ABSTRACT
Because greater Akt substrate of 160 kDa (AS160) phosphorylation has been reported in insulin-stimulated skeletal muscles without improved Akt activation several hours post-exercise, we hypothesized that prior exercise would result in attenuated AS160 dephosphorylation in insulin-stimulated rat skeletal muscle. Epitrochlearis muscles were isolated from rats that were sedentary (SED) or exercised 3 h earlier (3 h post-exercise; 3hPEX). Paired muscles were incubated with [(3)H]-2-deoxyglucose (2-DG) without insulin or with insulin. Lysates from other insulin-stimulated muscles from SED or 3hPEX rats were evaluated using AS160(Thr642) and AS160(Ser588) dephosphorylation assays. Prior exercise led to greater 2-DG uptake concomitant with greater AS160(Thr642) phosphorylation and a non-significant trend (P=0.087) for greater AS160(Ser588). Prior exercise also reduced AS160(Thr642) and AS160(Ser588) dephosphorylation rates. These results support the idea that attenuated AS160 dephosphorylation may favor greater AS160 phosphorylation post-exercise.
Subject(s)
GTPase-Activating Proteins/metabolism , Insulin/metabolism , Muscle, Skeletal/metabolism , Physical Conditioning, Animal/physiology , Proto-Oncogene Proteins c-akt/metabolism , Animals , Insulin/pharmacology , Male , Phosphorylation/drug effects , Phosphorylation/physiology , Rats , Rats, WistarABSTRACT
Bradykinin can enhance skeletal muscle glucose uptake (GU), and exercise increases both bradykinin production and muscle insulin sensitivity, but bradykinin's relationship with post-exercise insulin action is uncertain. Our primary aim was to determine if the B2 receptor of bradykinin (B2R) is essential for the post-exercise increase in GU by insulin-stimulated mouse soleus muscles. Wildtype (WT) and B2R knockout (B2RKO) mice were sedentary or performed 60 minutes of treadmill exercise. Isolated soleus muscles were incubated with [³H]-2-deoxyglucose +/-insulin (60 or 100 microU/ml). GU tended to be greater for WT vs. B2RKO soleus with 60 microU/ml insulin (P=0.166) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). Both genotypes had significant exercise-induced reductions (P<0.05) in glycemia and insulinemia, and the decrements for glucose (approximately 14 %) and insulin (approximately 55 %) were similar between genotypes. GU tended to be greater for exercised vs. sedentary soleus with 60 microU/ml insulin (P=0.063) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). There were no significant interactions between genotype and exercise for blood glucose, plasma insulin or GU. These results indicate that the B2R is not essential for the exercise-induced decrements in blood glucose or plasma insulin or for the post-exercise increase in GU by insulin-stimulated mouse soleus muscle.
Subject(s)
Blood Glucose/metabolism , Insulin, Regular, Human/metabolism , Muscle, Skeletal/metabolism , Physical Conditioning, Animal , Receptor, Bradykinin B2/genetics , Animals , Genotype , Humans , Insulin, Regular, Human/blood , Insulin, Regular, Human/pharmacology , Mice , Mice, Knockout , Receptor, Bradykinin B2/metabolismABSTRACT
Increased phosphorylation of Akt substrate of 160 kDa (AS160) is essential to trigger the full increase in insulin-stimulated glucose transport in skeletal muscle. The primary aim of this study was to characterize the time course for reversal of insulin-stimulated AS160 phosphorylation in rat skeletal muscle after insulin removal. The time courses for reversal of insulin effects both upstream (Akt phosphorylation) and downstream (glucose uptake) of AS160 were also determined. Epitrochlearis muscles were incubated in vitro using three protocols which differed with regard to insulin exposure: no insulin (never exposed to insulin), transient insulin (30 min with 1.8 nmol/l insulin, then incubation without insulin for 10, 20 or 40 min), or sustained insulin (continuously incubated with 1.8 nmol/l insulin). After removal of muscles from insulin, Akt and AS160 phosphorylation reversed rapidly, each with a half-time of <10 min and essentially full reversal by 20 min. Glucose uptake reversed more slowly (half time between 10 and 20 min with essentially full reversal by 40 min). Removal of muscles from insulin resulted in a rapid reversal of the increase in AS160 phosphorylation which preceded the reversal of the increase in glucose uptake, consistent with AS160 phosphorylation being essential for maintenance of insulin-stimulated glucose uptake.
Subject(s)
GTPase-Activating Proteins/metabolism , Insulin/metabolism , Muscle, Skeletal/metabolism , Animals , Biological Transport , Deoxyglucose/metabolism , Humans , In Vitro Techniques , Male , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Threonine , Time FactorsABSTRACT
AIMS AND BACKGROUND: Protein O-linked glycosylation is regulated in vivo by the concentration of hexosamine substrates. Calorie restriction (60% of ad libitum intake) for 20 days causes decreased UDP-N-acetylhexosamine levels and increased insulin-mediated glucose transport in rat skeletal muscle. Conversely, prolonged incubation (19 h) of muscle with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenyl-carbamate (PUGNAc; an inhibitor of N-acetyl-beta-D-glucosaminidase) is characterized by increased O-linked glycosylation and insulin resistance. We aimed to determine the calorie restriction effect on O-linked glycosylation and characterize the temporal relationship between PUGNAc-induced O-linked glycosylation and insulin resistance. HYPOTHESIS: A calorie restriction protocol characterized by decreased muscle hexosamine levels will result in a global reduction in O-linked glycosylated proteins in muscle, and PUGNAc-induced insulin resistance will coincide with increased O-linked glycosylation. METHODS: Plantaris muscle and liver from rats (ad libitum or calorie restricted) were analysed for O-linked glycosylation using two antibodies against different O-linked N-acetylglucosamine epitopes. Also, rat epitrochlearis muscles were incubated for 8.5 h +/- 100 mum PUGNAc prior to measurement of [(3)H]-3-O-methylglucose transport and O-linked glycosylation. RESULTS: Calorie restriction did not alter protein O-linked glycosylated levels in muscle or liver. Incubation with PUGNAc for 8.5 h resulted in increased in O-linked glycosylation but unaltered basal or insulin-stimulated glucose transport. CONCLUSIONS: The delay between O-linked glycosylation and insulin resistance in muscle incubated with PUGNAc suggests an indirect, relatively slow mechanism for insulin resistance. The effect of calorie restriction on insulin action in muscle is unlikely to be the direct result of a global change in protein O-linked glycosylation.
Subject(s)
Acetylglucosamine/analogs & derivatives , Acetylglucosamine/pharmacology , Caloric Restriction , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Oximes/pharmacology , Phenylcarbamates/pharmacology , Animals , Biological Transport, Active/drug effects , Biological Transport, Active/physiology , Electrophoresis, Polyacrylamide Gel , Glucose/metabolism , Glycosylation/drug effects , Insulin/pharmacology , Insulin Resistance , Liver/metabolism , Male , Muscle, Skeletal/drug effects , Rats , Rats, Inbred F344 , Tissue Culture Techniques , beta-N-Acetylhexosaminidases/antagonists & inhibitorsABSTRACT
Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.
Subject(s)
3-O-Methylglucose/metabolism , Bradykinin/analogs & derivatives , Insulin/pharmacology , Kallikreins/physiology , Kininogens/physiology , Muscle Contraction/physiology , Trypsin Inhibitors , Adamantane/analogs & derivatives , Adamantane/pharmacology , Animals , Aprotinin/pharmacology , Biological Transport/drug effects , Biological Transport/physiology , Blood Physiological Phenomena , Bradykinin/pharmacology , Bradykinin Receptor Antagonists , Drug Combinations , Glucose/pharmacology , Humans , Kallikreins/antagonists & inhibitors , Male , Plant Proteins/pharmacology , Rats , Rats, Wistar , Receptor, Bradykinin B2 , Serine Proteinase Inhibitors/pharmacology , Tromethamine/pharmacologyABSTRACT
The purpose of this study was to determine the influence of insulin receptor substrate-1 (IRS-1) expression on GLUT1 and GLUT4 glucose transporter protein abundance, contraction-stimulated glucose uptake, and contraction-induced glycogen depletion by skeletal muscle. Mice (6 months old) from three genotypes were studied: wild-type (IRS-1(+/+)), heterozygous (IRS-1(+/-)) for the null allele, and IRS-1 knockouts (IRS-1(-/-)) lacking a functional IRS-1 gene. In situ muscle contraction was induced (electrical stimulation of sciatic nerve) in one hindlimb using contralateral muscles as controls. Soleus and extensor digitorum longus were dissected and 2-deoxyglucose uptake was measured in vitro. 2-Deoxyglucose uptake was higher in basal muscles (no contractions) from IRS-1(-/-) vs. both other genotypes. Contraction-stimulated 2-deoxyglucose uptake and glycogen depletion did not differ among genotypes. Muscle IRS-1 protein was undetectable for IRS-1(-/-) mice, and values were approximately 40 % lower in IRS-1(+/-) than in IRS-1(+/+) mice. No difference was found in IRS-1(+/+) compared to IRS-1(-/-) groups regarding muscle abundance of GLUT1 and GLUT4. Substantial reduction or elimination of IRS-1 did not alter the hallmark effects of contractions on muscle carbohydrate metabolism--activation of glucose uptake and glycogen depletion.
Subject(s)
Deoxyglucose/metabolism , Monosaccharide Transport Proteins/analysis , Muscle Contraction/physiology , Muscle Proteins , Muscle, Skeletal/metabolism , Phosphoproteins/deficiency , Animals , Body Weight , Electric Stimulation , Female , Glucose Transporter Type 1 , Glucose Transporter Type 4 , Glycogen/metabolism , Insulin Receptor Substrate Proteins , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/anatomy & histology , Muscle, Skeletal/chemistry , Organ Size , Phosphoproteins/genetics , Phosphoproteins/physiology , Sciatic NerveABSTRACT
Insulin resistance is common in old age, and exercise training can improve insulin sensitivity. The purpose of this study was to determine the influence of age (6 vs 26 months) and exercise training (10 weeks of treadmill running) on insulin signaling protein abundance in skeletal muscle from male Fisher 344 rats. Muscle levels of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and Akt1, a serine-threonine kinase, were determined. IRS-1 was reduced with aging, IR and PI3K abundance was greater in old rats, and Akt1 was unchanged. IRS-1 was increased by training in old but not young rats, and IR was increased by training in young but not old rats. PI3K tended to increase and Akt1 did not change with training, regardless of age. Aging does not uniformly affect insulin signaling protein abundance, and exercise differentially alters IR and IRS-1 in young and old rats, thereby eliminating age-related differences in these proteins.
Subject(s)
Aging/metabolism , Muscle, Skeletal/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/analysis , Phosphoproteins/metabolism , Physical Conditioning, Animal , Receptor, Insulin/metabolism , Analysis of Variance , Animals , Immunoblotting , Insulin Receptor Substrate Proteins , Male , Models, Animal , Probability , Random Allocation , Rats , Rats, Inbred F344 , Receptor, Insulin/analysis , Reference Values , Sensitivity and SpecificityABSTRACT
Effects of genetic selection for high wheel-running activity (17th generation) and access to running wheels on skeletal muscle glucose uptake were studied in mice with the following treatments for 8 wk: 1) access to unlocked wheels; 2) same as 1, but wheels locked 48 h before glucose uptake measurement; or 3) wheels always locked. Selected mice ran more than random-bred (nonselected) mice (8-wk mean +/- SE = 8,243 +/- 711 vs. 3,719 +/- 233 revolutions/day). Body weight was 5-13% lower for selected vs. nonselected groups. Fat pad/body weight was ~40% lower for selected vs. nonselected and unlocked vs. locked groups. Insulin-stimulated glucose uptake and fat pad/body weight were inversely correlated for isolated soleus (r = -0.333; P < 0.005) but not extensor digitorum longus (EDL) or epitrochlearis muscles. Insulin-stimulated glucose uptake was higher in EDL (P < 0.02) for selected vs. nonselected mice. Glucose uptake did not differ by wheel group, and amount of running did not correlate with glucose uptake for any muscle. Wheel running by mice did not enhance subsequent glucose uptake by isolated muscles.
Subject(s)
Glucose/pharmacokinetics , Mice, Inbred ICR/genetics , Muscle, Skeletal/metabolism , Physical Exertion/physiology , Animals , Antimetabolites/pharmacokinetics , Blood Glucose/metabolism , Breeding , Deoxyglucose/pharmacokinetics , Female , Glycogen/metabolism , Hematocrit , Hypoglycemic Agents/blood , Hypoglycemic Agents/pharmacology , Insulin/blood , Insulin/pharmacology , Liver/anatomy & histology , Liver/metabolism , Male , Mice , Motor Activity/physiology , Muscle, Skeletal/anatomy & histology , Organ SizeABSTRACT
A moderate reduction in calorie intake (calorie restriction, CR) improves insulin-stimulated glucose transport in skeletal muscle. Therefore, we studied muscle insulin signalling in ad libitum (AL) and CR ( approximately 60% AL intake for 20 days) fed rats, which received a control injection (sterile water) or an insulin injection (30 U kg-1 body weight). In control (not insulin-treated) rats, there was no detectable tyrosine phosphorylation of insulin receptor (IR), regardless of diet; no diet effect on tyrosine phosphorylation of insulin receptor substrate-1 (IRS1) or IRS1-associated phosphatidylinositol 3-kinase (PI3K) protein and 21% higher IRS1-associated PI3K activity in AL vs. CR. In insulin-treated rats, tyrosine-phosphorylated IR was 79% higher for CR vs. AL; tyrosine-phosphorylated IRS1 was 109% higher for CR vs. AL; IRS1-associated PI3K protein and IRS1-associated PI3K activity were unaffected by diet. Calorie restriction amplifies early insulin signalling steps without changing IRS1-associated PI3K, suggesting enhanced glucose transport is mediated by altering: IRS1-PI3K localization, PI3K associated with proteins other than IRS1 or post-PI3K events.
Subject(s)
Food Deprivation/physiology , Hypoglycemic Agents/blood , Insulin/blood , Muscle, Skeletal/enzymology , Phosphoproteins/metabolism , Receptor, Insulin/metabolism , Animals , Body Weight , Energy Intake/physiology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Insulin Receptor Substrate Proteins , Male , Muscle, Skeletal/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Binding/physiology , Rats , Rats, Inbred F344 , Signal Transduction/physiology , Tyrosine/metabolismABSTRACT
Caloric restriction (CR), a reduction in calorie intake without malnutrition, improves insulin sensitivity in various species, including mice, rats, rhesus and cynomolgus monkeys, and humans. Skeletal muscle is quantitatively the most important tissue for blood glucose clearance. Therefore, we assessed the effect of 6 years of CR (30% reduction in calorie intake) in male rhesus monkeys (14-20 years old) on muscle expression of several proteins involved in insulin action. Whole body insulin sensitivity (assessed by Modified Minimal Model) was significantly increased in CR relative to Control monkeys. CR did not alter the expression of GLUT4 glucose transporter or phosphatidylinositol-3 kinase p85 subunit (PI3K). Insulin receptor substrate-1(IRS-1) abundance tended to be greater for CR compared to Control monkeys (p = .051), but correlational analysis revealed no association between IRS-1 and insulin sensitivity (r2 = .075, p = .271). These findings indicate that the CR-induced increase in insulin sensitivity in rhesus monkeys is unrelated to alterations in GLUT4, P13K, and IRS-1 abundance.
Subject(s)
Energy Intake , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , Muscle, Skeletal/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor, Insulin/metabolism , Aging/physiology , Animals , Glucose Transporter Type 4 , Insulin Resistance , Macaca mulatta , Male , Statistics, NonparametricABSTRACT
Previous studies have demonstrated enhanced insulin sensitivity in calorie-restricted [CR, fed 60% ad libitum (AL) one time daily] compared with AL-fed rats. To evaluate the effects of reduced food intake, independent of temporal differences in consumption, we studied AL (unlimited food access)-fed and CR (fed one time daily) rats along with groups temporally matched for feeding [fed 3 meals (M) daily]: MAL and MCR, eating 100 and 60% of AL intake, respectively. Insulin-stimulated glucose transport by isolated muscle was increased in MCR and CR vs. AL and MAL; there was no significant difference for MCR vs. CR or MAL vs. AL. Intramuscular triglyceride concentration, which is inversely related to insulin sensitivity in some conditions, did not differ among groups. Muscle concentration of UDP-N-acetylhexosamines [end products of the hexosamine biosynthetic pathway (HBP)] was lower in MCR vs. MAL despite unaltered glutamine-fructose-6-phosphate aminotransferase activity (rate-limiting enzyme for HBP). These results indicate that the CR-induced increase in insulin-stimulated glucose transport in muscle is attributable to an altered amount, not timing, of food intake and is independent of lower triglyceride concentration. They further suggest that enhanced insulin action might involve changes in HBP.
Subject(s)
Energy Intake , Hexosamines/metabolism , Insulin/pharmacology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , 3-O-Methylglucose/pharmacokinetics , Adipose Tissue/anatomy & histology , Animals , Blood Glucose/analysis , Body Weight , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Glycogen/metabolism , Hexoses/metabolism , Insulin/blood , Leptin/blood , Liver/anatomy & histology , Liver/metabolism , Male , Muscle Proteins/metabolism , Muscle, Skeletal/anatomy & histology , Organ Size , Rats , Rats, Inbred F344 , Triglycerides/metabolismABSTRACT
Calorie restriction (CR), even for brief periods (4-20 days), results in increased whole-body insulin sensitivity, in large part due to enhanced insulin-stimulated glucose transport by skeletal muscle. Evidence suggests that the cellular alterations leading to this effect are postreceptor steps in insulin signaling. To determine whether insulin receptor substrate (IRS)-1 is essential for the insulin-sensitizing effect of CR, we measured in vitro 2-deoxyglucose (2DG) uptake in the presence and absence of insulin by skeletal muscle isolated from wild-type (WT) mice and transgenic mice lacking IRS-1 (knockout [KO]) after either ad libitum (AL) feeding or 20 days of CR (60% of ad libitum intake). Three muscles (soleus, extensor digitorum longus [EDL], and epitrochlearis) from male and female mice (4.5-6 months old) were studied. In each muscle, insulin-stimulated 2DG uptake was not different between genotypes. For EDL and epitrochlearis, insulin-stimulated 2DG uptake was greater in CR compared to AL groups, regardless of sex. Soleus insulin-stimulated 2DG uptake was greater in CR compared with AL in males but not females. The diet effect on 2DG uptake was not different for WT and KO animals. Genotype also did not alter the CR-induced decrease in plasma constituents (glucose, insulin, and leptin) or body composition (body weight, fat pad/body weight ratio). Consistent with previous studies in rats, IRS-1 protein expression in muscle was reduced in WT-CR compared with WT-AL mice, and muscle IRS-2 abundance was unchanged by diet. Skeletal muscle IRS-2 protein expression was significantly lower in WT compared with KO mice. These data demonstrate that IRS-1 is not essential for the CR-induced increase in insulin-stimulated glucose transport in skeletal muscle, and the absence of IRS-1 does not modify any of the characteristic adaptations of CR that were evaluated.
Subject(s)
Food Deprivation , Insulin/pharmacology , Muscle, Skeletal/metabolism , Phosphoproteins/physiology , Receptor, Insulin/physiology , Adipose Tissue/metabolism , Animals , Blood Glucose/metabolism , Cells, Cultured , Deoxyglucose/pharmacokinetics , Energy Intake , Female , Insulin/blood , Insulin Receptor Substrate Proteins , Intracellular Signaling Peptides and Proteins , Leptin , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muscle, Skeletal/drug effects , Phosphoproteins/metabolism , Proteins/metabolism , Rats , Receptor, Insulin/metabolismABSTRACT
We evaluated the effects of 8 mo of calorie restriction [CR: 60% of ad libitum (AL) food intake] on glucose uptake by 14 tissues in unanesthetized, adult (12 mo) F344xBN rats. Glucose metabolism was assessed by the 2-[3H]deoxyglucose tracer technique at 1500 or 2100. Despite an approximately 60% decline in insulinemia with CR, plasma 2-[3H]deoxyglucose clearance for CR was greater than for AL at both times. A small, CR-related decrease in glucose metabolic index (R'g) occurred only at 1500 in the spleen and heart, and this decrease was reversed at 2100. In some tissues (cerebellum, lung, kidney, soleus, and diaphragm), R'g was unaffected by diet, regardless of time. In the other tissues (brown fat, 3 white fat pads, epitrochlearis, plantaris, and gastrocnemius), R'g was higher or tended to be higher for CR vs. AL at one or both times. These findings indicate that 8 mo of CR did not cause a continuous reduction in in vivo glucose uptake by any tissue studied, and, in several insulin-sensitive tissues, glucose uptake was at times greater for CR vs. AL rats.
Subject(s)
Blood Glucose/metabolism , Diet, Reducing , Energy Intake , Glucose/metabolism , Adipose Tissue , Adipose Tissue, Brown/metabolism , Animals , C-Peptide/blood , Cerebellum/metabolism , Deoxyglucose/pharmacokinetics , Fatty Acids, Nonesterified/blood , Insulin/blood , Lactates/blood , Male , Metabolic Clearance Rate , Muscle, Skeletal/metabolism , Organ Specificity , Rats , Rats, Inbred F344 , TritiumABSTRACT
Reduced calorie intake [calorie restriction (CR); 60% of ad libitum (AL)] leads to enhanced glucose transport without altering total GLUT-4 glucose transporter abundance in skeletal muscle. Therefore, we tested the hypothesis that CR (20 days) alters the subcellular distribution of GLUT-4. Cell surface GLUT-4 content was higher in insulin-stimulated epitrochlearis muscles from CR vs. AL rats. The magnitude of this increase was similar to the CR-induced increase in glucose transport, and GLUT-4 activity (glucose transport rate divided by cell surface GLUT-4) was unaffected by diet. The CR effect was specific to the insulin-mediated pathway, as evidenced by the observations that basal glucose transport and cell surface GLUT-4 content, as well as hypoxia-stimulated glucose transport, were unchanged by diet. CR did not alter insulin's stimulation of insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity. Muscle abundance of IRS-2 and p85 subunit of PI3K were unaltered by diet, but IRS-1 content was lower in CR vs. AL. These data demonstrate that, despite IRS-1-PI3K activity similar to AL, CR specifically increases insulin's activation of glucose transport by enhancing the steady-state proportion of GLUT-4 residing on the cell surface.
Subject(s)
Energy Intake , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , 3-O-Methylglucose/pharmacokinetics , Animals , Biological Transport/physiology , Blood Glucose/analysis , Cell Membrane/metabolism , Cohort Studies , Glucose/metabolism , Glucose Transporter Type 4 , Hypoxia/metabolism , Insulin/blood , Male , Rats , Rats, Inbred F344ABSTRACT
We studied the effect of a high physiologic concentration of palmitate (1mM) on in vitro 2-deoxy-D-glucose (2DG) uptake by flexor digitorum brevis (FDB) muscle from ad libitum fed rats (AL) and rats fed 60% of ad libitum intake (CR) for 20 days. CR did not alter muscle 2DG uptake in the absence of insulin, but relative to AL, CR significantly (p<0.01) increased 2DG uptake in the presence of 20,000 microU/ml insulin. This effect of CR persisted in the presence of 1mM palmitate. The presence of 1mM palmitate significantly (p<0.01) impaired 2DG glucose uptake, both in the presence and absence of insulin, to the same extent in AL and CR muscle, despite an 18% decrease in FABPpm expression with CR. Thus, although CR profoundly affects insulin-mediated muscle glucose uptake, it does not alter the ability of extracellular fatty acid to modulate glucose utilization by skeletal muscle.
Subject(s)
Deoxyglucose/pharmacokinetics , Food Deprivation , Muscles/metabolism , Neoplasm Proteins , Nerve Tissue Proteins , Palmitates/pharmacology , Animals , Blood Glucose/metabolism , Body Weight , Carrier Proteins/metabolism , Cell Membrane/metabolism , Energy Intake , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Fatty Acids, Nonesterified/blood , Fatty Acids, Nonesterified/pharmacology , Glucose/pharmacokinetics , Male , Muscles/drug effects , Myelin P2 Protein/metabolism , Rats , Rats, Inbred F344ABSTRACT
The aim of this study was to compare, in 19-month-old male Fischer 344 rats, the influence of brief (20 days) and prolonged (approximately 15 months) calorie restriction (CR; consuming approximately 60% of ad libitum, AL, intake) on circulating levels of glucose, insulin, C-peptide, and free fatty acids (FFA); age-matched AL rats were also studied. In the prolonged CR group, there was an approximately 85% decline in fat pad masses (epididymal and retroperitoneal) compared to AL and brief CR rats (these latter groups did not differ significantly). Compared to AL levels, glucose was 15% lower with prolonged CR (p < 0.05) while the brief CR values tended to be lower (10%) than AL; the CR groups did not differ significantly. Plasma FFA levels were significantly (p < 0.05) greater (85-106%) in the brief CR group compared to each of the other groups. Plasma insulin concentrations for the CR groups were lower (p < 0.05; approximately 50-60%) than AL levels. Plasma concentrations of C-peptide (an indicator of insulin secretion) were also lower for each CR group vs AL levels, and a high correlation was found between plasma insulin and C-peptide concentrations (r2 = 0.90; p < 0.001). The C-peptide/insulin ratios for the CR groups were similar, and the value of each CR group exceeded that for the AL rats. These results demonstrate that: the CR-induced reduction in plasma insulin is attributable in large part to reduced insulin secretion; these decreases in insulin secretion and concentration are essentially undiminished when brief CR is initiated rather late in life, and the reductions are independent of substantial reductions in body fat.
Subject(s)
Food Deprivation/physiology , Adipose Tissue/anatomy & histology , Aging/physiology , Animals , Blood Glucose/metabolism , Body Weight , C-Peptide/blood , Energy Intake , Fatty Acids, Nonesterified/blood , Insulin/blood , Insulin/metabolism , Insulin Secretion , Male , Rats , Rats, Inbred F344 , Time FactorsABSTRACT
Insulin resistance is a common syndrome that often precedes the development of noninsulin-dependent diabetes mellitus (NIDDM). Both diet and genetic factors are associated with insulin resistance. BTBR and C57BL/6J (B6) mice have normal insulin responsiveness and normal fasting plasma insulin levels. However, a cross between these two strains yielded male offspring with severe insulin resistance. Surprisingly, on a basal diet (6.5% fat), the insulin resistance was not associated with fasting hyperinsulinemia. However, a 15% fat diet produced significant hyperinsulinemia in the male mice (twofold at 10 weeks; P < .05). At 10 weeks of age, visceral fat contributed approximately 4.3% of the total body weight in the males versus 1.8% in females. In the males, levels of plasma triacylglycerol and total cholesterol increased 40% and 30%, respectively, compared to females. Plasma free fatty acid concentrations were unchanged. Oral glucose tolerance tests revealed significant levels of hyperglycemia and hyperinsulinemia 15 to 90 minutes after oral glucose administration in the male mice. This was particularly dramatic in males on a 15% fat diet. Glucose transport was examined in skeletal muscles in (BTBR x B6)F1 mice. In the nonhyperinsulinemic animals (females), insulin stimulated 2-deoxyglucose transport 3.5-fold in the soleus and 2.8-fold in the extensor digitorum longus muscles. By contrast, glucose transport was not stimulated in the hyperinsulinemic male mice. Hypoxia stimulates glucose transport through an insulin-independent mechanism. This is known to involve the translocation of GLUT4 from an intracellular pool to the plasma membrane. In the insulin-resistant male mice, hypoxia induced glucose transport as effectively as it did in the insulin-responsive mice. Thus, defective glucose transport in the (BTBR x B6)F1 mice is specific for insulin-stimulated glucose transport. This is similar to what has been observed in muscles taken from obese NIDDM patients. These animals represent an excellent genetic model for studying insulin resistance and investigating the transition from insulin resistance in the absence of hyperinsulinemia to insulin resistance with hyperinsulinemia.
Subject(s)
Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 2/genetics , Disease Models, Animal , Insulin Resistance/genetics , Mice, Inbred C57BL/genetics , Mice, Inbred Strains/genetics , Muscle Proteins , Adipose Tissue/pathology , Animals , Biological Transport, Active/drug effects , Body Weight , Crosses, Genetic , Deoxyglucose/metabolism , Dietary Fats/toxicity , Female , Genotype , Glucose Tolerance Test , Glucose Transporter Type 4 , Hyperlipidemias/blood , Hyperlipidemias/genetics , Hyperlipidemias/pathology , Hypoxia/blood , Insulin/blood , Insulin/pharmacology , Male , Mice , Monosaccharide Transport Proteins/metabolism , Muscle, Skeletal/metabolism , Obesity/blood , Obesity/genetics , Obesity/pathology , Organ Size , Sex CharacteristicsABSTRACT
The most rapid age-related decrease in insulin-stimulated glucose uptake in skeletal muscle occurs between 3 and 5 wk of age in rats. Therefore, we studied unstimulated, insulin-stimulated, and in vitro hypoxia-stimulated 2-deoxy-D-[G-3H]glucose (2-DG) uptake in isolated soleus, flexor digitorum brevis (FDB), and epitrochlearis muscles from rats at 21, 28, and 35 days of age. Age-related decrements in insulin- (approximately 40-60%) and hypoxia-stimulated (approximately 50%) 2-DG uptake occurred in all muscles, and most of the decline was evident by 28 days. Unstimulated 2-DG uptake declined significantly with advancing age in the epitrochlearis (73%) and FDB (60%) and tended to decrease in the soleus (38%). The time course and relative magnitude of these decrements were similar under unstimulated, insulin-stimulated, and hypoxic conditions. GLUT-4 protein concentration was unaltered by age in each muscle. These results indicate that a substantial age-related decrement in 2-DG uptake occurs in several limb muscles from rats at 21 vs. 28-35 days by a mechanism that is independent of GLUT-4 levels and not specific for the insulin-dependent pathway.
Subject(s)
Age Factors , Glucose/metabolism , Insulin/metabolism , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , Muscle, Skeletal/metabolism , Animals , Biological Transport , Blood Glucose/metabolism , Body Weight , Glucose Transporter Type 4 , Glycogen/metabolism , Hypoxia/metabolism , Male , Muscle, Skeletal/anatomy & histology , Organ Size , RatsABSTRACT
This study quantified the orthotropic elastic changes in cortical bone due to aging as well as determined any elastic changes after acute treatments of growth hormone (GH). Three groups of twenty rats represented three age groups of young adult (9 months), middle age (20 months), and old (31 months) rats. During a ten day period, half of the rats in each age group were given twice-daily doses of recombinant human GH while the remaining half were injected with a vehicle control (saline). The effects of aging and GH on the elastic characteristics of cortical bone were quantified via ultrasonic wave propagation. Propagation velocities of longitudinal and shear waves were measured through cubic cortical specimens from the posterior femoral diaphysis. Density was measured by Archimedes' technique. The normalized, orthotropic elastic properties of Young's moduli (Eii), shear moduli (Gij), and Poisson's ratios (Vij) were calculated and used to compare the groups (where i and j = 1, 2, or 3 reference the radial, circumferential, and longitudinal axes, respectively). Cortical elastic moduli consistently increased with age with the strongest effects demonstrated in radial dependent properties such as E11 (+ 25.3% from 9 to 31 months, p = 0.0004) and G12 (+ 12.6% from 20 to 31 months, p = 0.0419). The ratio of transverse to axial displacement (Poisson's ratio) typically decreased with age (9 to 31 months) as seen in V31 (-24.95%, p = 0.0134) and V32 (-20.7%, p = 0.0015). Overall, a ten day treatment with GH produced no global statistical change in elastic properties (p > 0.05). However, GH did minimize the age related differences that were measured for E22, E33, and V32 between the 9 and 31 month old groups essentially returning old bone to its youthful elastic state. These finding add orthotropic detail to the current understanding of changing cortical elastic properties during aging as well as providing a reference for further studies of GH.
Subject(s)
Aging/physiology , Bone Density/drug effects , Bone and Bones/physiology , Growth Hormone/pharmacology , Analysis of Variance , Animals , Anisotropy , Bone and Bones/diagnostic imaging , Bone and Bones/drug effects , Elasticity/drug effects , In Vitro Techniques , Male , Rats , Rats, Inbred F344 , Recombinant Proteins , Stress, Mechanical , UltrasonographyABSTRACT
The primary purpose of this study was to determine the impact of brief dietary restriction (DR; 5 or 20 days) on skeletal muscle glucose transport activity (GTA) of 24-month-old female Fischer 344 rats. Basal GTA of isolated epitrochlearis muscles was unaffected by DR. Insulin-stimulated GTA was significantly increased by DR only at 20 days (51%). We also assessed the influence of DR on energy sources (blood-borne and stored). An approximately 20% decline in glycemia occurred in each DR group, but plasma-free fatty acid and beta-hydroxybutyrate concentrations were unaffected. Plasma insulin was reduced by 50% after 20 days. Hepatic glycogen was rapidly mobilized (-69% at 5 days; -83% at 20 days). The depletions of visceral adipose stores was slower (no significant decline at 5 days; -30% at 20 days), but the eventual reduction accounts for a significant amount of energy. The results demonstrate that muscle from old rats can rapidly upregulate GTA in response to brief DR. The relative magnitude of this increase represents a substantial portion of the increases previously observed after prolonged DR.