Louise Chang

The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.

An inhibitor of the protein kinases TBK1 and IKK-ɛ improves obesity-related metabolic dysfunctions in mice

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKK-ɛ and TANK-binding kinase 1 (TBK1) are induced in liver and fat by NF-κB activation upon high-fat diet feeding and in turn initiate a program of counterinflammation that preserves energy storage. Here we report that amlexanox, an approved small-molecule therapeutic presently used in the clinic to treat aphthous ulcers and asthma, is an inhibitor of these kinases. Treatment of obese mice with amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis. Because of its record of safety in patients, amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.

The TC10-interacting protein CIP4/2 is required for insulin-stimulated Glut4 translocation in 3T3L1 adipocytes

The GTPase TC10 plays a critical role in insulin-stimulated glucose transport. We report here the identification of the TC10-interacting protein CIP4/2 (Cdc42-interacting protein 4/2) as an effector in this pathway. CIP4/2 localizes to an intracellular compartment under basal conditions and translocates to the plasma membrane on insulin stimulation. Overexpression of constitutively active TC10 brings CIP4/2 to the plasma membrane, whereas overexpression of an inhibitory form of TC10 blocks the translocation of CIP4/2 produced by insulin. Overexpression of mutant forms of CIP4/2 containing an N-terminal deletion or with diminished TC10 binding inhibits insulin-stimulated Glut4 translocation. These data suggest that CIP4/2 may play an important role in insulin-stimulated glucose transport as a downstream effector of TC10.

Inflammation produces catecholamine resistance in obesity via activation of PDE3B by the protein kinases IKKε and TBK1

Obesity produces a chronic inflammatory state involving the NFκB pathway, resulting in persistent elevation of the noncanonical IκB kinases IKKε and TBK1. In this study, we report that these kinases attenuate β-adrenergic signaling in white adipose tissue. Treatment of 3T3-L1 adipocytes with specific inhibitors of these kinases restored β-adrenergic signaling and lipolysis attenuated by TNFα and Poly (I:C). Conversely, overexpression of the kinases reduced induction of Ucp1, lipolysis, cAMP levels, and phosphorylation of hormone sensitive lipase in response to isoproterenol or forskolin. Noncanonical IKKs reduce catecholamine sensitivity by phosphorylating and activating the major adipocyte phosphodiesterase PDE3B. In vivo inhibition of these kinases by treatment of obese mice with the drug amlexanox reversed obesity-induced catecholamine resistance, and restored PKA signaling in response to injection of a β-3 adrenergic agonist. These studies suggest that by reducing production of cAMP in adipocytes, IKKε and TBK1 may contribute to the repression of energy expenditure during obesity. DOI: http://dx.doi.org/10.7554/eLife.01119.001

Metabolic Crosstalk: molecular links between glycogen and lipid metabolism in obesity

Glycogen and lipids are major storage forms of energy that are tightly regulated by hormones and metabolic signals. We demonstrate that feeding mice a high-fat diet (HFD) increases hepatic glycogen due to increased expression of the glycogenic scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels were augmented in mice and cells after activation of the mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target SREBP1. Deletion of the PTG gene in mice prevented HFD-induced hepatic glycogen accumulation. Of note, PTG deletion also blocked hepatic steatosis in HFD-fed mice and reduced the expression of numerous lipogenic genes. Additionally, PTG deletion reduced fasting glucose and insulin levels in obese mice while improving insulin sensitivity, a result of reduced hepatic glucose output. This metabolic crosstalk was due to decreased mTORC1 and SREBP activity in PTG knockout mice or knockdown cells, suggesting a positive feedback loop in which once accumulated, glycogen stimulates the mTORC1/SREBP1 pathway to shift energy storage to lipogenesis. Together, these data reveal a previously unappreciated broad role for glycogen in the control of energy homeostasis.

A subcutaneous adipose tissue-liver signalling axis controls hepatic gluconeogenesis.

The search for effective treatments for obesity and its comorbidities is of prime importance. We previously identified IKK-ε and TBK1 as promising therapeutic targets for the treatment of obesity and associated insulin resistance. Here we show that acute inhibition of IKK-ε and TBK1 with amlexanox treatment increases cAMP levels in subcutaneous adipose depots of obese mice, promoting the synthesis and secretion of the cytokine IL-6 from adipocytes and preadipocytes, but not from macrophages. IL-6, in turn, stimulates the phosphorylation of hepatic Stat3 to suppress expression of genes involved in gluconeogenesis, in the process improving glucose handling in obese mice. Preliminary data in a small cohort of obese patients show a similar association. These data support an important role for a subcutaneous adipose tissue–liver axis in mediating the acute metabolic benefits of amlexanox on glucose metabolism, and point to a new therapeutic pathway for type 2 diabetes.

TC10 and insulin-stimulated glucose transport

Insulin stimulates glucose uptake in insulin-responsive tissues by means of the translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane. Two pathways are required, one involving activation of a phosphatidylinositol 3-kinase (PI 3-kinase) and downstream protein kinases, and one involving activation of the Rho-family GTPase TC10. TC10 activation by insulin is catalyzed by the exchange factor C3G, which is translocated to lipid rafts along with its binding partner CrkII as a consequence of Cbl tyrosine phosphorylation by the insulin receptor. This activation of TC10 is dependent on localization of TC10 in the lipid raft subdomains of the plasma membrane. We describe experimental approaches using the insulin-responsive cell line 3T3-L1 adipocytes to study the role of TC10 in insulin-stimulated glucose transport.

TC10 Is Regulated by Caveolin in 3T3-L1 Adipocytes

Background TC10 is a small GTPase found in lipid raft microdomains of adipocytes. The protein undergoes activation in response to insulin, and plays a key role in the regulation of glucose uptake by the hormone. Methodology/Principal Findings TC10 requires high concentrations of magnesium in order to stabilize guanine nucleotide binding. Kinetic analysis of this process revealed that magnesium acutely decreased the nucleotide release and exchange rates of TC10, suggesting that the G protein may behave as a rapidly exchanging, and therefore active protein in vivo. However, in adipocytes, the activity of TC10 is not constitutive, indicating that mechanisms must exist to maintain the G protein in a low activity state in untreated cells. Thus, we searched for proteins that might bind to and stabilize TC10 in the inactive state. We found that Caveolin interacts with TC10 only when GDP-bound and stabilizes GDP binding. Moreover, knockdown of Caveolin 1 in 3T3-L1 adipocytes increased the basal activity state of TC10. Conclusions/Significance Together these data suggest that TC10 is intrinsically active in vivo, but is maintained in the inactive state by binding to Caveolin 1 in 3T3-L1 adipocytes under basal conditions, permitting its activation by insulin.

Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking

An inflammation-associated kinase also stimulates glucose uptake through plasma membrane translocation of GLUT4 in response to insulin. Kinase for both inflammatory and insulin responses After a meal, insulin released from the pancreas triggers glucose uptake by cells in part by promoting the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. GLUT4 translocation requires a protein complex called the exocyst, which tethers GLUT4-containing vesicles to the plasma membrane. Uhm et al. found that the kinase TBK1, which mediates inflammatory responses, also phosphorylated exocyst subunits. These phosphorylation events were required for the fusion of GLUT4-containing vesicles with the plasma membrane. Insulin increased glucose uptake in adipocytes from wild-type mice but not in those from TBK1-deficient mice. The authors speculate that the involvement of TBK1 in insulin responses may be a means to counter the catabolic effects of inflammation. Insulin stimulates glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane. The exocyst complex tethers GLUT4-containing vesicles to the plasma membrane, a process that requires the binding of the G protein (heterotrimeric guanine nucleotide–binding protein) RalA to the exocyst complex. We report that upon activation of RalA, the protein kinase TBK1 phosphorylated the exocyst subunit Exo84. Knockdown of TBK1 blocked insulin-stimulated glucose uptake and GLUT4 translocation; knockout of TBK1 in adipocytes blocked insulin-stimulated glucose uptake; and ectopic overexpression of a kinase-inactive mutant of TBK1 reduced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The phosphorylation of Exo84 by TBK1 reduced its affinity for RalA and enabled its release from the exocyst. Overexpression of a kinase-inactive mutant of TBK1 blocked the dissociation of the TBK1/RalA/exocyst complex, and treatment of 3T3-L1 adipocytes with specific inhibitors of TBK1 reduced the rate of complex dissociation. Introduction of phosphorylation-mimicking or nonphosphorylatable mutant forms of Exo84 blocked insulin-stimulated GLUT4 translocation. Thus, these data indicate that TBK1 controls GLUT4 vesicle engagement and disengagement from the exocyst, suggesting that exocyst components not only constitute a tethering complex for the GLUT4 vesicle but also act as “gatekeepers” controlling vesicle fusion at the plasma membrane.

An inhibitor of the protein kinases TBK1 and IKK-[epsiv] improves obesity-related metabolic dysfunctions in mice

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKK-ɛ and TANK-binding kinase 1 (TBK1) are induced in liver and fat by NF-κB activation upon high-fat diet feeding and in turn initiate a program of counterinflammation that preserves energy storage. Here we report that amlexanox, an approved small-molecule therapeutic presently used in the clinic to treat aphthous ulcers and asthma, is an inhibitor of these kinases. Treatment of obese mice with amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis. Because of its record of safety in patients, amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.