The effect of insulin on free amino acid pools and protein synthesis in rat skeletal muscle in vitro.
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ABSTRACT:
SUBMITTER: Hider RC
PROVIDER: S-EPMC1178179 | biostudies-other | 1971 Dec
REPOSITORIES: biostudies-other
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Project description:Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1-3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) alpha-subunit (Ser(52))]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser(240/244)) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h (P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2 alpha phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA (P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery.
| S-EPMC2867366 | biostudies-literature
Project description:Rates of muscle protein synthesis were measured in vivo in tissues of post-absorptive young rats that were given intravenous infusions of various combinations of insulin and amino acids. In the absence of amino acid infusion, there was a steady rise in muscle protein synthesis with plasma insulin concentration up to 158 mu units/ml, but when a complete amino acids mixtures was included maximal rates were obtained at 20 mu units/ml. The effect of the complete mixture could be reproduced by a mixture of essential amino acids or of branched-chain amino acids, but not by a non-essential mixture, alanine, methionine or glutamine. It is concluded that amino acids, particularly the branched-chain ones, increase the sensitivity of muscle protein synthesis to insulin.
| S-EPMC1135117 | biostudies-other
Project description:Abstract Amino acid transporters (AATs) provide a link between amino acid availability and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activation although the direct relationship remains unclear. Previous studies in various cell types have used high insulin concentrations to determine the role of insulin on mTORC1 signaling and AAT mRNA abundance. However, this approach may limit applicability to human physiology. Therefore, we sought to determine the effect of insulin on mTORC1 signaling and whether lower insulin concentrations stimulate AAT mRNA abundance in muscle cells. We hypothesized that lower insulin concentrations would increase mRNA abundance of select AAT via an mTORC1-dependent mechanism in C2C12 myotubes. Insulin (0.5 nmol/L) significantly increased phosphorylation of the mTORC1 downstream effectors p70 ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). A low rapamycin dose (2.5 nmol/L) significantly reduced the insulin-(0.5 nmol/L) stimulated S6K1 and S6 phosphorylation. A high rapamycin dose (50 nmol/L) further reduced the insulin-(0.5 nmol/L) stimulated phosphorylation of S6K1 and S6. Insulin (0.5 nmol/L) increased mRNA abundance of SLC38A2/SNAT2 (P ? 0.043) and SLC7A5/LAT1 (P ? 0.021) at 240 min and SLC36A1/PAT1 (P = 0.039) at 30 min. High rapamycin prevented an increase in SLC38A2/SNAT2 (P = 0.075) and SLC36A1/PAT1 (P ? 0.06) mRNA abundance whereas both rapamycin doses prevented an increase in SLC7A5/LAT1 (P ? 0.902) mRNA abundance. We conclude that a low insulin concentration increases SLC7A5/LAT1 mRNA abundance in an mTORC1-dependent manner in skeletal muscle cells.
| S-EPMC4002227 | biostudies-literature
Project description:BackgroundDecreased insulin availability and high blood glucose levels, the hallmark features of poorly controlled diabetes, drive disease progression and are associated with decreased skeletal muscle mass. We have shown that mice with β-cell dysfunction and normal insulin sensitivity have decreased skeletal muscle mass. This project asks how insulin deficiency impacts on the structure and function of the remaining skeletal muscle in these animals.MethodsSkeletal muscle function was determined by measuring exercise capacity and specific muscle strength prior to and after insulin supplementation for 28 days in 12-week-old mice with conditional β-cell deletion of the ATP binding cassette transporters ABCA1 and ABCG1 (β-DKO mice). Abca1 and Abcg1 floxed (fl/fl) mice were used as controls. RNAseq was used to quantify changes in transcripts in soleus and extensor digitorum longus muscles. Skeletal muscle and mitochondrial morphology were assessed by transmission electron microscopy. Myofibrillar Ca2+ sensitivity and maximum isometric single muscle fibre force were assessed using MyoRobot biomechatronics technology.ResultsRNA transcripts were significantly altered in β-DKO mice compared with fl/fl controls (32 in extensor digitorum longus and 412 in soleus). Exercise capacity and muscle strength were significantly decreased in β-DKO mice compared with fl/fl controls (P = 0.012), and a loss of structural integrity was also observed in skeletal muscle from the β-DKO mice. Supplementation of β-DKO mice with insulin restored muscle integrity, strength and expression of 13 and 16 of the dysregulated transcripts in and extensor digitorum longus and soleus muscles, respectively.ConclusionsInsulin insufficiency due to β-cell dysfunction perturbs the structure and function of skeletal muscle. These adverse effects are rectified by insulin supplementation.
| S-EPMC10834341 | biostudies-literature
Project description:To identify insulin responsive genes in humans, in the first protocol, skeletal muscle biopsies from six non-diabetic subjects were obtained before and after a two-hour of hyperinsulinaemic (infusion rate 40 mU/m2/min) euglycemic clamp. A variable infusion of glucose (180 g/l) enriched with tritiated glucose (100 μCi/500 ml) maintained euglycemia during insulin infusion, with monitoring of plasma glucose concentration every 5 to 10 min during the basal and clamp periods using an automated glucose oxidation method (Glucose Analyzer 2, Beckman Instruments, Fullerton, CA). In the second protocol, skeletal muscle biopsies from six non-diabetic subjects were obtained before and after a 3-hour hyperinsulinemic (infusion rate 40 mU/m2/min) euglycemic clamp in order to increase the effects of insulin on gene expression. A variable infusion of glucose (180 g/l) was used to maintain euglycemia during insulin infusion with monitoring of plasma glucose concentration every 5 to 10 min using an automated glucose oxidation method (Glucose Analyzer 2, Beckman Instruments, Fullerton, CA). Keywords: dose response The muscle biopsies were obtained from the vastus lateralis muscle under local anesthesia before and after hyperinsulinaemic (infusion rate 40 mU/m2/min) euglycemic clamp
2007-03-01 | E-GEOD-7146 | biostudies-arrayexpress
Project description:The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol-requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well-developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca2+ storage, release, and uptake to control muscle excitation-contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca2+ handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.-Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K. C., Liu, Y., Hiess, F., Knollmann, B. C., Chen, S. R. W., Tang, J., Chen, X.-Z., Agellon, L. B., Michalak, M. Two pools of IRE1α in cardiac and skeletal muscle cells.
| S-EPMC6662970 | biostudies-literature
Project description:BackgroundNutrition administered as intermittent bolus feeds rather than continuously promotes greater protein synthesis rates in skeletal muscle and enhances lean growth in a neonatal piglet model. The molecular mechanisms responsible remain unclear.ObjectivesWe aimed to identify the insulin- and/or amino acid-signaling components involved in the enhanced stimulation of skeletal muscle by intermittent bolus compared to continuous feeding in neonatal pigs born at term.MethodsTerm piglets (2-3 days old) were fed equal amounts of sow milk replacer [12.8 g protein and 155 kcal/(kg body weight · d)] by orogastric tube as intermittent bolus meals every 4 hours (INT) or by continuous infusion (CTS). After 21 days, gastrocnemius muscle samples were collected from CTS, INT-0 (before a meal), and INT-60 (60 minutes after a meal) groups (n = 6/group). Insulin- and amino acid-signaling components relevant to mechanistic target of rapamycin complex (mTORC) 1 activation and protein translation were measured.ResultsPhosphorylation of the insulin receptor, IRS-1, PDK1, mTORC2, pan-Akt, Akt1, Akt2, and TSC2 was 106% to 273% higher in the skeletal muscle of INT-60 piglets than in INT-0 and CTS piglets (P < 0.05), but phosphorylation of PTEN, PP2A, Akt3, ERK1/2, and AMPK did not differ among groups, nor did abundances of PHLPP, SHIP2, and Ubl4A. The association of GATOR2 with Sestrin1/2, but not CASTOR1, was 51% to 52% lower in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05), but the abundances of SLC7A5/LAT1, SLC38A2/SNAT2, SLC38A9, Lamtor1/2, and V-ATPase did not differ. Associations of mTOR with RagA, RagC, and Rheb and phosphorylation of S6K1 and 4EBP1, but not eIF2α and eEF2, were 101% to 176% higher in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05).ConclusionsThe enhanced rates of muscle protein synthesis and growth with intermittent bolus compared to continuous feeding in a neonatal piglet model can be explained by enhanced activation of both the insulin- and amino acid-signaling pathways that regulate translation initiation.
| S-EPMC8417931 | biostudies-literature
Project description:To identify insulin responsive genes in humans, in the first protocol, skeletal muscle biopsies from six non-diabetic subjects were obtained before and after a two-hour of hyperinsulinaemic (infusion rate 40 mU/m2/min) euglycemic clamp. A variable infusion of glucose (180 g/l) enriched with tritiated glucose (100 μCi/500 ml) maintained euglycemia during insulin infusion, with monitoring of plasma glucose concentration every 5 to 10 min during the basal and clamp periods using an automated glucose oxidation method (Glucose Analyzer 2, Beckman Instruments, Fullerton, CA). In the second protocol, skeletal muscle biopsies from six non-diabetic subjects were obtained before and after a 3-hour hyperinsulinemic (infusion rate 40 mU/m2/min) euglycemic clamp in order to increase the effects of insulin on gene expression. A variable infusion of glucose (180 g/l) was used to maintain euglycemia during insulin infusion with monitoring of plasma glucose concentration every 5 to 10 min using an automated glucose oxidation method (Glucose Analyzer 2, Beckman Instruments, Fullerton, CA). Keywords: dose response
2007-03-01 | GSE7146 | GEO
Project description:Transient insulin deprivation with concurrent hyperglucagonemia is a catabolic state that can occur in type 1 diabetes. To evaluate glucagon's catabolic effect in the setting of its glucogenic effect, we measured the regional exchanges of amino acid metabolites (amino-metabolites) across muscle and splanchnic beds in 16 healthy humans during either somatostatin followed by glucagon or saline infusion alone. Despite a twofold or greater increase in the regional exchange of amino-metabolites by glucagon, whole-body kinetics and concentrations of amino acids (AA) remained stable. Glucagon increased the splanchnic uptake of not only gluconeogenic but also essential (EAA) AA while increasing their release from the muscle bed. Regional tracer-based kinetics and 3-methylhistidine release indicate that EAA release from muscle is likely caused by reduced protein synthesis rather than increased protein degradation. Furthermore, many metabolites known to affect insulin action and metabolism were altered by hyperglucagonemia including increase in branched-chain AA and keto acids of leucine and isoleucine in arterial plasma. Further, an increase in arterial concentrations of α-aminoadipic acid arising from increased conversion from lysine in the splanchnic bed was noted. These results demonstrate that hyperglucagonemia during hypoinsulinemia increases net muscle protein catabolism and substantially increases the exchange of amino metabolites across splanchnic and muscle beds.
| S-EPMC9490357 | biostudies-literature
Project description:Sentrin-specific protease (SENP) 2 has been suggested as a possible novel drug target for the treatment of obesity and type 2 diabetes mellitus after observations of a palmitate-induced increase in SENP2 that lead to increased fatty acid oxidation and improved insulin sensitivity in skeletal muscle cells from mice. However, no precedent research has examined the role of SENP2 in human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism as well as insulin sensitivity in human skeletal muscle using cultured primary human myotubes. Acute (4 h) oleic acid oxidation was reduced in SENP2-knockdown (SENP2-KD) cells compared to control cells, with no difference in uptake. After prelabeling (24 h) with oleic acid, total lipid content and incorporation into triacylglycerol was decreased, while incorporation into other lipids, as well as complete oxidation and β-oxidation was increased in SENP2-KD cells. Basal glucose uptake (i.e., not under insulin-stimulated conditions) was higher in SENP2-KD cells, whereas oxidation was similar to control myotubes. Further, basal glycogen synthesis was not different in SENP2-KD myotubes, but both insulin-stimulated glycogen synthesis and AktSer473 phosphorylation was completely blunted in SENP2-KD cells. In conclusion, SENP2 plays an important role in fatty acid and glucose metabolism in human myotubes. Interestingly, it also appears to have a pivotal role in regulating myotube insulin sensitivity. Future studies should examine the role of SENP2 in regulation of insulin sensitivity in other tissues and in vivo, defining the potential for SENP2 as a drug target.
| S-EPMC8663970 | biostudies-literature