Negative Regulators of Insulin Signaling Revealed in a Genome-Wide Functional Screen

Type 2 diabetes develops due to a combination of insulin resistance and β-cell failure and current therapeutics aim at both of these underlying causes. Several negative regulators of insulin signaling are known and are the subject of drug discovery efforts. We sought to identify novel contributors to insulin resistance and hence potentially novel targets for therapeutic intervention.An arrayed cDNA library encoding 18,441 human transcripts was screened for inhibitors of insulin signaling and revealed known inhibitors and numerous potential novel regulators. The novel hits included proteins of various functional classes such as kinases, phosphatases, transcription factors, and GTPase associated proteins. A series of secondary assays confirmed the relevance of the primary screen hits to insulin signaling and provided further insight into their modes of action.Among the novel hits was PALD (KIAA1274, paladin), a previously uncharacterized protein that when overexpressed led to inhibition of insulin's ability to down regulate a FOXO1A-driven reporter gene, reduced upstream insulin-stimulated AKT phosphorylation, and decreased insulin receptor (IR) abundance. Conversely, knockdown of PALD gene expression resulted in increased IR abundance, enhanced insulin-stimulated AKT phosphorylation, and an improvement in insulin's ability to suppress FOXO1A-driven reporter gene activity. The present data demonstrate that the application of arrayed genome-wide screening technologies to insulin signaling is fruitful and is likely to reveal novel drug targets for insulin resistance and the metabolic syndrome

Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity

AIMS/HYPOTHESIS: Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake. MATERIALS AND METHODS: Insulin sensitivity in Pten heterozygous (Pten(+/−)) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten(+/−) mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3β (GSK3β), a substrate of PKB/Akt, was determined by western immunoblotting. RESULTS: Following i.p. insulin challenge, blood glucose levels in Pten(+/−) mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten(+/−) mice. Enhanced glucose uptake was observed both in Pten(+/−) myocytes and in skeletal muscle of Pten(+/−) mice by PET. PKB and GSK3β phosphorylation was enhanced and prolonged in Pten(+/−) myocytes. CONCLUSIONS/INTERPRETATION: Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten(+/−) mice

Reduced Serotonin Reuptake Transporter (SERT) Function Causes Insulin Resistance and Hepatic Steatosis Independent of Food Intake

Serotonin reuptake transporter (SERT) is a key regulator of serotonin neurotransmission and a major target of antidepressants. Antidepressants, such as selectively serotonin reuptake inhibitors (SSRIs), that block SERT function are known to affect food intake and body weight. Here, we provide genetic evidence that food intake and metabolism are regulated by separable mechanisms of SERT function. SERT-deficient mice ate less during both normal diet and high fat diet feeding. The reduced food intake was accompanied with markedly elevated plasma leptin levels. Despite reduced food intake, SERT-deficient mice exhibited glucose intolerance and insulin resistance, and progressively developed obesity and hepatic steatosis. Several lines of evidence indicate that the metabolic deficits of SERT-deficient mice are attributable to reduced insulin-sensitivity in peripheral tissues. First, SERT-deficient mice exhibited beta-cell hyperplasia and islet-mass expansion. Second, biochemical analyses revealed constitutively elevated JNK activity and diminished insulin-induced AKT activation in the liver of SERT-deficient mice. SERT-deficient mice exhibited hyper-JNK activity and hyperinsulinemia prior to the development of obesity. Third, enhancing AKT signaling by PTEN deficiency corrected glucose tolerance in SERT-deficient mice. These findings have potential implications for designing selective SERT drugs for weight control and the treatment of metabolic syndromes

Regulation of PTEN inhibition by the pleckstrin homology domain of P-REX2 during insulin signaling and glucose homeostasis

Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain–mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance