Effects of Exercise on AMPK Signaling and Downstream Components to PI3K in Rat with Type 2 Diabetes

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr³⁰⁸) and (Ser⁴⁷³) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.     

TRB3 Is Involved in Free Fatty Acid-Induced INS-1-Derived Cell Apoptosis via the Protein Kinase C δ Pathway

Chronic exposure to free fatty acids (FFAs) may induce β cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger β cell apoptosis is still unclear. Tribbles homolog 3 (TRB3) is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 β cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC) δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 β cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.     

Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ~50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.     

Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes

Insulin resistance is a primary characteristic of type 2 diabetes. Several lines of evidence suggest that accumulation of free fatty acids in skeletal muscle may at least in part contribute to insulin resistance and may be linked to mitochondrial dysfunction, leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits protein kinase B (Akt) activity in response to insulin. However, the role of Bax and Bcl-2 in regulating palmitate-induced apoptosis has not been well studied. Therefore, the purpose of this study was to determine whether palmitate-induced apoptosis in C(2)C(12) myotubes is dependent on Bax to Bcl-2 binding. An additional purpose of this study was to determine whether the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate-treated myoblasts. Apoptotic signaling proteins were examined in C(2)C(12) myotubes treated overnight with palmitate. Bax to Bcl-2 binding was determined through a coimmunoprecipitation assay that was performed in myotubes after 2 h of serum starvation, followed by 10 min of serum reintroduction. This experiment evaluated whether temporal Akt activity coincided with Bax to Bcl-2 binding. Last, the contribution of Bax to palmitate-induced apoptosis was determined by treatment with Bax siRNA. Palmitate treatment increased apoptosis in C(2)C(12) myotubes as shown by a twofold increase in DNA fragmentation, an approximately fivefold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. In addition, there was a fourfold reduction in Bax to Bcl-2 binding with palmitate treatment, which mirrored the reduction in Akt(Ser473) phosphorylation. Furthermore, treatment of the C(2)C(12) myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax-mediated apoptosis in C(2)C(12) myotubes and that this effect corresponds to reductions in Akt(Ser473) phosphorylation.     

Activation of Protein Kinase C α Is Necessary for Sorting the PDGF β-Receptor to Rab4a-dependent Recycling

Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) β-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) α as the critical signaling component that regulates the sorting of the PDGF β-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKCα, using myristoylated inhibitory peptides or by knockdown of PKCα with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF β-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF β-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF β-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF β-receptors on early endosomes is regulated by sequential activation of PKCα and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.     

Molecular basis of insulin-responsive GLUT4 trafficking systems revealed by single molecule imaging

Development of a 'static retention' property of GLUT4, the insulin-responsive glucose transporter, has emerged as being essential for achieving its maximal insulin-induced surface exposure. Herein, employing quantum-dot-based nanometrology of intracellular GLUT4 behavior, we reveal the molecular basis of its systematization endowed upon adipogenic differentiation of 3T3L1 cells. Specifically, (i) the endosomes-to-trans-Golgi network (TGN) retrieval system specialized for GLUT4 develops in response to sortilin expression, which requires an intricately balanced interplay among retromers, golgin-97 and syntaxin-6, the housekeeping vesicle trafficking machinery. (ii) The Golgin-97-localizing subdomain of the differentiated TGN apparently serves as an intermediate transit route by which GLUT4 can further proceed to the stationary GLUT4 storage compartment. (iii) AS160/Tbc1d4 then renders the 'static retention' property insulin responsive, i.e. insulin liberates GLUT4 from the static state only in the presence of functional AS160/Tbc1d4. (iv) Moreover, sortilin malfunction and the resulting GLUT4 sorting defects along with retarded TGN function might be etiologically related to insulin resistance. Together, these observations provide a conceptual framework for understanding maturation/retardation of the insulin-responsive GLUT4 trafficking system that relies on the specialized subdomain of differentiated TGN.     

Protein kinase Cdelta and calmodulin regulate epidermal growth factor receptor recycling from early endosomes through Arp2/3 complex and cortactin

The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR.     

Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts

We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε^−/−) mice to examine this mechanistically. PKCε^−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε^−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.     

Enhancement of TNF-α expression and inhibition of glucose uptake by nicotine in the presence of a free fatty acid in C2C12 skeletal myocytes

Smoking is a risk factor for insulin resistance and metabolic syndrome. However, mechanisms responsible for smoking-induced insulin resistance are unclear. We examined the combined effect of nicotine, a toxic substance in tobacco smoke, and palmitate in the serum physiological concentration range on tumor necrosis factor-α (TNF-α) expression and impairment of glucose uptake in C2C12 myotubes, since smokers do not have increased serum free fatty acid (FFA) concentrations with insulin resistance compared to nonsmokers. C2C12 myotubes were incubated for 24 h with nicotine (1 μmol/l) in the presence or absence of palmitate (200 μmol/l). RT-PCR and Western blotting showed increased TNF-α expression in C2C12 myotubes treated with nicotine in the presence of palmitate. Furthermore, stimulation with nicotine in the presence of palmitate enhanced the production of reactive oxygen species (ROS) and activated the protein kinase C-nuclear factor-κB (PKC-NF-κB) pathway, as detected by dihydroethidium staining and Western blotting, respectively. Consequently, the translocation of GLUT4 to the plasma membrane as well as insulin-stimulated Akt phosphorylation was impaired, and glucose uptake to the myocytes was blocked. In addition, the production of ROS was suppressed by 4-hydroxy-TEMPO, and inhibition of GLUT4 translocation to the plasma membrane was canceled. These results suggest that in C2C12 myotubes, nicotine in the presence of palmitate enhanced the production of ROS and the expression of TNF-α through the PKC-NF-κB pathway; suppressed GLUT4 translocation to the plasma membrane; and impaired glucose uptake to cells. This pathway represents a possible mechanism by which smoking induces insulin resistance in the body.     

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12 myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a >or=10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.     

Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate.

We have employed C2C12 myotubes to investigate lipid inhibition of insulin-stimulated signal transduction and glucose metabolism. Cells were preincubated for 18 h in the absence or presence of free fatty acids (FFAs) and stimulated with insulin, and the effects on glycogen synthesis and signaling intermediates were determined. While the unsaturated FFAs oleate and linoleate inhibited both basal and insulin-stimulated glycogen synthesis, the saturated FFA palmitate reduced only insulin-stimulated glycogen synthesis, and was found to inhibit insulin-stimulated phosphorylation of glycogen synthase kinase-3 and protein kinase B (PKB). However, no effect of palmitate was observed on tyrosine phosphorylation, p85 association, or phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates. In contrast, palmitate promoted phosphorylation of mitogen-activated protein MAP) kinases. Ceramide, a derivative of palmitate, has recently been associated with similar inhibition of PKB, and here, ceramide levels were found to be elevated 2-fold in palmitate-treated C2C12 cells. Incubation of C2C12 cells with ceramide closely reproduced the effects of palmitate, leading to inhibition of glycogen synthesis and PKB and to stimulation of MAP kinase. We conclude that palmitate-induced insulin resistance occurs by a mechanism distinct from that of unsaturated FFAs, and involves elevation of ceramide by de novo synthesis, leading to PKB inhibition without affecting IRS-1 function.     

TM-25659-Induced Activation of FGF21 Level Decreases Insulin Resistance and Inflammation in Skeletal Muscle via GCN2 Pathways

The TAZ activator 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma. TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet-induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the effects of TM-25659 on skeletal muscle functions in C2 myotubes and C57BL/6J mice. We studied the molecular mechanisms underlying the contribution of TM-25659 to palmitate (PA)-induced insulin resistance in C2 myotubes. TM-25659 improved PA-induced insulin resistance and inflammation in C2 myotubes. In addition, TM-25659 increased FGF21 mRNA expression, protein levels, and FGF21 secretion in C2 myotubes via activation of GCN2 pathways (GCN2-phosphoeIF2α-ATF4 and FGF21). This beneficial effect of TM-25659 was diminished by FGF21 siRNA. C57BL/6J mice were fed a HF diet for 30 weeks. The HF-diet group was randomly divided into two groups for the next 14 days: the HF-diet and HF-diet + TM-25659 groups. The HF diet + TM-25659-treated mice showed improvements in their fasting blood glucose levels, insulin sensitivity, insulin-stimulated Akt phosphorylation, and inflammation, but neither body weight nor food intake was affected. The HF diet + TM-25659-treated mice also exhibited increased expression of both FGF21 mRNA and protein. These data indicate that TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 in models of PA-induced insulin resistance and HF diet-induced insulin resistance.     

Expression of Protein Kinase C Isoforms in Pancreatic Islets and Liver of Male Goto-Kakizaki Rats,a Model of Type 2 Diabetes

Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.     

Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes

A strong correlation between intramyocellular lipid concentrations and the severity of insulin resistance has fueled speculation that lipid oversupply to skeletal muscle, fat, or liver may desensitize these tissues to the anabolic effects of insulin. To identify free fatty acids (FFAs) capable of inhibiting insulin action, we treated 3T3-L1 adipocytes or C2C12 myotubes with either the saturated FFA palmitate (C16:0) or the monounsaturated FFA oleate (C18:1), which were shown previously to be the most prevalent FFAs in rat soleus and gastrocnemius muscles. In C2C12 myotubes, palmitate, but not oleate, inhibited insulin-stimulation of glycogen synthesis, as well as its activation of Akt/Protein Kinase B (PKB), an obligate intermediate in the regulation of anabolic metabolism. Palmitate also induced the accrual of ceramide and diacylglycerol (DAG), two lipid metabolites that have been shown to inhibit insulin signaling in cultured cells and to accumulate in insulin resistant tissues. Interestingly, in 3T3-L1 adipocytes, neither palmitate nor oleate inhibited glycogen synthesis or Akt/PKB activation, nor did they induce ceramide or DAG synthesis. Using myotubes, we also tested whether other saturated fatty acids blocked insulin signaling while promoting ceramide and DAG accumulation. The long-chain fatty acids stearate (18:0), arachidate (20:0), and lignocerate (24:0) reproduced palmitate's effects on these events, while saturated fatty acids with shorter hydrocarbon chains [i.e., laurate (12:0) and myristate (14:0)] failed to induce ceramide accumulation or inhibit Akt/PKB activation. Collectively these findings implicate excess delivery of long-chain fatty acids in the development of insulin resistance resulting from lipid oversupply to skeletal muscle.