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GLUT4 glucose transporters are expressed nearly exclusively in adipose and muscle cells, where they cycle to and from the plasma membrane. In cells not stimulated with insulin, GLUT4 is targeted to specialized GLUT4 storage vesicles (GSVs), which sequester it away from the cell surface. Insulin acts within minutes to mobilize these vesicles, translocating GLUT4 to the plasma membrane to enhance glucose uptake. The mechanisms controlling GSV sequestration and mobilization are poorly understood. An insulin-regulated aminopeptidase that cotraffics with GLUT4, IRAP, is required for basal GSV retention and insulin-stimulated mobilization. TUG and Ubc9 bind GLUT4, and likely retain GSVs within unstimulated cells. These proteins may be components of a retention receptor, which sequesters GLUT4 and IRAP away from recycling vesicles. Insulin may then act on this protein complex to liberate GLUT4 and IRAP, discharging GSVs into a recycling pathway for fusion at the cell surface. How GSVs are anchored intracellularly, and how insulin mobilizes these vesicles, are the important topics for ongoing research. Regulation of GLUT4 trafficking is tissue-specific, perhaps in part because the formation of GSVs requires cell type-specific expression of sortilin. Proteins controlling GSV retention and mobilization can then be more widely expressed. Indeed, GLUT4 likely participates in a general mechanism by which the cell surface delivery of various membrane proteins can be controlled by extracellular stimuli. Finally, it is not known if defects in the formation or intracellular retention of GSVs contribute to human insulin resistance, or play a role in the pathogenesis of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Objective: Caloric restriction (CR) is known to enhance insulin sensitivity and reduce the risk of metabolic disorders; however, its molecular mechanisms are not fully understood. This study aims to elucidate specific proteins and pathways responsible for these benefits., Methods: We examined adipose tissue from participants in the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy Phase 2 (CALERIE 2) study, comparing proteomic profiles from individuals after 12 and 24 months of CR with baseline and an ad libitum group. Biochemical and cell-specific physiological approaches complemented these analyses., Results: Our data revealed that CR upregulates prostacyclin synthase (PTGIS) in adipose tissue, an enzyme crucial for producing prostacyclin (PGI2). PGI2 improves the ability of insulin to stimulate the tether-containing UBX domain for GLUT4 (TUG) cleavage pathway, which is essential for glucose uptake regulation. Additionally, iloprost, a PGI2 analog, was shown to increase insulin receptor density on cell membranes, increasing glucose uptake in human adipocytes. CR also reduces carbonylation of GLUT4, a modification that is detrimental to GLUT4 function., Conclusions: CR enhances insulin sensitivity by promoting PTGIS expression and stimulating the TUG cleavage pathway, leading to increased GLUT4 translocation to the cell surface and decreased GLUT4 carbonylation. These findings shed light on the complex molecular mechanisms through which CR favorably impacts insulin sensitivity and metabolic health., (© 2024 The Obesity Society.)

Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In response to insulin stimulation, fat and muscle cells mobilize GLUT4 glucose transporters to the cell surface to enhance glucose uptake. Ubiquitin-like processing of TUG (Aspscr1, UBXD9) proteins is a central mechanism to regulate this process. Here, recent advances in this area are reviewed. The data support a model in which intact TUG traps insulin-responsive "GLUT4 storage vesicles" at the Golgi matrix by binding vesicle cargoes with its N-terminus and matrix proteins with its C-terminus. Insulin stimulation liberates these vesicles by triggering endoproteolytic cleavage of TUG, mediated by the Usp25m protease. Cleavage occurs in fat and muscle cells, but not in fibroblasts or other cell types. Proteolytic processing of intact TUG generates TUGUL, a ubiquitin-like protein modifier, as the N-terminal cleavage product. In adipocytes, TUGUL modifies a single protein, the KIF5B kinesin motor, which carries GLUT4 and other vesicle cargoes to the cell surface. In muscle, this or another motor may be modified. After cleavage of intact TUG, the TUG C-terminal product is extracted from the Golgi matrix by the p97 (VCP) ATPase. In both muscle and fat, this cleavage product enters the nucleus, binds PPARγ and PGC-1α, and regulates gene expression to promote fatty acid oxidation and thermogenesis. The stability of the TUG C-terminal product is regulated by an Ate1 arginyltransferase-dependent N-degron pathway, which may create a feedback mechanism to control oxidative metabolism. Although it is now clear that TUG processing coordinates glucose uptake with other aspects of physiology and metabolism, many questions remain about how this pathway is regulated and how it is altered in metabolic disease in humans., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bogan.)

Insulin stimulates glucose uptake in adipocytes by triggering translocation of glucose transporter 4-containg vesicles to the plasma membrane. Under basal conditions, these vesicles (IRVs for insulin-responsive vesicles) are retained inside the cell via a "static" or "dynamic" mechanism. We have found that inhibitors of RNA and protein synthesis, actinomycin D and emetine, stimulate Glut4 translocation and glucose uptake in adipocytes without engaging conventional signaling proteins, such as Akt, TBC1D4, or TUG. Actinomycin D does not significantly affect endocytosis of Glut4 or recycling of transferrin, suggesting that it specifically increases exocytosis of the IRVs. Thus, the intracellular retention of the IRVs in adipocytes requires continuous RNA and protein biosynthesis de novo. These results point out to the existence of a short-lived inhibitor of IRV translocation thus supporting the "static" model., (© 2022. The Author(s).)

TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.

Pancreatic β cells secrete insulin in response to increased glucose concentrations. Müller et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010039) use 3D FIB-SEM to study the architecture of these cells and to elucidate how glucose stimulation remodels microtubules to control insulin secretory granule exocytosis., (© 2021 Bogan.)

Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The physiological importance of vasopressin inactivation has long been appreciated, but the mechanisms and potential pathophysiologic roles of this process remain active subjects of research. Human Placental Leucine Aminopeptidase (P-LAP, encoded by the LNPEP gene) is an important determinant of vasopressinase activity during pregnancy and is associated with gestational diabetes insipidus and preeclampsia. Insulin-Regulated Aminopeptidase (IRAP), the rodent homologue of P-LAP, is coregulated with the insulin-responsive glucose transporter, GLUT4, in adipose and muscle cells. Recently, the Tether containing a UBX domain for GLUT4 (TUG) protein was shown to mediate the coordinated regulation of water and glucose homeostasis. TUG sequesters IRAP and GLUT4 intracellularly in the absence of insulin. Insulin and other stimuli cause the proteolytic cleavage of TUG to mobilize these proteins to the cell surface, where IRAP acts to terminate the activity of circulating vasopressin. Intriguingly, genetic variation in LNPEP is associated with the vasopressin response and mortality during sepsis, and increased copeptin, a marker of vasopressin secretion, is associated with cardiovascular and metabolic disease. We propose that in the setting of insulin resistance in muscle, increased cell-surface IRAP and accelerated vasopressin degradation cause a compensatory increase in vasopressin secretion. The increased vasopressin concentrations present at the kidneys then contribute to hypertension in the metabolic syndrome. Further analyses of metabolism and of vasopressin and copeptin may yield novel insights into a unified pathophysiologic mechanism linking insulin resistance and hypertension, and potentially other components of the metabolic syndrome, in humans., (© 2020 Elsevier Inc. All rights reserved.)

Fat and muscle cells contain a specialized, intracellular organelle known as the GLUT4 storage vesicle (GSV). Insulin stimulation mobilizes GSVs, so that these vesicles fuse at the cell surface and insert GLUT4 glucose transporters into the plasma membrane. This example is likely one instance of a broader paradigm for regulated, non-secretory exocytosis, in which intracellular vesicles are translocated in response to diverse extracellular stimuli. GSVs have been studied extensively, yet these vesicles remain enigmatic. Data support the view that in unstimulated cells, GSVs are present as a pool of preformed small vesicles, which are distinct from endosomes and other membrane-bound organelles. In adipocytes, GSVs contain specific cargoes including GLUT4, IRAP, LRP1, and sortilin. They are formed by membrane budding, involving sortilin and probably CHC22 clathrin in humans, but the donor compartment from which these vesicles form remains uncertain. In unstimulated cells, GSVs are trapped by TUG proteins near the endoplasmic reticulum - Golgi intermediate compartment (ERGIC). Insulin signals through two main pathways to mobilize these vesicles. Signaling by the Akt kinase modulates Rab GTPases to target the GSVs to the cell surface. Signaling by the Rho-family GTPase TC10α stimulates Usp25m-mediated TUG cleavage to liberate the vesicles from the Golgi. Cleavage produces a ubiquitin-like protein modifier, TUGUL, that links the GSVs to KIF5B kinesin motors to promote their movement to the cell surface. In obesity, attenuation of these processes results in insulin resistance and contributes to type 2 diabetes and may simultaneously contribute to hypertension and dyslipidemia in the metabolic syndrome.

The Golgi is well known to act as center for modification and sorting of proteins for secretion and delivery to other organelles. A key sorting step occurs at the trans -Golgi network and is mediated by protein adapters. However, recent data indicate that sorting also occurs much earlier, at the cis -Golgi, and uses lipid acylation as a novel means to regulate anterograde flux. Here, we examine an emerging role of S-palmitoylation/acylation as a mechanism to regulate anterograde routing. We discuss the critical Golgi-localized DHHC S-palmitoyltransferase enzymes that orchestrate this lipid modification, as well as their diverse protein clients (e.g., MAP6, SNAP25, CSP, LAT, β-adrenergic receptors, GABA receptors, and GLUT4 glucose transporters). Critically, for integral membrane proteins, S-acylation can act as new a "self-sorting" signal to concentrate these cargoes in rims of Golgi cisternae, and to promote their rapid traffic through the Golgi or, potentially, to bypass the Golgi. We discuss this mechanism and examine its potential relevance to human physiology and disease, including diabetes and neurodegenerative diseases.

Insulin stimulates the exocytic translocation of specialized vesicles in adipocytes, which inserts GLUT4 glucose transporters into the plasma membrane to enhance glucose uptake. Previous results support a model in which TUG ( T ether containing a U BX domain for G LUT4) proteins trap these GLUT4 storage vesicles at the Golgi matrix and in which insulin triggers endoproteolytic cleavage of TUG to translocate GLUT4. Here, we identify the muscle splice form of Usp25 (Usp25m) as a protease required for insulin-stimulated TUG cleavage and GLUT4 translocation in adipocytes. Usp25m is expressed in adipocytes, binds TUG and GLUT4, dissociates from TUG-bound vesicles after insulin addition, and colocalizes with TUG and insulin-responsive cargoes in unstimulated cells. Previous results show that TUG proteolysis generates the ubiquitin-like protein, TUGUL (for TUGu biquitin- l ike). We now show that TUGUL modifies the kinesin motor protein, KIF5B, and that TUG proteolysis is required to load GLUT4 onto these motors. Insulin stimulates TUG proteolytic processing independently of phosphatidylinositol 3-kinase. In nonadipocytes, TUG cleavage can be reconstituted by transfection of Usp25m, but not the related Usp25a isoform, together with other proteins present on GLUT4 vesicles. In rodents with diet-induced insulin resistance, TUG proteolysis and Usp25m protein abundance are reduced in adipose tissue. These effects occur soon after dietary manipulation, prior to the attenuation of insulin signaling to Akt. Together with previous data, these results support a model whereby insulin acts through Usp25m to mediate TUG cleavage, which liberates GLUT4 storage vesicles from the Golgi matrix and activates their microtubule-based movement to the plasma membrane. This TUG proteolytic pathway for insulin action is independent of Akt and is impaired by nutritional excess., (© 2018 Habtemichael et al.)

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β-cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β-cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β-cells and contribute to β-cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β-cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single-granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose-stimulated fusion events, and modulated the proportion of full fusion and kiss-and-run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol-overloaded β-cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol-induced phosphatidylinositol 4,5-bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss-and-run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

A novel stress-inducible protein, Sestrin2 (Sesn2), declines in the heart with aging. AMPK has emerged as a pertinent stress-activated kinase that has been shown to have cardioprotective capabilities against myocardial ischemic injury. We identified the interaction between Sesn2 and AMPK in the ischemic heart. To determine whether ischemic AMPK activation-modulated by the Sesn2-AMPK complex in the heart-is impaired in aging that sensitizes the heart to ischemic insults, young C57BL/6 mice (age 3-4 mo), middle-aged mice (age 10-12 mo), and aged mice (age 24-26 mo) were subjected to left anterior descending coronary artery occlusion for in vivo regional ischemia. The ex vivo working heart system was used for measuring substrate metabolism. The protein level of Sesn2 in hearts was gradually decreased with aging. Of interest, ischemic AMPK activation was blunted in aged hearts compared with young hearts ( P < 0.05); the AMPK downstream glucose uptake and the rate of glucose oxidation were significantly impaired in aged hearts during ischemia and reperfusion ( P < 0.05 vs. young hearts). Myocardial infarction size was larger in aged hearts ( P < 0.05 vs. young hearts). Immunoprecipitation with Sesn2 Ab revealed that cardiac Sesn2 forms a complex with AMPK and upstream liver kinase B1 (LKB1) during ischemia. Of interest, the binding affinity between Sesn2 and AMPK upstream LKB1 is impaired in aged hearts during ischemia ( P < 0.05 vs. young hearts). Furthermore, Sesn2-knockout hearts demonstrate a cardiac phenotype and response to ischemic stress that is similar to wild-type aged hearts ( i.e., impaired ischemic AMPK activation and higher sensitivity to ischemia- and reperfusion- induced injury). Adeno-associated virus-Sesn2 was delivered to aged hearts via a coronary delivery approach and significantly rescued the protein level of Sesn2 and the ischemic tolerance of aged hearts; therefore, Sesn2 is a scaffold protein that mediates AMPK activation in the ischemic myocardium via an interaction with AMPK upstream LKB1. Decreased Sesn2 levels in aging lead to a blunted ischemic AMPK activation, alterations in substrate metabolism, and an increased sensitivity to ischemic insults-Quan, N., Sun, W., Wang, L., Chen, X., Bogan, J. S., Zhou, X., Cates, C., Liu, Q., Zheng, Y., Li J. Sestrin2 prevents age-related intolerance to ischemia and reperfusion injury by modulating substrate metabolism., (© FASEB.)

Aberrant mitochondrial fission plays a pivotal role in the pathogenesis of skeletal muscle insulin resistance. However, fusion-fission dynamics are physiologically regulated by inherent tissue-specific and nutrient-sensitive processes that may have distinct or even opposing effects with respect to insulin sensitivity. Based on a combination of mouse population genetics and functional in vitro assays, we describe here a regulatory circuit in which peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte master regulator and receptor for the thiazolidinedione class of antidiabetic drugs, controls mitochondrial network fragmentation through transcriptional induction of Bnip3. Short hairpin RNA-mediated knockdown of Bnip3 in cultured adipocytes shifts the balance toward mitochondrial elongation, leading to compromised respiratory capacity, heightened fatty acid β-oxidation-associated mitochondrial reactive oxygen species generation, insulin resistance, and reduced triacylglycerol storage. Notably, the selective fission/Drp1 inhibitor Mdivi-1 mimics the effects of Bnip3 knockdown on adipose mitochondrial bioenergetics and glucose disposal. We further show that Bnip3 is reciprocally regulated in white and brown fat depots of diet-induced obesity and leptin-deficient ob/ob mouse models. Finally, Bnip3(-/-) mice trade reduced adiposity for increased liver steatosis and develop aggravated systemic insulin resistance in response to high-fat feeding. Together, our data outline Bnip3 as a key effector of PPARγ-mediated adipose mitochondrial network fragmentation, improving insulin sensitivity and limiting oxidative stress., (© 2016 by the American Diabetes Association.)

Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation., (© 2016. Published by The Company of Biologists Ltd.)

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To fully understand skeletal muscle at the cellular level, it is essential to evaluate single muscle fibers. Accordingly, the major goals of this study were to determine if there are fiber type-related differences in single fibers from rat skeletal muscle for: 1) contraction-stimulated glucose uptake and/or 2) the abundance of GLUT4 and other metabolically relevant proteins. Paired epitrochlearis muscles isolated from Wistar rats were either electrically stimulated to contract (E-Stim) or remained resting (No E-Stim). Single fibers isolated from muscles incubated with 2-deoxy-d-[(3)H]glucose (2-DG) were used to determine fiber type [myosin heavy chain (MHC) isoform protein expression], 2-DG uptake, and abundance of metabolically relevant proteins, including the GLUT4 glucose transporter. E-Stim, relative to No E-Stim, fibers had greater (P < 0.05) 2-DG uptake for each of the isolated fiber types (MHC-IIa, MHC-IIax, MHC-IIx, MHC-IIxb, and MHC-IIb). However, 2-DG uptake for E-Stim fibers was not significantly different among these five fiber types. GLUT4, tethering protein containing a UBX domain for GLUT4 (TUG), cytochrome c oxidase IV (COX IV), and filamin C protein levels were significantly greater (P < 0.05) in MHC-IIa vs. MHC-IIx, MHC-IIxb, or MHC-IIb fibers. TUG and COX IV in either MHC-IIax or MHC-IIx fibers exceeded values for MHC-IIxb or MHC-IIb fibers. GLUT4 levels for MHC-IIax fibers exceeded MHC-IIxb fibers. GLUT4, COX IV, filamin C, and TUG abundance in single fibers was significantly (P < 0.05) correlated with each other. Differences in GLUT4 abundance among the fiber types were not accompanied by significant differences in contraction-stimulated glucose uptake., (Copyright © 2015 the American Physiological Society.)

The steroid receptor coactivator 1 (SRC1) regulates key metabolic pathways, including glucose homeostasis. SRC1(-/-) mice have decreased hepatic expression of gluconeogenic enzymes and a reduction in the rate of endogenous glucose production (EGP). We sought to determine whether decreasing hepatic and adipose SRC1 expression in normal adult rats would alter glucose homeostasis and insulin action. Regular chow-fed and high-fat-fed male Sprage-Dawley rats were treated with an antisense oligonucleotide (ASO) against SRC1 or a control ASO for 4 wk, followed by metabolic assessments. SRC1 ASO did not alter basal EGP or expression of gluconeogenic enzymes. Instead, SRC1 ASO increased insulin-stimulated whole body glucose disposal by ~30%, which was attributable largely to an increase in insulin-stimulated muscle glucose uptake. This was associated with an approximately sevenfold increase in adipose expression of lipocalin-type prostaglandin D2 synthase, a previously reported regulator of insulin sensitivity, and an approximately 70% increase in plasma PGD2 concentration. Muscle insulin signaling, AMPK activation, and tissue perfusion were unchanged. Although GLUT4 content was unchanged, SRC1 ASO increased the cleavage of tether-containing UBX domain for GLUT4, a regulator of GLUT4 translocation. These studies point to a novel role of adipose SRC1 as a regulator of insulin-stimulated muscle glucose uptake.

Insulin regulates glucose uptake by controlling the subcellular location of GLUT4 glucose transporters. GLUT4 is sequestered within fat and muscle cells during low-insulin states, and is translocated to the cell surface upon insulin stimulation. The TUG protein is a functional tether that sequesters GLUT4 at the Golgi matrix. To stimulate glucose uptake, insulin triggers TUG endoproteolytic cleavage. Cleavage accounts for a large proportion of the acute effect of insulin to mobilize GLUT4 to the cell surface. During ongoing insulin exposure, endocytosed GLUT4 recycles to the plasma membrane directly from endosomes, and bypasses a TUG-regulated trafficking step. Insulin acts through the TC10α GTPase and its effector protein, PIST, to stimulate TUG cleavage. This action is coordinated with insulin signals through AS160/Tbc1D4 and Tbc1D1 to modulate Rab GTPases, and with other signals to direct overall GLUT4 targeting. Data support the idea that the N-terminal TUG cleavage product, TUGUL, functions as a novel ubiquitin-like protein modifier to facilitate GLUT4 movement to the cell surface. The C-terminal TUG cleavage product is extracted from the Golgi matrix, which vacates an "anchoring" site to permit subsequent cycles of GLUT4 retention and release. Together, GLUT4 vesicle translocation and TUG cleavage may coordinate glucose uptake with physiologic effects of other proteins present in the GLUT4-containing vesicles, and with potential additional effects of the TUG C-terminal product. Understanding this TUG pathway for GLUT4 retention and release will shed light on the regulation of glucose uptake and the pathogenesis of type 2 diabetes.

Obesity is associated with a chronic, low-grade, systemic inflammation that may contribute to the development of insulin resistance and type 2 diabetes. Resveratrol, a natural compound with anti-inflammatory properties, is shown to improve glucose tolerance and insulin sensitivity in obese mice and humans. Here, we tested the effect of a 2-year resveratrol administration on proinflammatory profile and insulin resistance caused by a high-fat, high-sugar (HFS) diet in white adipose tissue (WAT) from rhesus monkeys. Resveratrol supplementation (80 and 480 mg/day for the first and second year, respectively) decreased adipocyte size, increased sirtuin 1 expression, decreased NF-κB activation, and improved insulin sensitivity in visceral, but not subcutaneous, WAT from HFS-fed animals. These effects were reproduced in 3T3-L1 adipocytes cultured in media supplemented with serum from monkeys fed HFS ± resveratrol diets. In conclusion, chronic administration of resveratrol exerts beneficial metabolic and inflammatory adaptations in visceral WAT from diet-induced obese monkeys., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure were increased by 12-13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.

Liver-specific thyroid hormone receptor-β (TRβ)-specific agonists are potent lipid-lowering drugs that also hold promise for treating nonalcoholic fatty liver disease and hepatic insulin resistance. We investigated the effect of two TRβ agonists (GC-1 and KB-2115) in high-fat-fed male Sprague-Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRβ- and liver-specific thyromimetic, also prevented hepatic steatosis but did not induce fasting hyperglycemia, increase basal EGP rate, or diminish hepatic insulin sensitivity. Surprisingly, insulin-stimulated peripheral glucose disposal was diminished because of a decrease in insulin-stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB-2115 treatment was associated with a decrease in GLUT4 protein content. Thus, although both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRβ agonists must consider the potential adverse effects on insulin sensitivity.

The formation of mature secretory granules is essential for proper storage and regulated release of hormones and neuropeptides. In pancreatic β cells, cholesterol accumulation causes defects in insulin secretion and may participate in the pathogenesis of type 2 diabetes. Using a novel cholesterol analog, we show for the first time that insulin granules are the major sites of intracellular cholesterol accumulation in live β cells. This is distinct from other, non-secretory cell types, in which cholesterol is concentrated in the recycling endosomes and the trans-Golgi network. Excess cholesterol was delivered specifically to insulin granules, which caused granule enlargement and retention of syntaxin 6 and VAMP4 in granule membranes, with concurrent depletion of these proteins from the trans-Golgi network. Clathrin also accumulated in the granules of cholesterol-overloaded cells, consistent with a possible defect in the last stage of granule maturation, during which clathrin-coated vesicles bud from the immature granules. Excess cholesterol also reduced the docking and fusion of insulin granules at the plasma membrane. Together, the data support a model in which cholesterol accumulation in insulin secretory granules impairs the ability of these vesicles to respond to stimuli, and thus reduces insulin secretion., (© 2012 John Wiley & Sons A/S.)

To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types.

To enhance glucose uptake into muscle and fat cells, insulin stimulates the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. This response requires the intersection of insulin signaling and vesicle trafficking pathways, and it is compromised in the setting of overnutrition to cause insulin resistance. Insulin signals through AS160/Tbc1D4 and Tbc1D1 to modulate Rab GTPases and through the Rho GTPase TC10α to act on other targets. In unstimulated cells, GLUT4 is incorporated into specialized storage vesicles containing IRAP, LRP1, sortilin, and VAMP2, which are sequestered by TUG, Ubc9, and other proteins. Insulin mobilizes these vesicles directly to the plasma membrane, and it modulates the trafficking itinerary so that cargo recycles from endosomes during ongoing insulin exposure. Knowledge of how signaling and trafficking pathways are coordinated will be essential to understanding the pathogenesis of diabetes and the metabolic syndrome and may also inform a wide range of other physiologies.

Skeletal muscle is a heterogeneous tissue. To further elucidate this heterogeneity, we probed relationships between myosin heavy chain (MHC) isoform composition and abundance of GLUT4 and four other proteins that are established or putative GLUT4 regulators [Akt substrate of 160 kDa (AS160), Tre-2/Bub2/Cdc 16-domain member 1 (TBC1D1), Tethering protein containing an UBX-domain for GLUT4 (TUG), and RuvB-like protein two (RUVBL2)] in 12 skeletal muscles or muscle regions from Wistar rats [adductor longus, extensor digitorum longus, epitrochlearis, gastrocnemius (mixed, red, and white), plantaris, soleus, tibialis anterior (red and white), tensor fasciae latae, and white vastus lateralis]. Key results were 1) significant differences found among the muscles (range of muscle expression values) for GLUT4 (2.5-fold), TUG (1.7-fold), RUVBL2 (2.0-fold), and TBC1D1 (2.7-fold), but not AS160; 2) significant positive correlations for pairs of proteins: GLUT4 vs. TUG (R = 0.699), GLUT4 vs. RUVBL2 (R = 0.613), TUG vs. RUVBL2 (R = 0.564), AS160 vs. TBC1D1 (R = 0.293), and AS160 vs. TUG (R = 0.246); 3) significant positive correlations for %MHC-I: GLUT4 (R = 0.460), TUG (R = 0.538), and RUVBL2 (R = 0.511); 4) significant positive correlations for %MHC-IIa: GLUT4 (R = 0.293) and RUVBL2 (R = 0.204); 5) significant negative correlations for %MHC-IIb vs. GLUT4 (R = -0.642), TUG (R = -0.626), and RUVBL2 (R = -0.692); and 6) neither AS160 nor TBC1D1 significantly correlated with MHC isoforms. In 12 rat muscles, GLUT4 abundance tracked with TUG and RUVBL2 and correlated with MHC isoform expression, but was unrelated to AS160 or TBC1D1. Our working hypothesis is that some of the mechanisms that regulate GLUT4 abundance in rat skeletal muscle also influence TUG and RUVBL2 abundance.

Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.

Insulin regulates the trafficking of GLUT4 glucose transporters in fat and muscle cells. In unstimulated cells, GLUT4 is sequestered intracellularly in small, insulin-responsive vesicles. Insulin stimulates the translocation of these vesicles to the cell surface, inserting the transporters into the plasma membrane to enhance glucose uptake. Formation of the insulin-responsive vesicles requires multiple interactions among GLUT4, IRAP, LRP1, and sortilin, as well as recruitment of GGA and ACAP1 adaptors and clathrin. Once formed, the vesicles are retained within unstimulated cells by the action of TUG, Ubc9, and other proteins. In addition to acting at other steps in vesicle recycling, insulin releases this retention mechanism to promote the translocation and fusion of the vesicles at the cell surface., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Objective: Insulin released by the beta-cell is thought to act locally to regulate glucagon secretion. The possibility that insulin might also act centrally to modulate islet glucagon secretion has received little attention., Research Design and Methods: Initially the counterregulatory response to identical hypoglycemia was compared during intravenous insulin and phloridzin infusion in awake chronically catheterized nondiabetic rats. To explore whether the disparate glucagon responses seen were in part due to changes in ventromedial hypothalamus (VMH) exposure to insulin, bilateral guide cannulas were inserted to the level of the VMH and 8 days later rats received a VMH microinjection of either 1) anti-insulin affibody, 2) control affibody, 3) artificial extracellular fluid, 4) insulin (50 microU), 5) insulin receptor antagonist (S961), or 6) anti-insulin affibody plus a gamma-aminobutyric acid A (GABA(A)) receptor agonist muscimol, prior to a hypoglycemic clamp or under baseline conditions., Results: As expected, insulin-induced hypoglycemia produced a threefold increase in plasma glucagon. However, the glucagon response was fourfold to fivefold greater when circulating insulin did not increase, despite equivalent hypoglycemia and C-peptide suppression. In contrast, epinephrine responses were not altered. The phloridzin-hypoglycemia induced glucagon increase was attenuated (40%) by VMH insulin microinjection. Conversely, local VMH blockade of insulin amplified glucagon twofold to threefold during insulin-induced hypoglycemia. Furthermore, local blockade of basal insulin levels or insulin receptors within the VMH caused an immediate twofold increase in fasting glucagon levels that was prevented by coinjection to the VMH of a GABA(A) receptor agonist., Conclusions: Together, these data suggest that insulin's inhibitory effect on alpha-cell glucagon release is in part mediated at the level of the VMH under both normoglycemic and hypoglycemic conditions.

Insulin regulates glucose uptake and storage in peripheral tissues, and has been shown to act within the hypothalamus to acutely regulate food intake and metabolism. The machinery for transduction of insulin signaling is also present in other brain areas, particularly in the hippocampus, but a physiological role for brain insulin outside the hypothalamus has not been established. Recent studies suggest that insulin may be able to modulate cognitive functions including memory. Here we report that local delivery of insulin to the rat hippocampus enhances spatial memory, in a PI-3-kinase dependent manner, and that intrahippocampal insulin also increases local glycolytic metabolism. Selective blockade of endogenous intrahippocampal insulin signaling impairs memory performance. Further, a rodent model of type 2 diabetes mellitus produced by a high-fat diet impairs basal cognitive function and attenuates both cognitive and metabolic responses to hippocampal insulin administration. Our data demonstrate that insulin is required for optimal hippocampal memory processing. Insulin resistance within the telencephalon may underlie the cognitive deficits commonly reported to accompany type 2 diabetes., (Published by Elsevier Inc.)

Membrane cholesterol modulates the ability of glucose to stimulate insulin secretion from pancreatic beta-cells. The molecular mechanism by which this occurs is not understood. Here, we show that in cultured beta-cells, cholesterol acts through phosphatidylinositol 4,5-bisphosphate (PIP(2)) to regulate actin dynamics, plasma membrane potential, and glucose-stimulated insulin secretion. Cholesterol-overloaded beta-cells exhibited decreased PIP(2) hydrolysis, with diminished glucose-induced actin reorganization, membrane depolarization, and insulin secretion. The converse findings were observed in cholesterol-depleted cells. These results support a model in which cholesterol depletion is coupled through PIP(2) to enhance both plasma membrane Ca2+ influx from the extracellular space, as well as inositol 1,4,5-triphosphate-stimulated Ca2+ efflux from intracellular stores. The inability to increase cytosolic Ca2+ may be the main underlying factor to account for impaired glucose-stimulated insulin secretion in cholesterol-overloaded beta-cells.

Peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1beta) is known to be a transcriptional coactivator for SREBP-1, the master regulator of hepatic lipogenesis. Here, we evaluated the role of PGC-1beta in the pathogenesis of fructose-induced insulin resistance by using an antisense oligonucletoide (ASO) to knockdown PGC-1beta in liver and adipose tissue. PGC-1beta ASO improved the metabolic phenotype induced by fructose feeding by reducing expression of SREBP-1 and downstream lipogenic genes in liver. PGC-1beta ASO also reversed hepatic insulin resistance induced by fructose in both basal and insulin-stimulated states. Furthermore, PGC-1beta ASO increased insulin-stimulated whole-body glucose disposal due to a threefold increase in glucose uptake in white adipose tissue. These data support an important role for PGC-1beta in the pathogenesis of fructose-induced insulin resistance and suggest that PGC-1beta inhibition may be a therapeutic target for treatment of NAFLD, hypertriglyceridemia, and insulin resistance associated with increased de novo lipogenesis.

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.

The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a beta-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and noted that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal diglycine ("GG") motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the "Ile-44 hydrophobic face" typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.

Acrp30/adiponectin is reduced in the serum of obese and diabetic individuals, and the genetic locus of adiponectin is linked to the metabolic syndrome. Recombinant adiponectin, administered to diet-induced obese mice, induced weight loss and improved insulin sensitivity. In muscle and liver, adiponectin stimulates AMP-activated protein kinase activation and fatty acid oxidation. To expression-clone molecules capable of binding adiponectin, we transduced a C2C12 myoblast cDNA retroviral expression library into Ba/F3 cells and panned infected cells on recombinant adiponectin linked to magnetic beads. We identified T-cadherin as a receptor for the hexameric and high-molecular-weight species of adiponectin but not for the trimeric or globular species. Only eukaryotically expressed adiponectin bound to T-cadherin, implying that posttranslational modifications of adiponectin are critical for binding. An adiponectin mutant lacking a conserved N-terminal cysteine residue required for formation of hexamer and high-molecular-weight species did not bind T-cadherin in coimmunoprecipitation studies. Although lacking known cellular functions, T-cadherin is expressed in endothelial and smooth muscle cells, where it is positioned to interact with adiponectin. Because T-cadherin is a glycosylphosphatidylinositol-anchored extracellular protein, it may act as a coreceptor for an as-yet-unidentified signaling receptor through which adiponectin transmits metabolic signals.

Insulin stimulates glucose uptake in fat and muscle by mobilizing the GLUT4 glucose transporter. GLUT4 is sequestered intracellularly in the absence of insulin, and is redistributed to the plasma membrane within minutes of insulin stimulation. But the trafficking mechanisms that control GLUT4 sequestration have remained elusive. Here we describe a functional screen to identify proteins that modulate GLUT4 distribution, and identify TUG as a putative tether, containing a UBX domain, for GLUT4. In truncated form, TUG acts in a dominant-negative manner to inhibit insulin-stimulated GLUT4 redistribution in Chinese hamster ovary cells and 3T3-L1 adipocytes. Full-length TUG forms a complex specifically with GLUT4; in 3T3-L1 adipocytes, this complex is present in unstimulated cells and is largely disassembled by insulin. Endogenous TUG is localized with the insulin-mobilizable pool of GLUT4 in unstimulated 3T3-L1 adipocytes, and is not mobilized to the plasma membrane by insulin. Distinct regions of TUG are required to bind GLUT4 and to retain GLUT4 intracellularly in transfected, non-adipose cells. Our data suggest that TUG traps endocytosed GLUT4 and tethers it intracellularly, and that insulin mobilizes this pool of retained GLUT4 by releasing this tether.

In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism.

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

Insulin stimulates adipose cells both to secrete proteins and to translocate the GLUT4 glucose transporter from an intracellular compartment to the plasma membrane. We demonstrate that whereas insulin stimulation of 3T3-L1 adipocytes has no effect on secretion of the alpha3 chain of type VI collagen, secretion of the protein hormone adipocyte complement related protein of 30 kD (ACRP30) is markedly enhanced. Like GLUT4, regulated exocytosis of ACRP30 appears to require phosphatidylinositol-3-kinase activity, since insulin-stimulated ACRP30 secretion is blocked by pharmacologic inhibitors of this enzyme. Thus, 3T3-L1 adipocytes possess a regulated secretory compartment containing ACRP30. Whether GLUT4 recycles to such a compartment has been controversial. We present deconvolution immunofluorescence microscopy data demonstrating that the subcellular distributions of ACRP30 and GLUT4 are distinct and nonoverlapping; in contrast, those of GLUT4 and the transferrin receptor overlap. Together with supporting evidence that GLUT4 does not recycle to a secretory compartment via the trans-Golgi network, we conclude that there are at least two compartments that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes: one for ACRP30 secretion and one for GLUT4 translocation.

The majority of cases of Cushing's disease are due to an underlying pituitary corticotroph microadenoma (< or = 10 mm). Corticotroph macroadenomas (> 10 mm) are a less common cause of Cushing's disease, and little is known about specific clinical and biochemical findings in such patients. To define further the clinical characteristics of patients with corticotroph macroadenomas, we performed a retrospective review of Cushing's disease due to macroadenomas seen at Massachusetts General Hospital between 1979 and 1995. Of 531 patients identified with a diagnostic code of Cushing's syndrome, 20 were determined to have Cushing's disease due to a macroadenoma based on radiographic evidence of pituitary adenoma greater than 10 mm and pathological confirmation of a pituitary adenoma. A comparison review of charts of 24 patients with Cushing's disease due to corticotroph microadenomas identified on the basis of radiographic evidence of a normal pituitary gland or a pituitary adenoma 10 mm or less in diameter was also performed. The mean ages of the patients (+/- SD) with macroadenomas and microadenomas were similar (39 +/- 12 and 38 +/- 14 yr, respectively). The baseline median 24-h urine free cortisol (UFC) excretion was 1341 nmol/day (range, 304-69,033 nmol/day) and 877 nmol/day (range, 293-2,558 nmol/day) for macroadenoma and microadenoma patients, respectively (P = 0.058). After the 48-h high dose dexamethasone suppression test, UFC decreased by 77 +/- 19% (mean +/- SD) and 91 +/- 7% in macroadenoma and microadenoma subjects, respectively (P = 0.04). Fifty-six percent of macroadenoma patients and 92% of microadenoma patients had greater than 80% suppression of UFC after high dose dexamethasone administration (P = 0.03). The baseline median 24-h urinary 17-hydroxysteroid (17-OHCS) excretion was 52 mumol/day (range, 25-786 mumol/day) and 44 mumol/day (range, 17-86 mumol/day) for macroadenoma and microadenoma subjects, respectively (P = 0.09). After the standard high dose dexamethasone suppression test, 17-OHCS excretion decreased by 46 +/- 33% and 72 +/- 22% for macroadenoma and microadenoma subjects, respectively (P = 0.02). Fifty-three percent of patients with macroadenomas and 86% of patients with microadenomas had greater than 50% suppression of 17-OHCS after high dose dexamethasone administration (P = 0.02). Baseline plasma ACTH values were above the normal range in 83.3% of macroadenoma patients and in 45% of microadenoma subjects (P = 0.05). Tumors were immunostained with the MIB-1 antibody for Ki-67 to investigate proliferation in the adenomas. There was a trend for a higher Ki-67 labeling index in corticotroph macroadenomas, and seven (44%) macroadenomas vs. three (18%) microadenomas had labeling indexes greater than 3%, but this was not statistically significant. In summary, corticotroph macroadenomas are often associated with less glucocorticoid suppressibility than the more frequently occurring microadenomas. Therefore, the lack of suppression of UFC or 17-OHCS after the administration of high dose dexamethasone in a patient with Cushing's disease does not necessarily imply the presence of ACTH-independent Cushing's syndrome and is more commonly seen in patients with corticotroph macroadenomas than in those with microadenomas. Increased plasma ACTH concentrations are typical of patients with corticotroph macroadenomas and may be a more sensitive indicator of neoplastic corticotrophs than the UFC or 17-OHCS response to standard high dose dexamethasone testing.

To facilitate studies of the SRY gene, a 4741-bp portion of the sex-determining region of the human Y chromosome was sequenced and characterized. Two RNAs were found to hybridize to this genomic segment, one transcript deriving from SRY and the second cross-hybridizing to a pseudogene located 2.5 kb 5' of the SRY open reading frame (ORF). Analysis of the SRY transcript using 3' and 5' rapid amplification and cloning of ends suggested that the entire SRY protein is encoded by a single exon. A 700-bp CpG island is located immediately 5' of the pseudogene (and 2 kb 5' of the SRY ORF). Within this CpG island lies the sequence CGCCCCGC, a potential binding site for the EGR-1/WT1 family of transcription factors, some of which appear to function in gonadal development.

A deletion map of the human Y chromosome was constructed by testing 96 individuals with partial Y chromosomes for the presence or absence of many DNA loci. The individuals studied included XX males, XY females, and persons in whom chromosome banding had revealed translocated, deleted, isodicentric, or ring Y chromosomes. Most of the 132 Y chromosomal loci mapped were sequence-tagged sites, detected by means of the polymerase chain reaction. These studies resolved the euchromatic region (short arm, centromere, and proximal long arm) of the Y chromosome into 43 ordered intervals, all defined by naturally occurring chromosomal breakpoints and averaging less than 800 kilobases in length. This deletion map should be useful in identifying Y chromosomal genes, in exploring the origin of chromosomal disorders, and in tracing the evolution of the Y chromosome.