Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase–1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.
Guoqiang Jiang, Zhihua Li, Franklin Liu, Kenneth Ellsworth, Qing Dallas-Yang, Margaret Wu, John Ronan, Christine Esau, Cain Murphy, Deborah Szalkowski, Raynald Bergeron, Thomas Doebber, Bei B. Zhang
Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in β cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and β cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced β cell mass. Overt diabetes did not ensue, because β cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced β cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in β cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis.
Agharul I. Choudhury, Helen Heffron, Mark A. Smith, Hind Al-Qassab, Allison W. Xu, Colin Selman, Marcus Simmgen, Melanie Clements, Marc Claret, Gavin MacColl, David C. Bedford, Kazunari Hisadome, Ivan Diakonov, Vazira Moosajee, Jimmy D. Bell, John R. Speakman, Rachel L. Batterham, Gregory S. Barsh, Michael L.J. Ashford, Dominic J. Withers
Central control of energy balance depends on the ability of proopiomelanocortin (POMC) or agouti-related protein (Agrp) hypothalamic neurons to sense and respond to changes in peripheral energy stores. Leptin and insulin have been implicated as circulating indicators of adiposity, but it is not clear how changes in their levels are perceived or integrated by individual neuronal subtypes. We developed mice in which a fluorescent reporter for PI3K activity is targeted to either Agrp or POMC neurons and used 2-photon microscopy to measure dynamic regulation of PI3K by insulin and leptin in brain slices. We show that leptin and insulin act in parallel to stimulate PI3K in POMC neurons but in opposite ways on Agrp neurons. These results suggest a new view of hypothalamic circuitry, in which the effects of leptin and insulin are integrated by anorexigenic but not by orexigenic neurons.
Allison Wanting Xu, Christopher B. Kaelin, Kiyoshi Takeda, Shizuo Akira, Michael W. Schwartz, Gregory S. Barsh
The capacity to adjust energy intake in response to changing energy requirements is a defining feature of energy homeostasis. Despite the identification of leptin as a key mediator of this process, the mechanism whereby changes of body adiposity are coupled to adaptive, short-term adjustments of energy intake remains poorly understood. To investigate the physiological role of leptin in the control of meal size and the response to satiety signals, and to identify brain areas mediating this effect, we studied Koletsky (fak/fak) rats, which develop severe obesity due to the genetic absence of leptin receptors. Our finding of markedly increased meal size and reduced satiety in response to the gut peptide cholecystokinin (CCK) in these leptin receptor–deficient animals suggests a critical role for leptin signaling in the response to endogenous signals that promote meal termination. To determine if the hypothalamic arcuate nucleus (ARC) (a key forebrain site of leptin action) mediates this leptin effect, we used adenoviral gene therapy to express either functional leptin receptors or a reporter gene in the area of the ARC of fak/fak rats. Restoration of leptin signaling to this brain area normalized the effect of CCK on the activation of neurons in the nucleus of the solitary tract and area postrema, key hindbrain areas for processing satiety-related inputs. This intervention also reduced meal size and enhanced CCK-induced satiety in fak/fak rats. These findings demonstrate that forebrain signaling by leptin, a long-term regulator of body adiposity, limits food intake on a meal-to-meal basis by regulating the hindbrain response to short-acting satiety signals.
Gregory J. Morton, James E. Blevins, Diana L. Williams, Kevin D. Niswender, Richard W. Gelling, Christopher J. Rhodes, Denis G. Baskin, Michael W. Schwartz
Hepatic insulin resistance is a critical component in the development of type 2 diabetes mellitus. In many cases, insulin resistance in liver is associated with reduced expression of both major insulin receptor substrate (IRS) proteins, IRS-1 and IRS-2. To investigate the specific functions of IRS-1 and IRS-2 in regulating liver function in vivo, we developed an adenovirus-mediated RNA interference technique in which short hairpin RNAs (shRNAs) are used to knock down IRS-1, IRS-2, or both, by 70–80% in livers of WT mice. The knockdown of IRS-1 resulted in an upregulation of the gluconeogenic enzymes glucose-6 phosphatase and phosphoenolpyruvate carboxykinase, as well as a marked increase in hepatic nuclear factor–4 α. Decreased IRS-1 was also associated with a decrease in glucokinase expression and a trend toward increased blood glucose, whereas knockdown of IRS-2 resulted in the upregulation of lipogenic enzymes SREBP-1c and fatty acid synthase, as well as increased hepatic lipid accumulation. The concomitant injection of IRS-1 and IRS-2 adenoviral shRNAs resulted in systemic insulin resistance, glucose intolerance, and hepatic steatosis. The alterations in the dual-knockdown mice were associated with defective Akt activation and Foxo1 phosphorylation. Taken together, our results demonstrate that hepatic IRS-1 and IRS-2 have complementary roles in the control of hepatic metabolism, with IRS-1 more closely linked to glucose homeostasis and IRS-2 more closely linked to lipid metabolism.
Cullen M. Taniguchi, Kohjiro Ueki, C. Ronald Kahn
Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c−/− mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c−/− cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c−/− cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.
Hajime Kanda, Yoshikazu Tamori, Hiroaki Shinoda, Mari Yoshikawa, Motoyoshi Sakaue, Jun Udagawa, Hiroki Otani, Fumi Tashiro, Jun-ichi Miyazaki, Masato Kasuga
Juan C. Molero, Thomas E. Jensen, Phil C. Withers, Michelle Couzens, Herbert Herzog, Christine B.F. Thien, Wallace Y. Langdon, Ken Walder, Maria A. Murphy, David D.L. Bowtell, Edna Hardeman, Majid Ghoddusi, David E. James, Gregory J. Cooney
Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the λ isoform of PKC in pancreatic β cells (βPKCλ–/– mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from βPKCλ–/– mice. Neither the β cell mass nor the islet insulin content of βPKCλ–/– mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3β was reduced in islets of βPKCλ–/– mice, and the expression of genes regulated by HNF3β was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3β expression by infection of islets from βPKCλ–/– mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKCλ plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for β cell function.
Naoko Hashimoto, Yoshiaki Kido, Tohru Uchida, Tomokazu Matsuda, Kazuhisa Suzuki, Hiroshi Inoue, Michihiro Matsumoto, Wataru Ogawa, Sakan Maeda, Hiroaki Fujihara, Yoichi Ueta, Yasuo Uchiyama, Kazunori Akimoto, Shigeo Ohno, Tetsuo Noda, Masato Kasuga
Concerted activation of different voltage-gated Ca2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type CaV2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in CaV2.3-knockout (CaV2.3–/–) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological CaV2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single CaV2.3–/– β cells. CaV2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the CaV2.3–/– mouse. We propose a specific role for CaV2.3 Ca2+ channels in second-phase insulin release, that of mediating the Ca2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.
Xingjun Jing, Dai-Qing Li, Charlotta S. Olofsson, Albert Salehi, Vikas V. Surve, José Caballero, Rosita Ivarsson, Ingmar Lundquist, Alexey Pereverzev, Toni Schneider, Patrik Rorsman, Erik Renström
Lipoprotein lipase (LPL) is thought to be the only enzyme responsible for the catabolism of triglycerides (TGs) associated with TG-rich lipoproteins in adipose tissue (AT). However, LPL deficiency in humans and induced mutant mice is not associated with decreased fat mass. We investigated whether endothelial lipase (EL), a recently discovered phospholipase, might represent an alternative mechanism for the uptake of phospholipid-derived fatty acids in murine lipoprotein-deficient AT. When LPL was expressed in AT and isolated murine adipocytes, EL mRNA was not detectable. In contrast, mouse AT and isolated adipocytes that lacked LPL expressed large amounts of EL mRNA. The cellular phospholipase activity in LPL-deficient fat pads was increased 4-fold compared with control fat pads and could be inhibited to control levels by a specific EL antibody. Fatty acids produced by EL activity were absorbed by adipocytes and incorporated into the TG moiety of AT. Our results suggest that EL activity in AT and other peripheral tissues might contribute to the tissue uptake of free fatty acids, which could have important implications for the metabolism of plasma lipoproteins.
Dagmar Kratky, Robert Zimmermann, Elke M. Wagner, Juliane G. Strauss, Weijun Jin, Gerhard M. Kostner, Guenter Haemmerle, Daniel J. Rader, Rudolf Zechner