Browsing by Subject "Insulin signaling"
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Item The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells(American Society for Biochemistry and Molecular Biology, 2017-11-17) Tunduguru, Ragadeepthi; Zhang, Jing; Aslamy, Arianne; Salunkhe, Vishal A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Thurmond, Debbie C.; Biochemistry and Molecular Biology, School of MedicineDefects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation.Item Insertion of a Synthetic Switch Into Insulin Provides Metabolite-Dependent Regulation of Hormone-Receptor Activation(Endocrine Society, 2021-05-03) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, Dodson M.; Weiss, Michael Aaron; Biochemistry and Molecular Biology, School of MedicineInsulin signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to large-scale structural reorganization. To test the coupling between insulin’s “opening” and receptor activation, we inserted an artificial ligand-dependent switch into insulin. Ligand binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the receptor. Similarly, metabolite-regulated signaling was not observed in control studies of (i) an unmodified insulin analog or (ii) an analog containing a diol sensor in the absence of internal tethering. Although as expected CD-detected secondary structure was unaffected by ligand binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and holoreceptor signaling. In addition to this basic finding, our results provide proof of principle for a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog designed to mitigate risk of hypoglycemia in the treatment of diabetes mellitus.Item Insulin Resistance Is Not Sustained Following Denervation in Glycolytic Skeletal Muscle(MDPI, 2021-05-06) McMillin, Shawna L.; Stanley, Erin C.; Weyrauch, Luke A.; Brault, Jeffrey J.; Kahn, Barbara B.; Witczak, Carol A.; Anatomy and Cell Biology, School of MedicineDenervation rapidly induces insulin resistance (i.e., impairments in insulin-stimulated glucose uptake and signaling proteins) in skeletal muscle. Surprisingly, whether this metabolic derangement is long-lasting is presently not clear. The main goal of this study was to determine if insulin resistance is sustained in both oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles following long-term (28 days) denervation. Mouse hindlimb muscles were denervated via unilateral sciatic nerve resection. Both soleus and EDL muscles atrophied ~40%. Strikingly, while denervation impaired submaximal insulin-stimulated [3H]-2-deoxyglucose uptake ~30% in the soleus, it enhanced submaximal (~120%) and maximal (~160%) insulin-stimulated glucose uptake in the EDL. To assess possible mechanism(s), immunoblots were performed. Denervation did not consistently alter insulin signaling (e.g., p-Akt (Thr308):Akt; p-TBC1D1 [phospho-Akt substrate (PAS)]:TBC1D1; or p-TBC1D4 (PAS):TBC1D4) in either muscle. However, denervation decreased glucose transporter 4 (GLUT4) levels ~65% in the soleus but increased them ~90% in the EDL. To assess the contribution of GLUT4 to the enhanced EDL muscle glucose uptake, muscle-specific GLUT4 knockout mice were examined. Loss of GLUT4 prevented the denervation-induced increase in insulin-stimulated glucose uptake. In conclusion, the denervation results sustained insulin resistance in the soleus but enhanced insulin sensitivity in the EDL due to increased GLUT4 protein levels.Item New Horizons: Next-Generation Insulin Analogues: Structural Principles and Clinical Goals(The Endocrine Society, 2022) Jarosinski, Mark A.; Chen, Yen-Shan; Varas, Nicolás; Dhayalan, Balamurugan; Chatterjee, Deepak; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineDesign of “first-generation” insulin analogues over the past 3 decades has provided pharmaceutical formulations with tailored pharmacokinetic (PK) and pharmacodynamic (PD) properties. Application of a molecular tool kit—integrating protein sequence, chemical modification, and formulation—has thus led to improved prandial and basal formulations for the treatment of diabetes mellitus. Although PK/PD changes were modest in relation to prior formulations of human and animal insulins, significant clinical advantages in efficacy (mean glycemia) and safety (rates of hypoglycemia) were obtained. Continuing innovation is providing further improvements to achieve ultrarapid and ultrabasal analogue formulations in an effort to reduce glycemic variability and optimize time in range. Beyond such PK/PD metrics, next-generation insulin analogues seek to exploit therapeutic mechanisms: glucose-responsive (“smart”) analogues, pathway-specific (“biased”) analogues, and organ-targeted analogues. Smart insulin analogues and delivery systems promise to mitigate hypoglycemic risk, a critical barrier to glycemic control, whereas biased and organ-targeted insulin analogues may better recapitulate physiologic hormonal regulation. In each therapeutic class considerations of cost and stability will affect use and global distribution. This review highlights structural principles underlying next-generation design efforts, their respective biological rationale, and potential clinical applications.Item Novel roles of sterol regulatory element-binding protein-1 in liver(2016-04-26) Jideonwo, Victoria N.; Morral, Núria; Considine, Robert V.; Elmendorf, Jeffrey S.; Hannon, Tamara; Herbert, Brittney-SheaSterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.Item ROCK1 deficiency preserves caveolar compartmentalization of signaling molecules and cell membrane integrity(Wiley, 2024-02-23) Shi, Jianjian; Wei, Lei; Pediatrics, School of MedicineIn this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb-shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin-1 and -3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β-adrenergic signaling molecules including β1-adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin-1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions.Item Structural principles of insulin formulation and analog design: A century of innovation(Elsevier, 2021-10) Jarosinski, Mark A.; Dhayalan, Balamurugan; Chen, Yen-Shan; Chatterjee, Deepak; Varas, Nicolás; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineBackground: The discovery of insulin in 1921 and its near-immediate clinical use initiated a century of innovation. Advances extended across a broad front, from the stabilization of animal insulin formulations to the frontiers of synthetic peptide chemistry, and in turn, from the advent of recombinant DNA manufacturing to structure-based protein analog design. In each case, a creative interplay was observed between pharmaceutical applications and then-emerging principles of protein science; indeed, translational objectives contributed to a growing molecular understanding of protein structure, aggregation and misfolding. Scope of review: Pioneering crystallographic analyses-beginning with Hodgkin's solving of the 2-Zn insulin hexamer-elucidated general features of protein self-assembly, including zinc coordination and the allosteric transmission of conformational change. Crystallization of insulin was exploited both as a step in manufacturing and as a means of obtaining protracted action. Forty years ago, the confluence of recombinant human insulin with techniques for site-directed mutagenesis initiated the present era of insulin analogs. Variant or modified insulins were developed that exhibit improved prandial or basal pharmacokinetic (PK) properties. Encouraged by clinical trials demonstrating the long-term importance of glycemic control, regimens based on such analogs sought to resemble daily patterns of endogenous β-cell secretion more closely, ideally with reduced risk of hypoglycemia. Major conclusions: Next-generation insulin analog design seeks to explore new frontiers, including glucose-responsive insulins, organ-selective analogs and biased agonists tailored to address yet-unmet clinical needs. In the coming decade, we envision ever more powerful scientific synergies at the interface of structural biology, molecular physiology and therapeutics.