Activation of p38 mitogen-activated protein kinase alpha and beta by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPKalpha and p38 MAPKbeta isoforms were found to be activated (2.1- and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPKalpha and p38 MAPKbeta. The activity of both isoforms was inhibited in vitro by 10 micromol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin- or contraction-stimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 micromol/l SB203580. These results show that p38 MAPKalpha and -beta isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.     

Glucagon-like peptide 1 recruits microvasculature and increases glucose use in muscle via a nitric oxide-dependent mechanism

Glucagon-like peptide 1 (GLP-1) increases tissue glucose uptake and causes vasodilation independent of insulin. We examined the effect of GLP-1 on muscle microvasculature and glucose uptake. After confirming that GLP-1 potently stimulates nitric oxide (NO) synthase (NOS) phosphorylation in endothelial cells, overnight-fasted adult male rats received continuous GLP-1 infusion (30 pmol/kg/min) for 2 h plus or minus NOS inhibition. Muscle microvascular blood volume (MBV), microvascular blood flow velocity (MFV), and microvascular blood flow (MBF) were determined. Additional rats received GLP-1 or saline for 30 min and muscle insulin clearance/uptake was determined. GLP-1 infusion acutely increased muscle MBV (P < 0.04) within 30 min without altering MFV or femoral blood flow. This effect persisted throughout the 120-min infusion period, leading to a greater than twofold increase in muscle MBF (P < 0.02). These changes were paralleled with increases in plasma NO levels, muscle interstitial oxygen saturation, hind leg glucose extraction, and muscle insulin clearance/uptake. NOS inhibition blocked GLP-1-mediated increases in muscle MBV, glucose disposal, NO production, and muscle insulin clearance/uptake. In conclusion, GLP-1 acutely recruits microvasculature and increases basal glucose uptake in muscle via a NO-dependent mechanism. Thus, GLP-1 may afford potential to improve muscle insulin action by expanding microvascular endothelial surface area.     

Nitric oxide synthase inhibition reduces glucose uptake during exercise in individuals with type 2 diabetes more than in control subjects

Nitric oxide (NO) synthase inhibition reduces leg glucose uptake during cycling without reducing leg blood flow (LBF) in young, healthy individuals. This study sought to determine the role of NO in glucose uptake during exercise in individuals with type 2 diabetes. Nine men with type 2 diabetes and nine control subjects matched for age, sex, peak pulmonary oxygen uptake (VO(2) peak), and weight completed two 25-min bouts of cycling exercise at 60 +/- 2% VO(2) peak, separated by 90 min. N(G)-monomethyl-L-arginine (L-NMMA) (total dose 6 mg/kg) or placebo was administered into the femoral artery for the final 15 min of exercise in a counterbalanced, blinded, crossover design. LBF was measured by thermodilution in the femoral vein, and leg glucose uptake was calculated as the product of LBF and femoral arteriovenous glucose difference. During exercise with placebo, glucose uptake was not different between control subjects and individuals with diabetes; however, LBF was lower and arterial plasma glucose and insulin levels were higher in individuals with diabetes. L-NMMA had no effect on LBF or arterial plasma glucose and insulin concentrations during exercise in both groups. L-NMMA significantly reduced leg glucose uptake in both groups, with a significantly greater reduction (P = 0.04) in the diabetic group (75 +/- 13%, 5 min after L-NMMA) compared with the control group (34 +/- 14%, 5 min after L-NMMA). These data suggest a greater reliance on NO for glucose uptake during exercise in individuals with type 2 diabetes compared with control subjects.     

Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation

Our laboratory has demonstrated that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity in vascular smooth muscle cells (VSMCs). In this study, we examined whether diabetes is accompanied by alterations in MBP activation and elucidated the components of the signaling pathway that mediate the effects of diabetes. VSMCs isolated from Goto-Kakizaki (GK) diabetic rats (a model for type 2 diabetes) exhibited marked impairment in MBP activation by insulin that was accompanied by failure of insulin to decrease the phosphorylation of a regulatory myosin-bound subunit (MBS) of MBP and inhibit Rho kinase activity resulting in increased myosin light-chain (MLC)20 phosphorylation and VSMC contraction. In VSMCs isolated from control rats, insulin inactivates Rho kinase and decreases MBS phosphorylation, leading to MBP activation. In addition to this pathway, insulin also appears to activate MBP by stimulating the phosphatidylinositol (PI) 3-kinase/nitric oxide (NO)/cGMP signaling pathway because treatment with the synthetic inhibitors of PI 3-kinase, NO synthase (NOS), and cGMP all blocked insulin's effect on MBP activation, whereas cGMP agonists and sodium nitroprusside (SNP) mimicked insulin's effect on MBP activation. VSMCs from diabetic GK rats exhibit reductions in insulin-mediated induction of inducible NOS protein expression and cGMP generation but normal MBP activation in response to SNP and cGMP agonist. This observation led us to examine the effect of diabetes on the activation status of the upstream insulin-signaling components. Although GK diabetes did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor or its content, insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was severely impaired. This was accompanied by marked reductions in IRS-1-associated PI 3-kinase activity. We conclude that insulin stimulates MBP via its regulatory subunit, MBS, by inactivating Rho kinase and stimulating NO/cGMP signaling via PI 3-kinase as part of a complex signaling network that controls MLC20 phosphorylation and VSMC contraction. Defective signaling via Rho kinase and the IRS-1/PI 3-kinase/NOS/cGMP pathway may mediate the inhibitory effects of hyperglycemia and diabetes on MBP activation in this experimental model.     

Glucose effects on skin keratinocytes: implications for diabetes skin complications.

Altered skin wound healing is a common cause of morbidity and mortality among diabetic patients. However, the molecular mechanisms whereby diabetes alters skin physiology have not been elucidated. In this study, we investigated the relative roles of hyperglycemia, insulin, and IGF-I, all of which are abnormal in diabetes, in primary murine skin keratinocytes. These cells proliferate and differentiate in vitro in a manner similar to skin in vivo. It was found that in the presence of high glucose (20 mmol/l), the glucose transport rate of primary proliferating or differentiating keratinocytes was downregulated, whereas at 2 mmol/l glucose, the transport rate was increased. These changes were associated with changes in the GLUT1 expression and with changes in the affinity constant (K(m)) of the transport. Exposure to high glucose was associated with changes in cellular morphology, as well as with decreased proliferation and enhancement of Ca(2+)-induced differentiation of keratinocytes. Furthermore, in the presence of high glucose, ligand-induced IGF-I receptor but not insulin receptor (IR) autophosphorylation was decreased. Consequently, in high glucose, the effects of IGF-I on glucose uptake and keratinocyte proliferation were inhibited. Interestingly, lack of IR expression in IR-null keratinocytes abolished insulin-induced glucose uptake and partially decreased insulin- and IGF-I-induced proliferation, demonstrating the direct involvement of the IR in these processes. Our results demonstrate that hyperglycemia and impaired insulin signaling might be directly involved in the development of chronic complications of diabetes by impairing glucose utilization of skin keratinocytes as well as skin proliferation and differentiation.     

Downregulation of protein kinase C and insulin-stimulated 2-deoxyglucose uptake in rat adipocytes by phorbol esters, glucose, and insulin

Phorbol esters translocatively activate and subsequently downregulate protein kinase C and insulin-stimulated glucose uptake in rat adipocytes. This study examined the possibility that other translocative activators of protein kinase C in rat adipocytes, e.g., insulin and glucose, provoke similar downregulating effects. Pretreatment of rat adipocytes for 20-24 h with phorbol esters, 3 nM insulin, 20 mM glucose, or 3 nM insulin plus 20 mM glucose resulted in concomitant decreases in protein kinase C and insulin-stimulated (or phorbol ester-stimulated) [3H]-2-deoxyglucose uptake. Downregulating effects of glucose on protein kinase C and insulin-stimulated [3H]-2-deoxyglucose uptake were also evident within 30 min in adipocytes freshly incubated in medium containing 5-20 mM, rather than 0, glucose. These findings confirm that protein kinase C is required during insulin-stimulated glucose uptake and raise the possibility that downregulation of protein kinase C by continued translocative activation of the enzyme may contribute (along with other factors) to impaired responsiveness of the glucose transport system after prolonged insulin and/or glucose treatment.     

A peroxovanadium compound stimulates muscle glucose transport as powerfully as insulin and contractions combined

Stimulation of glucose transport by insulin involves tyrosine phosphorylation of the insulin receptor (IR) and IR substrates (IRSs). Peroxovanadates inhibit tyrosine phosphatases, also resulting in tyrosine phosphorylation of the IRSs. Muscle contractions stimulate glucose transport by a mechanism independent of the insulin-signaling pathway. We found that the peroxovanadate compound bis-peroxovanadium,1,10-phenanthrolene [bpV(phen)] stimulates glucose transport to the same extent as the additive effects of maximal insulin and contraction stimuli. Translocation of GLUT4 to the cell surface mediates stimulation of glucose transport. There is evidence suggesting there are separate insulin- and contraction-stimulated pools of GLUT4-containing vesicles. We tested the hypothesis that bpV(phen) stimulates both the insulin- and the contraction-activated pathways. Stimulation of glucose transport and GLUT4 translocation by bpV(phen) was completely blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin and LY294002. The combined effect of bpV(phen) and contractions was no greater than that of bpV(phen) alone. Activation of the IRS-PI 3-K signaling pathway was much greater with bpV(phen) than with insulin. Our results suggest that the GLUT4 vesicles that are normally translocated in response to contractions but not insulin can respond to the signal generated via the IRS-PI 3-K pathway if it is sufficiently powerful.     

High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes

Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes.     

ACCUMULATED ENDOGENOUS NITRIC OXIDE SYNTHASE INHIBITORS IN INHIBITING URETHRAL RELAXATION FOLLOWING ESTROGEN SUPPLEMENTATION IN OVARIECTOMIZED RABBITS

PURPOSE: We investigated the possible role of the endogenous nitric oxide (NO) synthase (NOS) inhibitors N-monomethyl-L-arginine (L-NMMA) and asymmetrical N, N-dimethyl-L-arginine (ADMA) in inhibiting urethral relaxation following estrogen supplementation in ovariectomized rabbits. MATERIALS AND METHODS: A total of 16 mature Japanese White female rabbits were divided into 2 groups. In the control group rabbits were sacrificed 2 weeks after bilateral ovariectomy. In the estrogen group estradiol was administered subcutaneously for 2 weeks with the aid of sustained release pellet from 2 weeks after ovariectomy until sacrifice. Isolated urethra was cut into transverse strips for functional study and processed to determine endogenous NOS inhibitors, NOS activity, dimethylarginine dimethylaminohydrolase (DDAH) activity as a metabolizing enzyme of endogenous NOS inhibitors and cyclic guanosine monophosphate production. RESULTS: Electrical field stimulation produced NO mediated and neurogenic relaxation of the urethral strip in the presence of guanethidine and atropine under contraction with phenylephrine. Relaxation was significantly decreased in the estrogen group and accompanied by decreased cyclic guanosine monophosphate production. Sodium nitroprusside induced relaxation was not different between the 2 groups. The content of L-NMMA plus ADMA in the urethra was significantly increased in the estrogen group. Ca dependent NOS activity in the urethra remained unaffected. DDAH activity was significantly lower in the estrogen group. CONCLUSIONS: Estrogen supplementation leads to decreased NO mediated and neurogenic urethral relaxation through the accumulation of L-NMMA and ADMA in the urethra. The accumulation of NOS inhibitors is possibly brought about by impaired DDAH activity.     

Effects of diabetes,insulin and antioxidants on NO synthase abundance and NO interaction with reactive oxygen species

BACKGROUND: Earlier studies have provided evidence for increased production of reactive oxygen species (ROS) and altered nitric oxide (NO) metabolism in diabetes. This study was intended to explore the effect of type I diabetes and its treatment with insulin alone or insulin plus antioxidant-fortified diet on expression of NOS isoforms and ROS interactions with lipids, glucose and NO. METHODS: Rats with streptozotocin-induced diabetes were divided into once-daily insulin (ultralente)-treated, insulin plus antioxidant (vitamin E and vitamin C)-treated and untreated groups. After four weeks, plasma malondialdehyde (MDA) and tissue endothelial (eNOS), neuronal (nNOS) NO synthases, carboxymethyllysine (CML) and nitrotyrosine were determined. RESULTS: The untreated diabetic animals exhibited severe hyperglycemia, elevated blood pressure, increased plasma MDA, high tissue CML and reduced tissue nitrotyrosine denoting enhanced lipid, glucose and protein oxidation but reduced NO oxidation by ROS. This was coupled with significant reduction of eNOS and nNOS expression in renal cortex and eNOS in the left ventricle. Insulin therapy partially lowered blood pressure, tissue CML, plasma glucose and MDA, but significantly raised eNOS expression and nitrotyrosine abundance to supranormal levels. Combined insulin and antioxidant therapies resulted in normalization of blood pressure, plasma MDA, tissue CML and nitrotyrosine without affecting glucose level or NOS expression. CONCLUSION: Oxidative stress in untreated diabetes is associated with down-regulation of NOS isoforms and increased ROS-mediated oxidation of lipid and glucose, but not NO. Amelioration of hyperglycemia with once-daily insulin administration alone results in up-regulation of NOS isoforms, reduction of lipid and glucose oxidation and increased NO oxidation. However, insulin plus antioxidant supplementation can normalize all three parameters.     

Nitric oxide synthase inhibition reduces leg glucose uptake but not blood flow during dynamic exercise in humans.

Nitric oxide (NO) appears to play a role in contraction-stimulated glucose uptake in isolated rodent skeletal muscle; however, no studies have examined this question in humans. Seven healthy men completed two 30-min bouts of supine cycling exercise at 60 +/- 2% peak pulmonary oxygen uptake (VO2 peak), separated by 90 min of rest. The NO synthase inhibitor N(G)-monomethyl-L-arginine ([L-NMMA]; total dose 5 mg/kg body weight) or saline (control) were administered via the femoral artery for the final 20 min of exercise in a randomized blinded crossover design. L-Arginine (5 mg/kg body weight) was co-infused during the final 5 min of each exercise bout. Leg blood flow (LBF) was measured by thermodilution in the femoral vein, and leg glucose uptake was calculated as the product of LBF and femoral arteriovenous (AV) glucose difference. L-NMMA infusion significantly (P < 0.05) reduced leg glucose uptake compared with control (48 +/- 12% lower at 15 min, mean +/- SE). The reduction in glucose uptake was due solely to a decrease in AV glucose difference, as there was no effect of L-NMMA infusion on LBF during exercise. Co-infusion of L-arginine restored glucose uptake during L-NMMA infusion to levels similar to control. These results indicate that NO production contributes substantially to exercise-mediated skeletal muscle glucose uptake in humans independent of skeletal muscle blood flow.     

HIV protease inhibitors acutely impair glucose-stimulated insulin release

HIV protease inhibitors (PIs) acutely and reversibly inhibit the insulin-responsive glucose transporter Glut 4, leading to peripheral insulin resistance and impaired glucose tolerance. Minimal modeling analysis of glucose tolerance tests on PI-treated patients has revealed an impaired insulin secretory response, suggesting additional pancreatic beta-cell dysfunction. To determine whether beta-cell function is acutely affected by PIs, we assayed glucose-stimulated insulin secretion in rodent islets and the insulinoma cell line MIN6. Insulin release from MIN6 cells and rodent islets was significantly inhibited by the PI indinavir with IC(50) values of 1.1 and 2.1 micro mol/l, respectively. The uptake of 2-deoxyglucose in MIN6 cells was similarly inhibited (IC(50) of 2.0 micro mol/l), whereas glucokinase activity was unaffected at drug levels as high as 1 mmol/l. Glucose utilization was also impaired at comparable drug levels. Insulin secretogogues acting downstream of glucose transport mostly reversed the indinavir-mediated inhibition of insulin release in MIN6 cells. Intravenous infusion of indinavir during hyperglycemic clamps on rats significantly suppressed the first-phase insulin response. These data suggest that therapeutic levels of PIs are sufficient to impair glucose sensing by beta-cells. Thus, together with peripheral insulin resistance, beta-cell dysfunction likely contributes to altered glucose homeostasis associated with highly active antiretroviral therapy.     

Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase

Glucose transport in skeletal muscle is stimulated by two distinct stimuli, insulin and exercise. The mechanism by which exercise stimulates glucose transport is not known, although it is distinct from the insulin-mediated pathway. Recently, it has been shown that AMP-activated protein kinase (AMPK) is activated by exercise in skeletal muscle, whereas pharmacological activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) leads to increased glucose transport. It has been postulated, therefore, that AMPK may be the link between exercise and glucose transport. To address this, we have examined the signaling pathway involved in the stimulation of glucose uptake after activation of AMPK. Here we show that activation of AMPK by AICAR in rat muscle and mouse H-2Kb muscle cells activates glucose transport approximately twofold. AMPK in H-2Kb cells is also activated by hyperosmotic stress and the mitochondrial uncoupling agent, dinitrophenol, both of which lead to increased glucose transport. In contrast, insulin, which activates glucose transport two- to-threefold in both rat muscle and H-2Kb cells, has no effect on AMPK activity. A previous study has shown that AMPK phosphorylates and activates endothelial nitric oxide synthase (NOS). We show here that NOS activity in H-2Kb cells is activated after stimulation of AMPK by AICAR. Treatment of H-2Kb cells or rat muscle with NOS inhibitors completely blocks the increase in glucose transport after activation of AMPK. In addition, an inhibitor of guanylate cyclase also blocks activation of glucose transport by AICAR in H-2Kb cells. These results indicate that activation of AMPK in muscle cells stimulates glucose transport by activation of NOS coupled to downstream signaling components, including cyclic GMP.     

Hyperglycemia inhibits insulin activation of Akt/protein kinase B but not phosphatidylinositol 3-kinase in rat skeletal muscle

Sustained hyperglycemia impairs insulin-stimulated glucose utilization in the skeletal muscle of both humans and experimental animals--a phenomenon referred to clinically as glucose toxicity. To study how this occurs, a model was developed in which hyperglycemia produces insulin resistance in vitro. Rat extensor digitorum longus muscles were preincubated for 4 h in Krebs-Henseleit solution containing glucose or glucose + insulin at various concentrations, after which insulin action was studied. Preincubation with 25 mmol/l glucose + insulin (10 mU/ml) led to a 70% decrease in the ability of insulin (10 mU/ml) to stimulate glucose incorporation into glycogen and a 30% decrease in 2-deoxyglucose (2-DG) uptake, compared with muscles incubated with 0 mmol/l glucose. Glucose incorporation into lipid and its oxidation to CO2 were marginally diminished, if at all. The alterations of glycogen synthesis and 2-DG uptake were first evident after 1 h and were maximal after 2 h of preincubation; they were not observed in muscles preincubated with 25 mmol/l glucose + insulin for 5 min. Preincubation for 4 h with 25 mmol/l glucose in the absence of insulin produced a similar although somewhat smaller decrease in insulin-stimulated glycogen synthesis; however, it did not alter 2-DG uptake, glucose oxidation to CO2, or incorporation into lipids. Studies of insulin signaling in the latter muscles revealed that activation of Akt/protein kinase B (PKB) was diminished by 60%, compared with that of muscles preincubated in a glucose-free medium; whereas activation of phosphatidylinositol (PI) 3-kinase, an upstream regulator of Akt/PKB in the insulin-signaling cascade, and of mitogen-activated protein (MAP) kinase, a parallel signal, was unaffected. Immunoblots demonstrated that this was not due to a change in Akt/PKB abundance. The results indicate that hyperglycemia-induced insulin resistance can be studied in rat skeletal muscle in vitro. They suggest that impairment of insulin action in these muscles is related to inhibition of Akt/PKB by events that do not affect PI 3-kinase.     

Differing effects of N(G)-monomethyl L-arginine and 7-nitroindazole on detrusor activity

AIMS: Previous studies reported that nitric oxide (NO) synthase (NOS) inhibition decreases micturition volume threshold (MVT), the volume required to produce a centrally mediated micturition contraction, and that NO can be released from urothelium by means of certain stimuli. With elucidation of multiple isoforms of NOS, studies were performed to determine whether inhibition of specific isoforms of NOS altered MVT in different ways. METHODS: In naive, anesthetized cats, the urinary bladder was exposed by means of a midline abdominal incision and cannulated through a slit in the internal urethra approximately 4-5 cm distal to the neck of the bladder. The left renal artery and left radial vein were cannulated for the intra-arterial and intravenous administration of drugs, respectively. All nerves were left intact. A control MVT was determined by slowly infusing saline into the bladder at a rate of 0.018 mL/kg per minute. Varying doses of L-NMMA (N(G)-monomethyl-L-arginine) or 7-NI (7-nitro indazole) were administered and the MVT was again determined. RESULTS: Inhibition of endothelial NOS (eNOS), by L-NMMA, or neuronal NOS (nNOS), by 7-NI, produces varying effects on certain detrusor activities and that inhibition of different isoforms of NOS produces qualitatively different effects. L-NMMA significantly decreases MVT (up to 60% decrease), whereas 7-NI significantly increases MVT (over 300% increase). L-NMMA increases frequency and onset of small bladder contractions, whereas 7-NI produces opposite effects. CONCLUSIONS: The results suggest that detrusor relaxation and contractility may be modulated by NO levels and that NO released from the urothelium may be a mediator of detrusor relaxation during the storage phase of micturition.     

Bradykinin augments insulin-stimulated glucose transport in rat adipocytes via endothelial nitric oxide synthase-mediated inhibition of Jun NH2-terminal kinase

An increase in bradykinin has been suggested to contribute to the enhanced insulin sensitivity observed in the presence of ACE inhibitors. To investigate a potential direct, nonvascular effect on an insulin target tissue, the effect of bradykinin on glucose uptake and insulin signaling was studied in primary rat adipocytes. Whereas basal glucose uptake was not altered, bradykinin augmented insulin-stimulated glucose uptake twofold, which was blocked by HOE-140, a bradykinin B2 receptor antagonist. The bradykinin effect on glucose uptake was nitric oxide (NO) dependent, mimicked by NO donors and absent in adipocytes from endothelial NO synthase-/- mice. Investigation of insulin signaling revealed that bradykinin enhanced insulin receptor substrate-1 (IRS-1) Tyr phosphorylation, Akt/protein kinase B phosphorylation, and GLUT4 translocation. In contrast, insulin-stimulated extracellular signal-regulated kinase1/2 and Jun NH2-terminal kinase (JNK) activation were decreased in the presence of bradykinin, accompanied by decreased IRS-1 Ser307 phosphorylation. Furthermore, bradykinin did not enhance insulin action in the presence of the JNK inhibitor, SP-600125, or in adipocytes from JNK1-/- mice. These data indicate that bradykinin enhances insulin sensitivity in adipocytes via an NO-dependent pathway that acts by modulating the feedback inhibition of insulin signaling at the level of IRS-1.     

Insulin-stimulated glucose transport and insulin internalization share a common postbinding step in adipocytes

We recently demonstrated that chymotrypsin substrate analogues inhibit receptor-mediated insulin internalization in isolated rat adipocytes. In this study, the effect on glucose transport of inhibiting insulin internalization with these agents was examined. Glucose transport was assayed by measuring [3H]-2-deoxyglucose uptake, and internalized insulin was measured after rapidly dissociating surface-bound insulin with an acidic buffer. The chymotrypsin substrate analogue N-acetyl-Tyr ethyl ester inhibited insulin internalization by 85% while increasing surface-bound insulin by 80-110%. Under these conditions, ATP levels were minimally altered, and basal glucose transport was unchanged; however, insulin-stimulated glucose transport was decreased by 86%. The inhibition of insulin-stimulated glucose transport was not overcome by supramaximal concentrations (400 ng/ml) of insulin. When insulin internalization and insulin-stimulated glucose transport were measured in the presence of increasing concentrations of N-acetyl-Tyr ethyl ester (0.1-1 mM), a strong and highly significant correlation (r = .97, P less than .001) was found between inhibition of insulin internalization and inhibition of insulin-stimulated glucose uptake. Fragments of N-acetyl-Tyr ethyl ester that do not inhibit insulin internalization were also without effect on insulin-stimulated glucose transport. In addition to N-acetyl-Tyr ethyl ester, four other chymotrypsin substrate analogues that are effective inhibitors of insulin internalization also markedly inhibited insulin-stimulated glucose transport. These results indicate that insulin internalization and insulin-stimulated glucose transport share a common postbinding step in adipocytes and that this step is inhibitable by chymotrypsin substrate analogues.     

Glucose autoregulates its uptake in skeletal muscle: involvement of AMP-activated protein kinase

Preexposure to a low concentration of glucose upregulates glucose transport into skeletal muscle, whereas exposure to a high concentration of glucose has the opposite effect. This autoregulatory process occurs independently of insulin, and the mechanism by which it operates is incompletely understood. Activation of the energy-sensing enzyme AMP-activated protein kinase (AMPK) has been shown to increase insulin-independent glucose transport into skeletal muscle in response to such stimuli as exercise and hypoxia. In the present study, we examined whether AMPK could also mediate glucose autoregulation. The activity of the alpha2 isoform of AMPK and 2-deoxyglucose uptake were assessed in incubated rat extensor digitorum longus muscle after preincubation for 4 h in media containing 0, 3, 6, or 25 mmol/l glucose. The principal findings were as follows. First, AMPK activity was highest in muscles incubated with no added glucose, and it decreased as the concentration of glucose was increased. In keeping with these findings, the concentration of malonyl CoA was increased, and acetyl CoA carboxylase phosphorylation at serine 79 was decreased as the medium glucose concentration was raised. Second, decreases in AMPK activity at the higher glucose concentrations correlated closely with decreases in glucose transport (2-deoxyglucose uptake), measured during a subsequent 20-min incubation at 6 mmol/l glucose (r(2) = 0.93, P < 0.001). Third, the decrease in AMPK activity at the higher glucose concentrations was not associated with changes in whole-tissue concentrations of creatine phosphate or adenine nucleotides; however, it did correlate with increases in the rate of glycolysis, as estimated by lactate release. The results suggest that glucose autoregulates its own transport into skeletal muscle by a mechanism involving AMPK. They also suggest that this autoregulatory mechanism is not paralleled by changes in whole-tissue concentrations of creatine phosphate ATP, or AMP, but they leave open the possibility that alterations in a cytosolic pool of these compounds play a regulatory role.     

Effect of acute ketosis on the endothelial function of type 1 diabetic patients: the role of nitric oxide.

In type 1 diabetic patients, acute loss of metabolic control is associated with increased blood flow, which is believed to favor the development of long-term complications. The mechanisms for inappropriate vasodilation are partially understood, but a role of endothelium-derived nitric oxide (NO) production can be postulated. We assessed, in type 1 diabetic patients, the effect of the acute loss of metabolic control and its restoration on forearm endothelial function in 13 type 1 diabetic patients who were studied under conditions of mild ketosis on two different occasions. In study 1, after basal determination, a rapid amelioration of the metabolic picture was obtained by insulin infusion. In study 2, seven type 1 diabetic patients underwent the same experimental procedure, except that fasting plasma glucose was maintained constant throughout. Basal plasma venous concentrations of nitrites/nitrates (NO2- + NO3-) were determined both before and after intravenous insulin infusion. Endothelium-dependent and -independent vasodilation of the brachial artery was assessed by an intra-arterial infusion of N(G)-monomethyl-L-arginine (L-NMMA) and sodium nitroprusside (SNP), respectively. The same parameters were determined in 13 control subjects at baseline conditions and during a hyperinsulinemic-euglycemic glucose clamp. Baseline forearm blood flow (4.89 +/- 0.86 vs. 3.65 +/- 0.59 ml x (100 ml tissue)(-1) x min(-1)) and NO2- + NO3- concentration (30 +/- 8 vs. 24 +/- 3 micromol/l) were higher in type 1 diabetic patients than in control subjects (P < 0.05). Insulin infusion was associated with lower forearm blood flow and plasma (NO2- + NO3-) concentration (P < 0.05), irrespective of the prevailing glucose levels, as compared with patients under ketotic conditions. The responses to L-NMMA were significantly lower in type 1 diabetic patients during euglycemia and hyperglycemic hyperinsulinemia (-11 +/- 5 and -10 +/- 4%, respectively, of the ratio of the infused arm to the control arm) than in control subjects at baseline (-18 +/- 6%, P < 0.05) and during hyperinsulinemia (-32 +/- 11%, P < 0.01). We conclude that the acute loss of metabolic control is associated with a functional disturbance of the endothelial function characterized by hyperemia and increased NO release during ketosis and blunted NO-mediated vasodilatory response during restoration of metabolic control by intravenous insulin. This functional alteration is unlikely to be explained by hyperglycemia itself.