Ganoderma lucidum extract stimulates glucose uptake in L6 rat skeletal muscle cells

The effect of Ganoderma lucidum extract on glucose uptake was studied in L6 rat skeletal muscle cells. G. lucidum extract increased glucose uptake about 2-fold compared to control. The extract stimulated the activity of phosphatidylinositol (PI) 3-kinase which is a major regulatory molecule in the glucose uptake pathway. About 7-fold increased activity of a PI 3-kinase was observed after treatment with G. lucidum extract, whereas PI 3-kinase inhibitor, LY294002, blocked the G. lucidum extract-stimulated PI 3-kinase activity in L6 skeletal muscle cells. Protein kinase B, a downstream mediator of PI 3-kinase, was also activated by G. lucidum extract. We then assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in the glucose uptake pathway. G. lucidum extract increased the phosphorylation level of both AMPK alpha1 and alpha2. Activity of p38 MAPK, a downstream mediator of AMPK, was also increased by G. lucidum extract. Taken together, these results suggest that G. lucidum extract may stimulate glucose uptake, through both PI 3-kinase and AMPK in L6 skeletal muscle cells thereby contributing to glucose homeostasis.     

Impaired fatty acid metabolism in type 2 diabetic skeletal muscle cells is reversed by PPARgamma agonists

The impact of type 2 diabetes on the ability of muscle to accumulate and dispose of fatty acids and triglycerides was evaluated in cultured muscle cells from nondiabetic (ND) and type 2 diabetic (T2D) subjects. In the presence of 5 microM palmitate, T2D muscle cells accumulated less lipid than ND cells (11.5 +/- 1.2 vs. 15.1 +/- 1.4 nmol/mg protein, P < 0.05). Chronic treatment (4 days) with the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist troglitazone increased palmitate accumulation, normalizing uptake in T2D cells. There were no significant differences between groups with regard to the relative incorporation of palmitate into neutral lipid species. This distribution was also unaffected by troglitazone treatment. beta-Oxidation of both long-chain (palmitate) and medium-chain (octanoate) fatty acids in T2D muscle cells was reduced by approximately 40% compared with ND cells. Palmitate oxidation occurred primarily in mitochondrial ( approximately 40-50% of total) and peroxisomal (20-30%) compartments. The diabetes-related defect in palmitate oxidation was localized to the mitochondrial component. Both palmitate and octanoate oxidation were stimulated by a series of thiazolidinediones. Oxidation in T2D muscle cells was normalized after treatment. Troglitazone increased the mitochondrial component of palmitate oxidation. Skeletal muscle cells from T2D subjects express defects in free fatty acid metabolism that are retained in vitro, most importantly defects in beta-oxidation. These defects can be corrected by treatment with PPARgamma agonists. Augmentation of fatty acid disposal in skeletal muscle, potentially reducing intramyocellular triglyceride content, may represent one mechanism for the lipid-lowering and insulin-sensitizing effects of thiazolidinediones.     

Regulation of plasma long-chain fatty acid oxidation in relation to uptake in human skeletal muscle during exercise

In the present study, we investigated possible sites of regulation of long-chain fatty acid (LCFA) oxidation in contracting human skeletal muscle. Leg plasma LCFA kinetics were determined in eight healthy men during bicycling (60 min, 65% peak oxygen uptake) with either high (H-FOX) or low (L-FOX) leg fat oxidation (H-FOX: 1,098 +/- 140; L-FOX: 494 +/- 84 micromol FA/min, P < 0.001), which was achieved by manipulating preexercise muscle glycogen (H-FOX: 197 +/- 21; L-FOX: 504 +/- 25 mmol/kg dry wt, P < 0.001). Several blood metabolites and hormones were kept nearly similar between trials by allocating a preexercise meal and infusing glucose intravenously during exercise. During exercise, leg plasma LCFA fractional extraction was identical between trials (H-FOX: 17.8 +/- 1.6; L-FOX: 18.2 +/- 1.8%, not significant), suggesting similar LCFA transport capacity in muscle. On the contrary, leg plasma LCFA oxidation was 99% higher in H-FOX than in L-FOX (421 +/- 47 vs. 212 +/- 37 micromol/min, P < 0.001). Probably due to the slightly higher (P < 0.01) plasma LCFA concentration in H-FOX than in L-FOX, leg plasma LCFA uptake was nonsignificantly (P = 0.17) higher (25%) in H-FOX than in L-FOX, yet the fraction of plasma LCFA uptake oxidized was 61% higher (P < 0.05) in H-FOX than in L-FOX. Accordingly, the muscle content of several lipid-binding proteins did not differ significantly between trials, although fatty acid translocase/CD36 and caveolin-1 were elevated (P < 0.05) by the high-intensity exercise and dietary manipulation allocated on the day before the experimental trial. The present data suggest that, in contracting human skeletal muscle with different fat oxidation rates achieved by manipulating preexercise glycogen content, transsarcolemmal transport is not limiting plasma LCFA oxidation. Rather, the latter seems to be limited by intracellular regulatory mechanisms.     

Electrical stimulation alters fatty acid metabolism in isolated skeletal muscle

Little is known about the contribution of plasma free fatty acid (FFA) and intramuscular triacylglycerol (TG) as substrates for energy production during prolonged electrical stimulation of skeletal muscle. The purpose of this study was to investigate the effects of continuous and intermittent electrical stimulation protocols of different intensities on exogenous FFA oxidation, exogenous FFA incorporation into intracellular TG, and intracellular TG content in the isolated in vitro rat flexor digitorum brevis muscle preparation. Muscles were electrically stimulated for 0.5 h continuously at 0.2 Hz or intermittently (30 s on, 60 s off) at 0.2, 0.4, 0.8, and 5.0 Hz while incubated at 37 degrees C in 0.5 mM palmitate-3% bovine serum albumin medium (pH 7.4) in the presence of insulin (100 microU/ml) and glucose (11 mM). Control muscles were frozen immediately after excision or incubated for 0.5 h. At similar frequencies, less exogenous FFA esterification and more exogenous FFA oxidation occurred during continuous than during intermittent stimulation. As the frequency of intermittent stimulation increased, the amount of exogenous FFA esterified decreased and the amount of exogenous FFA oxidized increased. The data also indicate that at least a portion of TG was constantly being hydrolyzed during electrical stimulation. Under stimulation conditions in which exogenous FFA esterification was below the control (resting muscle) level, intramuscular TG content was significantly decreased compared with control TG content values. Thus both plasma FFA and intramuscular TG are substrates for energy production during electrical stimulation. However, the stimulation parameters employed affect the quantities utilized.     

Chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.     

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.     

Cytokine regulation of skeletal muscle fatty acid metabolism: effect of interleukin-6 and tumor necrosis factor-alpha

IL-6 and TNF-alpha have been associated with insulin resistance and type 2 diabetes. Furthermore, abnormalities in muscle fatty acid (FA) metabolism are strongly associated with the development of insulin resistance. However, few studies have directly examined the effects of either IL-6 or TNF-alpha on skeletal muscle FA metabolism. Here, we used a pulse-chase technique to determine the effect of IL-6 (50-5,000 pg/ml) and TNF-alpha (50-5,000 pg/ml) on FA metabolism in isolated rat soleus muscle. IL-6 (5,000 pg/ml) increased exogenous and endogenous FA oxidation by approximately 50% (P < 0.05) but had no effect on FA uptake or incorporation of FA into endogenous lipid pools. In contrast, TNF-alpha had no effect on FA oxidation but increased FA incorporation into diacylglycerol (DAG) by 45% (P < 0.05). When both IL-6 (5,000 pg/ml) and insulin (10 mU/ml) were present, IL-6 attenuated insulin's suppressive effect on FA oxidation, increasing exogenous FA oxidation (+37%, P < 0.05). Furthermore, in the presence of insulin, IL-6 reduced the esterification of FA to triacylglycerol by 22% (P < 0.05). When added in combination with IL-6 or leptin (10 microg/ml), the TNF-alpha-induced increase in DAG synthesis was inhibited. In conclusion, the results demonstrate that IL-6 plays an important role in regulating fat metabolism in muscle, increasing rates of FA oxidation, and attenuating insulin's lipogenic effects. In contrast, TNF-alpha had no effect on FA oxidation but increased FA incorporation into DAG, which may be involved in the development of TNF-alpha-induced insulin resistance in skeletal muscle.     

Regulation of skeletal muscle metabolism by enzyme binding.

The random diffusion mechanism is usually assumed in analyzing the energetics of specific pathways despite the findings that enzymes associate with each other and (or) with various membranous and contractile elements of the cell. Successive glycolytic enzymes have been shown to associate in the cytosol as enzyme complexes or bind to the thin filaments. Furthermore, the degree of glycolytic enzyme interactions have been shown to change with altered rates of carbon flux through the pathway. In particular, the proportions of aldolase, phosphofructokinase, and glyceraldehyde phosphate dehydrogenase bound to the contractile proteins have been found to increase with increased rates of glycolysis. In addition, decreasing pH and ionic strength are also associated with an increase in glycolytic enzyme interactions. The kinetics displayed by interacting enzymes generally serve to enhance their catalytic efficiencies. The associations of the glycolytic enzymes serve to enhance metabolite transfer rates, increase the local concentrations of intermediates, and provide for regulation of activity via effectors. Therefore these interactions provide an additional mechanism for regulating glycolytic flux in skeletal muscle.     

Design and synthesis of lupeol analogues and their glucose uptake stimulatory effect in L6 skeletal muscle cells

Structure modifications of lupeol at the isopropylene moiety have been described via allylic oxidation using selenium dioxide. The antidiabetic efficacy of lupeol analogues were evaluated in vitro as glucose uptake stimulatory effect in L6 skeletal muscle cells. From all tested compounds, 2, 3, 4b and 6b showed significant stimulation of glucose uptake with respective percent stimulation of 173.1 (p <0.001), 114.1 (p <0.001), 98.3 (p <0.001) and 107.3 (p <0.001) at 10 μM concentration. Stimulation of glucose uptake by these compounds is associated with enhanced translocation of glucose transporter 4 (GLUT4) and activation of IRS-1/PI3-K/AKT-dependent signaling pathway in L6 cells. Structure–activity relationship analysis of these analogues demonstrated that the integrity of α,β-unsaturated carbonyl and acetyl moieties were important in the retention of glucose uptake stimulatory effect. It is therefore proposed that naturally occurring lupeol and their analogues might reduce blood glucose, at least in part, through stimulating glucose utilization by skeletal muscles.     

Ca2+ effects on glucose transport and fatty acid oxidation in L6 skeletal muscle cell cultures

We examined the effect of Ca²⁺ on skeletal muscle glucose transport and fatty acid oxidation using L6 cell cultures. Ca²⁺ stimulation of glucose transport is controversial. We found that caffeine (a Ca²⁺ secretagogue) stimulation of glucose transport was only evident in a two-part incubation protocol (“post-incubation”). Caffeine was present in the first incubation, the media removed, and labeled glucose added for the second. Caffeine elicited a rise in Ca²⁺ in the first incubation that was dissipated by the second. This post-incubation procedure was insensitive to glucose concentrations in the first incubation. With a single, direct incubation system (all components present together) caffeine caused a slight inhibition of glucose transport. This was likely due to caffeine induced inhibition of phosphatidylinositol 3-kinase (PI3K), since nanomolar concentrations of wortmannin, a selective PI3K inhibitor, also inhibited glucose transport, and previous investigators have also found this action.We did find a Ca²⁺ stimulation (using either caffeine or ionomycin) of fatty acid oxidation. This was observed in the absence (but not the presence) of added glucose. We conclude that Ca²⁺ stimulates fatty acid oxidation at a mitochondrial site, secondary to malonyl CoA inhibition (represented by the presence of glucose in our experiments). In summary, the experiments resolve a controversy on Ca²⁺ stimulation of glucose transport by skeletal muscle, introduce an important experimental consideration for the measurement of glucose transport, and uncover a new site of action for Ca²⁺ stimulation of fatty acid oxidation.     

Thiazolidinediones upregulate impaired fatty acid uptake in skeletal muscle of type 2 diabetic subjects

We examined the regulation of free fatty acid (FFA, palmitate) uptake into skeletal muscle cells of nondiabetic and type 2 diabetic subjects. Palmitate uptake included a protein-mediated component that was inhibited by phloretin. The protein-mediated component of uptake in muscle cells from type 2 diabetic subjects (78 +/- 13 nmol. mg protein-1. min-1) was reduced compared with that in nondiabetic muscle (150 +/- 17, P < 0.01). Acute insulin exposure caused a modest (16 +/- 5%, P < 0.025) but significant increase in protein-mediated uptake in nondiabetic muscle. There was no significant insulin effect in diabetic muscle (+19 +/- 19%, P = not significant). Chronic (4 day) treatment with a series of thiazolidinediones, troglitazone (Tgz), rosiglitazone (Rgz), and pioglitazone (Pio) increased FFA uptake. Only the phloretin-inhibitable component was increased by treatment, which normalized this activity in diabetic muscle cells. Under the same conditions, FFA oxidation was also increased by thiazolidinedione treatment. Increases in FFA uptake and oxidation were associated with upregulation of fatty acid translocase (FAT/CD36) expression. FAT/CD36 protein was increased by Tgz (90 +/- 22% over control), Rgz (146 +/- 42%), and Pio (111 +/- 37%, P < 0.05 for all 3) treatment. Tgz treatment had no effect on fatty acid transporter protein-1 and membrane-associated plasmalemmal fatty acid-binding protein mRNA expression. We conclude that FFA uptake into cultured muscle cells is, in part, protein mediated and acutely insulin responsive. The basal activity of FFA uptake is impaired in type 2 diabetes. In addition, chronic thiazolidinedione treatment increased FFA uptake and oxidation into cultured human skeletal muscle cells in concert with upregulation of FAT/CD36 expression. Increased FFA uptake and oxidation may contribute to lower circulating FFA levels and reduced insulin resistance in skeletal muscle of individuals with type 2 diabetes following thiazolidinedione treatment.     

Eicosapentaenoic acid (20:5 n-3) increases fatty acid and glucose uptake in cultured human skeletal muscle cells

This study was conducted to evaluate the chronic effects of eicosapentaenoic acid (EPA) on fatty acid and glucose metabolism in human skeletal muscle cells. Uptake of [14C]oleate was increased >2-fold after preincubation of myotubes with 0.6 mM EPA for 24 h, and incorporation into various lipid classes showed that cellular triacylgycerol (TAG) and phospholipids were increased 2- to 3-fold compared with control cells. After exposure to oleic acid (OA), TAG was increased 2-fold. Insulin (100 nM) further increased the incorporation of [14C]oleate into all lipid classes for EPA-treated myotubes. Fatty acid beta-oxidation was unchanged, and complete oxidation (CO2) decreased in EPA-treated cells. Basal glucose transport and oxidation (CO2) were increased 2-fold after EPA, and insulin (100 nM) stimulated glucose transport and oxidation similarly in control and EPA-treated myotubes, whereas these responses to insulin were abolished after OA treatment. Lower concentrations of EPA (0.1 mM) also increased fatty acid and glucose uptake. CD36/FAT (fatty acid transporter) mRNA expression was increased after EPA and OA treatment compared with control cells. Moreover, GLUT1 expression was increased 2.5-fold by EPA, whereas GLUT4 expression was unchanged, and activities of the mitogen-activated protein kinase p38 and extracellular signal-regulated kinase were decreased after treatment with OA compared with EPA. Together, our data show that chronic exposure of myotubes to EPA promotes increased uptake and oxidation of glucose despite a markedly increased fatty acid uptake and synthesis of complex lipids.     

Oleic acid uptake by in vitro English sparrow skeletal muscle

Studies of prolonged avian flight have shown it to require large amounts of energy supplied mainly by free fatty acids (FFA). In the present study, the high levels of plasma ketone bodies found in sparrows (2.58 mmol l(-1)) are supportive of the use of fatty acids for flight. To determine the nature of fatty acid (oleic acid, OA) uptake, various pharmacological agents were used. The uptake of OA was examined using the extensor digitorum communis (EDC) muscle of English sparrows incubated in vitro. Initial studies demonstrated that radiolabeled OA uptake decreased in the presence of increasing unlabeled OA, suggesting that uptake occurred by a facilitative transport process. To further characterize OA uptake, EDC muscles were incubated with either: insulin (2 ng ml(-1)), insulin-like growth factor isoform-1 (IGF-I; 48 ng ml(-1)), 5'-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 2 mmol) or caffeine (5 mmol). Insulin, but not IGF-I, significantly increased OA uptake by avian EDC (P < 0.01). Caffeine and AICAR were ineffective at increasing OA uptake. A specific inhibitor of FFA transport by fatty acid transporters (FAT/CD36), sulfo-N-succinimidyl oleate (SSO; 500 micromoles), significantly decreased OA uptake at 2.5 min. The effectiveness of SSO suggests that a FAT/CD36-like protein is expressed in avian tissues. As uptake of OA was not completely blocked by SSO, it is likely that other mechanisms for FFA movement across membranes, such as diffusion, may be present.     

A null mutation in H-FABP only partially inhibits skeletal muscle fatty acid metabolism

The low-molecular-mass, cytosolic heart-type fatty acid-binding protein (H-FABP) is thought to be required for shuttling FA through the cytosol. Therefore, we examined the effects of an H-FABP-null mutation on FA and carbohydrate metabolism in isolated soleus muscle at rest and during a period of increased metabolic demand (30-min contraction). There were lower concentrations of creatine phosphate (-41%), ATP (-22%), glycogen (-34%), and lactate (-31%) (P < 0.05) in H-FABP-null soleus muscles, but no differences in citrate synthase and beta-3-hydroxyacyl-CoA dehydrogenase activities or in the intramuscular triacylglycerol (TAG) depots. There was a 43% increase in subsarcolemmal mitochondria in H-FABP-null solei. FA transport was reduced by 30% despite normal content of sarcolemmal long-chain fatty acid transporters fatty acid translocase/CD36 and plasma membrane-associated FABP transport proteins. Compared with wild-type soleus muscles, the H-FABP-null muscles at rest hydrolyzed less TAG (-22%), esterified less TAG (-49%), and oxidized less palmitate (-71%). The H-FABP-null soleus muscles retained a substantial capacity to increase FA metabolism during contraction (TAG esterification by +72%, CO2 production by +120%), although these rates remained lower (TAG esterification -26% and CO2 production -64%) than in contracting wild-type soleus muscles. Glycogen utilization during 30 min of contraction did not differ, whereas glucose oxidation was lower at rest (-24%) and during contraction (-32%) in H-FABP-null solei. Although these studies demonstrate that the absence of H-FABP alters rates of FA metabolism, it is also apparent that glucose oxidation is downregulated. The substantial increase in FA metabolism in contracting H-FABP-null muscle may indicate that other FABPs are also present, a possibility that we were not able to completely eliminate.     

Subsarcolemmal and intermyofibrillar mitochondria play distinct roles in regulating skeletal muscle fatty acid metabolism

Skeletal muscle contains two populations of mitochondria that appear to be differentially affected by disease and exercise training. It remains unclear how these mitochondrial subpopulations contribute to fiber type-related and/or training-induced changes in fatty acid oxidation and regulation of carnitine palmitoyltransferase-1 (CPT1), the enzyme that controls mitochondrial fatty acid uptake in skeletal muscle. To this end, we found that fatty acid oxidation rates were 8.9-fold higher in subsarcolemmal mitochondria (SS) and 5.3-fold higher in intermyofibrillar mitochondria (IMF) that were isolated from red gastrocnemius (RG) compared with white gastrocnemius (WG) muscle, respectively. Malonyl-CoA (10 µM), a potent inhibitor of CPT1, completely abolished fatty acid oxidation in SS and IMF mitochondria from WG, whereas oxidation rates in the corresponding fractions from RG were inhibited only 89% and 60%, respectively. Endurance training also elicited mitochondrial adaptations that resulted in enhanced fatty acid oxidation capacity. Ten weeks of treadmill running differentially increased palmitate oxidation rates 100% and 46% in SS and IMF mitochondria, respectively. In SS mitochondria, elevated fatty acid oxidation rates were accompanied by a 48% increase in citrate synthase activity but no change in CPT1 activity. Nonlinear regression analyses of mitochondrial fatty acid oxidation rates in the presence of 0–100 µM malonyl-CoA indicated that IC₅₀ values were neither dependent on mitochondrial subpopulation nor affected by exercise training. However, in IMF mitochondria, training reduced the Hill coefficient (P < 0.05), suggesting altered CPT1 kinetics. These results demonstrate that endurance exercise provokes subpopulation-specific changes in mitochondrial function that are characterized by enhanced fatty acid oxidation and modified CPT1-malonyl-CoA dynamics. endurance exercise training; CPT-1; fiber type; rat; mitochondrial subpopulations