Regulation of glucose transporter translocation in health and diabetes.

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.

Usp25m protease regulates ubiquitin-like processing of TUG proteins to control GLUT4 glucose transporter translocation in adipocytes.

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 (Tether containing a UBX domain for GLUT4) 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 TUGubiquitin-like). 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.

GLUT4 Storage Vesicles: Specialized Organelles for Regulated Trafficking.

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.

Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action.

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.

Sestrin2 prevents age-related intolerance to ischemia and reperfusion injury by modulating substrate metabolism.

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.

Excess cholesterol inhibits glucose-stimulated fusion pore dynamics in insulin exocytosis.

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 ß-cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from ß-cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in ß-cells and contribute to ß-cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs ß-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 ß-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.

Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

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.

Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle.

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.

Fiber type effects on contraction-stimulated glucose uptake and GLUT4 abundance in single fibers from rat skeletal muscle.

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.

Cholesterol accumulation increases insulin granule size and impairs membrane trafficking.

The formation of mature secretory granules is essential for proper storage and regulated release of hormones and neuropeptides. In pancreatic ß 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 ß 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.

Enhanced fasting glucose turnover in mice with disrupted action of TUG protein in skeletal muscle.

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.

Targeting steroid receptor coactivator 1 with antisense oligonucleotides increases insulin-stimulated skeletal muscle glucose uptake in chow-fed and high-fat-fed male rats.

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.

A proteolytic pathway that controls glucose uptake in fat and muscle.

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.

The ubiquitin regulatory X (UBX) domain-containing protein TUG regulates the p97 ATPase and resides at the endoplasmic reticulum-golgi intermediate compartment.

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.

Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation.

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.

Thyroid hormone receptor-ß agonists prevent hepatic steatosis in fat-fed rats but impair insulin sensitivity via discrete pathways.

Liver-specific thyroid hormone receptor-ß (TRß)-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 TRß 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 TRß- 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 TRß agonists must consider the potential adverse effects on insulin sensitivity.

The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL.

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.

Ubiquitin-like processing of TUG proteins as a mechanism to regulate glucose uptake and energy metabolism in fat and muscle.

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.

Granular detail of ß cell structures for insulin secretion.

Pancreatic ß 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.

Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake.

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.