Stimulatory Effect Of Insulin Research Articles

Insulin increases the expression of contractile phenotypic markers in airway smooth muscle

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.

Essential role of p38 MAPK for activation of skeletal muscle glucose transport by lithium

Lithium increases glucose transport and glycogen synthesis in insulin-sensitive cell lines and rat skeletal muscle, and has been used as a non-selective inhibitor of glycogen synthase kinase-3 (GSK-3). However, the molecular mechanisms underlying lithium action on glucose transport in mammalian skeletal muscle are unknown. Therefore, we examined the effects of lithium on glucose transport activity, glycogen synthesis, insulin signaling elements (insulin receptor (IR), Akt, and GSK-3β), and the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) in the absence or presence of insulin in isolated soleus muscle from lean Zucker rats. Lithium (10 mM LiCl) enhanced basal glucose transport by 62% (p < 0.05) and augmented net glycogen synthesis by 112% (p < 0.05). Whereas lithium did not affect basal IR tyrosine phosphorylation or Akt ser473 phosphorylation, it did enhance (41%, p < 0.05) basal GSK-3β ser9 phosphorylation. Lithium further enhanced (p < 0.05) the stimulatory effects of insulin on glucose transport (43%), glycogen synthesis (44%), and GSK-3β ser9 phosphorylation (13%). Lithium increased (p < 0.05) p38 MAPK phosphorylation both in the absence (37%) and presence (41%) of insulin. Importantly, selective inhibition of p38 MAPK (using 10 μM A304000) completely prevented the basal activation of glucose transport by lithium, and also significantly reduced (52%, p < 0.05) the lithium-induced enhancement of insulin-stimulated glucose transport. Theses results demonstrate that lithium enhances basal and insulin-stimulated glucose transport activity and glycogen synthesis in insulin-sensitive rat skeletal muscle, and that these effects are associated with a significant enhancement of GSK-3β phosphorylation. Importantly, we have documented an essential role of p38 MAPK phosphorylation in the action lithium on the glucose transport system in isolated mammalian skeletal muscle.

Molecular Basis of Insulin Action

Insulin is the main anabolic and anticatabolic hormone in mammals. The stimulatory effect of insulin on glucose uptake in muscle and adipose tissue is a consequence of the rapid translocation of GLUT4 glucose transporters from an intracellular site to the cell surface. The actions of insulin are initiated by hormone binding to its cell surface receptors. Insulin receptors are ligand-stimulated protein tyrosine kinases and phosphorylate a number of proteins, known as insulin receptor substrate proteins. Insulin resistance has been recognized as a main pathogenic factor in the development of type 2 diabetes, and has been associated with dyslipidemia, hypertension, endothelial dysfunction, inflammation and coagulative state. The current challenge is the study of impaired insulin signaling pathways leading to beta-cell dysfunction and its progression to type 2 diabetes, as well as control of chronic inflammation processes that may improve insulin action.

Metabolic Diapause in Pancreatic β-Cells Expressing a Gain-of-function Mutant of the Forkhead Protein Foxo1

Diabetes is associated with decreased pancreatic beta-cell function and mass. It is unclear whether diabetes treatment should aim at restoring beta-cell performance/mass or at inducing "beta-cell rest" to prevent further deterioration. The transcription factor Foxo1 protects beta-cells against oxidative stress induced by hyperglycemia and prevents beta-cell replication in insulin-resistant states. Here we show that these combined effects are associated with a concerted repression of genes involved in glycolysis, nitric-oxide synthesis, G protein-coupled receptor signaling, and ion transport. Conversely, Foxo1 increases expression of several neurotransmitter receptors and fails to regulate target genes predicted from Caenorhabditis elegans and Drosophila studies. Functional analyses show decreased glucose utilization and insulin secretion in beta-cells overexpressing Foxo1. We propose the definition of "metabolic diapause" for the changes induced by Foxo1 to protect beta-cells against oxidative stress. The data provide genetic underpinning for the concept of beta-cell rest as a treatment goal in diabetes.

Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.

Fine Tuning PDK1 Activity by Phosphorylation at Ser163

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.

Influence of 17β-estradiol and insulin on type II collagen and protein synthesis of articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Type II collagen is one main structural protein of articular cartilage matrix and its synthesis is increased by insulin in growth plate cartilage. Therefore, it was investigated if [ 3H]-proline incorporation and type II collagen synthesis (immunocytochemistry, ELISA) in female bovine articular chondrocytes are affected by 17β-estradiol and/or insulin. Articular chondrocytes were cultured in monolayers at 5% O 2 in medium containing serum for 5–9 days, followed by application of 10 −13 to 10 −9 M estradiol or 5 μg/ml insulin during a serum-free culture phase of 2–3 days. Immunostaining for type II collagen was strong in the serum-free culture phase whereas it was negative for type I collagen, indicating that cells did not dedifferentiate to fibroblast-like cells during culture in serum-free medium. Whereas insulin raised the proline incorporation and the type II collagen synthesis significantly, physiological doses of estradiol did not show significant effects. The stimulating effect of insulin on the [ 3H]-proline incorporation or the type II collagen synthesis was significantly suppressed after preincubation of cells with 10 −11 to 10 −9 M estradiol resembling an unfavorable effect for articular cartilage. The suppression was reversed if cells were incubated with 10 −11 to 10 −7 M tamoxifen or ICI 182,780 combined with 10 −11 or 10 −9 M estradiol followed by incubation with 5 μg/ml insulin, indicating an estrogen receptor-mediated process. Because the articular cartilage of diabetic patients is biomechanically less stable, further experiments are needed to clarify the role of estradiol and insulin in the metabolism of articular chondrocytes.

L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic β-cell line and protects from pro-inflammatory cytokine-induced apoptosis

Acute effects of nutrient stimuli on pancreatic beta-cell function are widely reported; however, the chronic effects of insulinotropic amino acids, such as L-alanine, on pancreatic beta-cell function and integrity are unknown. In the present study, the effects of prolonged exposure (24 h) to the amino acid L-alanine on insulin secretory function, gene expression and pro-inflammatory cytokine-induced apoptosis were studied using clonal BRIN-BD11 cells. Expression profiling of BRIN-BD11 cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the iNOS (inducible nitric oxide synthase) inhibitor NMA (N(G)-methyl-L-arginine acetate) or the iNOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL-1beta (interleukin-1beta), TNF-alpha (tumour necrosis factor-alpha) and IFN-gamma (interferon-gamma)] was additionally assessed by flow cytometry. Culture for 24 h with 10 mM L-alanine resulted in desensitization to the subsequent acute insulin stimulatory effects of L-alanine. This was accompanied by substantial changes in gene expression of BRIN-BD11 cells. Sixty-six genes were up-regulated >1.8-fold, including many involved in cellular signalling, metabolism, gene regulation, protein synthesis, apoptosis and the cellular stress response. Subsequent functional experiments confirmed that L-alanine provided protection of BRIN-BD11 cells from pro-inflammatory cytokine-induced apoptosis. Protection from apoptosis was mimicked by NMA or DPI suggesting L-alanine enhances intracellular antioxidant generation. These observations indicate important long-term effects of L-alanine in regulating gene expression, secretory function and the integrity of insulin-secreting cells. Specific amino acids may therefore play a key role in beta-cell function in vivo.

HIV Antiretroviral Agents Inhibit Protein Synthesis and Decrease Ribosomal Protein S6 and 4EBP1 Phosphorylation in C2C12 Myocytes

Combined antiretroviral drug regimens have promoted clinical, immunologic, and virologic improvements in AIDS patients. Nevertheless, these therapies are associated with derangements in lipid and carbohydrate metabolism. In this study, we examined the effects of a representative protease inhibitor (nelfinavir), a nonnucleoside reverse transcriptase inhibitor (nevirapine), and a nucleoside reverse transcriptase inhibitor (zidovudine) on protein synthesis in skeletal muscle cells. To examine these processes, C2C12 myocytes were treated with increasing concentrations of nelfinavir, nevirapine, or zidovudine for 1 or 2 days, and rates of protein synthesis were determined by measuring [35S]methionine/cysteine incorporation into cellular proteins. Treatment of myocytes with therapeutic concentrations of nelfinavir, nevirapine, or zidovudine for 48 hr decreased protein synthesis by 14-20%. An approximately 60% decline was observed in cells treated with higher concentrations of nevirapine or nelfinavir. In contrast, the basal rate of protein synthesis was not affected when cells were incubated with these compounds for 24 hr. Therapeutic concentrations of nelfinavir and nevirapine did not impair the anabolic effect of insulin on protein synthesis. However, zidovudine suppressed the stimulatory effect of insulin. The decreased protein synthesis induced by nelfinavir and zidovudine was associated with decreases in the phosphorylation of the S6 ribosomal protein (rpS6) and the repressor binding protein 4EBP1, while the inhibitory effect of nevirapine was mainly associated with a decline in phosphorylated 4EBP1. In conclusion, nelfinavir, nevirapine, and zidovudine treatments decreased protein synthesis in myocytes and this effect was correlated with a reduction in the phosphorylation level of proteins that regulate translation initiation.

Roles of Insulin Receptor Substrates in Insulin-Induced Stimulation of Renal Proximal Bicarbonate Absorption

Insulin resistance is frequently associated with hypertension, but the mechanism underlying this association remains speculative. Although insulin is known to modify renal tubular functions, little is known about roles of insulin receptor substrates (IRS) in the renal insulin actions. For clarifying these issues, the effects of insulin on the rate of bicarbonate absorption (JHCO3-) were compared in isolated renal proximal tubules from wild-type, IRS1-deficient (IRS1-/-), and IRS2-deficient (IRS2-/-) mice. In wild-type mice, physiologic concentrations of insulin significantly increased JHCO3-. This stimulation was completely inhibited by wortmannin and LY-294002, indicating that the phosphatidylinositol 3-kinase pathway mediates the insulin action. The stimulatory effect of insulin on JHCO3- was completely preserved in IRS1-/- mice but was significantly attenuated in IRS2-/- mice. Similarly, insulin-induced Akt phosphorylation was preserved in IRS1-/- mice but was markedly attenuated in IRS2-/- mice. Furthermore, insulin-induced tyrosine phosphorylation of IRS2 was more prominent than that of IRS1. These results indicate that IRS2 plays a major role in the stimulation of renal proximal absorption by insulin. Because defects at the level of IRS1 may underlie at least some forms of insulin resistance, sodium retention, facilitated by hyperinsulinemia through the IRS1-independent pathway, could be an important factor in pathogenesis of hypertension in insulin resistance.

Insulin signaling in rabbit preimplantation embryos

Addition of insulin during In-vitro-culture improves embryonic development by increasing cell proliferation and reducing apoptosis. The insulin receptor (IR) signaling involves two major pathways, the mitogenic-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3-K). In muscle and adipocyte cells insulin stimulates glucose uptake mainly by activation of the PI3-K pathway and translocation of glucose transporter (GLUT) 4. We have currently shown the expression of two insulin-responsive GLUT (4, 8) in rabbit embryos. The mechanism of insulin action on embryonic cells, however, is still not clear. In the present study the expression of IR-isoforms (IR-A, IR-B) and the activation of the PI3-kinase and the MAPK signaling pathways were studied in rabbit preimplantation embryos. The rabbit mRNA sequences for the coding region of IR-A and -B were cloned and sequenced. Using specific primer combinations for RT-PCR the expression of both isoforms was analyzed. The IR expression was detectable from the blastocyst stage onwards, with an IR-isoform specific pattern for embryoblast (Em) and trophoblast (Tr) cells. Whereas IR-A was found in both Em and Tr cells, the IR-B-isoform was detectable only in the trophoblast. In cultured blastocysts the effect of insulin stimulation (170 nM) was studied by analysis of Akt and Erk phosphorylation, GLUT4 translocation and measurement of 3-OMG glucose transport. Insulin stimulated the phosphorylation of the mitogenic activated protein kinase Erk1/2, but not the phosphorylation of the PI3-K dependent protein kinase Akt. Glucose uptake and GLUT4 localization were not influenced by insulin stimulation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control. The possible function of insulin-responsive GLUT isoforms and the cell specific expression of IR-A and -B in rabbit preimplantation embryos need further investigation.

Acute treatment with TNF-α attenuates insulin-stimulated protein synthesis in cultures of C2C12myotubes through a MEK1-sensitive mechanism

Insulin and TNF-alpha exert opposing effects on skeletal muscle protein synthesis that are mediated in part by the rapamycin-sensitive mammalian target of rapamycin (mTOR) pathway and the PD-98059-sensitive, extracellular signal-regulated kinase (ERK)1/2 pathway. The present study examined the separate and combined effects of insulin (INS), TNF, PD-98059, or dnMEK1 adenovirus on the translational control of protein synthesis in C(2)C(12) myotubes. Cultures were treated with INS, TNF, PD-98059, dnMEK1, or a combination of INS + TNF with PD-98059 or dnMEK1. INS stimulated protein synthesis, enhanced eIF4E.eIF4G association, and eIF4G phosphorylation and repressed eIF4E.4E-BP1 association vs. control. INS also promoted phosphorylation of ERK1/2, S6K1, and 4E-BP1 and dephosphorylation of eIF4E. TNF alone did not have an effect on protein synthesis (vs. control), eIF4E.eIF4G association, or the phosphorylation of eIF4G, S6K1, or 4E-BP1, although it transiently increased ERK1/2 and eIF4E phosphorylation. When myotubes were treated with TNF + INS, the cytokine blocked the insulin-induced stimulation of protein synthesis. This appeared to be due to an attenuation of insulin-stimulated eIF4E.eIF4G association, because other stimulatory effects of INS, e.g., phosphorylation of ERK1/2, 4E-BP1, S6K1, eIF4G, and eIF4E and eIF4E.4E-BP1 association, were unaffected. Finally, treatment of myotubes with PD-98059 or dnMEK1 adenovirus before TNF + INS addition resulted in a derepression of protein synthesis and the association of eIF4G with eIF4E. These findings suggest that TNF abrogates insulin-induced stimulation of protein synthesis in myotubes through a decrease in eIF4F complex assembly independently of S6K1 and 4E-BP1 signaling and dependently on a MEK1-sensitive signaling pathway.

The acute effects of insulin on the cardiorespiratory responses to hypoxia in streptozotocin‐induced diabetic rats

We examined whether or not streptozotocin (STZ)-induced diabetic rats, which have a lower heart rate (HR, beats min(-1)) than control rats, could maintain hypoxic ventilatory response. Twenty-six Wistar rats, which had been injected with STZ (60 mg kg(-1), EXP) or vehicle (0.1 m citrate buffer, CONT) intraperitoneally at 9 weeks of age, had their cardiorespiratory responses to normoxia and 12%O(2) examined after 5 weeks. Compared with CONT rats, EXP rats had a higher blood glucose [24 +/- 3 vs. 5 +/- 1 (mean +/- SD) mmol L(-1)], a lower body weight (320 +/- 23 vs. 432 +/- 24 g), lower HR (303 +/- 49 vs. 380 +/- 44 in normoxia, and 343 +/- 56 vs. 443 +/- 60 in hypoxia) and a lower mean arterial blood pressure (MAP) (89 +/- 6 vs. 102 +/- 10 mmHg in hypoxia). In contrast, both groups had similar values in ventilation (V(E)), V(E)-metabolic rate (MR) ratio and arterial blood gases (ABGs). In EXP rats, with an acute insulin supplement (i.v., 0.75 U h(-1) for 1.5-2 h), not only blood glucose, but also HR, and MAP were normalized as those obtained in CONT rats, and in hypoxia further increased without affecting V(E)-MR ratio and ABGs. Such acute cardiorespiratory stimulating effects of insulin could not be obtained in non-diabetic rats (n = 7, 355 +/- 24 g), in which euglycaemia (mean 6.4 mmol L(-1)) was maintained during the measurements. Our results suggest that, in STZ-induced diabetic rats: (1) ventilation is hardly suppressed by hyperglycaemia, (2) cardiorespiratory responses can be acutely stimulated by short insulin injection, and (3) the effects, including those through acute blood glucose normalization, are possibly specific for the diabetic impairments.

Effect of CCK-8 on insulin-induced hyperphagia and hypothalamic orexigenic neuropeptide expression in the rat

The influence of cholecystokinin octapeptide (CCK-8) on normal and insulin-induced feeding and expression of orexigenic hypothalamic neuropeptides was investigated in male rats. CCK-8, administered during meals (4 μg/kg) or continuously (32 μg/kg over 60 min), blunted the stimulating effect of insulin (50 IU/kg) on feeding by reducing meal size (−60%; P < 0.05 or −86%; P < 0.0001, respectively). Rats without access to food and injected with IP insulin (50 IU/kg) showed increased hypothalamic mRNA levels of orexin (+30%; P < 0.05) and melanin-concentrating hormone (+52%; P < 0.05), as compared with ad libitum-fed and saline-injected control rats. Continuous IP infusion of CCK-8 (32 μg/kg) blunted these increases. Our results suggest that both orexin and melanin-concentrating hormone participate in the response to insulin hypoglycemia without food being present; these neurons may be involved in mechanisms related to insulin-induced hyperphagia. Signals triggered by peripheral CCK-8 act to decrease the expression of orexin and melanin-concentrating hormone. This may be associated with a reduction in hyperphagia.

Insulin Inhibits Platelet-derived Growth Factor-induced Cell Proliferation

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

Role of calcium in the secretion of leptin from white adipocytes.

The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B, and W-13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus calcium appears to be specifically required for glucose utilization. On the other hand, (45)Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A-23187, and ATP) increased (45)Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A-23187, calcium, and thapsigargin inhibited ISLS, even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS, mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake, and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits ISLS at a level independent of glucose metabolism. Thus calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.

Attenuation of insulin-Mediated pressor effect and nitric oxide release in rats with fructose-Induced insulin resistance

Hyperinsulinemia and insulin resistance frequently coexist and have been implicated in the pathogenesis of hypertension. This study aimed to identify the specific effect of hyperinsulinemia on blood pressure and nitric oxide (NO) release in fructose-induced hyperinsulinemic, insulin-resistant rats. Male Sprague-Dawley rats were fed either standard chow or a 60% fructose-enriched diet for 8 weeks before acute study. After basal period, somatostatin (1.3 microg/kg/min) and a variable glucose infusion (to maintain euglycemia) were given intravenously to all groups of rats. In control rats (C) and fructose-fed rats (F) with insulin infusion, insulin (4 mU/kg/min) was given to create a similar hyperinsulinemic condition. In C and F without insulin infusion, a vehicle instead of insulin was infused to produce a hypoinsulinemic state. In C, somatostatin reduced plasma insulin level but did not alter mean arterial pressure (MAP) and heart rate. Insulin infusion significantly increased MAP and NOx (sum of nitrate and nitrite) levels after 90 min and thereafter the elevated MAP and NOx responses were sustained throughout the study. In the basal period, F exhibited significantly higher MAP and plasma insulin levels than C. The index of insulin sensitivity (M) was significantly lower in F than in C with insulin infusion. Somatostatin significantly reduced plasma insulin level but did not affect MAP and NOx level in F. The stimulatory effects of insulin on MAP and NO levels were significantly smaller in F than in C. In conclusion, acute insulin-induced pressor response and NO release were attenuated in rats with fructose-treated insulin resistance, suggesting that hyperinsulinemia-associated attenuation of NO production contributes, at least partly, to the development of fructose-induced hypertension in rats.

Stimulatory Effect of Insulin and Epidermal Growth Factor on Activity of Protein Kinase A, Glucose-6-Phosphate Dehydrogenase, and Glycogen Synthase in Skeletal Muscles of Chick Embryos and Chickens

Insulin is able to produce two types of regulatory effects on muscles—metabolic and growth stimulating. Study of the effect of insulin and epidermal growth factor (EGF) on activity of cAMP-dependent protein kinase (PKA), glucose-6-phosphate dehydrogenase (G-6PDH), a starting enzyme of pentosephosphate pathway (PPP), and glycogen synthase (GS), a key enzyme of the glycogen synthesis, has shown that both types of the insulin effects do not arise simultaneously in the course of embryogenesis. The growth-stimulating effect mediated by adenylyl cyclase—cAMP is revealed since the 10th embryonal day. It was established for the first time that insulin could participate in growth stimulation by activating PKA in vivo and in vitro in muscles of the 10–14-day old embryos and the 8–10-day old chickens. The stimulating effect of insulin on G-6PDH activity is revealed since the same embryonal period and gradually increases. Insulin in vivo and in vitro simulates the glycogen synthase activity by increasing its conversion from non-active to active (GS-I) form only in the 15-day old embryos and in chickens. The stimulating effects of insulin and EGF on both G-6PDH activity (in embryos and chickens) and GS (in chickens) was shown to blocked by selective inhibitors of tyrosine kinases, thyrphostin 47 and genestein, in the dose-dependent manner, which indicates involvement of receptor of the tyrosine kinase type in these effects. The complex of the established facts permits concluding that insulin at early embryonal stages stimulates in the chicken muscles the PKA and G-6PDH activities involved in action of this hormone on growth, which is especially pronounced at the stage of myoblast proliferation. Meanwhile, the metabolic insulin effect (stimulation of the glycogen synthase system) appears in the second half of embryonal period and coincides in time with the period of muscle cell differentiation and active muscle contractures.

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.

Insulin-Regulated Adenylyl Cyclase Signaling System in Cell Culture of Mouse Fibroblasts of L Line

In terms of the subsequent study of the earlier discovered adenylyl cyclase signaling mechanism of insulin action [1, 2], we studied participation in it of various isoforms of protein kinase C. As object of study, a culture of mouse fibroblast cells of the L line (LSM subline) was chosen. It has been shown that insulin and a non-hydrolyzed analog of GTP, Gpp[NH]p, stimulate the adenylyl cyclase (AC) activity in these cells both individually and in combination. Activators of phorbol-sensitive isoforms of protein kinase C, diacylglycerol and phorbol ester (phorbol-12-myristate-13-acetate) at their concentrations of 1–100 nM stimulate basal activity of AC. In their presence, a significant decrease of stimulating effects of insulin and Gpp[NH]p or their complete disappearance are observed. Calphostin C (1–100 nM), an inhibitor of both phorbol-sensitive and atypical, phorbol-insensitive isoforms of protein kinase C, somewhat increased the basal AC activity. However, the stimulating effects of insulin and Gpp[NH]p in the presence of calphostin C decreased markedly. On the whole, the obtained data allow us to suggest participation of various isoforms of protein kinase C (sensitive and insensitive to the phorbol esters) in regulation of the process of the insulin signal transduction in mouse fibroblasts through the adenylyl cyclase signaling mechanism. Thus, mechanisms of functioning of the insulin-regulated AC-system in fibroblasts, representatives of connective tissue cells, are similar to those that we described earlier in muscle tissues of vertebrate and invertebrate animals. Taken together, these data indicate the absence of tissue- and species-specificity in functioning of the insulin-regulated AC system and its wide spread in tissues of different animals.