Submaximal Insulin Concentrations Research Articles

P300 or CBP are required for calorie restriction-mediated enhancement of skeletal muscle insulin sensitivity

A calorie-restricted diet robustly improves skeletal muscle insulin sensitivity and is a cornerstone lifestyle intervention for preventing and treating clinical hyperglycemia. While studies demonstrate that insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3K) and RAC-beta serine/threonine-protein kinase (AKT2) are fundamental to enhanced muscle insulin sensitivity after calorie restriction (CR), the downstream signaling steps remain to be fully elucidated. We have previously demonstrated that the lysine acetyltransferases, E1A binding protein (p300) or cAMP response element binding protein binding protein (CBP) are required for insulin-stimulated glucose uptake by skeletal muscle, at both sub-maximal and maximal concentrations of insulin. Moreover, p300/CBP are phosphorylated by AKT, which increases their acetyltransferase activity. Considering these findings together, the objective of this study was to determine whether p300 or CBP are required for the ability of CR to enhance skeletal muscle insulin sensitivity. We hypothesized that combined inhibition of p300 and CBP acetyltransferase activity would prevent CR-induced enhancement of skeletal muscle insulin sensitivity. To address this objective, 8-week-old male C57BL/6NJ mice were either fed ad libitum (AL; n=16) or were fed a CR diet (60% of AL intake; n=16) diet for 20 days. Then, an ex vivo [3H]-2-deoxyglucose (2DOG) approach was used to assess basal and insulin-stimulated (60μU/mL [0.36nM]) 2DOG uptake in paired extensor digitorum longus (EDL) and soleus from fasted (4 h) mice. To define the role of p300/CBP acetyltransferase activity, paired muscles from AL- or CR-fed mice were pre-incubated for 60 minutes with either DMSO (experimental control; n=8/dietary group [AL-DMSO, CR-DMSO]) or the p300/CBP acetyltransferase activity inhibitor, ip300w (25mM; n=8/dietary group [AL-ip300w, CR-ip300w]). As expected, insulin-stimulated 2DOG uptake (i.e. Insulin 2DOG minus basal 2DOG) in DMSO incubated muscles was ~2.3-fold and ~3.3-fold higher in the soleus and EDL, respectively, from CR- vs. AL-fed mice. Remarkably, ip300w not only blocked insulin-stimulated 2DOG uptake in AL-fed mice, but it also abrogated the insulin-sensitizing effects of CR on muscle insulin sensitivity. To further define the importance of p300 and CBP to CR-induced enhancement of skeletal muscle insulin sensitivity, we studied mice with tamoxifen-inducible and skeletal muscle-specific knockout of CBP and heterozygous (HZ) loss of p300 (referred to as, i-mCKO/PHZ) or knockout of p300 and HZ loss of CBP (referred to as, i-mPKO/CHZ). In these mice, the AL diet or 20 days of CR was initiated 3 weeks after ~10 week-old mice were dosed with tamoxifen (5 consecutive days, 2 mg/day); Cre recombinase-negative littermates, who were also dosed with tamoxifen, were the experimental controls (referred to as wildtype [WT]). Interestingly, oral glucose tolerance and EDL insulin sensitivity were comparably enhanced by CR in WT and both i-mCKO/PHZ and i-mPKO/CHZ mice, thus suggesting that just one allele of p300 or CBP is suffcient for CR to enhance skeletal muscle insulin sensitivity. Taken together, these results demonstrate that p300 or CBP are essential for the ability of calorie restriction to enhance skeletal muscle insulin sensitivity. This work was supported by a grant from the National Institutes of Health (R56 DK1311211), and the Dr. Maryam Ahmadian Memorial Summer Fellowship Program (L.M.L.) and the UC San Diego Medical Scientist Training Program (L.S.F.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A high-throughput chemical–genetics screen in murine adipocytes identifies insulin-regulatory pathways

Pathways linking activation of the insulin receptor to downstream targets of insulin have traditionally been studied using a candidate gene approach. To elucidate additional pathways regulating insulin activity, we performed a forward chemical-genetics screen based on translocation of a glucose transporter 4 (Glut4) reporter expressed in murine 3T3-L1 adipocytes. To identify compounds with known targets, we screened drug-repurposing and natural product libraries. We identified, confirmed, and validated 64 activators and 65 inhibitors that acutely increase or rapidly decrease cell-surface Glut4 in adipocytes stimulated with submaximal insulin concentrations. These agents were grouped by target, chemical class, and mechanism of action. All groups contained multiple hits from a single drug class, and several comprised multiple structurally unrelated hits for a single target. Targets include the β-adrenergic and adenosine receptors. Agonists of these receptors increased and inverse agonists/antagonists decreased cell-surface Glut4 independently of insulin. Additional activators include insulin sensitizers (thiazolidinediones), insulin mimetics, dis-inhibitors (the mTORC1 inhibitor rapamycin), cardiotonic steroids (the Na+/K+-ATPase inhibitor ouabain), and corticosteroids (dexamethasone). Inhibitors include heterocyclic amines (tricyclic antidepressants) and 21 natural product supplements and herbal extracts. Mechanisms of action include effects on Glut4 trafficking, signal transduction, inhibition of protein synthesis, and dissipation of proton gradients. Two pathways that acutely regulate Glut4 translocation were discovered: de novo protein synthesis and endocytic acidification. The mechanism of action of additional classes of activators (tanshinones, dalbergiones, and coumarins) and inhibitors (flavonoids and resveratrol) remains to be determined. These tools are among the most sensitive, responsive, and reproducible insulin-activity assays described to date.

운동 강도 차이가 고지방식이 Sprague-Dawley Rat의 골격근 내 PGC-1α, PPAR-γ 및 인슐린 저항에 미치는 영향

This study investigated the effects of exercise intensity on PGC-1α, PPAR-γ, and insulin resistance in skeletal muscle of high fat diet-fed Sprague-Dawley rats. Forty rats were randomly divided into five groups: sedentary control group (SED), high fat diet group (HF), high fat diet+low-intensity exercise group (HFLE, 22 m/min, 60 min, 6 days/week), high fat diet+moderate-intensity exercise group (HFME, 26 m/min, 51 min), and high fat diet+high-intensity exercise group (HFHE, 30 m/min, 46 min). After 4 weeks of high fat diet and endurance exercise training, the lipid profiles, insulin, and glucose concentrations were determined in plasma. PGC-1α, PPAR-γ, and GLUT-4 contents were measured in plantaris muscle. The rate of glucose transport in soleus muscle was determined under submaximal insulin concentration (1,000 μIU/mL insulin, 20 min) during muscle incubation. Plasma glucose during oral glucose tolerance test in HF was significantly greater than that in SED, and plasma glucose levels in the three exercise (EX) groups were significantly lower that those in SED and HF at 30 and 60 min, respectively (P<0.05). Plasma insulin levels in the EX groups were significantly reduced by 60 min compared to that in HF (P<0.05). The protein expression level of PGC-1α as well as muscle glucose uptake were significantly higher in SED and HF than those in the three EX groups (P<0.05), and HFHE showed significantly higher levels than HFLE and HFME. Expression levels of GLUT-4 and PPAR-γ were significantly higher in the HFLE, HFME, and HFHE groups compared to the SED and HF (P<0.05). Therefore, the results of this study indicate that 4 weeks of high fat diet significantly developed whole body insulin resistance but did not affect PGC-1α, PPAR-γ, or the glucose transport rate in skeletal muscle, and exercise training was able to attenuate deteriorated whole body insulin resistance due to high fat diet. In addition, high intensity training significantly affected PGC-1α expression and the glucose transport rate of skeletal muscle in comparison with low and middle training intensities.

Effects of Korean red ginseng supplementation on muscle glucose uptake in high-fat fed rats

AbstractIt has been recognized that ginseng has anti-diabetic effects in skeletal muscle, but the mechanism has not been intensively investigated. The aim of this study was to investigate the effects of Korean red ginseng (Panax ginseng) supplementation on muscle glucose uptake in high-fat fed rats. Sixteen rats were randomly divided into two groups: a control group (CON, n = 8) and a Korean red ginseng group (KRG, n = 8). The KRG group ingested RG extract (1 g·kg−1, 6 days/week) mixed in water for two weeks. After the two-week treatment, plasma lipid profiles, and glucose and insulin concentrations were measured. The triglyceride (TG) and glucose transporter 4 (GLUT-4) contents were measured in the skeletal muscle and liver. The rate of glucose transport was determined under a submaximal insulin concentration during muscle incubation. Plasma FFA concentrations were significantly decreased in KRG (P < 0.05). Liver and muscle triglyceride concentrations were also decreased in the KRG treatment group (P < 0.05) compared to the CON group. In addition, resting plasma insulin and glucose levels were significantly lower after Korean red ginseng treatment (P < 0.05). However, muscle glucose uptake was not affected by Korean red ginseng treatment, as evidenced by the rate of glucose transport in the epitorchealis muscle under submaximal insulin concentrations. These results suggest that while KRG supplementation could improve whole body insulin resistance and plasma lipid profiles, it is unlikely to have an effect on the insulin resistance of skeletal muscle, which is the major tissue responsible for plasma glucose handling.

Hormone Sensitive Lipase knockout mice have higher Post Exercise Insulin Sensitivity despite accumulation of diacylglycerol

AimTo evaluate an accumulation of diacylglycerol (DAG) on post exercise insulin sensitivity.MethodInsulin‐stimulated 2‐deoxyglucose uptake, using a submaximal insulin concentration, was measured in incubated mouse soleus (SOL) and extensor digitorum longus (EDL) muscle from wildtype (WT) and HSL Knock Out (KO) mice 90 min after completion of 40 min treadmill running.ResultsPost exercise insulin stimulated glucose uptake was 14% and 25% higher in SOL and EDL respectively in HSL KO compared to WT. Intramyocellular triacylglycerol was reduced to the same extent in both WT and HSL KO (p&lt;0.01). Muscle DAG was 40% higher after exercise in HSL KO mice compared to WT (p&lt;0.05), there were no differences in muscle DAG levels at rest. Phosphorylation of AMPK thr172 and ACC ser212 were not increased post exercise in both genotypes. Insulin induced signaling (phosphorylation of AKT ser473 and AKT thr308) was similar in both genotypes in both SOL and EDL. Total GLUT 4 protein expression was 20% and 25% higher in SOL and EDL respectively (p&lt;0.05) in HSL KO compared to WT.ConclusionPost exercise insulin sensitivity was higher in HSL KO despite accumulation of DAG. Moreover, the data suggests that reduced fatty acid availability for mitochondrial oxidation plays a role in the observed increased post exercise insulin sensitivity.

Globular adiponectin activates Akt in cultured myocytes

The serine/threonine kinase Akt plays an important role in insulin-mediated glucose uptake. Adiponectin (Adp) is known to sensitize this process. The purpose of the current study is to investigate if Adp activates Akt independently from insulin; and if Adp synergizes with insulin on Akt phosphorylation in the rat skeletal muscle L6 cells. Differentiated L6 cells were serum-starved and exposed to various concentrations (0–100nM) of recombinant globular Adp (gAdp) and/or insulin for different time periods at 37°C. Phosphorylation of Akt was monitored by Western blot using an antiserum against pSer473 or pThr308 Akt. The results demonstrate that gAdp activates Akt in dose- and time-dependent manners. When L6 cells were treated with sub-maximal concentrations of both insulin (10nM) and gAdp (10nM) for 10min neither synergistic nor additive activation of Akt was observed. Similar non-synergistic or non-additive effect of gAdp on insulin-induced Akt activation was also observed in mouse C2C12 myocytes and rat vascular smooth muscle PAC cells. Moreover, pretreatment of the L6 cells with wortmannin (100nM) for 20min significantly reduced gAdp (100nM) induced and insulin (100nM) induced Akt activation by ∼80 and ∼70%, respectively. These data suggest that adiponectin stimulates Akt activation via the wortmannin sensitive pathway in L6 cells; and that its effects on Akt phosphorylation are not additive to those of insulin.

An amino acid mixture is essential to optimize insulin-stimulated glucose uptake and GLUT4 translocation in perfused rodent hindlimb muscle

The purpose of this study was to investigate whether an amino acid mixture increases glucose uptake across perfused rodent hindlimb muscle in the presence and absence of a submaximal insulin concentration, and if the increase in glucose uptake is related to an increase in GLUT4 plasma membrane density. Sprague-Dawley rats were separated into one of four treatment groups: basal, amino acid mixture, submaximal insulin, or amino acid mixture with submaximal insulin. Glucose uptake was greater for both insulin-stimulated treatments compared with the non-insulin-stimulated treatment groups but amino acids only increased glucose uptake in the presence of insulin. Phosphatidylinositol 3-kinase (PI 3-kinase) activity was greater for both insulin-stimulated treatments with amino acids having no additional impact. Akt substrate of 160 kDa (AS160) phosphorylation, however, was increased by the amino acids in the presence of insulin, but not in the absence of insulin. AMPK was unaffected by insulin or amino acids. Plasma membrane GLUT4 protein concentration was greater in the rats treated with insulin compared with no insulin in the perfusate. In the presence of insulin, amino acids increased GLUT4 density in the plasma membrane but had no effect in the absence of insulin. AS160 phosphorylation and plasma membrane GLUT4 density accounted for 76% of the variability in muscle glucose uptake. Collectively, these findings suggest that the beneficial effects of an amino acid mixture on skeletal muscle glucose uptake, in the presence of a submaximal insulin concentration, are due to an increase in AS160 phosphorylation and plasma membrane-associated GLUT4, but independent of PI 3-kinase and AMPK activation.

Identification of P-Rex1 as a Novel Rac1-Guanine Nucleotide Exchange Factor (GEF) That Promotes Actin Remodeling and GLUT4 Protein Trafficking in Adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.

Glucose Uptake-Enhancing Activity of the Ethyl Acetate Extract of Dennettia tripetala in 3T3-L1 Adipocytes

Glucose uptake-enhancing activity of the ethyl acetate extract of Dennettia tripetala (EDT) was investigated using 3T3-L1 adipocytes. EDT yielded 4.43 percent w/w dry matter, showed the presence of alkaloids and flavonoids, contained calcium, iron, zinc, magnesium, copper and manganese. Out of the five extracts of D. tripetala, EDT gave the highest activity (75 percent) in enhancing glucose uptake in 3T3-L1 adipocytes, which was not significantly (p > 0.05) different from insulin (340 nM), the standard drug. EDT caused concentration-dependent increase in glucose uptake with maximal effect at 5 μg/ml, but further increase beyond 10 μg/ml caused a shut down in glucose uptake. Optimal effect was achieved at 16 h post incubation. There was no synergistic effect with increasing doses (2.5, 5.0, 10 μg/ml) of sub maximal concentrations of insulin, rather there was significant (p < 0.05) decrease compared with insulin (340 nM) alone. Unlike other protein inhibitors used, brefeldin significantly (p < 0.05) inhibited EDT-induced glucose uptake by more than 40 percent. In conclusion, EDT enhanced glucose uptake partly by the mobilization of glucose uptake proteins from the interior of the cell to plasma membrane via the Golgi apparatus. Therefore, this mechanism may be responsible for the antihyperglycaemic effect of EDT reported in our previous study.

Resistance to the antilipolytic effect of insulin in adipocytes of African-American compared to Caucasian postmenopausal women

High fatty acid (FA) flux is associated with systemic insulin resistance, and African-American (AA) women tend to be more insulin resistant. We assessed possible depot and race difference in the antilipolytic effect of insulin in adipocytes isolated from abdominal (Abd) and gluteal (Glt) subcutaneous (sc) adipose tissue of overweight, postmenopausal AA and Caucasian (C) women. Percent body fat, fasting insulin, visceral adiposity, and adipocyte size was higher in AA women. Disinhibited lipolysis (presence of adenosine deaminase) per unit adipocyte surface area was similar in Abd and Glt and in AA and C. However, rates of 'basal' [submaximal phenylisopropyl adenosine (PIA)-suppressed] and insulin-suppressed lipolysis were higher in Abd of AA compared with C women even after adjustment for percent fat and visceral fat area. The race difference in rates of PIA- and insulin-suppressed lipolysis in AA were correlated with their hyperinsulinemia, but AA race, independent of fasting insulin, was associated with lower responsiveness (percent suppression) to submaximal insulin concentrations, although sensitivity (ED50) was not affected. Overall, these data are consistent with the hypothesis that decreased responsiveness of Abd adipocytes to antilipolytic effectors may contribute to higher FA availability and thereby to racial differences in insulin resistance.

Hyperglycemic diet and training alter insulin sensitivity, intramyocellular lipid content but not UCP3 protein expression in rat skeletal muscles

Intramyocellular lipids (IMCL) and mitochondrial uncoupling protein-3 (UCP3) have been implicated in the development of muscular insulin resistance. This study aimed to investigate the role of IMCL and UCP3 in the development of glucose intolerance and muscular insulin resistance during 12 weeks of an obesity-inducing 'cafeteria-style' diet alone (CAF), or in conjunction with exercise training from weeks 8-12 (CAFTR), in rats. At the end of the intervention period, gain in body weight was 20% higher in CAF (305+/-10 g) than controls (CON) (255+/-14 g; p<0.001) and CAFTR (253+/-7 g; p<0.001). Furthermore, compared with CON, the Matsuda insulin sensitivity index (ISI), assessed during a 2-h intravenous glucose tolerance test, was markedly lower in CAF (6.7+/-0.4) than in either CON (15.6+/-1.4; p<0.001) or CAFTR (11.2+/-1.1; p<0.001). Moreover, in CAF glucose transport at a submaximal insulin concentration (200 microU/ml) was reduced by approximately 60% (p<0.05) in both red and white gastrocnemius muscles, but not in m. soleus. However, glucose transport in CAFTR was similar to CON in red gastrocnemius. In CAF fiber-specific IMCL content determined in m. soleus and extensor digitorum longus (EDL), was higher than in CON (p<0.01) and CAFTR (p<0.001). Muscle UCP3 protein content was not changed by any of the interventions. Interestingly, within CAF and CAFTR, ISI closely negatively correlated with IMCL content in both type I (soleus, r=-0.93; EDL, r=-0.90; p<0.05) and IIa (EDL, r=-0.52, p<0.05) muscle fibers. These findings indicate that changes in IMCL content but not UCP3 content are implicated in short-term effects of cafeteria-style diet and exercise training on muscular insulin sensitivity in rats.

Effects of Endurance Exercise and High-Fat Diet on Insulin Resistance and Ceramide Contents of Skeletal Muscle in Sprague-Dawley Rats

BackgroundWe evaluated the effects of endurance exercise and a high-fat diet on insulin resistance and ceramide contents of skeletal muscle in Sprague-Dawley rats.MethodsWe randomly divided 32 rats into four groups: control (CON, n = 8), high fat diet (HF, n = 8), exercise (Ex, 24 m/min for 2 hours, 5 days/wk, n = 8), HF/Ex (n = 8). After 4-week treatments, plasma lipid profiles, glucose and insulin concentrations were measured. The triglycerides (TG), ceramide, and glucose transporter 4 (GLUT-4) contents were measured in the skeletal muscle. The rate of glucose transport was determined under submaximal insulin concentration during the muscle incubation.ResultsFree fatty acid levels were significantly higher in CON and HF than Ex (P = 0.032). Plasma glucose levels in HF were significantly higher than the two Ex groups (P = 0.002), and insulin levels were significantly higher in HF than in other three groups (P = 0.021). Muscular TG concentrations were significantly higher in HF than CON and Ex and also in HF/Ex than Ex, respectively (P = 0.005). Hepatic TG concentrations were significantly higher in HF than other three groups but Ex was significantly lower than HF/Ex (P = 0.000). Muscular ceramide content in HF was significantly greater than that in either Ex or HF/Ex (P = 0.031). GLUT-4 levels in CON and HF were significantly lower than those in Ex and HF/Ex (P = 0.009, P = 0.003). The glucose transport rate in submaximal insulin concentration was lower in CON than in either Ex or HF/Ex (P = 0.043), but not different from HF.ConclusionThis study suggests that high fat diet for 4 weeks selectively impairs insulin resistance, but not glucose transport rate, GLUT-4 and ceramide content in skeletal muscle per se. However, endurance exercise markedly affects the content of ceramide and insulin resistance in muscle.

The effect of 5 days of aspartate and asparagine supplementation on glucose transport activity in rat muscle

The consumption of protein supplements containing amino acids is increasing around the world. Aspartate (Asp) and asparagine (Asn) are amino acids metabolized by skeletal muscle. This metabolism involves biochemical pathways that are involved in increasing Krebs cycle activity via anaplerotic reactions, resulting in higher glutamine concentrations. A connection between amino acid supplementation, glycogen concentration, and glucose uptake has been previously demonstrated. The purpose of this study was to evaluate the effect of Asp and Asn supplementation on glucose uptake in rats using three different glycogen concentrations. The results indicate that Asp and Asn supplementation in rats with high glycogen concentrations (fed state) further increased the glycogen concentration in the muscle, and decreased in vitro 2-deoxyglucose (a glucose analog) uptake by the muscle at maximal insulin concentrations. When animals had a medium glycogen concentration (consumed lard for 3 days), glucose uptake was higher in the supplemented group at sub-maximal insulin concentrations. We conclude that supplementation of Asp and Asn reduced glucose transport in rat muscle only at higher levels of glycogen. The ingestion of lard for 3 days changed the responsiveness and sensitivity to insulin, and that group had higher levels of insulin sensitivity with Asp and Asn supplementation.

Inhibition of apoB secretion from HepG2 cells by insulin is amplified by naringenin, independent of the insulin receptor

Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins is characteristic of the dyslipidemia associated with insulin resistance. Recently, we demonstrated that the flavonoid naringenin, like insulin, decreased apoB secretion from HepG2 cells by activation of both the phosphoinositide-3-kinase (PI3-K) pathway and the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK(erk)) pathway. In the present study, we determined whether naringenin-induced signaling required the insulin receptor (IR) and sensitized the cell to the effects of insulin, and whether the kinetics of apoB assembly and secretion in cells exposed to naringenin were similar to those of insulin. Immunoblot analysis revealed that insulin stimulated maximal phosphorylation of IR and IR substrate-1 after 10 min, whereas naringenin did not affect either at any time point up to 60 min. The combination of naringenin and submaximal concentrations of insulin potentiated extracellular-regulated kinase 1/2 activation and enhanced upregulation of the LDL receptor, downregulation of microsomal triglyceride transfer protein expression, and inhibition of apoB-100 secretion. Multicompartmental modeling of apoB pulse-chase studies revealed that attenuation of secreted radiolabeled apoB in naringenin- or insulin-treated cells was similar under lipoprotein-deficient or oleate-stimulated conditions. Naringenin and insulin both stimulated intracellular apoB degradation via a kinetically defined rapid pathway. Therefore, naringenin, like insulin, inhibits apoB secretion through activation of both PI3-K and MAPK(erk) signaling, resulting in similar kinetics of apoB secretion. However, the mechanism for naringenin-induced signaling is independent of the IR. Naringenin represents a possible strategy for reduction of hepatic apoB secretion, particularly in the setting of insulin resistance.

Effects of 4 Weeks Recombinant Human Growth Hormone Administration on Insulin Resistance of Skeletal Muscle in Rats

PurposeEffect of recombinant human growth hormone (rhGH) administration on lipid storage, and its subsequent effect on insulin sensitivity have not yet been adequately examined. Thus, we investigated the effects of rhGH treatment on muscle triglyceride (TG) and ceramide content, and insulin sensitivity after 4 weeks of rhGH administration in rats.Materials and MethodsFourteen rats were randomly assigned to two groups: rhGH injection group (GH, n = 7) and saline injection group (CON, n = 7). GH received rhGH by subcutaneous injections (130 µg·kg-1·day-1, 6 days·week-1) for 4 weeks, while CON received saline injections that were equivalent in volume to GH group. Intramuscular TG and ceramide content and hepatic TG content were measured. To determine insulin sesitivity, oral glucose tolerance test (OGTT) and muscle incubation for glucose transport rate were performed in rats, and used as indicators of insulin sensitivity. We also examined plasm lipid profiles.ResultsAfter 4 weeks of rhGH treatment, the GH group had higher muscle and liver TG contents than the CON (p < 0.05). Ceramide content in GH was significantly greater than that in CON (p < 0.05). GH also had higher plasma levels of FFA (p < 0.05), glucose and insulin responses during OGTT (p < 0.05), and lower glucose transport rates in submaximal insulin concentration (p < 0.05) as compared with CON. Results indicate that rhGH treatment is associated with insulin resistance in rats.ConclusionrhGH treatment elevated muscle TG and ceramide content, and hepatic TG content. Thus, elevation of these compounde by rhGH treatment could contribute to the development of insulin resistance in rats.

The Effects of Growth Hormone Administration and Endurance Exercise Training on Insulin Resistance in Sprague-Dawley Rat

This study investigated the effects of endurance exercise on hGH-induced insulin resistance of skeletal muscle in rats. Forty rats were randomly divided into 4 groups: CON (saline injection group, n=10), GH (hGH injection group, n=10), Ex (Exercise group, n=10), GH/Ex (Growth hormone administration/ Exercise group, n=10). GH, GH/Ex animals received hGH by subcutaneous injections (0.4 IU/kg/day, 6 days/week) for 8 weeks and CON, Ex animals received saline injections with the same volume as GH. The exercise regimen was designed in a 8% grade treadmill, 24m/min training speed with 5 times/week. After 8 weeks of treatment, the plasma lipids profiles, triglyceride contents in muscle, muscle glucose transport rates, and OGTT were measured in these rats. After 8-week treatment, plasma Insulin & glucose levels in GH were significantly increased than other 3 groups during OGTT. The rate of glucose transport under submaximal insulin concentrations (1000 uU/ml) was significantly lower in GH being 1.02±0.10 mol/ml/hr as opposed to 1.42±0.12 mol/ml/hr for the CON. However, there was not a significant difference between GH/Ex (1.31±0.10 mol/ ml/hr) and CON. Plasma FFA levels were significantly higher in GH (0.40±0.04 mmol/l), GH/Ex (0.42±0.05 mmol/l) than CON (0.29±0.03 mmol/l). Triglyceride contents in plantaris muscle was significantly greater in GH (0.52±0.06 mol/g) than CON (0.36±0.01 mol/g), Ex (0.39±0.02 mol/g), However GH injection did not significantly affect plasma TC, HDL-C concentrations. In conclusion, regular exercise training attenuates GH-induced insulin resistance in skeletal muscle of rats.

Contrasting insulin dose-dependent defects in activation of atypical protein kinase C and protein kinase B/Akt in muscles of obese diabetic humans

Insulin-stimulated glucose transport in muscle is impaired in obesity and type 2 diabetes, but alterations in levels of relevant signalling factors, i.e. atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt), are still uncertain. Clamp studies using maximal insulin concentrations have revealed defects in activation of aPKC, but not PKB, in both obese non-diabetic and obese diabetic subjects. In contrast, clamp studies using submaximal insulin concentrations revealed defects in PKB activation/phosphorylation in obese non-diabetic and diabetic subjects, but changes in aPKC were not reported. The aim of this study was to test the hypothesis that dose-related effects of insulin may account for the reported differences in insulin signalling to PKB in diabetic muscle. We compared enzymatic activation of aPKC and PKB, and PKB phosphorylation (threonine-308 and serine-473) during hyperinsulinaemic-euglycaemic clamp studies using both submaximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin levels in non-diabetic control and obese diabetic subjects. In lean control subjects, the submaximal insulin concentration increased aPKC activity and glucose disposal to approximately 50% of the maximal level and PKBbeta activity to 25% of the maximal level, but PKBalpha activity was not increased. In these subjects, phosphorylation of PKBalpha and PKBbeta was increased to near-maximal levels at submaximal insulin concentrations. In obese diabetic subjects, whereas aPKC activation was defective at submaximal and maximal insulin concentrations, PKBbeta activation and the phosphorylation of PKBbeta and PKBalpha were defective at submaximal, but not maximal, insulin concentrations. Defective PKBbeta activation/phosphorylation, seen on submaximal insulin stimulation in diabetic muscle, may largely reflect impaired activation of insulin signalling factors present in concentrations greater than those needed for full PKB activation/phosphorylation. Defective aPKC activation, seen at all insulin levels, appears to reflect, at least partly, an impaired action of distal factors needed for aPKC activation, or poor aPKC responsiveness.

GLUT4 Distribution between the Plasma Membrane and the Intracellular Compartments Is Maintained by an Insulin-modulated Bipartite Dynamic Mechanism

The GLUT4 glucose transporter is predominantly retained inside basal fat and muscle cells, and it is rapidly recruited to the plasma membrane with insulin stimulation. There is controversy regarding the mechanism of basal GLUT4 retention. One model is that GLUT4 retention is dynamic, based on slow exocytosis and rapid internalization of the entire pool of GLUT4 (Karylowski, O., Zeigerer, A., Cohen, A., and McGraw, T. E. (2004) Mol. Biol. Cell 15, 870-882). In this model, insulin increases GLUT4 in the plasma membrane by modulating GLUT4 exocytosis and endocytosis. The second model is that GLUT4 retention is static, with approximately 90% of GLUT4 stored in compartments that are not in equilibrium with the cell surface in basal conditions (Govers, R., Coster, A. C., and James, D. E. (2004) Mol. Cell Biol. 24, 6456-6466). In this model, insulin increases GLUT4 in the plasma membrane by releasing it from the static storage compartment. Here we show that under all experimental conditions examined, basal GLUT4 retention is by a bipartite dynamic mechanism involving slow efflux and rapid internalization. To establish that the dynamic model developed in studies of the extreme conditions of >100 nm insulin and no insulin also describes GLUT4 behavior at more physiological insulin concentrations, we characterized GLUT4 trafficking in 0.5 nm insulin. This submaximal insulin concentration promotes an intermediate effect on both GLUT4 exocytosis and endocytosis, resulting in an intermediate degree of redistribution to the plasma membrane. These data establish that changes in the steady-state surface/total distributions of GLUT4 are the result of gradated, insulin-induced changes in GLUT4 exocytosis and endocytosis rates.

S-15261, a new anti-hyperglycemic agent, reduces hepatic glucose production through direct and insulin-sensitizing effects

S-15261 is a new oral anti-hyperglycemic agent that increases insulin sensitivity in various insulin-resistant animal models. The aim of this study was to determine the short- and long-term effects of S-15261 and its metabolites (S–15511 and Y-415) on fatty acid and glucose metabolism in hepatocytes isolated from 24-h starved rats. During short-term exposure (1 h) neither S-15261 nor its metabolites affected fatty acid oxidation whatever the concentration used. By contrast, S-15261 and its two metabolites reduced the rates of glucose production from lactate/pyruvate and dihydroxyacetone. Using crossover plot analysis, it was shown that Y-415 reduced hepatic gluconeogenesis upstream the formation of dihydroxyacetone phosphate. After 48 h in culture, S-15261 and its two metabolites reduced the rates of glucose production from lactate/pyruvate secondarily to a decrease in PEPCK and Glc-6-Pase mRNA levels. A part of these effects on gene expression could be due to a drug-induced reduction in PGC-1 gene expression. When hepatocytes were cultured in the presence of a submaximal concentration of insulin (10 −9 M), S-15261, through its metabolite S-15511, enhanced insulin sensitivity both on gene expression (PEPCK, Glc-6-Pase, PGC-1) and on gluconeogenesis. Furthermore, S-15261 and S-15511 induced the expression of GK and FAS genes as the result of an increased in SREBP-1c mRNA levels. Finally, S-15511 enhanced the stimulatory effect of insulin on GK mRNA level through an additional increase in SREBP-1c gene expression. In conclusion, this work reveals that S-15261 via its metabolites reduces hepatic glucose production through direct and insulin-sensitizing effects on genes encoding regulatory proteins of hepatic glucose metabolism.

Hyperglycemia Potentiates H2O2 Production in Adipocytes and Enhances Insulin Signal Transduction: Potential Role for Oxidative Inhibition of Thiol-Sensitive Protein-Tyrosine Phosphatases

Insulin signal transduction in adipocytes is accompanied by a burst of cellular hydrogen peroxide (H(2)O(2)) that facilitates insulin signaling by inhibiting thiol-dependent protein-tyrosine phosphatases (PTPs) that are negative regulators of insulin action. As hyperglycemia is associated with increased cellular reactive oxygen species, we postulated that high glucose conditions might potentiate the H(2)O(2) generated by insulin and modulate insulin-stimulated protein phosphorylation. Basal H(2)O(2) generation was increased threefold in differentiated 3T3-L1 adipocytes by growth in 25 mM glucose versus 5 mM glucose. High glucose increased the sensitivity of the insulin-stimulated H(2)O(2) signal to lower concentrations of insulin. Basal endogenous total PTP activity and the activity of PTP1B, a PTP implicated in the negative regulation of insulin signaling, were reduced in high glucose conditions, and their further reduction by insulin stimulation was more enhanced in high versus low glucose medium. Phosphorylation of the insulin receptor, IRS-1, and Akt in response to insulin was also significantly enhanced in high glucose conditions, especially at submaximal insulin concentrations. In primary rat adipocytes, high glucose increased insulin-stimulated H(2)O(2) production and potentiated the oxidative inhibition of total PTP and PTP1B activity; however, insulin signaling was not enhanced in the primary cells in high glucose apparently due to cross-regulation of insulin-stimulated protein phosphorylation by activation of protein kinase C (PKC). These studies indicate that high glucose can enhance insulin stimulated H(2)O(2) generation and augment oxidative PTP inhibition in cultured and primary adipocytes, but the overall balance of insulin signal transduction is determined by additional signal effects in high glucose, including the activation of PKC.