Mark A. Magnuson

Tissue-Specific Knockout of the Insulin Receptor in Pancreatic β Cells Creates an Insulin Secretory Defect Similar to that in Type 2 Diabetes

Dysfunction of the pancreatic beta cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the beta cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the beta cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic beta cell and suggest that defects in insulin signaling at the level of the beta cell may contribute to the observed alterations in insulin secretion in type 2 diabetes.

Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.

Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxPtechnology in combination with gene targeting to perform global, β cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. β cell- and hepatocyte-specific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a β cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in β cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the β cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both β cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-γ (PPARγ, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct–specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg flox/flox mice. Deletion of collecting duct Pparg decreased renal Na+ avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na+ absorption and Scnn1g mRNA (encoding the epithelial Na+ channel ENaCγ) expression through a PPARγ-dependent pathway. These studies identify Scnn1g as a PPARγ target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

Impaired insulin secretion and β-cell loss in tissue-specific knockout mice with mitochondrial diabetes

Mitochondrial dysfunction is an important contributor to human pathology and it is estimated that mutations of mitochondrial DNA (mtDNA) cause approximately 0.5–1% of all types of diabetes mellitus. We have generated a mouse model for mitochondrial diabetes by tissue-specific disruption of the nuclear gene encoding mitochondrial transcription factor A (Tfam, previously mtTFA; ref. 7) in pancreatic β-cells. This transcriptional activator is imported to mitochondria, where it is essential for mtDNA expression and maintenance. The Tfam-mutant mice developed diabetes from the age of approximately 5 weeks and displayed severe mtDNA depletion, deficient oxidative phosphorylation and abnormal appearing mitochondria in islets at the ages of 7–9 weeks. We performed physiological studies of β-cell stimulus–secretion coupling in islets isolated from 7–9-week-old mutant mice and found reduced hyperpolarization of the mitochondrial membrane potential, impaired Ca2+-signalling and lowered insulin release in response to glucose stimulation. We observed reduced β-cell mass in older mutants. Our findings identify two phases in the pathogenesis of mitochondrial diabetes; mutant β-cells initially display reduced stimulus–secretion coupling, later followed by β-cell loss. This animal model reproduces the β-cell pathology of human mitochondrial diabetes and provides genetic evidence for a critical role of the respiratory chain in insulin secretion.

Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein–coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2–/–). EP2–/– mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2–/– mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2–/– mice developed profound systolic hypertension, whereas wild–type mice showed no change in systolic blood pressure. Analysis of wild–type and EP2–/– mice on day 5 of pregnancy indicated that the reduced litter size of EP2–/– mice is due to a pre–implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt–sensitive hypertension, and also plays an essential part in female fertility.

Deletion of PPARγ in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance

Peroxisome proliferator-activated receptor γ (PPARγ) plays a crucial role in adipocyte differentiation, glucose metabolism, and other physiological processes. To further explore the role of PPARγ in adipose tissues, we used a Cre/loxP strategy to generate adipose-specific PPARγ knockout mice. These animals exhibited marked abnormalities in the formation and function of both brown and white adipose tissues. When fed a high-fat diet, adipose-specific PPARγ knockout mice displayed diminished weight gain despite hyperphagia, had diminished serum concentrations of both leptin and adiponectin, and did not develop glucose intolerance or insulin resistance. Characterization of in vivo glucose dynamics pointed to improved hepatic glucose metabolism as the basis for preventing high-fat diet-induced insulin resistance. Our findings further illustrate the essential role for PPARγ in the development of adipose tissues and suggest that a compensatory induction of hepatic PPARγ may stimulate an increase in glucose disposal by the liver.

β-cell–specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter β-cell mass

Regulation of glucose homeostasis by insulin depends on the maintenance of normal β-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function, but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on β-cells. Igf1 has been shown to influence β-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r+/−) are bred with mice lacking insulin receptor substrate 2 (Irs2−/−), the resulting compound knockout mice show a reduction in mass of β-cells similar to that observed in pancreas of Igf1r−/− mice (ref. 11), suggesting a role for Igf1r in growth of β-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a β-cell–specific knockout of Igf1r (βIgf1r−/−). These mice show normal growth and development of β-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in β-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, Igf1r is not crucial for islet β-cell development, but participates in control of differentiated function.

Identification of Protor as a novel Rictor-binding component of mTOR complex-2

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.

Liver-specific deletion of negative regulator Pten results in fatty liver and insulin hypersensitivity [corrected].

In the liver, insulin controls both lipid and glucose metabolism through its cell surface receptor and intracellular mediators such as phosphatidylinositol 3-kinase and serine-threonine kinase AKT. The insulin signaling pathway is further modulated by protein tyrosine phosphatase or lipid phosphatase. Here, we investigated the function of phosphatase and tension homologue deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, by targeted deletion of Pten in murine liver. Deletion of Pten in the liver resulted in increased fatty acid synthesis, accompanied by hepatomegaly and fatty liver phenotype. Interestingly, Pten liver-specific deletion causes enhanced liver insulin action with improved systemic glucose tolerance. Thus, deletion of Pten in the liver may provide a valuable model that permits the study of the metabolic actions of insulin signaling in the liver, and PTEN may be a promising target for therapeutic intervention for type 2 diabetes.

mTOR Complex 2 Is Required for the Development of Prostate Cancer Induced by Pten Loss in Mice

mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice. Furthermore, we find that Rictor is a haploinsufficient gene and that deleting one copy protects Pten heterozygous mice from prostate cancer. Finally, we show that the development of prostate cancer caused by Pten deletion specifically in prostate epithelium requires mTORC2, but that for normal prostate epithelial cells, mTORC2 activity is nonessential. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility.