N − 3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells

Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N − 3 polyunsaturated fatty acids (n − 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n − 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 μM of PAL without or with 50 μM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase Cθ activation. EPA and DHA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n − 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n − 3PUFA, but only EPA and DHA increased PAL β-oxidation, decreased PAL incorporation into DG and reduced protein kinase Cθ activation. EPA and DHA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial β-oxidation.     

Reciprocal inhibition of in vitro substrate movement into avian skeletal muscle

Plasma glucose and ketone concentrations are much higher in birds than in humans and birds exhibit resistance to insulin-mediated glucose uptake into muscle. Therefore, birds may offer a model in which to examine the effects of high plasma glucose and free fatty acid (FFA) concentrations on substrate preference. The present study examined the uptake of radiolabeled oleic acid (OA; C18:1) and radiolabeled glucose by skeletal muscle isolated from the forewing of English sparrows (Passer domesticus). In dose–response studies, unlabeled glucose and OA (20 mM each) inhibited the uptake of their respective radiolabeled counterparts. To examine the effects of glucose on OA uptake, muscles were incubated for 60 min in a buffer containing 20 mM glucose with the addition of radiolabeled OA. This level of glucose significantly decreased radiolabeled OA uptake by 36%. Using the same methodology, 20 mM OA significantly decreased radiolabeled glucose transport by 49%. Comparing control values for glucose (0.952 ± 0.04 μM/mg muscle) and OA uptake (2.20 ± 0.29 μM/mg muscle), it is evident that OA is preferentially taken up by avian skeletal muscle. As FFAs provide a greater amount of energy per mole (146 ATP/OA) than carbohydrates (36 ATP/glucose), storing and utilizing fats may be more energy-efficient for birds. As studies in mammals have shown that FFAs may impair glucose uptake pathways, it is suspected that high FFA uptake by avian skeletal muscle may induce their notably lower glucose transport.     

Glyceollin improves endoplasmic reticulum stress-induced insulin resistance through CaMKK-AMPK pathway in L6 myotubes

Glyceollin has been shown to have antidiabetic properties, although its molecular mechanism is not known. Here, we have investigated the metabolic effects of glyceollin in animal models of insulin resistance and in endoplasmic reticulum (ER) stress-responsive muscle cells. db/db mice were treated with glyceollin for 4 weeks and triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were measured. Glyceollin reduced serum insulin and triglycerides and increased HDL levels in db/db mice. Furthermore, glyceollin caused a significant improvement in glucose homeostasis without altering body weight and food intake in db/db mice. In muscle cells, glyceollin increased the activity of AMP-activated protein kinase (AMPK) as well as cellular glucose uptake. Fatty acid oxidation was also increased. In parallel, phosphorylation of acetyl-CoA carboxylase (ACC) at Ser-79 was increased, consistent with decreased ACC activity. An insulin-resistant state was induced by exposing cells to 5 μg/ml of tunicamycin as indicated by decreased insulin-mediated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Inhibition of insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake under ER stress was prevented by glyceollin. Strikingly, glyceollin reduced ER stress-induced, c-Jun NH₂-terminal kinase activation and subsequently increased insulin signaling via stimulation of AMPK activity in L6 myotubes. Pharmacologic inhibition or knockdown of Ca²⁺/calmodulin-dependent protein kinase kinase blocked glyceollin-increased AMPK phosphorylation and insulin sensitivity under ER stress conditions. Taken together, these results indicate that glyceollin-mediated enhancement of insulin sensitivity under ER stress conditions is predominantly accomplished by activating AMPK, thereby having beneficial effects on hyperglycemia and insulin resistance.     

Hypoglycemic effect of aspalathin,a rooibos tea component from Aspalathus linearis,in type 2 diabetic model db/db mice

Effects of aspalathin, a green rooibos tea component, on glucose metabolism were studied in vitro and in vivo. We first examined the effect of aspalathin on glucose uptake by cultured L6 myotubes and on insulin secretion from cultured RIN-5F pancreatic β-cells in vitro, and then investigated the effect of dietary aspalathin on fasting blood glucose level and conducted an intraperitoneal glucose tolerance test (IPGTT) using type 2 diabetes model mice in vivo. Aspalathin dose-dependently and significantly increased glucose uptake by L6 myotubes at concentrations 1–100 μM. It also significantly increased insulin secretion from cultured RIN-5F cells at 100 μM. Dietary aspalathin (0.1–0.2%) suppressed the increase in fasting blood glucose levels of db/db mice for 5 weeks. In IPGTT, aspalathin improved impaired glucose tolerance at 30, 60, 90, and 120 min in db/db mice. These results suggest that aspalathin has beneficial effects on glucose homeostasis in type 2 diabetes through stimulating glucose uptake in muscle tissues and insulin secretion from pancreatic β-cells.     

In vitro anti-hyperglycemic activity of 4-hydroxyisoleucine derivatives

The nonproteinogenic amino acid, 4-hydroxyisoleucine (1) has been isolated in large quantities from the fenugreek (T. foenum-graecum) seeds. Few novel derivatives (3–11 and 13–18) were prepared from the naturally occurring 4-hydroxyisoleucine (1) and screened for their in vitro glucose uptake stimulatory effect in L-6 skeletal muscle cells. The derivatives 6, 7, 8, 10 and 11 exhibited better glucose uptake stimulatory activity than parent compound, 4-hydroxyisoleucine at 5 and 10 µM concentrations and compounds 7 and 11 enhanced translocation of insulin sensitive glucose transporters-4 in skeletal muscle cells.     

Increased proton leak and SOD2 expression in myotubes from obese non-diabetic subjects with a family history of type 2 diabetes

Muscle insulin resistance is linked to oxidative stress and decreased mitochondrial function. However, the exact cause of muscle insulin resistance is still unknown. Since offspring of patients with type 2 diabetes mellitus (T2DM) are susceptible to developing insulin resistance, they are ideal for studying the early development of insulin resistance. By using primary muscle cells derived from obese non-diabetic subjects with (FH +) or without (FH ?) a family history of T2DM, we aimed to better understand the link between mitochondrial function, oxidative stress, and muscle insulin resistance. Insulin-stimulated glucose uptake and glycogen synthesis were normal in FH + myotubes. Resting oxygen consumption rate was not different between groups. However, proton leak was higher in FH + myotubes. This was associated with lower ATP content and decreased mitochondrial membrane potential in FH + myotubes. Surprisingly, mtDNA content was higher in FH + myotubes. Oxidative stress level was not different between FH + and FH ? groups. Reactive oxygen species content was lower in FH + myotubes when differentiated in high glucose/insulin (25 mM/150 pM), which could be due to higher oxidative stress defenses (SOD2 expression and uncoupled respiration). The increased antioxidant defenses and mtDNA content in FH + myotubes suggest the existence of compensatory mechanisms, which may provisionally prevent the development of insulin resistance.     

The effect of acute exercise on GLUT4 levels in peripheral blood mononuclear cells of sled dogs

Using sled dogs as exercise model, our objectives of this study were to (1) assess the effects of one acute bout of high-intensity exercise on surface GLUT4 concentrations on easily accessible peripheral blood mononuclear cells (PBMC) and (2) compare our findings with published research on exercise induced GLUT4 in skeletal muscle. During the exercise bout, dogs ran 5 miles at approximately 90% of VO₂ max. PMBC were collected before exercise (baseline), immediately after exercise and after 24 h recovery.GLUT4 was measured via ELISA. Acute exercise resulted in a significant increase on surface GLUT4 content on PBMC. GLUT4 was increased significantly immediately after exercise (~50%; p<0.05) and reduced slightly by 24 h post-exercise as compared to baseline (~22%; p>0.05). An effect of acute exercise on GLUT4 levels translocated to the cell membrane was observed, with GLUT4 levels not yet returned to baseline after 24 h post-exercise. In conclusion, the present investigation demonstrated that acute high-intensity exercise increased GLUT4 content at the surface of PBMC of sled dogs as it has been reported in skeletal muscle in other species. Our findings underline the potential use of peripheral blood mononuclear cell GLUT4 protein content as minimally invasive proxy to investigate relationships between insulin sensitivity, insulin resistance, GLUT4 expression and glucose metabolism.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Glucagon increases insulin levels by stimulating insulin secretion without effect on insulin clearance in mice

Circulating insulin is dependent on a balance between insulin appearance through secretion and insulin clearance. However, to what extent changes in insulin clearance contribute to the increased insulin levels after glucagon administration is not known. This study therefore assessed and quantified any potential effect of glucagon on insulin kinetics in mice. Prehepatic insulin secretion in mice was first estimated following glucose (0.35 g/kg i.v.) and following glucose plus glucagon (10 μg/kg i.v.) using deconvolution of plasma C-peptide concentrations. Plasma concentrations of glucose, insulin, and glucagon were then measured simultaneously in individual mice following glucose alone or glucose plus glucagon (pre dose and at 1, 5, 10, 20 min post). Using the previously determined insulin secretion profiles and the insulin concentration-time measurements, a population modeling analysis was applied to estimate the one-compartment kinetics of insulin disposition with and without glucagon. Glucagon with glucose significantly enhanced prehepatic insulin secretion (Cmax and AUC_0-20) compared to that with glucose alone (p < 0.0001). From the modeling analysis, the population mean and between-animal SD of insulin clearance was 6.4 ± 0.34 mL/min for glucose alone and 5.8 ± 1.5 mL/min for glucagon plus glucose, with no significant effect of glucagon on mean insulin clearance. Therefore, we conclude that the enhancement of circulating insulin after glucagon administration is solely due to stimulated insulin secretion.     

Escherichia coli HGT: Engineered for high glucose throughput even under slowly growing or resting conditions

Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase. The strain offers about three-fold higher rates of cell-specific glucose uptake under nitrogen-limitation (0.6 g_Glc g_CDW⁻¹ h⁻¹) compared to that of wild type, with a maximum glucose uptake rate of about 1.8 g_Glc g_CDW⁻¹ h⁻¹ already at a 0.3 h⁻¹ specific growth rate. The surplus of imported glucose is almost completely available via pyruvate and is used to fuel pyruvate and lactate formation. Thus, E. coli HGT represents a novel chassis as a host for pyruvate-derived products.     

Insulin-releasing and cytotoxic properties of the frog skin peptide,tigerinin-1R: a structure–activity study

The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH₂) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by l-arginine, l-lysine and l-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P < 0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01 nM compared with 0.1 nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3 μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P < 0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM indicating that plasma membrane integrity had been preserved. [A5W] tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC₅₀ = 265 ± 16 μM) and inhibited growth of Escherichia coli (MIC = 500 μM) and Staphylococcus aureus (MIC = 250 μM). The circular dichroism spectra of tigerinin-1R and [A5W] tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R] tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P < 0.05) enhanced insulin release and improved glucose tolerance over a 60 min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.     

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.     

Lower dipeptidyl peptidase-4 following exercise training plus weight loss is related to increased insulin sensitivity in adults with metabolic syndrome

Dipeptidyl peptidase-4 (DPP-4) is a circulating glycoprotein that impairs insulin-stimulated glucose uptake and is linked to obesity and metabolic syndrome. However, the effect of exercise on plasma DPP-4 in adults with metabolic syndrome is unknown. Therefore, we determined the effect of exercise on DPP-4 and its role in explaining exercise-induced improvements in insulin sensitivity. Fourteen obese adults (67.9 ± 1.2 years, BMI: 34.2 ± 1.1 kg/m²) with metabolic syndrome (ATP III criteria) underwent a 12-week supervised exercise intervention (60 min/day for 5 days/week at ∼85% HR_max). Plasma DPP-4 was analyzed using an enzyme-linked immunosorbent assay. Insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp (40 mU/m²/min) and estimated by HOMA-IR. Visceral fat (computerized tomography), 2-h glucose levels (75 g oral glucose tolerance), and basal fat oxidation as well as aerobic fitness (indirect calorimetry) were also determined before and after exercise. The intervention reduced visceral fat, lowered blood pressure, glucose and lipids, and increased aerobic fitness (P < 0.05). Exercise improved clamp-derived insulin sensitivity by 75% (P < 0.001) and decreased HOMA-IR by 15% (P < 0.05). Training decreased plasma DPP-4 by 10% (421.8 ± 30.1 vs. 378.3 ± 32.5 ng/ml; P < 0.04), and the decrease in DPP-4 was associated with clamp-derived insulin sensitivity (r = −0.59; P < 0.04), HOMA-IR (r = 0.59; P < 0.04) and fat oxidation (r = −0.54; P < 0.05). Increased fat oxidation also correlated with lower 2-h glucose levels (r = −0.64; P < 0.02). Exercise training reduces plasma DPP-4, which may be linked to elevated insulin sensitivity and fat oxidation. Maintaining low plasma DPP-4 concentrations is a potential mechanism whereby exercise plus weight loss prevents/delays the onset of type 2 diabetes in adults with metabolic syndrome.     

Chronic insulin infusion induces reversible glucose intolerance in lean rats yet ameliorates glucose intolerance in obese rats

BackgroundAlthough insulin resistance (IR) is a key factor in the pathogenesis of type 2 diabetes (T2D), the precise role of insulin in the development of IR remains unclear. Therefore, we investigated whether chronic basal insulin infusion is causative in the development of glucose intolerance.MethodsNormoglycemic lean rats surgically instrumented with i.v. catheters were infused with insulin (3 mU/kg/min) or physiological saline for 6 weeks. At infusion-end, plasma insulin levels along with glucose tolerance were assessed.ResultsSix weeks of insulin infusion induced glucose intolerance and impaired insulin response in healthy rats. Interestingly, the effects of chronic insulin infusion were completely normalized following 24 h withdrawal of exogenous insulin and plasma insulin response to glucose challenge was enhanced, suggesting improved insulin secretory capacity. As a result of this finding, we assessed whether the effects of insulin therapy followed by a washout could ameliorate established glucose intolerance in obese rats. Obese rats were similarly instrumented and infused with insulin or physiological saline for 7 days followed by 24 h washout. Seven day-insulin therapy in obese rats significantly improved glucose tolerance, which was attributed to improved insulin secretory capacity and improved insulin signaling in liver and skeletal muscle.ConclusionModerate infusion of insulin alone is sufficient to cause glucose intolerance and impair endogenous insulin secretory capacity, whereas short-term, intensive insulin therapy followed by insulin removal effectively improves glucose tolerance, insulin response and peripheral insulin sensitivity in obese rats.General significanceNew insight into the link between insulin and glucose intolerance may optimize T2D management.     

Insulinotropic effect of cinnamaldehyde on transcriptional regulation of pyruvate kinase,phosphoenolpyruvate carboxykinase,and GLUT4 translocation in experimental diabetic rats

Diabetes mellitus is a chronic metabolic disorder affecting about 6% of population worldwide with its complications and is rapidly reaching epidemic scale. Cinnamomum zeylanicum is widely used in alternative system of medicine for treatment of diabetes. In the present study, we have performed bioassay guided fractionation of chloroform extract of C. zeylaniucm and identified cinnamaldehyde (CND) as an active principle against diabetes. In continuation to it, a detailed study was undertaken to elucidate its mode of antidiabetic action in STZ induced diabetic rats. Oral administration of CND (20 mg/kg bw) to diabetic rats for 2 months showed significant improvement (p < 0.001) in muscle and hepatic glycogen content. In vitro incubation of pancreatic islets with CND enhanced the insulin release compared to glibenclamide. The insulinotropic effect of CND was found to increase the glucose uptake through glucose transporter (GLUT4) translocation in peripheral tissues. The treatment also showed a significant improvement in altered enzyme activities of pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and their mRNA expression levels.Furthermore, the median lethal dose (LD₅₀) of CND could not be obtained even at 20 times (0.4 g/kg bw) of its effective dose. With the high margin of safety of CND, it can be developed as a potential therapeutic candidate for the treatment of diabetes.     

Effect of pain location on spatial reorganisation of muscle activity

IntroductionWe aimed to determine whether the changes in muscle activity (in terms of both gross electromyography (EMG) and motor unit (MU) discharge characteristics) observed during pain are spatially organized with respect to pain location within a muscle which is the main contributor of the task.MethodsSurface and fine-wire EMG was recorded during matched low-force isometric plantarflexion from soleus (from four quadrants with fine-wire EMG and from the medial/lateral sides with surface EMG), both gastrocnemii heads, peroneus longus, and tibialis anterior. Four conditions were tested: two control conditions that each preceded contractions with pain induced in either the lateral (Pain_L) or medial (Pain_M) side of soleus.ResultsNeither the presence (p = 0.28) nor location (p = 0.19) of pain significantly altered gross muscle activity of any location (lateral/medial side of soleus, gastrocnemii, peroneus longus and tibialis anterior). Group data from 196 MUs show redistribution of MU activity throughout the four quadrants of soleus, irrespective of pain location. The significant decrease of MU discharge rate during pain (p < 0.0001; Pain_L: 7.3 ± 0.9–6.9 ± 1.1 Hz, Pain_M: 7.0 ± 1.1 to 6.6 ± 1.1 Hz) was similar for all quadrants of the soleus (p = 0.43), regardless of the pain location (p = 0.98). There was large inter-participant variation in respect to the characteristics of the altered MU discharge with pain.ConclusionResults from both surface and fine-wire EMG recordings do not support the hypothesis that muscle activity is reorganized in a simple systematic manner with respect to pain location.     

Relación del factor de crecimiento de fibroblastos 21 con indicadores de masa y función muscular en personas mayores con sobrepeso u obesidad. Estudio exploratorio

BackgroundAge-related decreases in muscle mass and function are associated with the development of metabolic impairments, particularly in the context of obesity. Fibroblast growth factor 21 (FGF-21) has been suggested as a common mediator of both processes. No known studies have examined the association between FGF-21 and muscle mass and function in overweight or obese older adults. With this in mind, this study aimed to investigate the association between plasma levels of FGF-21 and muscle mass and function outcomes in overweight or obese older adults.Materials and methodsExploratory study, which included 39 adults of 60-70 years old with body mass indexes > 25 kg/m². As study outcomes, measurements were made of appendicular muscle mass (AMM), grip strength, 5 times sit-to-stand test (5xSTT), as well as plasma levels of FGF-21, fasting glucose, and insulin. The homeostatic model assessment index (HOMA-IR) was also calculated to determine the presence of insulin resistance.ResultsSignificant relationships were found between plasma levels of FGF-21 vs 5xSTT (rho = 0.49; P < .05). Moreover, FGF-21 levels were significantly higher in those with insulin resistance (P < .05), as well as with having lower levels of AMM (P < .05).ConclusionThere is a relationship between the plasma levels of FGF-21 and muscle function outcomes in overweight or obese older adults. Future studies should investigate the potential causalities between these relationships.