Differential effects of insulin-like growth factor I (IGF-I) and IGF-binding protein-1 on protein metabolism in human skeletal muscle cells.

Insulin-like growth factor-binding protein-1 (BP-1) is a multifunctional protein that binds IGF-I in solution and integrins on the cell surface. BP-1 is overexpressed during catabolic illnesses, and the protein accumulates in skeletal muscle. To define a potential physiological role for BP-1 in regulating muscle protein balance, we have examined the effect of IGF-I and BP-1 on protein synthesis and degradation in human skeletal muscle cells. IGF-I-stimulated protein synthesis by 20%, and this was completely inhibited by either phosphorylated or nonphosphorylated BP-1. Half-maximal inhibition of protein synthesis occurred at a molar ratio of BP-1 to IGF-I of 1.5:1. BP-1 failed to form a complex with a truncated form of IGF-I (desIGF-I), and consequently, BP-1 failed to inhibit the ability of desIGF-I to stimulate protein synthesis. IGF-I and BP-1 dose-dependently inhibited protein degradation individually, and both BP-1 phosphovariants failed to block the ability of IGF-I to do the same. Blocking integrin receptor occupancy with the integrin antagonist echistatin blunted the ability of BP-1 to inhibit protein degradation, but had no significant effect on IGF-I-mediated changes in protein synthesis or degradation. The extracellular matrix protein vitronectin also inhibited protein degradation, but vitronectin receptor antibodies failed to block BP-1 action. In contrast, antibodies to the beta1 integrin subunit blocked BP-1-mediated inhibition of protein degradation. Rapamycin inhibited IGF-I-dependent protein synthesis, but not the ability of IGF-I to inhibit proteolysis. In contrast, rapamycin completely blocked the ability of BP-1 to inhibit proteolysis. Our results demonstrate that BP-1 inhibits IGF-I-mediated protein synthesis by binding to IGF-I. BP-1, acting independently of IGF-I, inhibits protein degradation. The IGF-independent response occurs via beta1 integrin binding and stimulation of a rapamycin-sensitive signal transduction pathway.     

Effects of the long-acting insulin analog insulin glargine on cultured human skeletal muscle cells: comparisons to insulin and IGF-I.

The aim of this study was to determine whether the long-acting insulin analog, insulin glargine, behaves like human insulin for metabolic and mitogenic responses in differentiated cultured human skeletal muscle cells from nondiabetic and diabetic subjects. Human insulin and insulin glargine were equipotent in their ability to compete for [(125)I]insulin binding. Insulin glargine displaced [(125)I]IGF-I from the IGF-I-binding site with approximately 0.5% the potency of IGF-I. In nondiabetic muscle cells, all three ligands stimulated glucose uptake similarly, whereas the sensitivity of glucose uptake was greatest in response to IGF-I and lower and equal for human insulin and insulin glargine. In diabetic muscle cells, the final responsiveness of glucose uptake was greatest for IGF-I and equivalent for human insulin and insulin glargine; sensitivities were the same as those for nondiabetic cells. Thymidine uptake into DNA was stimulated foremost by IGF-I, whereas human insulin and insulin glargine showed equivalent, but greatly reduced, sensitivities and potencies (<1% IGF-I). Stimulation of Akt phosphorylation was slightly more responsive to IGF-I compared with human insulin and insulin glargine, with sensitivities similar to glucose uptake stimulation. We conclude that in human skeletal muscle cells, insulin glargine is equivalent to human insulin for metabolic responses and does not display augmented mitogenic effects.     

Differential effects of IGF-I, IGF-II and insulin in human preadipocytes and adipocytes--role of insulin and IGF-I receptors

We compared insulin and IGF effects in adipocytes expressing IR (insulin receptors), and preadipocytes expressing IR and IGF-IR (IGF-I receptors). Treatment of adipocytes with insulin, IGF-II or IGF-I resulted in phosphorylation of IR. Order of potency was insulin>IGF-II>IGF-I. In preadipocytes IR, IGF-IR and insulin/IGF-I hybrid receptors (HR) were detected. Treatment of preadipocytes with IGF-I and IGF-II 10(-8)M resulted in activation of IGF-IR and IR whereas insulin was more potent in activating IR, with no effect on IGF-IR. In adipocytes glucose transport was 100-fold more sensitive to insulin than to IGFs and the maximal effect was higher with insulin. In preadipocytes glucose accumulation and DNA synthesis was equally sensitive to insulin and IGFs but the maximal effect was higher with IGF-I. In conclusion, insulin and IGF-I activate their cognate receptors and IGF-I also HR. IGF-II activates IR, IGF-IR and HR. Insulin and IGF-I are partial agonists to each other's receptors.     

IGF-I and insulin induce different intracellular calcium signals in skeletal muscle cells

We studied the effect of IGF-I and insulin on intracellular Ca(2+) in primary cultured myotubes. IGF-I induced a fast and transient Ca(2+) increase, measured as fluo-3 fluorescence. This response was blocked by both genistein and AG538. IGF-I induced a fast inositol-1,4,5-trisphosphate (IP(3)) increase, kinetically similar to the Ca(2+) rise. The Ca(2+) signal was blocked by inhibitors of the IP(3) pathway. On the other hand, insulin produced a fast (<1 s) and transient Ca(2+) increase. Insulin-induced Ca(2+) increase was blocked in Ca(2+)-free medium and by either nifedipine or ryanodine. In the normal muscle NLT cell line, the Ca(2+ )signals induced by both hormones resemble those of primary myotubes. GLT cells, lacking the alpha1-subunit of dihydropyridine receptor (DHPR), responded to IGF-I but not to insulin, while GLT cells transfected with the alpha1-subunit of DHPR reacted to both hormones. Moreover, dyspedic muscle cells, lacking ryanodine receptors, responded to IGF-I as NLT cells, however they show no insulin-induced calcium increase. Moreover, G-protein inhibitors, pertussis toxin (PTX) and GDPbetaS, blocked the insulin-induced Ca(2+) increase without major modification of the response to IGF-I. The different intracellular Ca(2+) patterns produced by IGF-I and insulin may improve our understanding of the early action mechanisms for these hormones in skeletal muscle cells.     

Impaired insulin action on skeletal muscle metabolism in essential hypertension

Previous studies have shown that essential hypertension is frequently associated with insulin resistance. The tissues responsible for this metabolic alteration have not been defined. We tested the hypothesis that skeletal muscle is the site of insulin resistance of essential hypertension with the use of the perfused forearm technique. Eight hypertensive (age 42 +/- 3 years, body mass index 27 +/- 1 kg/m2, intra-arterial mean blood pressure 126 +/- 4 mm Hg) and seven normotensive (age 48 +/- 3 years, body mass index 26 +/- 1 kg/m2, mean blood pressure 95 +/- 4 mm Hg) male volunteers were studied. After glucose ingestion (40 g/m2), normal glucose tolerance in the patients was maintained at the expense of a heightened plasma insulin response, suggesting the presence of insulin resistance. During graded, local (intra-arterial) hyperinsulinemia encompassing the physiological range (12-120 milliunits/l), glucose uptake by forearm tissues was significantly (p less than 0.03) reduced in the hypertensive subjects as compared with the controls at each of five insulin steps, by 43% on the average. In addition, forearm lactate and pyruvate release were significantly less stimulated in the hypertensive than in the normotensive group (p less than 0.01 for both), presumably as a consequence of the decreased glucose influx. Forearm exchange of oxygen, carbon dioxide, lipid substrates (free fatty acids, glycerol, and beta-hydroxybutyrate), and potassium were similar in the hypertensive and normotensive groups in the basal state. Insulin had no effect on oxygen consumption, carbon dioxide production, and respiratory quotient in either study group, whereas it stimulated free fatty acids, glycerol, and potassium uptake to the same extent in the hypertensive and normotensive groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of receptors for IGF-I and insulin; evidence for hybrid insulin/IGF-I receptor in human coronary artery endothelial cells.

OBJECTIVE: Coronary artery disease is a prevalent cause of morbidity and mortality in diabetes. Little is known about insulin-like growth factor-I receptors (IGF-IR) and insulin receptors (IR) in human coronary endothelium. Our aim was to characterize IGF-IR and IR in human coronary artery endothelial cells (HCAEC). DESIGN: Cultured human coronary artery endothelial cells were used. Gene expression was measured by quantitative real-time RT-PCR analysis and receptor affinity by ligand binding. Receptor protein, phosphorylation of IGF-IR and IR beta-subunit as well as the presence of hybrid insulin receptor/Insulin-like growth factor-I receptor (Hybrid IR/IGF-IR) was analyzed by immunoprecipitation and Western blot. Postreceptor effects of insulin and IGF-I were assed by (3)H-thymidine incorporation. RESULTS: The gene expression of IGF-IR was several folds higher than that of IR. and insulin receptor isoform A (IR-A) was 20-fold more expressed than insulin receptor isoform B (IR-B) in HCAEC. The specific binding of (125)I-IGF-I was higher than that of (125)I-insulin. Insulin and the new long acting insulin analog, glargine, interacted with the IGF-IR with over thousand and 100-fold less potency than IGF-I itself, whereas IGF-II had 6 times lower potency than IGF-I. Phosphorylation of the IGF-IR beta-subunit was obtained by concentrations of 10(-10)-10(-8)M IGF-I, 10(-6)M of insulin, inconsistently by 10(-8)M insulin and not at all by 10(-10)-10(-9)M insulin. The IR beta-subunit was phosphorylated by insulin and IGF-I at concentrations of 10(-9)-10(-8)M. When immunoprecipitating with specific monoclonal anti-IR or anti-IGF-IR alpha-subunit antibodies we found bands situated in slightly different positions suggesting the presence of Hybrid IR/IGF-IR. IGF-I, IGF-II and insulin (10(-9)-10(-7)M) had no significant effect on (3)H-thymidine incorporation into DNA. CONCLUSIONS: Human coronary endothelial cells express more IGF-IR than IR, mainly IR-A, and also Hybrid IR/IGF-IR. Both IGF-I and insulin phosphorylate their receptors, but only IGF-I seems to phosphorylate Hybrid IR/IGF-IR. Our study provides experimental evidence for a possible role of IGF-IR, IR and Hybrid IR/IGF-IR in human coronary artery endothelial cells.     

Intracellular fatty acid metabolism in skeletal muscle and insulin resistance

Triglyceride accumulation in skeletal muscle is increased in subjects with insulin resistance. Increased intracellular lipolysis from stored triglyceride may induce insulin resistance in skeletal muscle by activating the glucose-fatty acid cycle. However, inconsistent with this hypothesis, intracellular lipolysis from skeletal muscle is decreased in high fat-fed, insulin resistant rats. Therefore, it is suggested that an increase in triglyceride accumulation is the result of decreased mitochondrial fatty acid oxidation in the cells. As evidence, fenofibrate (a PPARalpha activator), rosiglitazone (a PPARgamma activator) and alpha-lipoic acid completely prevented the development of diabetes in obese diabetes-prone rats. All three drugs increased fatty acid oxidation and decreased triglyceride accumulation in skeletal muscle. Administration of ALA activated AMPK and increased fatty acid oxidation. It is suggested that decreased fatty acid oxidation in skeletal muscle is one of the major factors leading to an accumulation of lipid metabolites and insulin resistance.     

Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo

The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.     

Effect of thyroxine administration on the IGF/IGF binding protein system in neonatal and adult thyroidectomized rats

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.     

Potential role of skeletal muscle glucose metabolism on the regulation of insulin secretion

Pancreatic beta cells sense glucose flux and release as much insulin as required in order to maintain glycaemia within a narrow range. Insulin secretion is regulated by many factors including glucose, incretins, and sympathetic and parasympathetic tones among other physiological factors. To identify the mechanisms linking obesity‐related insulin resistance with impaired insulin secretion represents a central challenge. Recently, it has been argued that a crosstalk between skeletal muscle and the pancreas may regulate insulin secretion. Considering that skeletal muscle is the largest organ in non‐obese subjects and a major site of insulin‐ and exercise‐stimulated glucose disposal, it appears plausible that muscle might interact with the pancreas and modulate insulin secretion for appropriate peripheral intracellular glucose utilization. There is growing evidence that muscle can secrete so‐called myokines that can have auto/para/endocrine actions. Although it is unclear in which direction they act, interleukin‐6 seems to be a possible muscle‐derived candidate protein mediating such inter‐organ communication. We herein review some of the putative skeletal muscle‐derived factors mediating this interaction. In addition, the evidence coming from in vitro, animal and human studies that support such inter‐organ crosstalk is thoroughly discussed.     

Bioactives of Artemisia dracunculus L enhance cellular insulin signaling in primary human skeletal muscle culture

An alcoholic extract of Artemisia dracunculus L (PMI 5011) has been shown to decrease glucose and improve insulin levels in animal models, suggesting an ability to enhance insulin sensitivity. We sought to assess the cellular mechanism by which this botanical affects carbohydrate metabolism in primary human skeletal muscle culture. We measured basal and insulin-stimulated glucose uptake, glycogen accumulation, phosphoinositide 3 (PI-3) kinase activity, and Akt phosphorylation in primary skeletal muscle culture from subjects with type 2 diabetes mellitus incubated with or without various concentrations of PMI 5011. We also analyzed the abundance of insulin receptor signaling proteins, for example, IRS-1, IRS-2, and PI-3 kinase. Glucose uptake was significantly increased in the presence of increasing concentrations of PMI 5011. In addition, glycogen accumulation, observed to be decreased with increasing free fatty acid levels, was partially restored with PMI 5011. PMI 5011 treatment did not appear to significantly affect protein abundance for IRS-1, IRS-2, PI-3 kinase, Akt, insulin receptor, or Glut-4. However, PMI 5011 significantly decreased levels of a specific protein tyrosine phosphatase, that is, PTP1B. Time course studies confirmed that protein abundance of PTP1B decreases in the presence of PMI 5011. The cellular mechanism of action to explain the effects by which an alcoholic extract of A dracunculus L improves carbohydrate metabolism on a clinical level may be secondary to enhancing insulin receptor signaling and modulating levels of a specific protein tyrosine phosphatase, that is, PTP1B.     

Supraphysiological hyperinsulinaemia is necessary to stimulate skeletal muscle protein anabolism in older adults: evidence of a true age-related insulin resistance of muscle protein metabolism

Aims/hypothesis  The physiological increase in muscle protein anabolism induced by insulin is blunted in healthy, glucose-tolerant older adults. We hypothesised that the age-related defect in muscle protein anabolism is a true insulin resistance state and can be overridden by supraphysiological hyperinsulinaemia. Methods  We used dye dilution, stable isotopic and immunoblotting techniques to measure leg blood flow, muscle protein synthesis, protein kinase B/mammalian target of rapamycin (Akt/mTOR) signalling, and amino acid kinetics in 14 healthy, glucose-tolerant older volunteers at baseline, and during an insulin infusion at postprandial (PD, 0.15 mU min⁻¹ 100 ml⁻¹) or supraphysiologically high (HD, 0.30 mU min⁻¹ 100 ml⁻¹) doses. Results  Leg blood flow, muscle protein synthesis, and Akt/mTOR signalling were not different at baseline. During hyperinsulinaemia, leg blood flow (p < 0.01) and muscle protein synthesis increased in the HD group only (PD [%/h]: from 0.063 ± 0.006 to 0.060 ± 0.005; HD [%/h]: from 0.061 ± 0.007 to 0.098 ± 0.007; p < 0.01). Muscle Akt phosphorylation increased in both groups, but the increase tended to be greater in the HD group (p = 0.07). The level of p70 ribosomal S6 kinase 1 (S6K1) phosphorylation increased in the HD group only (p < 0.05). Net amino acid balance across the leg improved in both groups, but a net anabolic effect was observed only in the HD group (p < 0.05). Conclusions/interpretation  We conclude that supraphysiological hyperinsulinaemia is necessary to stimulate muscle protein synthesis and anabolic signalling in healthy older individuals, suggesting the existence of a true age-related insulin resistance of muscle protein metabolism.     

Tissue composition affects measures of postabsorptive human skeletal muscle metabolism: comparison across genders

Despite clear anthropomorphic differences, gender differences in human skeletal muscle protein and carbohydrate metabolism have not been carefully examined. We compared postabsorptive forearm glucose, oxygen, and lactate balances and forearm protein kinetics between 40 male and 36 female subjects. Forearm composition was measured in a subset of 17 subjects (8 males and 9 females) using multislice magnetic resonance imaging. Oxygen uptake, net phenylalanine release, and estimated rates of forearm protein synthesis and degradation were greater in male than in female subjects when expressed as the rate per 100 mL forearm volume (P < 0.05). In males, however, muscle accounted for 58% of forearm volume, compared with 46% in females (P < 0.001). When phenylalanine balance, protein degradation and synthesis, and glucose and oxygen uptake were expressed per 100 mL forearm muscle, there were no significant differences across gender. Likewise, the extraction fractions for oxygen, glucose, phenylalanine, and labeled phenylalanine were comparable in males and females. We conclude that cross-gender comparisons of metabolic variables must accommodate differences in tissue composition. These data indicate that in the postabsorptive state, skeletal muscle metabolism of glucose, protein, and oxygen do not differ by gender in healthy young humans.     

Effect of glucose and insulin on triacylglycerol metabolism in isolated normal and diabetic skeletal muscle

The effects of insulin and glucose on triacylglycerol (TG) metabolism in normal and diabetic isolated skeletal muscle were investigated in this study. Intracellular TG was continuously synthesized and hydrolyzed in both normal and diabetic skeletal muscle. In the absence of insulin and glucose, normal and diabetic skeletal muscle TG content and synthesis were decreased. In contrast, in the presence of insulin and glucose, the normal and diabetic TG contents were unchanged and triacylglycerol synthesis was increased as compared with the respective control values. However, insulin and glucose increased intramuscular TG content to a greater extent than could be accounted for by their stimulation of TG synthesis, indicating that insulin and glucose appear to inhibit TG hydrolysis in diabetic muscle, as well as in normal muscle. In addition, these data suggest that diabetes causes a defect in the ability of insulin and glucose to stimulate TG synthesis, as the increase in diabetic muscle TG synthesis in the presence of insulin and glucose was less than in normal muscle.     

Relative effects of pregnancy and corticosterone administration on skeletal muscle metabolism in the rat

The effects of pregnancy and corticosterone treatment on skeletal muscle metabolism were compared using a noncyclically perfused rat hindlimb preparation. Animal groups studied included 3-week-pregnant animals, rats injected with corticosterone (7.5 mg/100 g BW . day) for 3 days, and age-matched control rats. All were fasted 24 h before perfusion. In baseline perfusions devoid of insulin, no differences were observed among the groups with respect to muscle glucose uptake and the release of lactate, pyruvate, and glycerol. Baseline alanine and phenylalanine release were significantly increased in corticosterone-treated rats compared to values observed in control and pregnant animals. In perfusions with 100 or 500 microU/ml insulin, glucose uptake was increased 5-fold above baseline uptake in the control group. At 100 microU/ml insulin concentrations, glucose uptake in pregnant and corticosterone-treated rats achieved only 50% of the increase seen in control experiments. With 500 microU/ml insulin, glucose uptake was decreased 20% in pregnant animals and 40% in rats receiving corticosterone relative to control values. Alanine release was significantly reduced below baseline after the administration of 500 microU/ml insulin in control rats. In these rats, phenylalanine release, an index of net protein degradation, also was reduced with both 100 and 500 microU/ml insulin. Similar insulin concentrations did not suppress the efflux of either amino acid below baseline in pregnant or corticosterone-treated groups. Corticosterone administration to nonpregnant rats appears to duplicate changes in skeletal muscle metabolism that occur during late rat pregnancy. Insulin resistance in both states is manifested by a decrease in insulin-stimulated glucose uptake and an inability of insulin to suppress net muscle proteolysis. Since plasma free glucocorticoid concentrations are increased in late human as well as rat gestation, these steroids may have an important role in the development of insulin resistance at skeletal muscle sites in this state.