Browsing by Author "Yoder, Stephanie M."

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    A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells
    (Elsevier, 2013) Kalwat, Michael A.; Yoder, Stephanie M.; Wang, Zhanxiang; Thurmond, Debbie C.; Biochemistry and Molecular Biology, School of Medicine
    Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ∼80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release.
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    Changes in Skeletal Muscle PAK1 Levels Regulate Tissue Crosstalk to Impact Whole Body Glucose Homeostasis
    (Frontiers, 2022-02-10) Merz, Karla E.; Tunduguru, Ragadeepthi; Ahn, Miwon; Salunkhe, Vishal A.; Veluthakal, Rajakrishnan; Hwang, Jinhee; Bhattacharya, Supriyo; McCown, Erika M.; Garcia, Pablo A.; Zhou, Chunxue; Oh, Eunjin; Yoder, Stephanie M.; Elmendorf, Jeffrey S.; Thurmond, Debbie C.; Anatomy, Cell Biology and Physiology, School of Medicine
    Skeletal muscle accounts for ~80% of insulin-stimulated glucose uptake. The Group I p21–activated kinase 1 (PAK1) is required for the non-canonical insulin-stimulated GLUT4 vesicle translocation in skeletal muscle cells. We found that the abundances of PAK1 protein and its downstream effector in muscle, ARPC1B, are significantly reduced in the skeletal muscle of humans with type 2 diabetes, compared to the non-diabetic controls, making skeletal muscle PAK1 a candidate regulator of glucose homeostasis. Although whole-body PAK1 knockout mice exhibit glucose intolerance and are insulin resistant, the contribution of skeletal muscle PAK1 in particular was unknown. As such, we developed inducible skeletal muscle-specific PAK1 knockout (skmPAK1-iKO) and overexpression (skmPAK1-iOE) mouse models to evaluate the role of PAK1 in skeletal muscle insulin sensitivity and glucose homeostasis. Using intraperitoneal glucose tolerance and insulin tolerance testing, we found that skeletal muscle PAK1 is required for maintaining whole body glucose homeostasis. Moreover, PAK1 enrichment in GLUT4-myc-L6 myoblasts preserves normal insulin-stimulated GLUT4 translocation under insulin resistance conditions. Unexpectedly, skmPAK1-iKO also showed aberrant plasma insulin levels following a glucose challenge. By applying conditioned media from PAK1-enriched myotubes or myoblasts to β-cells in culture, we established that a muscle-derived circulating factor(s) could enhance β-cell function. Taken together, these data suggest that PAK1 levels in the skeletal muscle can regulate not only skeletal muscle insulin sensitivity, but can also engage in tissue crosstalk with pancreatic β-cells, unveiling a new molecular mechanism by which PAK1 regulates whole-body glucose homeostasis.
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    Munc18c: a controversial regulator of peripheral insulin action
    (Elsevier, 2014-11) Ramalingam, Latha; Yoder, Stephanie M.; Oh, Eunjin; Thurmond, Debbie C.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Insulin resistance, a hallmark of impaired glucose tolerance and type 2 diabetes (T2D), arises from dysfunction of insulin action and subsequent glucose uptake by peripheral tissues, predominantly skeletal muscle and fat. Exocytosis of glucose transporter (GLUT4)-containing vesicles facilitated by soluble NSF (N-ethylmaleimide-sensitive factor) attachment receptor (SNARE) protein isoforms, and Munc18c (mammalian homolog of Unc-18c) mediates this glucose uptake. Emerging evidences, including recent human clinical studies, point to pivotal roles for Munc18c in peripheral insulin action in adipose and skeletal muscle. Intriguing new advances are also initiating debates regarding the molecular mechanism(s) controlling Munc18c action. The objective of this review is therefore to present a balanced perspective of new continuities and controversies surrounding the regulation and requirement for Munc18c in the regulation of peripheral insulin action.
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    The p21-activated kinase (PAK1) is involved in diet-induced beta cell mass expansion and survival in mice and human islets
    (Springer, 2016-10) Ahn, Miwon; Yoder, Stephanie M.; Wang, Zhanxiang; Oh, Eunjin; Ramalingam, Latha; Tunduguru, Ragadeepthi; Thurmond, Debbie C.; Department of Pediatrics, IU School of Medicine
    AIMS/HYPOTHESIS: Human islets from type 2 diabetic donors are reportedly 80% deficient in the p21 (Cdc42/Rac)-activated kinase, PAK1. PAK1 is implicated in beta cell function and maintenance of beta cell mass. We questioned the mechanism(s) by which PAK1 deficiency potentially contributes to increased susceptibility to type 2 diabetes. METHODS: Non-diabetic human islets and INS 832/13 beta cells cultured under diabetogenic conditions (i.e. with specific cytokines or under glucolipotoxic [GLT] conditions) were evaluated for changes to PAK1 signalling. Combined effects of PAK1 deficiency with GLT stress were assessed using classic knockout (Pak1 (-/-) ) mice fed a 45% energy from fat/palmitate-based, 'western' diet (WD). INS 832/13 cells overexpressing or depleted of PAK1 were also assessed for apoptosis and signalling changes. RESULTS: Exposure of non-diabetic human islets to diabetic stressors attenuated PAK1 protein levels, concurrent with increased caspase 3 cleavage. WD-fed Pak1 knockout mice exhibited fasting hyperglycaemia and severe glucose intolerance. These mice also failed to mount an insulin secretory response following acute glucose challenge, coinciding with a 43% loss of beta cell mass when compared with WD-fed wild-type mice. Pak1 knockout mice had fewer total beta cells per islet, coincident with decreased beta cell proliferation. In INS 832/13 beta cells, PAK1 deficiency combined with GLT exposure heightened beta cell death relative to either condition alone; PAK1 deficiency resulted in decreased extracellular signal-related kinase (ERK) and B cell lymphoma 2 (Bcl2) phosphorylation levels. Conversely, PAK1 overexpression prevented GLT-induced cell death. CONCLUSIONS/INTERPRETATION: These findings suggest that PAK1 deficiency may underlie an increased diabetic susceptibility. Discovery of ways to remediate glycaemic dysregulation via altering PAK1 or its downstream effectors offers promising opportunities for disease intervention.
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    YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet β cell
    (ASBMB, 2014-04-18) Yoder, Stephanie M.; Dineen, Stacey L.; Wang, Zhanxiang; Thurmond, Debbie C.; Department of Pediatrics, IU School of Medicine
    Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.