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Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance

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Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance Diabetes Volume 65, June 2016 1577 Reema P. Vazirani,1 Akanksha Verma,2 L. Amanda Sadacca,1 Melanie S. Buckman,3 Belen Picatoste,1 Muheeb Beg,1 Christopher Torsitano,1 Joanne H. Bruno,1 Rajesh T. Patel,1 Kotryna Simonyte,4 Joao P. Camporez,5 Gabriela Moreira,5 Domenick J. Falcone,6 Domenico Accili,7 Olivier Elemento,2 Gerald I. Shulman,6,8 Barbara B. Kahn,4 and Timothy E. McGraw1,3 Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance Diabetes 2016;65:1577–1589 | DOI: 10.2337/db15-1128 Insulin controls glucose uptake into adipose and muscle of the precise control of insulin-regulated trafficking cells by regulating the amount of GLUT4 in the plasma in adipocytes. membrane. The effect of insulin is to promote the trans- location of intracellular GLUT4 to the plasma mem- brane. The small Rab GTPase, Rab10, is required for Insulin regulation of the amount of the GLUT4 in the insulin-stimulated GLUT4 translocation in cultured 3T3- plasma membrane of muscle and adipose cells has a major METABOLISM L1 adipocytes. Here we demonstrate that both insulin- role in the control of postprandial blood glucose levels. stimulated glucose uptake and GLUT4 translocation In the basal (unstimulated) state, GLUT4 is retained in- totheplasmamembranearereducedbyabouthalf tracellularly through rapid endocytosis and slow recycling fi in adipocytes from adipose-speci c Rab10 knockout back to the plasma membrane. The exclusion of GLUT4 (KO) mice. These data demonstrate that the full effect from the plasma membrane limits basal glucose flux into of insulin on adipose glucose uptake is the integrated fat and muscle cells. Insulin stimulation alters the kinetics effect of Rab10-dependent and Rab10-independent of GLUT4 trafficking by accelerating exocytosis and slowing pathways, establishing a divergence in insulin signal fi endocytosis, inducing a net redistribution of GLUT4 to transduction to the regulation of GLUT4 traf cking. In – fi theplasmamembrane(1 3). The accumulation of GLUT4 in adipose-speci c Rab10 KO female mice, the partial in- fl hibition of stimulated glucose uptake in adipocytes in- the plasma membrane results in increased glucose ux into duces insulin resistance independent of diet challenge. cells and the postprandial disposal of glucose. The effect of During euglycemic-hyperinsulinemic clamp, there is no insulin on glucose uptake into muscle and adipose can be suppression of hepatic glucose production despite accounted for by altered GLUT4 localization (4,5). Studies fi normal insulin suppression of plasma free fatty acids. of genetically modi ed mice have established that the role The impact of incomplete disruption of stimulated ad- of GLUT4 in insulin-stimulated glucose uptake into muscle ipocyte GLUT4 translocation on whole-body glucose (6) and adipose (7) are essential for the maintenance of homeostasis is driven by a near complete failure of glucose homeostasis. Furthermore, defective GLUT4 trans- insulin to suppress hepatic glucose production rather location is a hallmark of insulin resistance and type 2 di- than a significant inhibition in muscle glucose uptake. abetes in humans (8–10). Consequently, to fully understand These data underscore the physiological significance insulin action and its disruption in metabolic disease, 1Department of Biochemistry, Weill Cornell Medical College, New York, NY Corresponding author: Timothy E. McGraw, [email protected]. 2 Department of Physiology and Biophysics, Weill Cornell Medical College, New York, Received 12 August 2015 and accepted 21 March 2016. NY This article contains Supplementary Data online at http://diabetes 3Department of Cardiothoracic Surgery, Weill Cornell Medical College, New York, NY .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1128/-/DC1. 4Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 5Departments of Internal Medicine and Cellular & Molecular Physiology, Yale School © 2016 by the American Diabetes Association. Readers may use this article as of Medicine, Yale University, New Haven, CT long as the work is properly cited, the use is educational and not for profit, and 6Department of Pathology, Weill Cornell Medical College, New York, NY the work is not altered. 7Department of Medicine and Naomi Berrie Diabetes Center, Columbia University, New York, NY 8Howard Hughes Medical Institute, Yale University, New Haven, CT 1578 Rab10 Deletion in Fat Causes Insulin Resistance Diabetes Volume 65, June 2016 it is essential to describe how the insulin signal is trans- remove the selection cassette and generate carriers of a duced to the regulation of GLUT4 trafficking at the molec- conditional Rab10 allele containing loxP sites flanking ular level. exon 1 of Rab10. The colony was backcrossed to C57BL/6J fl Studies of the cultured 3T3-L1 adipocyte cell line have mice four times. Heterozygotes with (Rab10 /wt + cre) fl established that the AS160-Rab10 signaling module is and without (Rab10 /wt) the adiponectin-cre transgene essential for stimulated GLUT4 translocation to the plasma were bred to generate mice for the majority of experi- fl fl membrane (11–15). Insulin activation of the Akt serine- ments. Homozygote flox females (Rab10 / ) and AR10KO fl fl threonine protein kinase leads to the phosphorylation males (Rab10 / + cre) were bred to generate mice for the and inactivation of the AS160 (TBC1D4) Rab GTPase ac- euglycemic-hyperinsulinemic clamp. Control data were de- tivating protein (16). Although AS160 has activity toward rived from littermate controls. The metabolic phenotypes fl fl a number of Rab proteins (17), Rab10 is the AS160 target of wild-type, cre, and flox (Rab10 / )micedidnotvarywhile Rab involved in the regulation of GLUT4 trafficking in eating a low-fat diet (LFD) (Supplementary Figs. 2 and 3). cultured adipocytes. In cultured muscle cells, Rab8a, but Mice were maintained on a standard 12-h light/dark cycle, not Rab10, mediates GLUT4 trafficking. In unstimulated and had ad libitum access to water and food. Mice were 3T3-L1 adipocytes, the GTPase activity of AS160 main- fed standard rodent chow except where noted that mice tains Rab10 in the inactive guanosine diphosphate–bound were fed a high-fat diet (HFD) or LFD (catalog #D12492 state. Insulin-stimulated inactivation of AS160 leads to or #D12450B; Research Diets). Unless otherwise noted, the Rab10-dependent translocation of GLUT4 to the all experiments were performed using female mice. All plasma membrane (11). Rab10 knockdown with small in- protocols were approved by the Weill Cornell Medical terfering RNA (siRNA) reduces insulin-stimulated GLUT4 College and Yale Medical School Institutional Animal translocation by about half in 3T3-L1 adipocytes, sug- Care and Use Committees. gesting that multiple signaling pathways downstream of the insulin receptor are required for the stimulation of Genotyping GLUT4 translocation (13). It is unknown whether there Tail DNA retrieved through proteinase K digestion was used – are both Rab10-dependent and Rab10-independent path- in touchdown PCR (at 65 55°C, followed by 30 cycles at ways in primary adipocytes. This phenomenon described 94°C for 30 min, at 55°C for 30 min, and at 72°C for 60 min) Rab10 9 in studies of cultured cells could be a consequence of in- with the following primer pairs: (5 -GGTAAAGG 9 9 complete Rab10 depletion by siRNA knockdown, which CAAGTAGATGTCCATG-3 ,5-GAAGAGCAATTAAACACTG 9 Cre Recombinase 9 would allow residual Rab10 activity, or it could be a char- CATGC-3 )and (5 -ATGTCCAATTTACTG 9 9 9 acteristic of the cultured 3T3-L1 adipocyte line. Knowing ACCG-3 ,5-CGCCGCATAACCAGTGAAAC-3 ). how the insulin signal is transduced to GLUT4 in vivo is Tissue Immunoblotting key for understanding the physiological activity of insulin Tissues were processed using a multisample pulverizer and its disruption in disease. and were prepared in lysis buffer (catalog #9803S; Cell fi We created adipose-speci c Rab10 knockout (KO) mice Signaling Technology) containing HALT inhibitor cocktail to evaluate the contribution of Rab10 to insulin regula- (catalog #78442; Thermo Scientific). An equivalent amount fi tion of GLUT4 traf cking in primary adipocytes. These of protein was used for all samples in each gel. Membranes studies demonstrate that Rab10 is an essential compo- were incubated with primary antibodies at 4°C overnight, nent of the machinery responsible for insulin stimulation as follows: Rab10 (1:500; catalog #4262; Cell Signaling fi of GLUT4 traf cking to the plasma membrane of primary Technology), GLUT4 (1:25,000; catalog #2213; Cell Signal- adipocytes. Approximately half of the insulin signal to ing Technology), phosphorylated AS160 T642 (1:1,000; GLUT4 requires Rab10, establishing that the full effect of catalog #8881; Cell Signaling Technology), AS160 insulin on GLUT4 requires both Rab10-dependent and (1:5,000; catalog #07–741; Millipore), Rab8a (1:1,000; cat- fi Rab10-independent pathways. Adipose-speci c Rab10 KO alog #610844; BD Biosciences), phosphorylated Akt S473 mice on a normal chow diet are insulin intolerant, and (1:2,000; catalog #9271; Cell Signaling Technology), and Akt during euglycemic-hyperinsulinemic clamp they have se- (1:1,000; catalog #9272; Cell Signaling Technology). verely blunted inhibition of hepatic glucose production. The demonstration that a partial defect in adipocyte GLUT4 Adipocyte Glucose Uptake and Size Measurement translocation can induce insulin resistance establishes the Primary adipocyte glucose uptake and size measurement importance of fully intact insulin regulation of adipocyte was performed as previously described (7), except for the glucose transport in glucose homeostasis. use of tracer amounts of 3H-2-deoxyglucose (50 nmol/L; catalog #NET549250UC; PerkinElmer) diluted in 5 mmol/L RESEARCH DESIGN AND METHODS glucose. Glucose uptake using a higher concentration of AR10KO Mice 2-deoxyglucose has been shown to be linear for up to Embryonic stem cells containing a Rab10 conditional KO 30 min in primary adipocytes (18). Briefly, dissociated adi- construct (Knockout Mouse Project CSD44891) were pocytes from female mice were treated with insulin for microinjected to generate chimera mice. Offspring with 20 min prior to the addition of 3H-2-deoxyglucose.
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