Browsing by Author "Brozinick, Joseph T."

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    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 Medicine
    Defects 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.
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    An early, reversible cholesterolgenic etiology of diet-induced insulin resistance
    (Elsevier, 2023) Covert, Jacob D.; Grice, Brian A.; Thornburg, Matthew G.; Kaur, Manpreet; Ryan, Andrew P.; Tackett, Lixuan; Bhamidipati, Theja; Stull, Natalie D.; Kim, Teayoun; Habegger, Kirk M.; McClain, Donald A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Anatomy, Cell Biology and Physiology, School of Medicine
    Objective: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance. Methods: C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet. Results: Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM. Conclusions: These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.
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    Excess membrane cholesterol is an early contributing reversible aspect of skeletal muscle insulin resistance in C57BL/6NJ mice fed a Western-style high-fat diet
    (American Physiological Society, 2019-08-06) Grice, Brian A.; Barton, Kelly J.; Covert, Jacob D.; Kreilach, Alec M.; Tackett, Lixuan; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Anatomy and Cell Biology, School of Medicine
    Skeletal muscle insulin resistance manifests shortly after high-fat feeding, yet mechanisms are not known. Here we set out to determine whether excess skeletal muscle membrane cholesterol and cytoskeletal derangement known to compromise glucose transporter (GLUT)4 regulation occurs early after high-fat feeding. We fed 6-wk-old male C57BL/6NJ mice either a low-fat (LF, 10% kcal) or a high-fat (HF, 45% kcal) diet for 1 wk. This HF feeding challenge was associated with an increase, albeit slight, in body mass, glucose intolerance, and hyperinsulinemia. Liver analyses did not reveal signs of hepatic insulin resistance; however, skeletal muscle immunoblots of triad-enriched regions containing transverse tubule membrane showed a marked loss of stimulated GLUT4 recruitment. An increase in cholesterol was also found in these fractions from HF-fed mice. These derangements were associated with a marked loss of cortical filamentous actin (F-actin) that is essential for GLUT4 regulation and known to be compromised by increases in membrane cholesterol. Both the withdrawal of the HF diet and two subcutaneous injections of the cholesterol-lowering agent methyl-β-cyclodextrin at 3 and 6 days during the 1-wk HF feeding intervention completely mitigated cholesterol accumulation, cortical F-actin loss, and GLUT4 dysregulation. Moreover, these beneficial membrane/cytoskeletal changes occurred concomitant with a full restoration of metabolic responses. These results identify skeletal muscle membrane cholesterol accumulation as an early, reversible, feature of insulin resistance and suggest cortical F-actin loss as an early derangement of skeletal muscle insulin resistance.
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    Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western‐style high‐fat diet
    (Wiley, 2017-08-15) Ambery, Ashley G.; Tackett, Lixuan; Penque, Brent A.; Brozinick, Joseph T.; Elmendorf, Jeffrey S.; Cellular and Integrative Physiology, School of Medicine
    Insulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F‐actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low‐fat (LF, 10% kCal) or high‐fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham‐exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF‐feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, “pre‐intervention” glucose tolerance tests were performed on all animals and demonstrated that HF‐fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham‐exercised HF‐fed mice showed a significant increase in PM cholesterol, loss of cortical F‐actin, and decrease in insulin‐stimulated glucose transport compared to sham‐exercised LF‐fed mice. These HF‐fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed.
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    IFN-α Induces Heterogenous ROS Production in Human β-Cells
    (bioRxiv, 2025-02-20) Wagner, Leslie E.; Melnyk, Olha; Turner, Abigail; Duffett, Bryce E.; Muralidharan, Charanya; Martinez-Irizarry, Michelle M.; Arvin, Matthew C.; Orr, Kara S.; Manduchi, Elisabetta; Kaestner, Klaus H.; Brozinick, Joseph T.; Linnemann, Amelia K.; Biochemistry and Molecular Biology, School of Medicine
    Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors, including viral infection. We investigated the impact of interferon alpha (IFN-α), a cytokine produced during the immune response to viral infection or the presence of un-edited endogenous double-stranded RNAs, on human β-cell physiology. Intravital microscopy on transplanted human islets using a β-cell-selective reactive oxygen species (ROS) biosensor (RIP1-GRX1-roGFP2), revealed a subset of human β-cells that acutely produce ROS in response to IFN-α. Comparison to Integrated Islet Distribution Program (IIDP) phenotypic data revealed that healthier donors had more ROS accumulating cells. In vitro IFN-α treatment of human islets similarly elicited a heterogenous increase in superoxide production that originated in the mitochondria. To determine the unique molecular signature predisposing cells to IFN-α stimulated ROS production, we flow sorted human islets treated with IFN-α. RNA sequencing identified genes involved in inflammatory and immune response in the ROS-producing cells. Comparison with single cell RNA-Seq datasets available through the Human Pancreas Analysis Program (HPAP) showed that genes upregulated in ROS-producing cells are enriched in control β-cells rather than T1D donors. Combined, these data suggest that IFN-α stimulates mitochondrial ROS production in healthy human β-cells, potentially predicting a more efficient antiviral response.
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    Membrane cholesterol balance in exercise and insulin resistance
    (2009-10) Habegger, Kirk M.; Elmendorf, Jeffrey S.; Roach, Peter J.; Brozinick, Joseph T.; Sturek, Michael S.; Considine, Robert V.
    Study has shown that plasma membrane (PM) cholesterol and cortical filamentous actin (F-actin) influence skeletal muscle glucose transport. Of fundamental and clinical interest is whether diabetogenic insults promote membrane/cytoskeletal dysfunction amendable for therapy. As exposure to excess fatty acid (FA)s induce glucose intolerance by mechanisms imperfectly understood, we tested if PM cholesterol/F-actin changes could contribute to FA-induced glucose transporter GLUT4 dysregulation in skeletal muscle. High-fat fed, insulin-resistant animals displayed elevated levels of skeletal muscle PM cholesterol and a loss in cortical F-actin, compared to normal-chow fed animals. Consistent with a PM cholesterol component of glucose intolerance, human skeletal muscle biopsies revealed an inverse correlation between PM cholesterol and whole-body glucose disposal. Mechanistically, exposure of L6 myotubes to the saturated FA palmitate induced an increase in PM cholesterol that destabilized actin filaments and decreased insulin-stimulated PM GLUT4 and glucose transport, which could be reversed with cholesterol lowering. Next, study tested if the lipid-lowering action of the antidiabetic AMP-activated protein kinase (AMPK) had a beneficial influence on PM cholesterol balance. Consistent with AMPK inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, a rate-limiting enzyme of cholesterol synthesis, we found that AMPK activation promoted a significant reduction in PM cholesterol and amplified basal and insulin-stimulated GLUT4 translocation. A similar loss of PM cholesterol induced by β-cyclodextrin caused an analogous enhancement of GLUT4 regulation. Interestingly, PM cholesterol replenishment abrogated the AMPK effect on insulin, but not basal, regulation of GLUT4 translocation. Conversely, AMPK knockdown prevented the enhancement of both basal and insulin-stimulated GLUT4 translocation. As a whole these studies show PM cholesterol accrual and cortical F-actin loss uniformly in skeletal muscle from glucose-intolerant mice, swine, and humans. In vivo and in vitro dissection demonstrated this membrane/cytoskeletal derangement induces insulin resistance and is promoted by excess FAs. Parallel studies unveiled that the action of AMPK entailed lowering PM cholesterol that enhanced the regulation of GLUT4/glucose transport by insulin. In conclusion, these data are consistent with PM cholesterol regulation being an unappreciated aspect of AMPK signaling that benefits insulin-stimulated GLUT4 translocation during states of nutrient excess promoting PM cholesterol accrual.
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    Regulation of skeletal muscle insulin sensitivity by PAK1
    (2016-012) Tunduguru, Ragadeepthi; Thurmond, Debbie C.; Elmendorf, Jeffrey S.; Atkinson, Simon J.; Brozinick, Joseph T.; Gunst, Susan J.
    Insulin-stimulated glucose uptake into skeletal muscle cells requires translocation of the glucose transporter-4 (GLUT4) from the cell interior to the plasma membrane. Insulin-stimulated GLUT4 vesicle translocation is dysregulated in Type 2 diabetes (T2D). The Group I p21–activated kinase (PAK1) is a required element in insulin-stimulated GLUT4 vesicle translocation in mouse skeletal muscle in vivo, although its placement and function(s) in the canonical insulin signaling cascade in skeletal muscle cells, remain undetermined. Therefore, the objective of my project is to determine the molecular mechanism(s) underlying the requirement for PAK1 in the process of insulin-stimulated GLUT4 vesicle translocation and subsequent glucose uptake by skeletal muscle cells. Toward this, my studies demonstrate that the pharmacological inhibition of PAK1 activation blunts insulin-stimulated GLUT4 translocation and subsequent glucose uptake into L6-GLUT4myc skeletal myotubes. Inhibition of PAK1 activation also ablates insulin-stimulated F-actin cytoskeletal remodeling, a process known to be required for mobilizing GLUT4 vesicles to the plasma membrane. Consistent with this mechanism, PAK1 activation was also required for the activation of cofilin, another protein implicated in F-actin remodeling. Interestingly, my studies reveal a novel molecular mechanism involving PAK1 signaling to p41-ARC, a regulatory subunit of the cytoskeletal Arp2/3 complex, and its interactions with another cytoskeletal factor, N-WASP, to elicit the insulin-stimulated F-actin remodeling in skeletal muscle cells. Pharmacological inactivation of N-WASP fully abrogated insulin-stimulated GLUT4 vesicle translocation to the cell surface, coordinate with blunted F-actin remodeling. Furthermore, my studies revealed new insulin-induced interactions amongst N WASP, actin, p41-ARC and PAK1; inactivation of PAK1 signaling blocked these dynamic interactions. Taken together, the above studies demonstrate the significance of PAK1 and its downstream signaling to F-actin remodeling in insulin-stimulated GLUT4 vesicle translocation and glucose uptake, revealing new signaling elements that may prove to be promising targets for future therapeutic design.