We investigated the chronic in vivo effect of resistin on insulin sensitivity and glucose metabolism by overexpressing resistin protein in male Wistar rats using intravenous administration of an adenovirus encoding mouse resistin. After 7 days of elevated resistin levels at a supraphysiological concentration, the animals displayed glucose intolerance and hyperinsulinemia during glucose tolerance tests, and insulin tolerance tests demonstrated an impaired glucose-lowering effect of insulin. The glucose clamp studies were performed at submaximal (4 mU/kg/min) and maximal (25 mU/kg/min) insulin infusion rates and demonstrated the presence of insulin resistance induced by elevated resistin levels. Indeed, the insulin-stimulated glucose infusion rate was decreased by 12–31%; suppression of hepatic glucose output was attenuated by 28–55%; and insulin suppression of circulating FFA levels was inhibited by 7%. Insulin receptor substrate–1 and –2 phosphorylation and Akt activation were impaired in muscle and adipose tissue. Interestingly, activation of AMP-activated protein kinase in skeletal muscle, liver, and adipose tissue was also significantly downregulated. Together, these results indicate that chronic “hyper-resistinemia” leads to whole-body insulin resistance involving impaired insulin signaling in skeletal muscle, liver, and adipose tissue, resulting in glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. Thus elevated resistin levels in normal rats fed a regular chow diet produce many of the features of human syndrome X.
Hiroaki Satoh, M.T. Audrey Nguyen, Philip D.G. Miles, Takeshi Imamura, Isao Usui, Jerrold M. Olefsky
Resistin is an adipose-derived hormone postulated to link adiposity to insulin resistance. To determine whether resistin plays a causative role in the development of diet-induced insulin resistance, we lowered circulating resistin levels in mice by use of a specific antisense oligodeoxynucleotide (ASO) directed against resistin mRNA and assessed in vivo insulin action by the insulin-clamp technique. After 3 weeks on a high-fat (HF) diet, mice displayed severe insulin resistance associated with an approximately 80% increase in plasma resistin levels. In particular, the rate of endogenous glucose production (GP) increased more than twofold compared with that in mice fed a standard chow. Treatment with the resistin ASO for 1 week normalized the plasma resistin levels and completely reversed the hepatic insulin resistance. Importantly, in this group of mice, the acute infusion of purified recombinant mouse resistin, designed to acutely elevate the levels of circulating resistin up to those observed in the HF-fed mice, was sufficient to reconstitute hepatic insulin resistance. These results provide strong support for a physiological role of resistin in the development of hepatic insulin resistance in this model.
Evan D. Muse, Silvana Obici, Sanjay Bhanot, Brett P. Monia, Robert A. McKay, Michael W. Rajala, Philipp E. Scherer, Luciano Rossetti
Glycerol, a product of adipose tissue lipolysis, is an important substrate for hepatic glucose synthesis. However, little is known about the regulation of hepatic glycerol metabolism. Here we show that several genes involved in the hepatic metabolism of glycerol, i.e., cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase (GPDH), glycerol kinase, and glycerol transporters aquaporin 3 and 9, are upregulated by fasting in wild-type mice but not in mice lacking PPARα. Furthermore, expression of these genes was induced by the PPARα agonist Wy14643 in wild-type but not PPARα−null mice. In adipocytes, which express high levels of PPARγ, expression of cytosolic GPDH was enhanced by PPARγ and β/δ agonists, while expression was decreased in PPARγ+/– and PPARβ/δ–/– mice. Transactivation, gel shift, and chromatin immunoprecipitation experiments demonstrated that cytosolic GPDH is a direct PPAR target gene. In line with a stimulating role of PPARα in hepatic glycerol utilization, administration of synthetic PPARα agonists in mice and humans decreased plasma glycerol. Finally, hepatic glucose production was decreased in PPARα-null mice simultaneously fasted and exposed to Wy14643, suggesting that the stimulatory effect of PPARα on gluconeogenic gene expression was translated at the functional level. Overall, these data indicate that PPARα directly governs glycerol metabolism in liver, whereas PPARγ regulates glycerol metabolism in adipose tissue.
David Patsouris, Stéphane Mandard, Peter J. Voshol, Pascal Escher, Nguan Soon Tan, Louis M. Havekes, Wolfgang Koenig, Winfried März, Sherrie Tafuri, Walter Wahli, Michael Müller, Sander Kersten
Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2′-methoxyethyl–modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic β cell function.
Kyle W. Sloop, Julia Xiao-Chun Cao, Angela M. Siesky, Hong Yan Zhang, Diane M. Bodenmiller, Amy L. Cox, Steven J. Jacobs, Julie S. Moyers, Rebecca A. Owens, Aaron D. Showalter, Martin B. Brenner, Achim Raap, Jesper Gromada, Brian R. Berridge, David K. B. Monteith, Niels Porksen, Robert A. McKay, Brett P. Monia, Sanjay Bhanot, Lynnetta M. Watts, M. Dodson Michael
Elevated FFA concentrations have been shown to reproduce some of the metabolic abnormalities of obesity. It has been hypothesized that visceral adipose tissue lipolysis releases excess FFAs into the portal vein, exposing the liver to higher FFA concentrations. We used isotope dilution/hepatic vein catheterization techniques to examine whether intra-abdominal fat contributes a greater portion of hepatic FFA delivery in visceral obesity. Obese women (n = 24) and men (n = 20) with a range of obesity phenotypes, taken together with healthy, lean women (n = 12) and men (n = 12), were studied. Systemic, splanchnic, and leg FFA kinetics were measured. The results showed that plasma FFA concentrations were approximately 20% greater in obese men and obese women. The contribution of splanchnic lipolysis to hepatic FFA delivery ranged from less than 10% to almost 50% and increased as a function of visceral fat in women (r = 0.49, P = 0.002) and in men (r = 0.52, P = 0.002); the slope of the relationship was greater in women than in men (P < 0.05). Leg and splanchnic tissues contributed a greater portion of systemic FFA release in obese men and women than in lean men and women. We conclude that the contribution of visceral adipose tissue lipolysis to hepatic FFA delivery increases with increasing visceral fat in humans and that this effect is greater in women than in men.
Soren Nielsen, ZengKui Guo, C. Michael Johnson, Donald D. Hensrud, Michael D. Jensen
E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.
Ainhoa Iglesias, Matilde Murga, Usua Laresgoiti, Anouchka Skoudy, Irantzu Bernales, Asier Fullaondo, Bernardino Moreno, José Lloreta, Seth J. Field, Francisco X. Real, Ana M. Zubiaga
We explored the effects of bile acids on triglyceride (TG) homeostasis using a combination of molecular, cellular, and animal models. Cholic acid (CA) prevents hepatic TG accumulation, VLDL secretion, and elevated serum TG in mouse models of hypertriglyceridemia. At the molecular level, CA decreases hepatic expression of SREBP-1c and its lipogenic target genes. Through the use of mouse mutants for the short heterodimer partner (SHP) and liver X receptor (LXR) α and β, we demonstrate the critical dependence of the reduction of SREBP-1c expression by either natural or synthetic farnesoid X receptor (FXR) agonists on both SHP and LXRα and LXRβ. These results suggest that strategies aimed at increasing FXR activity and the repressive effects of SHP should be explored to correct hypertriglyceridemia.
Mitsuhiro Watanabe, Sander M. Houten, Li Wang, Antonio Moschetta, David J. Mangelsdorf, Richard A. Heyman, David D. Moore, Johan Auwerx
We evaluated the effects of E2F1 on glucose homeostasis using E2F1–/– mice. E2F1–/– mice show an overall reduction in pancreatic size as the result of impaired postnatal pancreatic growth. Furthermore, these animals have dysfunctional β cells, linked to impaired PDX-1 activity. Because of the disproportionate small pancreas and dysfunctional islets, E2F1–/– mice secrete insufficient amounts of insulin in response to a glucose load, resulting in glucose intolerance. Despite this glucose intolerance, E2F1–/– mice do not develop overt diabetes mellitus because they have insulin hypersensitivity, which is secondary to a diminished adipose tissue mass and altered adipocytokine levels, which compensates for the defect in insulin secretion. These data demonstrate that factors controlling cell proliferation, such as E2F1, determine pancreatic growth and function, subsequently affecting metabolic homeostasis.
Lluis Fajas, Jean-Sébastien Annicotte, Stéphanie Miard, David Sarruf, Mitsuhiro Watanabe, Johan Auwerx
IGF-1 has been associated with the pathogenesis of diabetic retinopathy, although its role is not fully understood. Here we show that normoglycemic/normoinsulinemic transgenic mice overexpressing IGF-1 in the retina developed most alterations seen in human diabetic eye disease. A paracrine effect of IGF-1 in the retina initiated vascular alterations that progressed from nonproliferative to proliferative retinopathy and retinal detachment. Eyes from 2-month-old transgenic mice showed loss of pericytes and thickening of basement membrane of retinal capillaries. In mice 6 months and older, venule dilatation, intraretinal microvascular abnormalities, and neovascularization of the retina and vitreous cavity were observed. Neovascularization was consistent with increased IGF-1 induction of VEGF expression in retinal glial cells. In addition, IGF-1 accumulated in aqueous humor, which may have caused rubeosis iridis and subsequently adhesions between the cornea and iris that hampered aqueous humor drainage and led to neovascular glaucoma. Furthermore, all transgenic mice developed cataracts. These findings suggest a role of IGF-1 in the development of ocular complications in long-term diabetes. Thus, these transgenic mice may be used to study the mechanisms that lead to diabetes eye disease and constitute an appropriate model in which to assay new therapies.
Jesús Ruberte, Eduard Ayuso, Marc Navarro, Ana Carretero, Víctor Nacher, Virginia Haurigot, Mónica George, Cristina Llombart, Alba Casellas, Cristina Costa, Assumpció Bosch, Fatima Bosch
In the current studies we generated transgenic mice that overexpress human Insig-1 in the liver under a constitutive promoter. In cultured cells Insig-1 and Insig-2 have been shown to block lipid synthesis in a cholesterol-dependent fashion by inhibiting proteolytic processing of sterol regulatory element–binding proteins (SREBPs), membrane-bound transcription factors that activate lipid synthesis. Insig’s exert this action in the ER by binding SREBP cleavage-activating protein (SCAP) and preventing it from escorting SREBPs to the Golgi apparatus where the SREBPs are processed to their active forms. In the livers of Insig-1 transgenic mice, the content of all nuclear SREBPs (nSREBPs) was reduced and declined further upon feeding of dietary cholesterol. The nuclear content of the insulin-induced SREBP isoform, SREBP-1c, failed to increase to a normal extent upon refeeding on a high-carbohydrate diet. The nSREBP deficiency produced a marked reduction in the levels of mRNAs encoding enzymes required for synthesis of cholesterol, fatty acids, and triglycerides. Plasma cholesterol levels were strongly reduced, and plasma triglycerides did not exhibit their normal rise after refeeding. These results provide in vivo support for the hypothesis that nSREBPs are essential for high levels of lipid synthesis in the liver and indicate that Insig’s modulate nSREBP levels by binding and retaining SCAP in the ER.
Luke J. Engelking, Hiroshi Kuriyama, Robert E. Hammer, Jay D. Horton, Michael S. Brown, Joseph L. Goldstein, Guosheng Liang