonine-specific protein kinase B (AKT) activation. Feeding with a WD increased body weight and fasting insulin level and decreased oral glucose tolerance in C57Bl/6J and IE-irKO mice. However, although the overall responses of the IE-irKO mice were not different from those of Villin-Cre (Vil-Cre):IRfl/+ and IRfl/fl controls, profound differences were found for female control Vil-Cre mice, which demonstrated reduced food intake, body weight, jejunal glucose transport, oral glucose tolerance, and fasting insulin and cholesterol levels. Vil-Cre mice also had smaller intestines compared with those of IE-irKO and IRfl/fl mice and greater insulin-mediated activation of jejunal IR and AKT. In summary, gain- and loss-of-function studies, with and without caloric overload, indicate that insulin did not exert remarkable effects on intestinal metabolic or morphologic phenotype except for a small effect on the colon. However, the transgenic control Vil-Cre mice displayed a distinct phenotype compared with other control and knockout animals, emphasizing the importance of thoroughly characterizing genetically modified mouse models.

Quantitative Proteomics of Intestinal Mucosa From Male Mice Lacking Intestinal Epithelial Insulin Receptors.

The goal of the present study was to determine whether loss of the insulin receptor alters the molecular landscape of the intestinal mucosa, using intestinal-epithelial insulin receptor knockout (IE-irKO) mice and both genetic (IRfl/fl and Villin-cre) controls. Quantitative proteomic analysis by liquid chromatography mass spectrometry was applied to jejunal and colonic mucosa from mice fed a normal chow diet and mice fed a Western diet (WD). Jejunal mucosa from IE-irKO mice demonstrated alterations in all intestinal cell lineages: Paneth, goblet, absorptive, and enteroendocrine cells. Only goblet and absorptive cells were affected in the colon. Also, a marked effect of WD consumption was found on the gut proteome. A substantial reduction was detected in Paneth cell proteins with antimicrobial activity, including lysozyme C-1, angiogenin-4, cryptdin-related sequence 1C-3 and -2, α-defensin 17, and intelectin-1a. The key protein expressed by goblet cells, mucin-2, was also reduced in the IE-irKO mice. Proteins involved in lipid metabolism, including aldose reductase-related protein 1, 15-hydroxyprostaglandin dehydrogenase, apolipoprotein A-II, and pyruvate dehydrogenase kinase isozyme 4, were increased in the mucosa of WD-fed IE-irKO mice compared with controls. In contrast, expression of the nutrient-responsive gut hormones, glucose-dependent insulinotropic polypeptide and neurotensin, was reduced in the jejunal mucosa of IE-irKO mice, and the expression of proteins of the P-type adenosine triphosphatases and the solute carrier-transporter family was reduced in the colon of WD-fed IE-irKO mice. In conclusion, IE-irKO mice display a distinct molecular phenotype, suggesting a biological role of insulin and its receptor in determining differentiated cell specificity in the intestinal epithelium.

Adipose weight gain during chronic insulin treatment of mice results from changes in lipid storage without affecting de novo synthesis of palmitate.

Insulin treatment is associated with increased adipose mass in both humans and mice. However, the underlying dynamic basis of insulin induced lipid accumulation in adipose tissue remains elusive. To assess this, young female C57BL6/J mice were fed a low fat diet for 3 weeks, treated subsequently with 7 days of constant subcutaneous insulin infusion by osmotic minipumps and compared to mice with only buffer infused. To track changes in lipid deposition during insulin treatment, metabolic labeling was conducted with heavy water for the final 4 days. Blood glucose was significantly lowered within one hour after implantation of insulin loaded mini pumps and remained lower throughout the study. Insulin treated animals gained significantly more weight during treatment and the mean weight of the subcutaneous adipose depots was significantly higher with the highest dose of insulin. Surprisingly, de novo palmitate synthesis within the subcutaneous and the gonadal depots was not affected significantly by insulin treatment. In contrast insulin treatment caused accumulation of triglycerides in both depots due to either deposition of newly synthesised triglycerides (subcutaneous depot) or inhibition of lipolysis (gonadal depot).

Recombinant adiponectin does not lower plasma glucose in animal models of type 2 diabetes.

AIMS/HYPOTHESIS: Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats. METHODS: Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10-30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5-15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days. RESULTS: Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active. CONCLUSIONS/INTERPRETATION: Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.

Free fatty acid-induced PP2A hyperactivity selectively impairs hepatic insulin action on glucose metabolism.

In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is "selective", in the sense that the insulin-mediated decrease of glucose production is blunted while insulin's effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity.

Improved insulin sensitivity and islet function after PPARdelta activation in diabetic db/db mice.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Several reports have shown that PPARdelta is involved in lipid metabolism, increasing fat oxidation and depleting lipid accumulation. Whether PPARdelta is involved in the regulation of glucose metabolism is not completely understood. In this study, we examined effects of long-term PPARdelta activation on glycemic control, islet function and insulin sensitivity in diabetic db/db mice. Male db/db mice were administered orally once daily with a selective and partial PPARdelta agonist (NNC 61-5920, 30 mg/kg) for eight weeks; control mice received vehicle. Fasting and non-fasting plasma glucose were reduced, reflected in reduced hemoglobinA(1c) (3.6+/-1.6% vs. 5.4+/-1.8 in db/db controls, P<0.05) and furthermore, the AUC(glucose) after oral glucose (3g/kg) was reduced by 67% (P<0.05) after long-term PPARdelta activation. Following intravenous glucose (1g/kg), glucose tolerance was improved after PPARdelta activation (K(G) 1.3+/-0.6 vs. -0.05+/-0.7 %/min, P=0.048). Insulin sensitivity, measured as the glucose clearance after intravenous injection of glucose (1g/kg) and insulin (0.75 or 1.0 U/kg), during inhibition of endogenous insulin secretion by diazoxide (25mg/kg), was improved (K(G) 2.9+/-0.6 vs. 1.3+/-0.3 %/min in controls, P<0.05) despite lower insulin levels. Furthermore, islets isolated from PPARdelta agonist treated mice demonstrated improved glucose responsiveness as well as improved cellular topography. In conclusion, PPARdelta agonism alleviates insulin resistance and improves islet function and topography, resulting in improved glycemia in diabetic db/db mice. This suggests that activation of PPARdelta improves glucose metabolism and may therefore potentially be target for treatment of type 2 diabetes.

The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading.

The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the alpha2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in alpha1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-beta and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that alpha2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken.

The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism.

Metformin, a drug widely used to treat type 2 diabetes, was recently shown to activate the AMP-activated protein kinase (AMPK) in intact cells and in vivo. In this study we addressed the mechanism for this effect. In intact cells, metformin stimulated phosphorylation of the key regulatory site (Thr-172) on the catalytic (alpha) subunit of AMPK. It did not affect phosphorylation of this site by either of two upstream kinases in cell-free assays, although we were able to detect an increase in upstream kinase activity in extracts of metformin-treated cells. Metformin has been reported to be an inhibitor of complex 1 of the respiratory chain, but we present evidence that activation of AMPK in two different cell types is not a consequence of depletion of cellular energy charge via this mechanism. Whereas we have not established the definitive mechanism by which metformin activates AMPK, our results show that the mechanism is different from that of the existing AMPK-activating agent, 5-aminoimidazole-4-carboxamide (AICA) riboside. Metformin therefore represents a useful new tool to study the consequences of AMPK activation in intact cells and in vivo. Our results also show that AMPK can be activated by mechanisms other than changes in the cellular AMP-to-ATP ratio.