Phosphorylation Of TBC1D1 Research Articles

KLF3 impacts insulin sensitivity and glucose uptake in skeletal muscle.

Skeletal muscle is one of the predominant sites involved in glucose disposal, accounting for approximately 80% of postprandial glucose uptake, and plays a critical role in maintaining glycemic homeostasis. Dysregulation of energy metabolism in skeletal muscle is involved in developing insulin resistance and type 2 diabetes (T2D). Transcriptomic responses of skeletal muscle to exercise found that the expression of Klf3 was increased in T2D Goto-Kakizaki (GK) rats and decreased after exercise with improved hyperglycemia and insulin resistance, implying that Klf3 might be associated with insulin sensitivity and glucose metabolism. We also found that knockdown of Klf3 promoted basal and insulin-stimulated glucose uptake in L6 myotubes, while overexpression of Klf3 resulted in the opposite. Through pairwise comparisons of L6 myotubes transcriptome, we identified 2256 and 1988 differentially expressed genes in Klf3 knockdown and overexpression groups, respectively. In insulin signaling, the expression of Slc2a4, Akt2, Insr and Sorbs1 was significantly increased by Klf3 knockdown and decreased with klf3 overexpression; Ptprf and Fasn were markedly downregulated in klf3 reduced group and upregulated in klf3 overexpressed group. Moreover, downregulation of Klf3 promoted the expression of GLUT4 and AKT proteins, as well as the translocation of GLUT4 to the cell membrane in the basal situation, and enhanced insulin sensitivity, characterized by increased insulin-stimulated GLUT4 translocation and AKT, TBC1D1 and TBC1D4 phosphorylation, while overexpression of Klf3 showed contrary results. These results suggest that Klf3 affects glucose uptake and insulin sensitivity via insulin signal transduction and intracellular metabolism, offering a novel potential treatment strategy for T2D.

Picalm, a novel regulator of GLUT4-trafficking in adipose tissue

ObjectivePicalm (phosphatidylinositol-binding clathrin assembly protein), a ubiquitously expressed clathrin-adapter protein, is a well-known susceptibility gene for Alzheimer's disease, but its role in white adipose tissue (WAT) function has not yet been studied. Transcriptome analysis revealed differential expression of Picalm in WAT of diabetes-prone and diabetes-resistant mice, hence we aimed to investigate the potential link between Picalm expression and glucose homeostasis, obesity-related metabolic phenotypes, and its specific role in insulin-regulated GLUT4 trafficking in adipocytes. MethodsPicalm expression and epigenetic regulation by microRNAs (miRNAs) and DNA methylation were analyzed in WAT of diabetes-resistant (DR) and diabetes-prone (DP) female New Zealand Obese (NZO) mice and in male NZO after time-restricted feeding (TRF) and alternate-day fasting (ADF). PICALM expression in human WAT was evaluated in a cross-sectional cohort and assessed before and after weight loss induced by bariatric surgery. siRNA-mediated knockdown of Picalm in 3T3-L1-cells was performed to elucidate functional outcomes on GLUT4-translocation as well as insulin signaling and adipogenesis. ResultsPicalm expression in WAT was significantly lower in DR compared to DP female mice, as well as in insulin-sensitive vs. resistant NZO males, and was also reduced in NZO males following TRF and ADF. Four miRNAs (let-7c, miR-30c, miR-335, miR-344) were identified as potential mediators of diabetes susceptibility-related differences in Picalm expression, while 11 miRNAs (including miR-23a, miR-29b, and miR-101a) were implicated in TRF and ADF effects. Human PICALM expression in adipose tissue was lower in individuals without obesity vs. with obesity and associated with weight-loss outcomes post-bariatric surgery. siRNA-mediated knockdown of Picalm in mature 3T3-L1-adipocytes resulted in amplified insulin-stimulated translocation of the endogenous glucose transporter GLUT4 to the plasma membrane and increased phosphorylation of Akt and Tbc1d4. Moreover, depleting Picalm before and during 3T3-L1 differentiation significantly suppressed adipogenesis, suggesting that Picalm may have distinct roles in the biology of pre- and mature adipocytes. ConclusionsPicalm is a novel regulator of GLUT4-translocation in WAT, with its expression modulated by both genetic predisposition to diabetes and dietary interventions. These findings suggest a potential role for Picalm in improving glucose homeostasis and highlight its relevance as a therapeutic target for metabolic disorders.

Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

TBC1D4-S711 Controls Skeletal Muscle Insulin Sensitization After Exercise and Contraction.

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.

Characterizing Adrenergic Regulation of Glucose Transporter 4-Mediated Glucose Uptake and Metabolism in the Heart

Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac β2 adrenoreceptor (β2AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac β2AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of β2AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.

MICAL1 facilitates pancreatic cancer proliferation, migration, and invasion by activating WNT/β-catenin pathway

BackgroundMICAL1 is involved in the malignant processes of several types of cancer; however, the role of MICAL1 in pancreatic cancer (PC) has not been well-characterized. This study aimed to investigate the expression and function of MICAL1 in PC.MethodsRT-qPCR and immunohistochemistry were used to detect MICAL1 expression in PC and adjacent nontumor tissues. Cell Counting Kit-8, EdU, clone formation, wound healing, and Transwell assays as well as animal models were used to investigate the effects of overexpression or inhibition of MICAL1 expression on the proliferation, invasion, and metastasis of PC cells. RNA-seq was used to explore the main pathway underlying the functions of MICAL1. Proteomics, mass spectrometry, and co-immunoprecipitation assays were used to investigate the interaction of proteins with MICAL1. Rescue experiments were conducted to validate these findings.ResultsBoth MICAL1 mRNA and protein levels were upregulated in PC tissues compared with matched adjacent nontumor tissues. The expression level of MICAL1 was associated with the proliferative and metastatic status of PC. Repression of MICAL1 significantly inhibited PC cell growth, migration, and invasion in vitro and in vivo. RNA sequencing analysis indicated that MICAL1 was closely correlated with the WNT pathway. Overexpression of MICAL1 (1) promoted the phosphorylation of TBC1D1 at the Ser660 site, (2) facilitated the distribution of FZD7 on the cytomembrane, (3) inhibited the degradation of FZD7 in the lysosome, and (4) activated the WNT pathway.ConclusionsMICAL1 was upregulated in PC and involved in stimulating the progression of PC cells by activating the WNT/β-catenin signaling pathway. Therefore, MICAL1 is a potential therapeutic target for PC.

Cancer causes dysfunctional insulin signaling and glucose transport in a muscle-type-specific manner.

Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation are not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5nM). Glucose transport was measured using 2-[3 H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389, and ribosomal-S6ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent on muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, the skeletal muscle could be a target for managing metabolic dysfunction in cancer.

Differential DNA Methylation and Expression of miRNAs in Adipose Tissue From Twin Pairs Discordant for Type 2 Diabetes.

The prevalence of type 2 diabetes (T2D) is increasing worldwide, but current treatments have limitations. miRNAs may play a key role in the development of T2D and can be targets for novel therapies. Here, we examined whether T2D is associated with altered expression and DNA methylation of miRNAs using adipose tissue from 14 monozygotic twin pairs discordant for T2D. Four members each of the miR-30 and let-7-families were downregulated in adipose tissue of subjects with T2D versus control subjects, which was confirmed in an independent T2D case-control cohort. Further, DNA methylation of five CpG sites annotated to gene promoters of differentially expressed miRNAs, including miR-30a and let-7a-3, was increased in T2D versus control subjects. Luciferase experiments showed that increased DNA methylation of the miR-30a promoter reduced its transcription in vitro. Silencing of miR-30 in adipocytes resulted in reduced glucose uptake and TBC1D4 phosphorylation; downregulation of genes involved in demethylation and carbohydrate/lipid/amino acid metabolism; and upregulation of immune system genes. In conclusion, T2D is associated with differential DNA methylation and expression of miRNAs in adipose tissue. Downregulation of the miR-30 family may lead to reduced glucose uptake and altered expression of key genes associated with T2D.

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis–Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM ∼6 μM) and Thr649 (KM ∼25 μM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 μM), Ser711 (KM ∼79 μM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.

Benzyl Isothiocyanate Ameliorates High-Fat Diet-Induced Hyperglycemia by Enhancing Nrf2-Dependent Antioxidant Defense-Mediated IRS-1/AKT/TBC1D1 Signaling and GLUT4 Expression in Skeletal Muscle.

Obesity caused lipotoxicity, which results in insulin resistance. We studied whether benzyl isothiocyanate (BITC) improved insulin resistance in muscle. BITC was studied in vivo in mice fed a high-fat diet (HFD) and in vitro in C2C12 myotubes treated with palmitic acid (PA). In C2C12 cells, BITC mitigated PA inhibition of glucose uptake and phosphorylation of IRS-1, AKT, and TBC1D1 in response to insulin. BITC upregulated the expression of HO-1, GSTP, and GCLM mRNA and protein as well as GSH contents, which suppressed oxidative damage. Knockdown of Nrf2 abrogated BITC enhancement of antioxidant defense and subsequently reversed BITC protection against PA-induced insulin resistance. Moreover, BITC upregulated the expression of GLUT4, PPARγ, and C/EBPα. In HFD-fed mice, plasma total cholesterol, nonesterified fatty acid, and glucose levels and HOMA-IR were dose-dependently decreased with 0.05 or 0.1% BITC administration. In gastrocnemius muscle, compared with the HFD group, BITC increased the phosphorylation of AKT and TBC1D1, GSH contents, and the expression of antioxidant enzymes as well as GLUT4. These results indicate that BITC ameliorates obesity-induced hyperglycemia by enhancing insulin sensitivity in muscle. This is partly attributed to its inhibition of lipotoxicity-induced oxidative insult and upregulation of GLUT4 expression.

Effect of post-exercise lactate administration on glycogen repletion and signaling activation in different types of mouse skeletal muscle.

Lactate is not merely a metabolic intermediate that serves as an oxidizable and glyconeogenic substrate, but it is also a potential signaling molecule. The objectives of this study were to investigate whether lactate administration enhances post-exercise glycogen repletion in association with cellular signaling activation in different types of skeletal muscle. Eight-week-old male ICR mice performed treadmill running (20 m/min for 60 min) following overnight fasting (16 h). Immediately after the exercise, animals received an intraperitoneal injection of phosphate-buffered saline or sodium lactate (equivalent to 1 g/kg body weight), followed by oral ingestion of water or glucose (2 g/kg body weight). At 60 min of recovery, glucose ingestion enhanced glycogen content in the soleus, plantaris, and gastrocnemius muscles. In addition, lactate injection additively increased glycogen content in the plantaris and gastrocnemius muscles, but not in the soleus muscle. Nevertheless, lactate administration did not significantly alter protein levels related to glucose uptake and oxidation in the plantaris muscle, but enhanced phosphorylation of TBC1D1, a distal protein regulating GLUT4 translocation, was observed in the soleus muscle. Muscle FBP2 protein content was significantly higher in the plantaris and gastrocnemius muscles than in the soleus muscle, whereas MCT1 protein content was significantly higher in the soleus muscle than in the plantaris and gastrocnemius muscles. The current findings suggest that an elevated blood lactate concentration and post-exercise glucose ingestion additively enhance glycogen recovery in glycolytic phenotype muscles. This appears to be associated with glyconeogenic protein content, but not with enhanced glucose uptake, attenuated glucose oxidation, or lactate transport protein.

Sex and fiber type independently influence AMPK, TBC1D1, and TBC1D4 at rest and during recovery from high-intensity exercise in humans.

Women and men present different metabolic responses to exercise, yet whether this phenomenon results from differences in fiber type (FT) composition or other sex-specific factors remains unclear. Therefore, our aim was to examine the effects of sex and FT independently on AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), Tre-2/BUB2/CDC1 domain family (TBC1D)1, and TBC1D4 in response to acute exercise. Segregated pools of myosin heavy chain (MHC) I and MHC IIa fibers were prepared from vastus lateralis biopsies of young trained men and women at rest and during recovery (0 min, 45 min, 90 min, or 180 min) from high-intensity interval exercise (6 × 1.5 min at 95% maximum oxygen uptake). In resting MHC I vs. IIa fibers, AMPKα2, AMPKγ3, and TBC1D1 were higher and TBC1D4 expression was lower in both sexes, along with higher phospho (p)-TBC1D1Ser660 and lower p-TBC1D4Thr642. Women expressed higher ACC than men in MHC IIa fibers and higher AMPKβ1, AMPKβ2, TBC1D1, and TBC1D4 in both FTs. Immediately after exercise, p-AMPKαThr172 increased only in MHC IIa fibers, whereas p-ACCSer221 increased in both FTs, with no change in p-TBC1D1Ser660 or p-TBC1D4Thr642. During recovery, delayed responses were observed for p-AMPKαThr172 in MHC I (45 min), p-TBC1D4Thr642 in both FTs (45 min), and p-TBC1D1Ser660 (180 min). FT-specific phosphorylation responses to exercise were similar between men and women. Data indicate that sex and FT independently influence expression of AMPK and its substrates. Thus failing to account for sex or FT may reduce accuracy and precision of metabolic protein measurements and conceal key findings.NEW & NOTEWORTHY This investigation is the first to compare muscle fiber type (FT)-specific analysis of proteins between the sexes, providing comprehensive data on AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), Tre-2/BUB2/CDC1 domain family (TBC1D)1, and TBC1D4 before and in the hours following high-intensity interval exercise (HIIT). Expression and phosphorylation of specific AMPK isoforms, ACC, TBC1D1, and TBC1D4 were shown to be FT dependent, sex dependent, or both, and TBC1D1 showed an unexpected delay in FT-dependent phosphorylation in the time period following HIIT.

Lipid oversupply induces CD36 sarcolemmal translocation via dual modulation of PKCζ and TBC1D1: an early event prior to insulin resistance.

Lipid oversupply may induce CD36 sarcolemmal translocation to facilitate fatty acid transport, which in turn causes dyslipidemia and type 2 diabetes. However, the underlying mechanisms of CD36 redistribution are still yet to be unraveled.Methods: High fat diet fed mice and palmitate/oleic acid-treated L6 cells were used to investigate the initial events of subcellular CD36 recycling prior to insulin resistance. The regulation of CD36 sarcolemmal translocation by lipid oversupply was assessed by insulin tolerance test (ITT), oral glucose tolerance test (OGTT), glucose/fatty acid uptake assay, surface CD36 and GLUT4 detection, and ELISA assays. To elucidate the underlying mechanisms, specific gene knockout, gene overexpression and/or gene inhibition were employed, followed by Western blot, co-immunoprecipitation, immunostaining, and kinase activity assay.Results: Upon lipid/fatty acid overload, PKCζ activity and TBC1D1 phosphorylation were enhanced along with increased sarcolemmal CD36. The inhibition of PKCζ or TBC1D1 was shown to block fatty acid-induced CD36 translocation and was synergistic in impairing CD36 redistribution. Mechanically, we revealed that AMPK was located upstream of PKCζ to control its activity whereas Rac1 facilitated PKCζ translocation to the dorsal surface of the cell to cause actin remodeling. Furthermore, AMPK phosphorylated TBC1D1 to release retained cytosolic CD36. The activated PKCζ and phosphorylated TBC1D1 resulted in a positive feedback regulation of CD36 sarcolemmal translocation.Conclusion: Collectively, our study demonstrated exclusively that lipid oversupply induced CD36 sarcolemmal translocation via dual modulation of PKCζ and TBC1D1, which was as an early event prior to insulin resistance. The acquired data may provide potential therapy targets to prevent lipid oversupply-induced insulin resistance.

TBC1D4 Is Necessary for Enhancing Muscle Insulin Sensitivity in Response to AICAR and Contraction.

Muscle insulin sensitivity for stimulating glucose uptake is enhanced in the period after a single bout of exercise. We recently demonstrated that AMPK is necessary for AICAR, contraction, and exercise to enhance muscle and whole-body insulin sensitivity in mice. Correlative observations from both human and rodent skeletal muscle suggest that regulation of the phosphorylation status of TBC1D4 may relay this insulin sensitization. However, the necessity of TBC1D4 for this phenomenon has not been proven. Thus, the purpose of this study was to determine whether TBC1D4 is necessary for enhancing muscle insulin sensitivity in response to AICAR and contraction. We found that immediately after contraction and AICAR stimulation, phosphorylation of AMPKα-Thr172 and downstream targets were increased similarly in glycolytic skeletal muscle from wild-type and TBC1D4-deficient mice. In contrast, 3 h after contraction or 6 h after AICAR stimulation, enhanced insulin-stimulated glucose uptake was evident in muscle from wild-type mice only. The enhanced insulin sensitivity in muscle from wild-type mice was associated with improved insulin-stimulated phosphorylation of TBC1D4 (Thr649 and Ser711) but not of TBC1D1. These results provide genetic evidence linking signaling through TBC1D4 to enhanced muscle insulin sensitivity after activation of the cellular energy sensor AMPK.

AMPK and TBC1D1 Regulate Muscle Glucose Uptake After, but Not During, Exercise and Contraction.

Exercise increases glucose uptake in skeletal muscle independently of insulin signaling. This makes exercise an effective stimulus to increase glucose uptake in insulin-resistant skeletal muscle. AMPK has been suggested to regulate muscle glucose uptake during exercise/contraction, but findings from studies of various AMPK transgenic animals have not reached consensus on this matter. Comparing methods used in these studies reveals a hitherto unappreciated difference between those studies reporting a role of AMPK and those that do not. This led us to test the hypothesis that AMPK and downstream target TBC1D1 are involved in regulating muscle glucose uptake in the immediate period after exercise/contraction but not during exercise/contraction. Here we demonstrate that glucose uptake during exercise/contraction was not compromised in AMPK-deficient skeletal muscle, whereas reversal of glucose uptake toward resting levels after exercise/contraction was markedly faster in AMPK-deficient muscle compared with wild-type muscle. Moreover, muscle glucose uptake after contraction was positively associated with phosphorylation of TBC1D1, and skeletal muscle from TBC1D1-deficient mice displayed impaired glucose uptake after contraction. These findings reconcile previous observed discrepancies and redefine the role of AMPK activation during exercise/contraction as being important for maintaining glucose permeability in skeletal muscle in the period after, but not during, exercise/contraction.

Four weeks of exercise early in life reprograms adult skeletal muscle insulin resistance caused by a paternal high-fat diet.

A paternal high-fat diet/obesity before mating can negatively influence the metabolism of offspring. Exercise only early in life has a remarkable effect with respect to reprogramming adult rat offspring exposed to detrimental insults before conception. Exercise only early in life normalized adult whole body and muscle insulin resistance as a result of having a high-fat fed/obese father. Unlike the effects on the muscle, early exercise did not normalize the reduced adult pancreatic beta cell mass as a result of having a high-fat fed/obese father. Early-life exercise training may be able to reprogram an individual whose father was obese, inducing long-lasting beneficial effects on health. A paternal high-fat diet (HFD) impairs female rat offspring glucose tolerance, pancreatic morphology and insulin secretion. We examined whether only 4weeks of exercise early in life could reprogram these negative effects. Male Sprague-Dawley rats consumed a HFD for 10weeks before mating with chow-fed dams. Female offspring remained sedentary or performed moderate intensity treadmill exercise (5daysweek-1 , 60minday-1 , 20mmin-1 ) from 5 to 9weeks of age. Paternal HFD impaired (P< 0.05) adult offspring whole body insulin sensitivity (i.p. insulin sensitivity test), as well as skeletal muscle ex vivo insulin sensitivity and TBC1D4 phosphorylation. It also lowered β-cell mass and reduced in vivo insulin secretion in response to an i.p. glucose tolerance test. Early-life exercise in offspring reprogrammed the negative effects of a paternal HFD on whole body insulin sensitivity, skeletal muscle ex vivo insulin-stimulated glucose uptake and TBC1D4 phosphorylation and also increased glucose transporter 4 protein. However, early exercise did not normalize the reduced pancreatic β-cell mass or insulin secretion. In conclusion, only 4weeks of exercise early in life in female rat offspring reprograms reductions in insulin sensitivity in adulthood caused by a paternal HFD without affecting pancreatic β-cell mass or insulin secretion.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

Chemical denervation using botulinum toxin increases Akt expression and reduces submaximal insulin-stimulated glucose transport in mouse muscle

Botulinum toxin A (botox) is a toxin used for spasticity treatment and cosmetic purposes. Botox blocks the excitation of skeletal muscle fibers by preventing the release of acetylcholine from motor nerves, a process termed chemical denervation. Surgical denervation is associated with increased expression of the canonical insulin-activated kinase Akt, lower expression of glucose handling proteins GLUT4 and hexokinase II (HKII) and insulin resistant glucose uptake, but it is not known if botox has a similar effect. To test this, we performed a time-course study using supra-maximal insulin-stimulation in mouse soleus ex vivo. No effect was observed in the glucose transport responsiveness at day 1, 7 and 21 after intramuscular botox injection, despite lower expression of GLUT4, HKII and expression and phosphorylation of TBC1D4. Akt protein expression and phosphorylation of the upstream kinase Akt were increased by botox treatment at day 21. In a follow-up study, botox decreased submaximal insulin-stimulated glucose transport. The marked alterations of insulin signaling, GLUT4 and HKII and submaximal insulin-stimulated glucose transport are a potential concern with botox treatment which merit further investigation in human muscle. Furthermore, the botox-induced chemical denervation model may be a less invasive alternative to surgical denervation.

GLUT12 promotes prostate cancer cell growth and is regulated by androgens and CaMKK2 signaling.

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5'-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.

PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca2+ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca2+ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis.