Acute vascular and metabolic actions of the green tea polyphenol epigallocatechin 3-gallate in rat skeletal muscle

Epidemiological studies show a dose-dependent relationship between green tea consumption and reduced risk for type 2 diabetes and cardiovascular disease. Bioactive compounds in green tea including the polyphenol epigallocatechin 3-gallate (EGCG) have insulin-mimetic actions on glucose metabolism and vascular function in isolated cell culture studies. The aim of this study is to explore acute vascular and metabolic actions of EGCG in skeletal muscle of Sprague–Dawley rats. Direct vascular and metabolic actions of EGCG were investigated using surgically isolated constant-flow perfused rat hindlimbs. EGCG infused at 0.1, 1, 10 and 100 μM in 15 min step-wise increments caused dose-dependent vasodilation in 5-hydroxytryptamine pre-constricted hindlimbs. This response was not impaired by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or the AMP-kinase inhibitor Compound C. The nitric oxide synthase (NOS) inhibitor N^G-Nitro-l-Arginine Methyl Ester (L-NAME) completely blocked EGCG-mediated vasodilation at 0.1–10 μM, but not at 100 μM. EGCG at 10 μM did not alter muscle glucose uptake nor did it augment insulin-stimulated muscle glucose uptake. The acute metabolic and vascular actions of 10 μM EGCG in vivo were investigated in anaesthetised rats during a hyperinsulinemic-euglycemic clamp (10 mU min⁻¹ kg⁻¹ insulin). EGCG and insulin both stimulated comparable increases in muscle microvascular blood flow without an additive effect. EGCG-mediated microvascular action occurred without altering whole body or muscle glucose uptake. We concluded that EGCG has direct NOS-dependent vasodilator actions in skeletal muscle that do not acutely alter muscle glucose uptake or enhance the vascular and metabolic actions of insulin in healthy rats.     

Leptin administration improves skeletal muscle insulin responsiveness in diet-induced insulin-resistant rats

In addition to suppressing appetite, leptin may also modulate insulin secretion and action. Leptin was administered here to insulin-resistant rats to determine its effects on secretagogue-stimulated insulin release, whole body glucose disposal, and insulin-stimulated skeletal muscle glucose uptake and transport. Male Wistar rats were fed either a normal (Con) or a high-fat (HF) diet for 3 or 6 mo. HF rats were then treated with either vehicle (HF), leptin (HF-Lep, 10 mg. kg(-1). day(-1) sc), or food restriction (HF-FR) for 12-15 days. Glucose tolerance and skeletal muscle glucose uptake and transport were significantly impaired in HF compared with Con. Whole body glucose tolerance and rates of insulin-stimulated skeletal muscle glucose uptake and transport in HF-Lep were similar to those of Con and greater than those of HF and HF-FR. The insulin secretory response to either glucose or tolbutamide (a pancreatic beta-cell secretagogue) was not significantly diminished in HF-Lep. Total and plasma membrane skeletal muscle GLUT-4 protein concentrations were similar in Con and HF-Lep and greater than those in HF and HF-FR. The findings suggest that chronic leptin administration reversed a high-fat diet-induced insulin-resistant state, without compromising insulin secretion.     

Effect of metformin on the vascular and glucose metabolic actions of insulin in hypertensive rats

We investigated the long-term effect of metformin treatment on blood pressure, insulin sensitivity, and vascular responses to insulin in conscious spontaneously hypertensive rats (SHR). The rats were instrumented with intravascular catheters and pulsed Doppler flow probes to measure blood pressure, heart rate, and blood flow. Insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp technique. Two groups of SHR received metformin (100 or 300 mg x kg(-1) x day(-1)) for 3 wk while another group of SHR and a group of Wistar Kyoto (WKY) rats were left untreated. We found that vasodilation of skeletal muscle and renal vasculatures by insulin is impaired in SHR. Moreover, a reduced insulin sensitivity was detected in vivo and in vitro in isolated soleus and extensor digitorum longus muscles from SHR compared with WKY rats. Three weeks of treatment with metformin improves the whole-body insulin-mediated glucose disposal in SHR but has no blood pressure-lowering effect and no influence on vascular responses to insulin (4 mU x kg(-1) x min(-1)). An improvement in insulin-mediated glucose transport activity was detected in isolated muscles from metformin-treated SHR, but in the absence of insulin no changes in basal glucose transport activity were observed. It is suggested that part of the beneficial effect of metformin on insulin resistance results from a potentiation of the hormone-stimulating effect on glucose transport in peripheral tissues (mainly skeletal muscle). The results argue against a significant antihypertensive or vascular effect of metformin in SHR.     

Acclimation temperature affects the metabolic response of amphibian skeletal muscle to insulin

Frog skeletal muscle mainly utilizes the substrates glucose and lactate for energy metabolism. The goal of this study was to determine the effect of insulin on the uptake and metabolic fate of lactate and glucose at rest in skeletal muscle of the American bullfrog, Lithobates catesbeiana, under varying temperature regimens. We hypothesize that lactate and glucose metabolic pathways will respond differently to the presence of insulin in cold versus warm acclimated frog tissues, suggesting an interaction between temperature and metabolism under varying environmental conditions. We employed radiolabeled tracer techniques to measure in vitro uptake, oxidation, and incorporation of glucose and lactate into glycogen by isolated muscles from bullfrogs acclimated to 5 °C (cold) or 25 °C (warm). Isolated bundles from Sartorius muscles were incubated at 5 °C, 15 °C, or 25 °C, and in the presence and absence of 0.05 IU/mL bovine insulin. Insulin treatment in the warm acclimated and incubated frogs resulted in an increase in glucose incorporation into glycogen, and an increase in intracellular [glucose] of 0.5 μmol/g (P<0.05). Under the same conditions lactate incorporation into glycogen was reduced (P<0.05) in insulin-treated muscle. When compared to the warm treatment group, cold acclimation and incubation resulted in increased rates of glucose oxidation and glycogen synthesis, and a reduction in free intracellular glucose levels (P<0.05). When muscles from either acclimation group were incubated at an intermediate temperature of 15 °C, insulin's effect on substrate metabolism was attenuated or even reversed. Therefore, a significant interaction between insulin and acclimation condition in controlling skeletal muscle metabolism appears to exist. Our findings further suggest that one of insulin's actions in frog muscle is to increase glucose incorporation into glycogen, and to reduce reliance on lactate as the primary metabolic fuel.     

Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle.

Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBS(Thr695)) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBS(Thr695) of MBP and caused MBP activation. Preexposure to N(G)-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBS(Thr695) phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBS(Thr695). Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBS(Thr695) phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.     

The vascular endothelial cell mediates insulin transport into skeletal muscle

The pathways by which insulin exits the vasculature to muscle interstitium have not been characterized. In the present study, we infused FITC-labeled insulin to trace morphologically (using confocal immunohistochemical methods) insulin transport into rat skeletal muscle. We biopsied rectus muscle at 0, 10, 30, and 60 min after beginning a continuous (10 mU x min(-1) x kg(-1)), intravenous FITC-insulin infusion (with euglycemia maintained). The FITC-insulin distribution was compared with that of insulin receptors (IR), IGF-I receptors (IGF-IR), and caveolin-1 (a protein marker for caveolae) in skeletal muscle vasculature. We observed that muscle endothelium stained strongly for FITC-insulin within 10 min, and this persisted to 60 min. Endothelium stained more strongly for FITC-insulin than any other cellular elements in muscle. IR, IGF-IR, and caveolin-1 were also detected immunohistochemically in muscle endothelial cells. We further compared their intracellular distribution with that of FITC-insulin in cultured bovine aortic endothelial cells (bAECs). Considerable colocalization of IR or IGF-IR with FITC-insulin was noted. There was some but less overlap of IR or IGF-IR or FITC-insulin with caveolin-1. Immunoprecipitation of IR coprecipitated caveolin-1, and conversely the precipitation of caveolin-1 brought down IR. Furthermore, insulin increased the tyrosine phosphorylation of caveolin-1, and filipin (which inhibits caveolae formation) blocked insulin uptake. Finally, the ability of insulin, IGF-I, and IGF-I-blocking antibody to diminish insulin transport across bAECs grown on transwell plates suggested that IGF-IR, in addition to IR, can also mediate transendothelial insulin transit. We conclude that in vivo endothelial cells rapidly take up and concentrate insulin relative to plasma and muscle interstitium and that IGF-IR, like IR, may mediate insulin transit through endothelial cells in a process involving caveolae.     

Chronic renin inhibition with aliskiren improves glucose tolerance, insulin sensitivity, and skeletal muscle glucose transport activity in obese Zucker rats

We have demonstrated previously that overactivity of the renin-angiotensin system (RAS) is associated with whole body and skeletal muscle insulin resistance in obese Zucker (fa/fa) rats. Moreover, this obesity-associated insulin resistance is reduced by treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor (type 1) blockers. However, it is currently unknown whether specific inhibition of renin itself, the rate-limiting step in RAS functionality, improves insulin action in obesity-associated insulin resistance. Therefore, the present study assessed the effect of chronic, selective renin inhibition using aliskiren on glucose tolerance, whole body insulin sensitivity, and insulin action on the glucose transport system in skeletal muscle of obese Zucker rats. Obese Zucker rats were treated for 21 days with either vehicle or aliskiren (50 mg/kg body wt ip). Renin inhibition was associated with a significant lowering (10%, P < 0.05) of resting systolic blood pressure and induced reductions in fasting plasma glucose (11%) and free fatty acids (46%) and homeostatic model assessment for insulin resistance (13%). Glucose tolerance (glucose area under the curve) and whole body insulin sensitivity (inverse of the glucose-insulin index) during an oral glucose tolerance test were improved by 15% and 16%, respectively, following chronic renin inhibition. Moreover, insulin-stimulated glucose transport activity in isolated soleus muscle of renin inhibitor-treated animals was increased by 36% and was associated with a 2.2-fold greater Akt Ser(473) phosphorylation. These data provide evidence that chronic selective inhibition of renin activity leads to improvements in glucose tolerance and whole body insulin sensitivity in the insulin-resistant obese Zucker rat. Importantly, chronic renin inhibition is associated with upregulation of insulin action on skeletal muscle glucose transport, and it may involve improved Akt signaling. These data support the strategy of targeting the RAS to improve both blood pressure regulation and insulin action in conditions of insulin resistance.     

Skeletal muscle reprogramming by activation of calcineurin improves insulin action on metabolic pathways

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow- versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-gamma co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.     

Selective inhibition of PKR improves vascular inflammation and remodelling in high fructose treated primary vascular smooth muscle cells

Background and objectiveDouble-stranded RNA dependent protein kinase (PKR) is reported to play a critical role in the pathogenesis of diabetes and associated vascular complications. Increased PKR activity is observed in metabolic disorders. Increased PKR activity is reported to induce inflammation and oxidative stress. Inflammation and oxidative stress are implicated in the pathogenesis of vascular disease. There are no studies done so far about the role of PKR in vascular smooth muscle cells (VSMCs) and the underlying molecular mechanism. Thus the aim of the present study is to investigate the role of PKR in high fructose treated VSMCs. Moreover, a selective PKR inhibitor, imoxin (C16) was used to investigate the underlying molecular mechanism.MethodsVSMCs were isolated by enzymatic digestion method from thoracic aorta of rats and incubated with high fructose (HF) and PKR inhibitor. Immunocytochemistry and Western blotting were performed for PKR and its downstream markers of inflammation, apoptosis and phenotypic transition (AGEs, MMP-9, and ERK_1/2). Oxidative stress was measured using flow cytometry. Cellular hypertrophy and proliferative index were determined by haematoxylin and eosin staining, MTT assay, BrdU labelling assay and agarose gel electrophoresis. Scratch test was done for migratory behaviour. Alizarin red staining was performed for assessing vascular calcification. Mitochondrial membrane potential and chromatin condensation was determined by rhodamine 6G and DAPI staining.ResultsPKR expression was significantly increased in HF treated VSMCs which was accompanied by increase in levels of gene markers of inflammation, oxidative stress and apoptosis. Moreover, increase in cellular proliferation, phenotypic switch and decrease in membrane potential was observed in HF treated VSMCs. All these effects of HF were attenuated by selective PKR inhibitor, imoxin (C16).ConclusionIn conclusion PKR activation plays an important role in the pathogenesis of vascular inflammation and remodelling, and therapeutically targeting PKR could be an effective approach to treat the abnormalities associated with vascular complications.     

Effects of exercise training and ACE inhibition on insulin action in rat skeletal muscle.

Our laboratory has demonstrated (Steen MS, Foianini KR, Youngblood EB, Kinnick TR, Jacob S, and Henriksen EJ, J Appl Physiol 86: 2044-2051, 1999) that exercise training and treatment with the angiotensin-converting enzyme (ACE) inhibitor trandolapril interact to improve insulin action in insulin-resistant obese Zucker rats. The present study was undertaken to determine whether a similar interactive effect of these interventions is manifest in an animal model of normal insulin sensitivity. Lean Zucker (Fa/-) rats were assigned to either a sedentary, trandolapril-treated (1 mg. kg(-1). day(-1) for 6 wk), exercise-trained (treadmill running for 6 wk), or combined trandolapril-treated and exercise-trained group. Exercise training alone or in combination with trandolapril significantly (P < 0.05) increased peak oxygen consumption by 26-32%. Compared with sedentary controls, exercise training alone or in combination with ACE inhibitor caused smaller areas under the curve for glucose (27-37%) and insulin (41-44%) responses during an oral glucose tolerance test. Exercise training alone or in combination with trandolapril also improved insulin-stimulated glucose transport in isolated epitrochlearis (33-50%) and soleus (58-66%) muscles. The increases due to exercise training alone or in combination with trandolapril were associated with enhanced muscle GLUT-4 protein levels and total hexokinase activities. However, there was no interactive effect of exercise training and ACE inhibition observed on insulin action. These results indicate that, in rats with normal insulin sensitivity, exercise training improves oral glucose tolerance and insulin-stimulated muscle glucose transport, whereas ACE inhibition has no effect. Moreover, the beneficial interactive effects of exercise training and ACE inhibition on these parameters are not apparent in lean Zucker rats and, therefore, are restricted to conditions of insulin resistance.     

Role of cGMP in relaxation of vascular and other smooth muscle

The hypothesis that the relaxant action of many drugs on vascular and other smooth muscle is mediated by increases in intracellular cGMP, the "cGMP hypothesis," is gaining wide acceptance. While much information supporting this idea can be found in the literature, there is also a significant amount of information indicating that an elevation in the tissue content of cGMP is by itself insufficient to cause smooth muscle relaxation. The literature is reviewed with reference to the criteria that need to be fulfilled to consider cGMP as the second messenger mediating relaxation of smooth muscle by a drug; i.e., activation of guanylate cyclase, elevation of tissue content of cGMP, potentiation by phosphodiesterase inhibitors, antagonism by inhibitors of cGMP synthesis, and production of relaxation by cGMP analogues. For each criterion, key observations supporting the hypothesis are considered, followed by examples of important observations not consistent with the hypothesis. It is concluded that in some smooth muscles, for example, rat myometrium and vas deferens, cGMP is not a mediator of drug-induced relaxation. In other smooth muscles, including vascular smooth muscle, cGMP appears to play an important role in the relaxation process; but current evidence suggests that other factors are also important and that the cGMP hypothesis may need to be modified.     

Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase.

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.     

Immobilization depresses insulin signaling in skeletal muscle

Prolonged immobilization depresses insulin-induced glucose transport in skeletal muscle and leads to a catabolic state in the affected areas, with resultant muscle wasting. To elucidate the altered intracellular mechanisms involved in the insulin resistance, we examined insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit (IR-beta) and insulin receptor substrate (IRS)-1 and activation of its further downstream molecule, phosphatidylinositol 3-kinase (PI 3-K), after unilateral hindlimb immobilization in the rat. The contralateral hindlimb served as control. After 7 days of immobilization of the rat, insulin was injected into the portal vein, and tibialis anterior muscles on both sides were extracted. Immobilization reduced insulin-stimulated tyrosine phosphorylation of IR-beta and IRS-1. Insulin-stimulated binding of IRS-1 to p85, the regulatory subunit of PI 3-K, and IRS-1-associated PI 3-K activity were also decreased in the immobilized hindlimb. Although IR-beta and p85 protein levels were unchanged, IRS-1 protein expression was downregulated by immobilization. Thus prolonged immobilization may cause depression of insulin-stimulated glucose transport in skeletal muscle by altering insulin action at multiple points, including the tyrosine phosphorylation, protein expression, and activation of essential components of insulin signaling pathways.     

Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.