ABSTRACT
Trodusquemine (MSI-1436) causes rapid and reversible weight loss in genetic models of obesity. To better predict the potential effects of trodusquemine in the clinic, we investigated the effects of trodusquemine treatment in a murine model of diet-induced obesity (DIO). Trodusquemine suppressed appetite, reduced body weight (BW) in a fat-specific manner, and improved plasma insulin and leptin levels in mice. Screening assays revealed that trodusquemine selectively inhibited protein-tyrosine phosphatase 1B (PTP1B), a key enzyme regulating insulin and leptin signaling. Trodusquemine significantly enhanced insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) beta and STAT3, direct targets of PTP1B, in HepG2 cells in vitro and/or hypothalamic tissue in vivo. These data establish trodusquemine as an effective central and peripheral PTP1B inhibitor with the potential to elicit noncachectic fat-specific weight loss and improve insulin and leptin levels.
Subject(s)
Appetite/drug effects , Body Composition/drug effects , Cholestanes/pharmacology , Hypolipidemic Agents/pharmacology , Obesity/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Spermine/analogs & derivatives , Weight Loss/drug effects , Animals , Diet , Disease Models, Animal , Hep G2 Cells , Humans , Hypothalamus/drug effects , Insulin/blood , Leptin/blood , Male , Mice , Mice, Inbred AKR , Mice, Obese , Obesity/metabolism , Phosphorylation , Receptor, Insulin/metabolism , STAT3 Transcription Factor/metabolism , Spermine/pharmacologyABSTRACT
Foxa1 (formerly hepatic nuclear factor 3alpha) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1-/- pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1-/- islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1-/- beta-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1-/- islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic beta-cells.
Subject(s)
Hepatocyte Nuclear Factor 3-alpha/deficiency , Insulin-Secreting Cells/physiology , Insulin/metabolism , Ion Channels/physiology , Islets of Langerhans/metabolism , Mitochondrial Proteins/physiology , Oxidative Phosphorylation/drug effects , Animals , Glucagon/metabolism , Glucose/pharmacology , Glyburide/pharmacology , Insulin Secretion , Mice , Uncoupling Protein 2ABSTRACT
The forkhead gene family, named after the founding gene member in Drosophila, is characterized by a unique DNA-binding domain. This so-called forkhead box encodes a winged-helix DNA-binding motif, the name of which describes the structure of the domain when bound to DNA. The three Fox (forkhead box) group A genes, Foxa1, Foxa2 and Foxa3, are expressed in embryonic endoderm, the germ layer that gives rise to the digestive system, and contribute to the specification of the pancreas and the regulation of glucose homoeostasis. Deletion of the Foxa2 gene in pancreatic beta-cells in mice results in a phenotype resembling PHHI (persistent hyperinsulinaemic hypoglycaemia of infancy). Molecular analyses have demonstrated that Foxa2 is an important regulator of the genes encoding Sur1, Kir6.2 and Schad (short chain L-3-hydroxyacyl-CoA dehydrogenase), mutation of which causes PHHI in humans. Foxa1 was shown to be an essential activator of glucagon gene expression in vivo. An additional winged-helix protein, Foxo1, contributes to pancreatic beta-cell function by regulating the Pdx1 gene, which is required for pancreatic development in cooperation with Foxa2.
Subject(s)
Embryonic Development/physiology , Gene Expression Regulation, Developmental , Mammals/embryology , Pancreas/embryology , Trans-Activators/physiology , Animals , DNA-Binding Proteins/metabolism , Diabetes Mellitus/embryology , Diabetes Mellitus/metabolism , Forkhead Box Protein O1 , Forkhead Transcription Factors , Hepatocyte Nuclear Factor 3-beta , Homeodomain Proteins/metabolism , Humans , Mice , Nerve Tissue Proteins , Nuclear Proteins/metabolism , Rats , Trans-Activators/metabolism , Transcription Factors/metabolismABSTRACT
The regulation of insulin secretion by pancreatic beta cells is perturbed in several diseases, including adult-onset (type 2) diabetes and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). The first mouse model for PHHI has a conditional deletion of the gene encoding the winged-helix transcription factor Foxa2 (Forkhead box a2, formerly Hepatocyte nuclear factor 3beta) in pancreatic beta cells. Using isolated islets, we found that Foxa2 deficiency resulted in excessive insulin release in response to amino acids and complete loss of glucose-stimulated insulin secretion. Most PHHI cases are associated with mutations in SUR1 (Sulfonylurea receptor 1) or KIR6.2 (Inward rectifier K(+) channel member 6.2), which encode the subunits of the ATP-sensitive K(+) channel, and RNA in situ hybridization of mutant mouse islets revealed that expression of both genes is Foxa2 dependent. We utilized expression profiling to identify additional targets of Foxa2. Strikingly, one of these genes, Hadhsc, encodes short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase, deficiency of which has been shown to cause PHHI in humans. Hadhsc is a direct target of Foxa2, as demonstrated by cotransfection as well as in vivo chromatin immunoprecipitation experiments using isolated islets. Thus, we have established Foxa2 as an essential activator of genes that function in multiple pathways governing insulin secretion.
Subject(s)
ATP-Binding Cassette Transporters , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Insulin/metabolism , Islets of Langerhans/metabolism , Nuclear Proteins/metabolism , Trans-Activators/genetics , Transcription Factors , 3-Hydroxyacyl CoA Dehydrogenases/deficiency , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Amino Acids/pharmacology , Animals , Cell Line , Cricetinae , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Dose-Response Relationship, Drug , Drug Interactions , Gene Deletion , Glucose/pharmacology , Hepatocyte Nuclear Factor 3-beta , Insulin Secretion , Islets of Langerhans/cytology , Kidney/cytology , Luciferases/metabolism , Mice , Mice, Inbred Strains , Mice, Knockout , Mice, Transgenic , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Promoter Regions, Genetic , Receptors, Drug , Sulfonylurea Receptors , Transcriptional ActivationABSTRACT
The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed "PancChip 4.0," which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research.
