GLUT4 Exocytosis Research Articles

Dynamin-2 mutations linked to neonatal-onset centronuclear myopathy impair exocytosis and endocytosis in adrenal chromaffin cells.

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.

Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from α-SNAP/NSF-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

GLUT4 dispersal at the plasma membrane of adipocytes: a super-resolved journey.

In adipose tissue, insulin stimulates glucose uptake by mediating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In 2010, insulin was revealed to also have a fundamental impact on the spatial distribution of GLUT4 within the plasma membrane, with the existence of two GLUT4 populations at the plasma membrane being defined: (1) as stationary clusters and (2) as diffusible monomers. In this model, in the absence of insulin, plasma membrane-fused GLUT4 are found to behave as clusters. These clusters are thought to arise from exocytic events that retain GLUT4 at their fusion sites; this has been proposed to function as an intermediate hub between GLUT4 exocytosis and re-internalisation. By contrast, insulin stimulation induces the dispersal of GLUT4 clusters into monomers and favours a distinct type of GLUT4-vesicle fusion event, known as fusion-with-release exocytosis. Here, we review how super-resolution microscopy approaches have allowed investigation of the characteristics of plasma membrane-fused GLUT4 and further discuss regulatory step(s) involved in the GLUT4 dispersal machinery, introducing the scaffold protein EFR3 which facilitates localisation of phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) to the cell surface. We consider how dispersal may be linked to the control of transporter activity, consider whether macro-organisation may be a widely used phenomenon to control proteins within the plasma membrane, and speculate on the origin of different forms of GLUT4-vesicle exocytosis.

1674-P: Increased Glucose Uptake in Brown Fat Promotes Energy Expenditure

Brown and beige fat dissipate energy as heat in response to β3-adrenergic receptor (β3AR) stimulation. Strategies to facilitate this response may be employed to promote weight loss and improve health in patients with metabolic disease. Previously, we discovered that GTPase RalA and its GAP complex RalGAP regulate exocytosis of glucose transporter GLUT4. We demonstrated in vivo, that adipose-tissue specific knockout of RalGAP subunit RalGAPB activates RalA and increases glucose uptake 7 times in brown fat (BAT), leading to improved glucose handling on high fat diet (HFD). Here we seek to test the effect of increased glucose uptake in BAT on energy expenditure (EE), using mice with BAT-specific knockout of RalGAPB (the KO mice). After 8 weeks on 45% HFD, the KO mice and control littermates (Ctr) were injected with β3AR agonist CL-316,243 (CL) or PBS for 8 days. Mice assigned to CL were placed in metabolic cages. Respiratory quotient was increased in the KO mice, indicating increased glucose oxidation (likely by BAT, due to increased glucose uptake). EE at room temperature was elevated in the KO mice. This was compensated by increased food uptake, explaining similar body weights between the KO and Ctr mice. CL injections, as expected, induced spikes in EE. Acute response to CL (arbitrarily defined as EE during 2 hours after CL injection) was consistent between daily CL injections in Ctr mice, and was increased twice in the KO mice compared to Ctr. In Ctr mice, 1st CL injection increased average day and night EE; this response gradually declined with consecutive CL injections. In the KO mice, 1st CL injection elicited similar to Ctr mice increase in average day and night EE; and the response to consecutive CL injections was preserved. As anticipated, CL reduced adiposity in Ctr mice, which was further augmented in the KO mice, consistent with finding on EE. We will further test the role of increased glucose uptake by BAT in EE of the KO mice, and will determine the mechanism of sustained CL responsiveness in the KO mice. Disclosure F.Rezayat: None. J.A.Resnick: None. I.M.Farah: None. Y.Skorobogatko: None. A.Saltiel: Board Member; Elgia Therapeutics. Funding National Institutes of Health (P30DK063491)

Assembly-promoting protein Munc18c stimulates SNARE-dependent membrane fusion through its SNARE-like peptide

Intracellular vesicle fusion requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cognate Sec1/Munc18 (SM) proteins. How SM proteins act in concert with trans-SNARE complexes to promote membrane fusion remains incompletely understood. Munc18c, a broadly distributed SM protein, selectively regulates multiple exocytotic pathways, including GLUT4 exocytosis. Here, using an in vitro reconstituted system, we discovered a SNARE-like peptide (SLP), conserved in Munc18-1 of synaptic exocytosis, is crucial to the stimulatory activity of Munc18c in vesicle fusion. The direct stimulation of the SNARE-mediated fusion reaction by SLP further supported the essential role of this fragment. Interestingly, we found SLP strongly accelerates the membrane fusion rate when anchored to the target membrane but not the vesicle membrane, suggesting it primarily interacts with t-SNAREs in cis to drive fusion. Furthermore, we determined the SLP fragment is competitive with the full-length Munc18c protein and specific to the cognate v-SNARE isoforms, supporting how it could resemble Munc18c’s activity in membrane fusion. Together, our findings demonstrate that Munc18c facilitates SNARE-dependent membrane fusion through SLP, revealing that the t-SNARE-SLP binding mode might be a conserved mechanism for the stimulatory function of SM proteins in vesicle fusion.

Abstract 11989: Cardiac Muscle-Specific Disruption of the Exocyst Trafficking Complex Results in Heart Failure

Increased myocardial glycolysis is protective against ischemic injury. Glucose uptake via glucose transporter GLUT4 is the rate-limiting step of glycolysis in cardiomyocytes. GLUT4 delivery to the cardiomyocyte membrane can be triggered by insulin or by increased ATP-demand (i.e. during ischemia) via the AMP-activated protein kinase (AMPK). Previous studies of adipocytes and our recent study in skeletal muscle demonstrated that the exocyst trafficking complex is critical for GLUT4 exocytosis in response to insulin. But it is not known if the insulin-insulin receptor-exocyst-GLUT4 signaling axis is conserved in the cardiomyocytes, or if the exocyst also regulates cardiac glucose uptake via AMPK-induced GLUT4 translocation. Investigating glucose transporter trafficking in cardiomyocytes will be key to better understanding the mechanism of cardiac glucose metabolism under normal and disease conditions. We hypothesized that the exocyst is necessary for myocardial glucose uptake and metabolism and its activity protects the heart from ischemic injury. We have generated tamoxifen-inducible cardiac muscle-specific knockout mice of the central exocyst subunit Exoc5 (Exoc5-CMKO) to evaluate the role of the exocyst in cardiac fuel metabolism in vivo . Unexpectedly we found that cardiac muscle-specific Exoc5 knockout shortened the lifespan of Exoc5-CMKO mice (median survival after tamoxifen treatment was 60.5 days). Echocardiography showed cardiac dysfunction in Exoc5-CMKO mice, with reduced ejection fraction (Ctrl: 0.77; CMKO: 0.43; p<0.0001) and fractional shortening (Ctrl: 45.57; CMKO:21.62; p<0.0001), and increased systolic and diastolic LV diameters (LVID s (mm): Ctrl: 1.81; CMKO: 3.32; LVID d (mm): Ctrl: 3.47; CMKO:4.2; p<0.0001) compared to controls. Histological analysis revealed increased fibrosis in Exoc5-CMKO hearts, while isolated knockout primary cardiomyocytes showed decreased contractility and altered calcium handling. Our findings suggest that the exocyst is necessary for proper cardiac muscle function. Future studies will investigate how impaired exocyst-mediated membrane trafficking leads to heart failure in our Exoc5-CMKO mice.

296-OR: Identification and Characterization of New Players in Insulin-Controlled GLUT4 Trafficking

The anabolic hormone insulin promotes glucose uptake by triggering the translocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. Upon the termination of insulin signaling, GLUT4 gradually returns to intracellular storage compartments through endocytosis. Defects in GLUT4 translocation are a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). Our team has performed genome-scale genetic screens to systematically dissect GLUT4 exocytosis and endocytosis. The screens recovered known GLUT4 trafficking regulators but most of the hits were not previously linked to the GLUT4 pathway. A regulator of GLUT4 endocytosis identified in the screens is AAGAB, a soluble factor of 34 kDa. AAGAB controls the stepwise assembly of AP2 adaptor, a central cargo adaptor in clathrin-mediated endocytosis. Unexpectedly, we found that AAGAB also binds to Munc18c and directly inhibits SNARE-Munc18-mediated GLUT4 vesicle fusion. Thus, AAGAB plays a dual role in GLUT4 trafficking. I will also discuss OSBPL8 and OSBPL10, two negative regulators of GLUT4 translocation identified in our screens. OSBPL8 and OSBPL10 are putative lipid transfer proteins operating between intracellular organelles. We propose that OSBPL8 and OSBPL10 regulate lipid dynamics required for insulin signaling, thereby influencing the activities of GLUT4 exocytic regulators. These genetic studies will likely facilitate the identification of new therapeutic targets for IR and T2D. Disclosure J. Shen: None. Funding American Diabetes Association (1-19-IBS-305); National Institutes of Health (DK095367, GM126960)

The exocyst complex regulates insulin-stimulated glucose uptake of skeletal muscle cells.

Skeletal muscle handles ~80-90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.

Intracellular Trafficking Network and Autophagy of PHBHHx Nanoparticles and their Implications for Drug Delivery

3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx), which is naturally generated by biodegradable polyhydroxyalkanoates synthesized by bacteria, is an attractive material for drug delivery due to its controllable physical properties, non-toxicity, environmental friendliness, degradable properties and good biocompatibility. However, the intracellular trafficking network pathways, especially the autophagy mechanism of PHBHHx nanoparticles (NPs), have rarely been investigated. In this paper, we successfully prepared the NPs used solvent displacement method and investigated the autophagy pathways and other intracellular trafficking mechanisms based on NPs with the assistance of Rab proteins. We found that NPs were internalized in cells mainly via clathrin endocytosis and caveolin endocytosis. Beside the classical pathways, we discovered two new pathways: the micropinocytosis early endosome (EEs)-micropinocytosis-lysosome pathway and the EEs-liposome-lysosome pathway. NPs were delivered to cells through endocytosis recycling vesicles and GLUT4 exocytosis vesicles. Similar to other nanoparticles, NPs also induced intracellular autophagy and were then degraded via endolysosomal pathways. 3-MA and CQ were used as autophagy inhibitors to avoid the degradation of NPs through lysosomes by blocking endolysosomal pathways. Tumor volumes and weights were significantly decreased when autophagy inhibitors and chemical drugs packaged in NPs were cooperatively used.

191-OR: New Regulators of Insulin-Stimulated GLUT4 Exocytosis

Insulin promotes glucose uptake by triggering the translocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. Defects in GLUT4 exocytosis are a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). To develop effective and safe treatments for IR and T2D, it is crucial to gain a comprehensive understanding of insulin-stimulated GLUT4 exocytosis at the molecular level. Recently, our group developed new genetic screen platforms to systematically dissect insulin-stimulated GLUT4 exocytosis, taking advantage of the revolutionary CRISPR-Cas9 genome editing system. Our genome-wide CRISPR screens recovered known GLUT4 exocytic regulators but most of the hits were not previously linked to the GLUT4 exocytic pathway. Here, I will focus on AAGAB, a negative regulator of GLUT4 translocation identified in the screens. We discovered that AAGAB plays a dual role in GLUT4 trafficking: 1) it promotes the endocytosis of GLUT4, and 2) it negatively regulates the SNARE-Munc18-mediated GLUT4 vesicle fusion. In addition, I will discuss OSBPL8 and OSBPL10, another two negative regulators identified in our screens. OSBPL8 and OSBPL10 are putative lipid transfer proteins operating between intracellular organelles. We propose that OSBPL8 and OSBPL10 regulate lipid dynamics required for insulin signaling, thereby influencing the activities of GLUT4 exocytic regulators. These genetic studies will facilitate the identification of new therapeutic targets for IR and T2D. Disclosure J. Shen: None. Funding National Institutes of Health

RalA controls glucose homeostasis by regulating glucose uptake in brown fat

Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo.

Identifying New Regulators of Insulin‐Stimulated GLUT4 Exocytosis

GLUT4 is an insulin responsive glucose transporter responsible for the maintenance of blood glucose homeostasis. At basal conditions GLUT4 is sequestered inside the cell in specialized storage vesicles. After a meal, insulin signaling causes translocation of GLUT4 from these storage vesicles to the plasma membrane where it aids in the removal of glucose from the blood. Misregulation of this system is a major contributor to the global rise in rates of insulin resistance and metabolic disease. While the physiological importance of this pathway is obvious, the underlying molecular mechanisms remain poorly understood. This difficulty in elucidating the pathway is due to several factors, such as the complicated nature of vesicle transport pathways and technical barriers that have existed with using mammalian cells for genetic screens. The advent of CRISPR‐Cas9 genome editing has allowed us to overcome this second obstacle. Many labs have shown its utility in dissecting pathways for cell viability and growth; our lab has adapted it for vesicle trafficking studies. Using the CRISPR‐Cas9 system in conjunction with our trafficking assay, we performed an unbiased genome wide knockout screen to search for previously unknown regulators of this complex and important metabolic pathway. Specifically, the screen was designed to find regulators whose knockout causes a decrease in GLUT4 translocation after insulin stimulation. The screen identified several known regulators of GLUT4 exocytosis, such as RABIF and Rab10, suggesting that the screen was successful. Many of the hits, however, are not known to be involved in the GLUT4 exocytic pathway, such as TRAF3 and ANTRXL.Support or Funding InformationR01 DK095367This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Absence of Carbohydrate Response Element Binding Protein in Adipocytes Causes Systemic Insulin Resistance and Impairs Glucose Transport

Lower adipose-ChREBP and de novo lipogenesis (DNL) are associated with insulin resistance in humans. Here, we generated adipose-specific ChREBP knockout (AdChREBP KO) mice with negligible sucrose-induced DNL in adipose tissue (AT). Chow-fed AdChREBP KO mice are insulin resistant with impaired insulin action in the liver, muscle, andAT and increased AT inflammation. HFD-fed AdChREBP KO mice are also more insulin resistantthan controls. Surprisingly, adipocytes lacking ChREBP display a cell-autonomous reduction in insulin-stimulated glucose transport that is mediated by impaired Glut4 translocation and exocytosis, not lower Glut4 levels. AdChREBP KO mice have lower levels of palmitic acid esters of hydroxy stearic acids (PAHSAs) in serum, and AT. 9-PAHSA supplementation completely rescues their insulin resistance and AT inflammation. 9-PAHSA also normalizes impaired glucose transport and Glut4 exocytosis in ChREBP KO adipocytes. Thus, lossof adipose-ChREBP is sufficient to cause insulin resistance, potentially by regulating AT glucose transport and flux through specific lipogenic pathways.

RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis

Rab GTPases are switched from their GDP-bound inactive conformation to a GTP-bound active state by guanine nucleotide exchange factors (GEFs). The first putative GEFs isolated for Rabs are RABIF (Rab-interacting factor)/MSS4 (mammalian suppressor of Sec4) and its yeast homolog DSS4 (dominant suppressor of Sec4). However, the biological function and molecular mechanism of these molecules remained unclear. In a genome-wide CRISPR genetic screen, we isolated RABIF as a positive regulator of exocytosis. Knockout of RABIF severely impaired insulin-stimulated GLUT4 exocytosis in adipocytes. Unexpectedly, we discovered that RABIF does not function as a GEF, as previously assumed. Instead, RABIF promotes the stability of Rab10, a key Rab in GLUT4 exocytosis. In the absence of RABIF, Rab10 can be efficiently synthesized but is rapidly degraded by the proteasome, leading to exocytosis defects. Strikingly, restoration of Rab10 expression rescues exocytosis defects, bypassing the requirement for RABIF. These findings reveal a crucial role of RABIF in vesicle transport and establish RABIF as a Rab-stabilizing holdase chaperone, a previously unrecognized mode of Rab regulation independent of its GDP-releasing activity. Besides Rab10, RABIF also regulates the stability of two other Rab GTPases, Rab8 and Rab13, suggesting that the requirement of holdase chaperones is likely a general feature of Rab GTPases.

Investigation and intervention of autophagy to guide cancer treatment with nanogels.

Cancer cells use autophagy to resist poor survival environmental conditions such as low PH, poor nutrients as well as chemical therapy. Nanogels have been used as efficient chemical drug carriers for cancer treatment. However, the effect of nanogels on autophagy is still unknown. Here, we used Rab proteins as the marker of multiple trafficking vesicles in endocytosis and LC3 as the marker of autophagy to investigate the intracellular trafficking network of Rhodamine B (Rho)-labeled nanogels. The nanogels were internalized by the cells through multiple protein dependent endocytosis and micropinocytosis. After inception by the cells, the nanogels were transported into multiple Rab positive vesicles including early endosomes (EEs), late endosomes (LEs), recycling endosomes (REs) and lipid droplets. Finally, these Rab positive vesicles were transported to lysosome. In addition, GLUT4 exocytosis vesicles could transport the nanogels out of the cells. Moreover, nanogels could induce autophagy and be sequestered in autophagosomes. The crosstalk between autophagosomes and Rab positive vesicles were investigated, we found that autophagosomes may receive nanogels through multiple Rab positive vesicles. Co-delivery of autophagy inhibitors such as chloroquine (CQ) and the chemotherapeutic drug doxorubicin (DOX) by nanogels blocked the autophagy induced by DOX greatly decreasing both of the volume and weight of the tumors in mice tumor models. Investigation and intervention of the autophagy pathway could provide a new method to improve the therapeutic effect of anticancer nanogels.

Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.

Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy.

The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine.

Syntaxin 4 Overexpression Ameliorates Effects of Aging and High-Fat Diet on Glucose Control and Extends Lifespan

Indirect evidence suggests that improved insulin sensitivity may contribute to improved lifespan of mice in which aging has been slowed by mutations, drugs, or dietary means, even in stocks of mice that do not show signs of late-life diabetes. Peripheral responses to insulin can be augmented by overexpression of Syntaxin 4 (Syn4), a plasma-membrane-localized SNARE protein. We show here that Syn4 transgenic (Tg) mice with high level expression of Syn4 had a significant extension of lifespan (33% increase in median) and showed increased peripheral insulin sensitivity, even at ages where controls exhibited age-related insulin resistance. Moreover, skeletal muscle GLUT4 and islet insulin granule exocytosis processes were fully protected in Syn4 Tg mice challenged with a high-fat diet. Hence, high-level expressing Syn4 Tg mice may exert better glycemic control, which slows multiple aspects of aging and extends lifespan, even in non-diabetic mice.

Knockdown of Exocyst Sec10 in Mouse Skeletal Muscle Cells and Analysis of Insulin‐Stimulated GLUT4 Exocytosis

Type II diabetes is a metabolic disorder characterized by insulin resistance and high glucose in the blood. The GLUT4 receptor is the major glucose transporter that allows insulin‐stimulated glucose uptake into metabolic tissues such as adipose and skeletal muscle. In cultured adipocyte 3T3‐L1 cells, the eight‐protein exocyst complex has been shown to exocytose GLUT4 vesicles to the plasma membrane when stimulated by insulin. Our lab has extensively studied the activity of the exocyst trafficking complex in renal epithelial cells, and we have successfully generated a conditional knockout mouse for the exocyst subunit Sec10. Previous studies have been limited to cultured adipocytes, but skeletal muscles are responsible for taking in a higher percentage of glucose, and it has been shown that exercise helps maintain glucose levels to prevent the onset of type II diabetes.To evaluate whether the exocyst also plays a role in GLUT4 regulation in skeletal muscle cells, we used shRNA to generate stable Sec10 knockdowns in C2C12 mouse skeletal muscle cells. After confirming the knockdown, we measured glucose uptake using two different methods. First, we measured the amount of glucose in the media of Sec10 knockdown and control cells over the course of 2 weeks. Second, we compared insulin‐stimulated glucose uptake using a fluorescent glucose analog, 2‐NBDG. Based on preliminary findings, knockdown of Sec10 in mouse skeletal muscle cells results in decreased glucose uptake. Ongoing work with skeletal muscle‐specific Sec10 knockout with our new mouse model will help with better characterization of the role of the exocyst in insulin signaling and GLUT4 exocytosis that may reveal new therapeutic targets for human type II diabetes and pre‐diabetic insulin resistance.

Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling.

Akt2 and its downstream effectors mediate insulin-stimulated GLUT4-storage vesicle (GSV) translocation and fusion with the plasma membrane (PM). Using mass spectrometry, we identify actin-capping protein Tropomodulin 3 (Tmod3) as an Akt2-interacting partner in 3T3-L1 adipocytes. We demonstrate that Tmod3 is phosphorylated at Ser71 on insulin-stimulated Akt2 activation, and Ser71 phosphorylation is required for insulin-stimulated GLUT4 PM insertion and glucose uptake. Phosphorylated Tmod3 regulates insulin-induced actin remodelling, an essential step for GSV fusion with the PM. Furthermore, the interaction of Tmod3 with its cognate tropomyosin partner, Tm5NM1 is necessary for GSV exocytosis and glucose uptake. Together these results establish Tmod3 as a novel Akt2 effector that mediates insulin-induced cortical actin remodelling and subsequent GLUT4 membrane insertion. Our findings suggest that defects in cytoskeletal remodelling may contribute to impaired GLUT4 exocytosis and glucose uptake.