Role of the Protein Kinase TBK1 in Insulin-Stimulated Glucose Transport.

Insulin stimulates glucose uptake in muscle and fat by promoting translocation of the facilitative transporter GLUT4 from intracellular compartments to the plasma membrane. While the pathways involved in GLUT4 vesicle trafficking are not completely understood, numerous studies have shown that small G proteins critically integrate signaling with the trafficking machineries in this process. Among the targets of these G proteins is the exocyst complex, which facilitates the tethering of GLUT4 vesicles to the plasma membrane. GLUT4 translocation requires both the assembly and recognition of the exocyst for targeted exocytosis, and G proteins mediate both of these processes. Exocyst assembly is controlled by activation of the Rho subfamily G protein TC10, while exocyst recognition is mediated by the G protein RalA. However, how GLUT4 vesicles dissociate from the G protein after binding is unclear, and the sequence of events that disengage GLUT4 vesicles from the individual subunits of the exocyst remain uncertain. Here I report that the protein kinase TBK1 is required for insulin-stimulated glucose transport and GLUT4 translocation in parallel with the Akt signaling pathway. Upon activation of RalA, TBK1 directly phosphorylates the exocyst subunit Exo84, a crucial step in insulin-stimulated glucose uptake. Knockdown of TBK1 blocks insulin-stimulated glucose uptake and GLUT4 translocation, and ectopic overexpression of a kinase-inactive mutant of TBK1 reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The phosphorylation of Exo84 on multiple sites by TBK1 reduces its affinity for RalA, and allows its release from the exocyst. Therefore, the interaction of TBK1/RalA/exocyst complex is dissociated upon Exo84 phosphorylation by TBK1 but overexpression of a kinase-inactive mutant of TBK1 blocks the dissociation of the complex, and treatment of 3T3-L1 adipocytes with specific inhibitors of TBK1 reduces the rate of complex dissociation. Introduction of mutant forms of Exo84 that prevent or mimic phosphorylation blocks insulin-stimulated GLUT4 translocation. Thus, these data indicate that TBK1 controls GLUT4 vesicle engagement and disengagement from the exocyst, suggesting that the exocyst is more than just a tethering complex for the GLUT4 vesicle, but also a ‘gatekeeper’ controlling vesicle fusion at the plasma membrane.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/113634/1/maeran_1.pd

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake

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

Otopetrin 1 protects mice from obesity-associated metabolic dysfunction through attenuating adipose tissue inflammation.

Chronic low-grade inflammation is emerging as a pathogenic link between obesity and metabolic disease. Persistent immune activation in white adipose tissue (WAT) impairs insulin sensitivity and systemic metabolism, in part, through the actions of proinflammatory cytokines. Whether obesity engages an adaptive mechanism to counteract chronic inflammation in adipose tissues has not been elucidated. Here we identified otopetrin 1 (Otop1) as a component of a counterinflammatory pathway that is induced in WAT during obesity. Otop1 expression is markedly increased in obese mouse WAT and is stimulated by tumor necrosis factor-α in cultured adipocytes. Otop1 mutant mice respond to high-fat diet with pronounced insulin resistance and hepatic steatosis, accompanied by augmented adipose tissue inflammation. Otop1 attenuates interferon-γ (IFN-γ) signaling in adipocytes through selective downregulation of the transcription factor STAT1. Using a tagged vector, we found that Otop1 physically interacts with endogenous STAT1. Thus, Otop1 defines a unique target of cytokine signaling that attenuates obesity-induced adipose tissue inflammation and plays an adaptive role in maintaining metabolic homeostasis in obesity

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

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake

TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue

The noncanonical IKK family member TANK-binding kinase 1 (TBK1) is activated by pro-inflammatory cytokines, but its role in controlling metabolism remains unclear. Here, we report that the kinase uniquely controls energy metabolism. Tbk1 expression is increased in adipocytes of HFD-fed mice. Adipocyte-specific TBK1 knockout (ATKO) attenuates HFD-induced obesity by increasing energy expenditure; further studies show that TBK1 directly inhibits AMPK to repress respiration and increase energy storage. Conversely, activation of AMPK under catabolic conditions can increase TBK1 activity through phosphorylation, mediated by AMPK's downstream target ULK1. Surprisingly, ATKO also exaggerates adipose tissue inflammation and insulin resistance. TBK1 suppresses inflammation by phosphorylating and inducing the degradation of the IKK kinase NIK, thus attenuating NF-κB activity. Moreover, TBK1 mediates the negative impact of AMPK activity on NF-κB activation. These data implicate a unique role for TBK1 in mediating bidirectional crosstalk between energy sensing and inflammatory signaling pathways in both over- and undernutrition

Non-Invasive Blood Glucose Monitoring (Semester 1 of Unknown), IPRO 331: Non-Invasive Blood Glucose Monitoring IPRO 331 IPRO Day Presentation Sp04

IPRO 331’s objective was to create a design for a non-invasive blood-glucose monitoring system based on the following: simple concept, user-friendliness (especially for children), a design that does not hamper the user’s lifestyle, cost effectiveness and portability.Deliverable

Non-Invasive Blood Glucose Monitoring (Semester 1 of Unknown), IPRO 331: Non-Invasive Blood Glucose Monitoring IPRO 331 Midterm Report Sp04

IPRO 331’s objective was to create a design for a non-invasive blood-glucose monitoring system based on the following: simple concept, user-friendliness (especially for children), a design that does not hamper the user’s lifestyle, cost effectiveness and portability.Deliverable