Insulin-stimulated Glucose Uptake In Adipocytes Research Articles

Altered Adipocyte Cell Size Distribution Prior to Weight Loss in the R6/2 Model of Huntington's Disease.

Metabolic alterations contribute to disease onset and prognosis of Huntington's disease (HD). Weight loss in the R6/2 mouse model of HD is a consistent feature, with onset in mid-to-late stage of disease. In the present study, we aimed to investigate molecular and functional changes in white adipose tissue (WAT) that occur at weight loss in R6/2 mice. We further elaborated on the effect of leptin-deficiency and early obesity in R6/2 mice. We performed analyses at 12 weeks of age; a time point that coincides with the start of weight loss in our R6/2 mouse colony. Gonadal (visceral) and inguinal (subcutaneous) WAT depot weights were monitored, as well as adipocyte size distribution. Response to isoprenaline-stimulated glycerol release and insulin-stimulated glucose uptake in adipocytes from gonadal WAT was assessed. In R6/2 mice, WAT depot weights were comparable to wildtype (WT) mice, and the response to insulin and isoprenaline in gonadal adipocytes was unaltered. Leptin-deficient R6/2 mice exhibited distinct changes compared to leptin-deficient WT mice. At 12 weeks, female leptin-deficient R6/2 mice had reduced body weight accompanied by an increased proportion of smaller adipocytes, while in contrast; male mice displayed a shift towards larger adipocyte sizes without a significant body weight reduction at this timepoint. We here show that there are early sex-specific changes in adipocyte cell size distribution in WAT of R6/2 mice and leptin-deficient R6/2 mice.

25-Hydroxyvitamin D Increases Insulin-Stimulated Glucose Uptake by Enhancing Adipocyte Differentiation.

Vitamin D and its receptor (vitamin D receptor; VDR) regulate calcium homeostasis in mammals. Recently, studies have shown that serum concentrations of 25-hydroxyvitamin D (25VD) are negatively associated with insulin resistance and the incidence of type 2 diabetes. In adipose tissues, glucose transporter 4 (GLUT4) contributes to insulin-stimulated glucose uptake; however, the effect of 25VD on glucose uptake in adipocytes remains unclear. We examined the role of 25VD in glucose uptake and the differentiation of adipose-derived stromal cells. Insulin-stimulated glucose uptake in adipocytes was increased by treatment with 25VD and decreased by VDR knockdown. The expression levels of GLUT4 were upregulated by 25VD treatment. 25VD exposure increased the expression of adipocyte differentiation-related genes including peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins through VDR, thereby enhancing the formation of mature adipocytes. Moreover, 25VD increased the expression levels of 11β-hydroxysteroid dehydrogenase 1 (HSD11B1), which catalyzes the conversion of cortisone to cortisol in a concentration-dependent manner. 25VD-stimulated adipocyte differentiation was suppressed by HSD11B1 knockdown. Cortisone together with 25VD enhanced adipocyte differentiation, whereas synthesized glucocorticoid dexamethasone-induced adipocyte differentiation is not promoted by 25VD. Overall, these results indicate that 25VD stimulates adipocyte differentiation through the induction of HSD11B1 expression, leading to increased insulin-induced glucose uptake in adipocytes.

Distinct biological activities of isomers from several families of branched fatty acid esters of hydroxy fatty acids (FAHFAs)

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids with antidiabetic and anti-inflammatory effects. Each FAHFA family consists of esters with different acyl chains and multiple isomers with branch points at different carbons. Some FAHFAs, including palmitic acid hydroxy stearic acids (PAHSAs), improve insulin sensitivity and glucose tolerance in mice by enhancing glucose-stimulated insulin secretion (GSIS), insulin-stimulated glucose transport, and insulin action to suppress hepatic glucose production and reducing adipose tissue inflammation. However, little is known about the biological effects of other FAHFAs. Here, we investigated whether PAHSAs, oleic acid hydroxy stearic acid, palmitoleic acid hydroxy stearic acid, and stearic acid hydroxy stearic acid potentiate GSIS in β-cells and human islets, insulin-stimulated glucose uptake in adipocytes, and anti-inflammatory effects in immune cells. We also investigated whether they activate G protein–coupled receptor 40, which mediates the effects of PAHSAs on insulin secretion and sensitivity in vivo. We show that many FAHFAs potentiate GSIS, activate G protein–coupled receptor 40, and attenuate LPS-induced chemokine and cytokine expression and secretion and phagocytosis in immune cells. However, fewer FAHFAs augment insulin-stimulated glucose uptake in adipocytes. S-9-PAHSA, but not R-9-PAHSA, potentiated GSIS and glucose uptake, while both stereoisomers had anti-inflammatory effects. FAHFAs containing unsaturated acyl chains with higher branching from the carboxylate head group are more likely to potentiate GSIS, whereas FAHFAs with lower branching are more likely to be anti-inflammatory. This study provides insight into the specificity of the biological actions of different FAHFAs and could lead to the development of FAHFAs to treat metabolic and immune-mediated diseases.

Psidium guajava L. leaf extract inhibits adipocyte differentiation and improves insulin sensitivity in 3T3-L1 cells.

BACKGROUND/OBJECTIVESPsidium guajava L. (guava) leaves have been shown to exhibit hypoglycemic and antidiabetic effects in rodents. This study investigated the effects of guava leaf extract on adipogenesis, glucose uptake, and lipolysis of adipocytes to examine whether the antidiabetic properties are mediated through direct effects on adipocytes.MATERIALS/METHODS3T3-L1 cells were treated with 25, 50, 100 µg/mL of methanol extract from guava leaf extract (GLE) or 0.1% dimethyl sulfoxide as a control. Lipid accumulation was evaluated with Oil Red O Staining and AdipoRed assay. Immunoblotting was performed to measure the expression of adipogenic transcription factors, fatty acid synthase (FAS), and AMP-activated protein kinase (AMPK). Glucose uptake under basal or insulin-stimulated condition was measured using a glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose. Lipolysis from fully differentiated adipocytes was measured by free fatty acids release into the culture medium in the presence or absence of epinephrine.RESULTSOil Red O staining and AdipoRed assay have shown that GLE treatment reduced lipid accumulation during adipocyte differentiation. Mitotic clonal expansion, an early essential event for adipocyte differentiation, was inhibited by GLE treatment. GLE inhibited the expression of transcription factors involved in adipocyte differentiation, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein-1c (SREBP-1c). FAS expression was also decreased while the phosphorylation of AMPK was increased by GLE treatment. In addition, GLE increased insulin-induced glucose uptake into adipocytes. In lipid-filled mature adipocytes, GLE enhanced epinephrine-induced lipolysis but reduced basal lipolysis dose-dependently.CONCLUSIONSThe results show that GLE inhibits adipogenesis and improves adipocyte function by reducing basal lipolysis and increasing insulin-stimulated glucose uptake in adipocytes, which can be partly associated with antidiabetic effects of guava leaves.

LNK deficiency decreases obesity-induced insulin resistance by regulating GLUT4 through the PI3K-Akt-AS160 pathway in adipose tissue.

In recent years, LNK, an adapter protein, has been found to be associated with metabolic diseases, including hypertension and diabetes. We found that the expression of LNK in human adipose tissue was positively correlated with serum glucose and insulin in obese people. We examined the role of LNK in insulin resistance and systemic energy metabolism using LNK-deficient mice (LNK-/-). With consumption of a high-fat diet, wild type (WT) mice accumulated more intrahepatic triglyceride, higher serum triglyceride (TG), free fatty acid (FFA) and high sensitivity C-reactive protein (hsCRP) compared with LNK-/- mice. However, there was no significant difference between LNK-/- and WT mice under normal chow diet. Meanwhile, glucose transporter 4 (GLUT4) expression in adipose tissue and insulin-stimulated glucose uptake in adipocytes were increased in LNK-/- mice. LNK-/- adipose tissue showed activated reactivity for IRS1/PI3K/Akt/AS160 signaling, and administration of a PI3K inhibitor impaired glucose uptake. In conclusion, LNK plays a pivotal role in adipose glucose transport by regulating insulin-mediated IRS1/PI3K/Akt/AS160 signaling.

1757-P: Tyrosine Phosphorylation of Phospholipase C in Human Skeletal Muscle Insulin Resistance

Insulin resistance in skeletal muscle is among the primary contributors to the development of type 2 diabetes (T2D), a leading cause of heart disease, stroke, etc. Over 26 million U.S. adults have T2D. Molecular mechanisms for skeletal muscle insulin resistance remain elusive, limiting strategies to prevent/treat T2D. Phospholipase C, gamma 1 (PLCG1, aka PLC-γ1) serves as a signal transducer of tyrosine kinases and regulates various downstream signaling pathways in vitro. PLCG1 has been shown to interact with insulin receptor (IR) via its pleckstrin homology domain and plays a significant role in insulin-stimulated glucose uptake in adipocytes. Moreover, phosphorylation of tyrosine residue 771 (pTyr771) of PLCG1 has been shown to regulate PLCG1 activity. However, no prior publication shows that PLCG1 pTyr771 is linked with insulin resistance and T2D. Previously, we observed that insulin-stimulated PLCG1 pTyr771 is impaired in skeletal muscle from subjects with T2D compared to lean controls. In the present work, we mutated Tyr771 of PLCG1 to phenylalanine, and observed reduced insulin-stimulated AKT and AS160 phosphorylation in primary human skeletal muscle cells derived from an insulin sensitive participant. To identify the upstream regulator of PLCG1, we carried out in vitro kinase assay by incubating PLCG1 with insulin receptor. PLCG1 was expressed and purified from E. coli. The insulin receptor (active cytoplasmic terminus) was expressed and purified from insect cells. After incubating PLCG1 with insulin receptor in vitro for 30 min in the presence of ATP, PLCG1 phosphorylation was determined by HPLC-ESI-MS/MS. The result demonstrated that insulin receptor phosphorylated Tyr771 of PLCG1. Western blotting using pTyr771 specific antibodies confirmed these findings. In summary, these results suggest that reduced insulin-stimulated pTyr771 may contribute to the development of insulin resistance and T2D, and the insulin receptor is an upstream regulator of PLCG1 pTyr771. Disclosure X. Zhang: None. A.H. Mestareehi: None. B. Seyoum: None. Z. Yi: None. Funding American Diabetes Association (1-13-TS-27 to Z.Y.); National Institutes of Health (R01DK107666, R01DK081750 to Z.Y.), (T32HL120822 to A.H.M.); DOTS Scholars (to A.H.M., Z.Y.)

The IRE1\u03b1-XBP1s pathway promotes insulin-stimulated glucose uptake in adipocytes by increasing PPAR\u03b3 activity

The peroxisome proliferator-activated receptor-γ (PPARγ) improves whole-body insulin sensitivity by regulating the adipogenic and metabolic functions of mature adipocytes. We have previously demonstrated that an active splice variant of X-box binding protein 1 (XBP1s) enhances PPARγ expression during adipogenesis. In this study, we investigated the role of XBP1s, particularly with respect to PPARγ, in the mechanisms underlying insulin sensitivity in mature adipocytes. Insulin was able to stimulate XBP1s generation by activating inositol-requiring enzyme 1 (IRE1) α and was also able to increase its transcriptional activity by inducing nuclear translocation. XBP1s also upregulated the levels of phosphorylated IRS1 and AKT, demonstrating a positive feedback regulatory mechanism linking insulin and XBP1s. XBP1s enhanced the expression of fibroblast growth factor 21 and, in turn, increased PPARγ activity, translocation of GLUT4 to the cell surface, and glucose uptake rate in adipocytes. In addition, XBP1s abolished palmitate-induced insulin resistance in adipocytes by increasing adiponectin secretion, repressing the secretion of pro-inflammatory adipokines such as leptin, monocyte chemoattractant protein 1, and tumor necrosis factor α, and decreasing fatty acid release. These findings provide a novel mechanism by which XBP1s stimulate insulin sensitivity in adipocytes through fibroblast growth factor 21 induction and PPARγ activation.

Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.

Sortilin is a multiligand sorting receptor responsible for the anterograde transport of lysosomal enzymes and substrates. Here we demonstrate that sortilin is also involved in retrograde protein traffic. In cultured 3T3-L1 adipocytes, sortilin together with retromer rescues Glut4 from degradation in lysosomes and retrieves it to the TGN, where insulin--responsive vesicles are formed. Mechanistically, the luminal Vps10p domain of sortilin interacts with the first luminal loop of Glut4, and the cytoplasmic tail of sortilin binds to retromer. Ablation of the retromer does not affect insulin signaling but decreases the stability of sortilin and Glut4 and blocks their entry into the small vesicular carriers. As a result, Glut4 cannot reach the insulin-responsive compartment, and insulin-stimulated glucose uptake in adipocytes is suppressed. We suggest that sortilin- and retromer-mediated Glut4 retrieval from endosomes may represent a step in the Glut4 pathway vulnerable to the development of insulin resistance and diabetes.

Insulin-Like Growth Factor (IGF) Binding Protein-2, Independently of IGF-1, Induces GLUT-4 Translocation and Glucose Uptake in 3T3-L1 Adipocytes.

Insulin-like growth factor binding protein-2 (IGFBP-2) is the predominant IGF binding protein produced during adipogenesis and is known to increase the insulin-stimulated glucose uptake (GU) in myotubes. We investigated the IGFBP-2-induced changes in basal and insulin-stimulated GU in adipocytes and the underlying mechanisms. We further determined the role of insulin and IGF-1 receptors in mediating the IGFBP-2 and the impact of IGFBP-2 on the IGF-1-induced GU. Fully differentiated 3T3-L1 adipocytes were treated with IGFBP-2 in the presence and absence of insulin and IGF-1. Insulin, IGF-1, and IGFBP-2 induced a dose-dependent increase in GU. IGFBP-2 increased the insulin-induced GU after long-term incubation. The IGFBP-2-induced impact on GU was neither affected by insulin or IGF-1 receptor blockage nor by insulin receptor knockdown. IGFBP-2 significantly increased the phosphorylation of PI3K, Akt, AMPK, TBC1D1, and PKCζ/λ and induced GLUT-4 translocation. Moreover, inhibition of PI3K and AMPK significantly reduced IGFBP-2-stimulated GU. In conclusion, IGFBP-2 stimulates GU in 3T3-L1 adipocytes through activation of PI3K/Akt, AMPK/TBC1D1, and PI3K/PKCζ/λ/GLUT-4 signaling. The stimulatory effect of IGFBP-2 on GU is independent of its binding to IGF-1 and is possibly not mediated through the insulin or IGF-1 receptor. This study highlights the potential role of IGFBP-2 in glucose metabolism.

Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake.

Mice deficient for zinc transporter 7 protein (ZnT7) are mildly zinc deficient with low body weight gain and body fat accumulation. To investigate the underlying mechanism of ZnT7 deficiency in body adiposity, we examined fatty acid composition and insulin sensitivity in visceral (epididymal) and subcutaneous fat pads from Znt7 knockout and control mice. We showed that ZnT7 deficiency had adverse effects on fatty acid metabolism and insulin action in subcutaneous fat but not in epididymal fat in mice, consistent with the ZnT7 protein expression pattern in adipose tissues. Importantly, we found that the expression of ZnT7 protein was induced by lipogenic differentiation and reached a peak when the adipocyte was fully differentiated in mouse 3T3-L1 adipocytes. We demonstrated, using Znt7 knockdown (Znt7KD) 3T3-L1 adipocytes, that reduction in Znt7 expression blunted activations of the signal transduction pathways that regulated both basal and insulin-stimulated glucose uptake in adipocytes, resulting in low glucose uptake and lipid accumulation. The expression of the signaling mediators critical for the initiation of pre-adipocyte differentiation, including Pparγ and C/Ebpα, appeared not to be affected by Znt7KD in 3T3-L1 adipocytes. These findings strongly suggest a role for ZnT7 in adipocyte lipogenesis.

TUSC5 regulates insulin-mediated adipose tissue glucose uptake by modulation of GLUT4 recycling.

ObjectiveFailure to properly dispose of glucose in response to insulin is a serious health problem, occurring during obesity and is associated with type 2 diabetes development. Insulin-stimulated glucose uptake is facilitated by the translocation and plasma membrane fusion of vesicles containing glucose transporter 4 (GLUT4), the rate-limiting step of post-prandial glucose disposal.MethodsWe analyzed the role of Tusc5 in the regulation of insulin-stimulated Glut4-mediated glucose uptake in vitro and in vivo. Furthermore, we measured Tusc5 expression in two patient cohorts.ResultsHerein, we report that TUSC5 controls insulin-stimulated glucose uptake in adipocytes, in vitro and in vivo. TUSC5 facilitates the proper recycling of GLUT4 and other key trafficking proteins during prolonged insulin stimulation, thereby enabling proper protein localization and complete vesicle formation, processes that ultimately enable insulin-stimulated glucose uptake. Tusc5 knockout mice exhibit impaired glucose disposal and TUSC5 expression is predictive of glucose tolerance in obese individuals, independent of body weight. Furthermore, we show that TUSC5 is a PPARγ target and in its absence the anti-diabetic effects of TZDs are significantly blunted.ConclusionsCollectively, these findings establish TUSC5 as an adipose tissue-specific protein that enables proper protein recycling, linking the ubiquitous vesicle traffic machinery with tissue-specific insulin-mediated glucose uptake into adipose tissue and the maintenance of a healthy metabolic phenotype in mice and humans.

Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes.

ObjectiveDefective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis.MethodsWe assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients.ResultsOur data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance.ConclusionsWe show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

Zinc Finger Protein 407 (ZFP407) Regulates Insulin-stimulated Glucose Uptake and Glucose Transporter 4 (Glut4) mRNA

The glucose transporter GLUT4 facilitates insulin-stimulated glucose uptake in peripheral tissues including adipose, muscle, and heart. GLUT4 function is impaired in obesity and type 2 diabetes leading to hyperglycemia and an increased risk of cardiovascular disease and neuropathy. To better understand the regulation of GLUT4 function, a targeted siRNA screen was performed and led to the discovery that ZFP407 regulates insulin-stimulated glucose uptake in adipocytes. The decrease in insulin-stimulated glucose uptake due to ZFP407 deficiency was attributed to a reduction in GLUT4 mRNA and protein levels. The decrease in GLUT4 was due to both decreased transcription of Glut4 mRNA and decreased efficiency of Glut4 pre-mRNA splicing. Interestingly, ZFP407 coordinately regulated this decrease in transcription with an increase in the stability of Glut4 mRNA, resulting in opposing effects on steady-state Glut4 mRNA levels. More broadly, transcriptome analysis revealed that ZFP407 regulates many peroxisome proliferator-activated receptor (PPAR) γ target genes beyond Glut4. ZFP407 was required for the PPARγ agonist rosiglitazone to increase Glut4 expression, but was not sufficient to increase expression of a PPARγ target gene reporter construct. However, ZFP407 and PPARγ co-overexpression synergistically activated a PPARγ reporter construct beyond the level of PPARγ alone. Thus, ZFP407 may represent a new modulator of the PPARγ signaling pathway.

Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes.

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic-mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase-Akt-mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake.

Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action. DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a (-/-) (Doc2aKO), Doc2b (-/-) (Doc2bKO) and Doc2a (-/-)/Doc2b (-/-) (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy. Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice. Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.

De novo duplication of 17p13.1–p13.2 in a patient with intellectual disability and obesity

17p13.1 Deletion encompassing TP53 has been described as a syndrome characterized by intellectual disability and dysmorphic features. Only one case with a 17p13.1 duplication encompassing TP53 has been reported in a patient with intellectual disability, seizures, obesity, and diabetes mellitus. Here, we present a patient with a 17p13.1 duplication who exhibited obesity and intellectual disability, similar to the previous report. The 9-year-old proposita was referred for the evaluation of intellectual disability and obesity. She also exhibited insulin resistance and liver dysfunction. She had wide palpebral fissures, upturned nostrils, a long mandible, short and slender fingers, and skin hyperpigmentation. Array comparative genomic hybridization (array CGH) detected a 3.2 Mb duplication of 17p13.1-p13.2 encompassing TP53, FXR2, NLGN2, and SLC2A4, which encodes the insulin-responsive glucose transporter 4 (GLUT4) associated with insulin-stimulated glucose uptake in adipocytes and muscle. We suggest that 17p13.1 duplication may represent a clinically recognizable condition characterized partially by a characteristic facial phenotype, developmental delay, and obesity.

Discovery of a Novel Target for the Dysglycemic Chromogranin A Fragment Pancreastatin: Interaction with the Chaperone GRP78 to Influence Metabolism

RationaleThe chromogranin A-derived peptide pancreastatin (PST) is a dysglycemic, counter-regulatory peptide for insulin action, especially in liver. Although previous evidence for a PST binding protein has been reported, such a receptor has not been identified or sequenced.Methods and ResultsWe used ligand affinity to purify the PST target, with biotinylated human PST (hCHGA273–301-amide) as “bait” and mouse liver homogenate as “prey”, and identified GRP78 (a.k.a. “78 kDa Glucose Regulated Protein”, HSPA5, BIP) as a major interacting partner of PST. GRP78 belongs to the family of heat shock proteins (chaperones), involved in several cellular processes including protein folding and glucose metabolism. We analyzed expression of GRP78 in the absence of PST in a mouse knockout model lacking its precursor CHGA: hepatic transcriptome data revealed global over-expression of not only GRP78 but also other heat shock transcripts (of the “adaptive UPR”) in CHGA(−/−) mice compared to wild-type (+/+). By contrast, we found a global decline in expression of hepatic pro-apoptotic transcripts in CHGA(−/−) mice. GRP78's ATPase enzymatic activity was dose-dependently inhibited by PST (IC50∼5.2 µM). PST also inhibited the up-regulation of GRP78 expression during UPR activation (by tunicamycin) in hepatocytes. PST inhibited insulin-stimulated glucose uptake in adipocytes, and increased hepatic expression of G6Pase (the final step in gluconeogenesis/glycogenolysis). In hepatocytes not only PST but also other GRP78-ATPase inhibitors (VER-155008 or ADP) increased G6Pase expression. GRP78 over-expression inhibited G6Pase expression in hepatocytes, with partial restoration by GRP78-ATPase inhibitors PST, VER-155008, or ADP.ConclusionsOur results indicate that an unexpected major hepatic target of PST is the adaptive UPR chaperone GRP78. PST not only binds to GRP78 (in pH-dependent fashion), but also inhibits GRP78's ATPase enzymatic activity, and impairs its biosynthetic response to UPR activation. PST decreases insulin-stimulated cellular glucose uptake, and PST as well as other chaperone ATPase activity inhibitors augment expression of G6Pase; GRP78 over-expression antagonizes this PST action. Analysis of the novel PST/GRP78 interaction may provide a new avenue of investigation into cellular glycemic control as well as dysglycemia.

Overexpression of C10orf116 promotes proliferation, inhibits apoptosis and enhances glucose transport in 3T3-L1 adipocytes

Data from our previous study demonstrated that C10orf116 is an adipocyte lineage-specific nuclear factor, which regulates master adipogenesis transcription factors during early differentiation. However, the precise functional properties of this gene have yet to be identified and further investigation is required. In the present study, we report the effects of C10orf116 expression on cell proliferation and apoptosis invitro and observed that the overexpression of C10orf116 stimulates proliferation and inhibits apoptosis in preadipocytes. Furthermore, we investigated the effects of C10orf116 on glucose uptake and demonstrated that the ectopic expression of C10orf116 significantly increases insulin-stimulated glucose uptake in adipocytes by increasing glucose transporter type4 (GLUT4) expression levels. Collectively, these data further support the hypothesis that C10orf116 is important in regulating glucose transport in adipocytes as well as the number of preadipocytes. The results of the present study may also provide insights into the complex mechanisms involved in the development of obesity.

Endopeptidase Cleavage Generates a Functionally Distinct Isoform of C1q/Tumor Necrosis Factor-related Protein-12 (CTRP12) with an Altered Oligomeric State and Signaling Specificity

Adipose tissue-derived adipokines are an important class of secreted metabolic regulators that mediate tissue cross-talk to control systemic energy balance. We recently described C1q/TNF-related protein-12 (CTRP12), a novel insulin-sensitizing adipokine that regulates glucose metabolism in liver and adipose tissue. However, the biochemical properties of CTRP12 and its naturally occurring cleaved isoform have not been characterized. Here, we show that CTRP12 is a secreted hormone subjected to multiple functionally relevant posttranslational modifications at highly conserved residues. For example, Asn(39) is glycosylated, whereas Cys(85) mediates the assembly of higher order oligomeric structure. Endopeptidase cleavage at Lys(91) generates a cleaved globular gCTRP12 isoform, the expression of which is increased by insulin. PCSK3/furin was identified as the major proprotein convertase expressed by adipocytes that mediates the endogenous cleavage of CTRP12. Cleavage at Lys(91) is context-dependent: mutation of the charged Arg(93) to Ala on the P2' position enhanced cleavage, and triple mutations (K90A/K91A/R93A) abolished cleavage. Importantly, the two isoforms of CTRP12 differ in oligomeric structures and are functionally distinct. The full-length protein forms trimers and larger complexes, and the cleaved isoform consisted of predominantly dimers. Whereas full-length fCTRP12 strongly activated Akt signaling in H4IIE hepatocytes and 3T3-L1 adipocytes, gCTRP12 preferentially activated MAP kinase (ERK1/2 and p38 MAPK) signaling. Further, only fCTRP12 improved insulin-stimulated glucose uptake in adipocytes. These results reveal a novel mechanism controlling signaling specificity and function of a hormone via cleavage-dependent alteration in oligomeric state.