help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0745
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kashyap, S. R.
Right arrow Articles by DeFronzo, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kashyap, S. R.
Right arrow Articles by DeFronzo, R. A.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Diabetes
Related Collections
Right arrow Diabetes and Insulin
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1100-1105
Copyright © 2005 by The Endocrine Society

Insulin Resistance Is Associated with Impaired Nitric Oxide Synthase Activity in Skeletal Muscle of Type 2 Diabetic Subjects

Sangeeta R. Kashyap1, Linda J. Roman1, Jennifer Lamont, Bettie Sue S. Masters, Mandeep Bajaj, Swangjit Suraamornkul, Renata Belfort, Rachele Berria, Dean L. Kellogg, Jr., Yanjuan Liu and Ralph A. DeFronzo

Diabetes Division (S.R.K., M.B., S.S., R.Bel., R.Ber., R.A.D.) and Departments of Biochemistry (L.J.R., J.L., B.S.S.M.) and Geriatrics (D.L.K., Y.L.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229

Address all correspondence and requests for reprints to: Ralph A. DeFronzo, M.D., The University of Texas Health Science Center at San Antonio, Diabetes Division, 7703 Floyd Curl Drive, San Antonio, Texas 78229.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Type 2 diabetes is an insulin-resistant state characterized by hyperinsulinemia and accelerated atherosclerosis. In vitro and in vivo studies in rodents have suggested that nitric oxide generation plays an important role in glucose transport and insulin action. We determined nitric oxide synthase (NOS) activity in skeletal muscle of 10 type 2 diabetic (hemoglobin A1C = 6.8 ± 0.1%) and 11 control subjects under basal conditions and during an 80 mU/m2·min euglycemic insulin clamp performed with vastus lateralis muscle biopsies before and after 4 h of insulin. In diabetics, insulin-stimulated glucose disposal (Rd) was reduced by 50%, compared with controls (5.4 ± 0.3 vs. 10.4 ± 0.5 mg/kg·min, P < 0.01). Basal NOS activity was markedly reduced in the diabetic group (101 ± 33 vs. 457 ± 164 pmol/min·mg protein, P < 0.05). In response to insulin, NOS activity increased 2.5-fold in controls after 4 h (934 ± 282 pmol/min·mg protein, P < 0.05 vs. basal), whereas insulin failed to stimulate NOS activity in diabetics (86 ± 28 pmol/min·mg protein, P = NS from basal). Basal NOS protein content in muscle was similar in controls and diabetics and did not change following insulin. In controls, insulin-stimulated NOS activity correlated inversely with fasting plasma insulin concentration (r = –0.58, P = 0.05) and positively with Rd (r = 0.71, P = 0.03). In control and diabetic groups collectively, Rd correlated with insulin-stimulated NOS activity (r = 0.52, P = 0.02). We conclude that basal and insulin-stimulated muscle NOS activity is impaired in well-controlled type 2 diabetic subjects, and the defect in insulin-stimulated NOS activity correlates closely with the severity of insulin resistance. These results suggest that impaired NOS activity may play an important role in the insulin resistance in type 2 diabetic individuals.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES MELLITUS (T2DM) is an insulin-resistant state associated with accelerated atherosclerosis, hypertension, dyslipidemia, and abnormal vasomotor responses to insulin (1, 2, 3). Endothelial dysfunction represents the earliest abnormality in the pathogenesis of atherosclerosis (4), and a central feature of endothelial dysfunction is loss of nitric oxide (NO) generation (5). Mice with gene disruption of endothelial nitric oxide synthase (eNOS) (6) or neuronal NOS (nNOS) (7) exhibit insulin resistance, hypertension, and dyslipidemia, whereas induction of inducible NO synthase (iNOS) by endotoxin is associated with impaired insulin-stimulated muscle glucose uptake (8). Conversely, targeted disruption of iNOS protects against obesity-linked insulin resistance in muscle (9). In humans the use of pharmacologic inhibitors of NO synthase (NOS) has provided indirect evidence that arterial NO production is diminished in insulin-resistant states including T2DM, obesity, hypertension, and dyslipidemia (10, 11, 12, 13).

Considerable controversy exists concerning the effect of insulin on NOS activity and NO generation. Studies with cultured endothelial cells suggest that insulin stimulates NO formation, whereas elevated glucose levels inhibit NO formation (14, 15). In humans, stimulation of skeletal muscle blood flow by insulin has been inferred to be NO dependent based on studies employing inhibitors of NOS (16, 17, 18). Skeletal muscle is a major target for insulin action in man, and impaired insulin-stimulated glucose transport is a characteristic defect in type 2 diabetic patients (19). In vivo studies have shown that both eNOS and nNOS are present in skeletal muscle (8), and targeted disruption of both eNOS (6, 7) and nNOS (7) in mice causes insulin resistance in skeletal muscle. Similarly, the NOS inhibitor, NG-nitro-L-arginine methyl ester, decreases insulin-stimulated glucose disposal (Rd) in skeletal and cardiac muscle in vivo in rats (20). Conversely, the NO donor sodium nitroprusside stimulates 2-deoxyglucose uptake in rat soleus muscle in the absence and presence of insulin (21, 22). The stimulatory effect of NO on glucose transport in muscle (23, 24, 25) and adipocytes (26) is mediated via mechanisms distinct from the insulin signaling and contraction pathways. Although some contradictory results have been reported (27, 28), most evidence supports a role for NO in the regulation of insulin action in skeletal muscle.

In the present study, we determined, for the first time, NOS activity in skeletal muscle of type 2 diabetic and healthy control subjects and related these findings to whole-body (muscle) insulin-mediated Rd and known biological markers of cardiovascular disease, vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Eleven type 2 diabetic subjects and 10 healthy age-, ethnicity- and weight-matched controls participated. Their clinical characteristics are shown in Table 1Go. Normal glucose tolerance was confirmed in all control subjects by a 75-g oral glucose tolerance test using American Diabetes Association criteria. The diabetic subjects had increased systolic/diastolic blood pressure, higher plasma triglyceride and free fatty acid (FFA) concentrations, and slightly decreased high-density lipoprotein (HDL) cholesterol levels, compared with the control group. The diabetic group was in reasonably good glycemic control, as reflected by the mean hemoglobin A1c (HbA1c) of 6.8%. The diabetic patients were being treated with diet (n = 8) or sulfonylureas (n = 3). Sulfonylurea drugs have been shown to have no direct effects on insulin sensitivity (29). No diabetic subject had received treatment with metformin, thiazolidinedione, or insulin. The mean duration of diabetes was less than 2 yr. Five diabetic subjects had a normal fasting plasma glucose concentration and were diagnosed with an oral glucose tolerance test (2-h plasma glucose > 200 mg/dl). Oral agents were discontinued 24 h before the study. Other than diabetes, none of the subjects had any medical problems, and none were taking any medications (other than sulfonylureas in three diabetic subjects) known to affect glucose metabolism. None of the participants smoked. None of the women were on hormonal replacement therapy. The purpose, nature, and potential risks of the study were explained to all subjects, and written consent was obtained before their participation. The protocol was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Subject characteristics

 
Study design

All studies were conducted in the General Clinical Research Center of the University of Texas Health Science Center at San Antonio and began at 0700 h after a 12-h overnight fast. Before the start of the euglycemic insulin clamp study, an antecubital vein was cannulated for infusion of all test substances. A second catheter was inserted retrogradely into a vein on the dorsum of the hand, and the hand was placed in a heated box (60 C) to obtain arterialized blood samples. A prime (25 µCi)-continuous infusion (0.25 µCi/min) of 3-[3H]glucose was started, and 2 h (3 h for diabetic subjects) were allowed for isotopic equilibration. The priming dose of tritiated glucose was increased in the diabetics in proportion to the increase in fasting plasma glucose concentration. Sixty minutes before the start of the insulin clamp, a percutaneous biopsy of the vastus lateralis muscle was obtained for determination of NOS activity and protein content (30). Muscle biopsy specimens (100–200 mg) were immediately blotted free of blood, frozen, and stored in liquid nitrogen until use. At the end of the tracer equilibration period, a primed-continuous infusion (80 mU/m2·min) of insulin was started and the plasma glucose was measured every 5 min with a glucose oxidase analyzer (Beckman Instruments, Fullerton, CA). Based on the negative feedback principle, a variable infusion of 20% glucose was adjusted to maintain the plasma glucose concentration constant at each subject’s fasting glucose level in the control group. In diabetic subjects, the plasma glucose concentration was allowed to decrease during the insulin infusion to 100 mg/dl, at which level it was maintained. At 30 and 240 min after the start of the insulin infusion, repeat vastus lateralis muscle biopsies were obtained from a site 4 cm distal to the first. At time 0 and at 180 and 240 min after the start of insulin, plasma was obtained for ICAM-1 and VCAM determination from standard ELISA kits. The insulin infusion was continued for a total of 240 min.

Methods

Plasma glucose-specific activity was determined on barium hydroxide/zinc sulfate extracts of plasma. Plasma insulin concentration was determined by RIA (Diagnostic Products, Los Angeles, CA). Plasma total cholesterol was determined on LX-20 Synchron automated system (Beckman Instruments, Brea, CA). Plasma HDL-cholesterol was determined by homogeneous liquid selective detergent HDLD (Beckman). Plasma triglycerides were determined by enzymatic GPO (Beckman). Plasma low-density lipoprotein-cholesterol was calculated from the Friedwald equation. The amount of NOS activity produced by skeletal muscle was measured by the NOS Detect assay kit as specified by the manufacturer (Stratagene, La Jolla, CA) and is based on the conversion of [14C]L-arginine (PerkinElmer Life Sciences, Norwalk, CT) to [14C]L-citrulline. Muscle tissue (20–70 mg) was resuspended in a 10x volume of ice-cold homogenization buffer (200–700 µl) (Stratagene). Tissue was homogenized by hand using a Kontes 0.2-ml microtissue grinder and centrifuged in a microcentrifuge at top speed for 5 min. The supernatant was transferred to a fresh tube and kept on ice until assayed for NOS activity. The protein content of the supernatant was determined using the micro-BCA assay (Pierce, Rockford, IL). Production of NO was assayed using 10 µl of tissue extract for 60 min at room temperature. Nonspecific production of L-citrulline was monitored by performing the reaction in the presence of N-{omega}-nitro-L-arginine, a NOS inhibitor. In the presence of N-{omega}-nitro-L-arginine, no significant production of NO was observed. NOS activity data were normalized by the absolute amount of protein present.

NOS protein content

The amount of NOS present in the tissue homogenates was determined by immunoblot analysis using an anti-nNOS polyclonal antibody raised in goat by one of the investigators (B.S.S.M.). This antibody cross-reacts strongly with all NOS isoforms and cannot be used to distinguish which isoforms are present. Immunoblotting was performed as described (31) and enzyme-linked immunodetection of goat antibody was performed using a rabbit antigoat IgG secondary antibody conjugated with alkaline phosphatase (Zymed, San Francisco, CA). Bands were developed with nitro blue tetrazolium (0.3 mg/ml) and 5-bromo-4-chloro-3-indolyl-phosphate (0.15 mg/ml) (Bio-Rad Laboratories, Hercules, CA).

Calculations

During the postabsorptive period, the rate of glucose appearance equals the rate of glucose disappearance and was calculated as the tritiated glucose infusion rate (dpm per minute) divided by the plasma tritiated glucose-specific activity (dpm per milligram). During the euglycemic insulin clamp, non-steady-state conditions prevail and rates of glucose appearance and disappearance were calculated with Steele’s non-steady-state equation, using a glucose distribution volume of 0.65. The rate of endogenous (primarily hepatic) glucose production during the insulin clamp was calculated by subtracting the exogenous glucose infusion rate from the tracer-derived rate of glucose appearance. The rate of insulin-stimulated Rd was calculated by adding the rate of residual endogenous glucose production to the cold glucose infusion rate.

Statistical methods

All data are presented as the mean ± SE. Differences between control and diabetic groups were compared using the unpaired two-tailed t test. Differences between basal and insulin-stimulated values within groups were compared using the paired t test. Correlation analysis was performed by the Pearson product moment method using StatView software (version 4.0; SAS Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Euglycemic insulin clamp

The plasma insulin concentration was significantly higher in diabetic vs. control subjects (13 ± 1 vs. 6 ± 1 µU/ml, P < 0.01). During the insulin clamp similar steady-state plasma insulin concentrations were obtained in diabetic and control groups (133 ± 7 vs. 127 ± 8 µU/ml, P = NS). The fasting plasma FFA concentration (750 ± 62 vs. 596 ± 43 µmol/liter, P < 0.05), as well as the plasma FFA concentration during the insulin clamp (137 ± 10 vs. 117 ± 8 µmol/liter, P < 0.05), was significantly higher in the diabetic vs. control group. Basal endogenous glucose production was increased in diabetic subjects, compared with controls (2.21 ± 0.22 vs. 1.93 ± 0.10 mg/kg·min, P < 0.05). During the euglycemic insulin clamp, the rate of insulin-stimulated Rd was reduced by 50% (5.4 ± 0.3 vs.10.4 ± 0.5 mg/kg·min, P < 0.01) in the diabetic group, and the suppression of endogenous glucose production was impaired as well (0.41 ± 0.08 vs. 0.09 ± 0.03 mg/kg·min, P <0.01).

In diabetic subjects NOS activity during the fasting state was markedly reduced, compared with control subjects (101 ± 33 vs. 457 ± 164 pmol/min·mg protein, P < 0.05) (Fig. 1Go). In control subjects, insulin increased NOS activity 2.5-fold at 4 h (934 ± 282 pmol/min·mg protein, P < 0.01 vs. basal), whereas NOS activity did not change (86 ± 28 pmol/min·mg protein, P = NS from basal) in diabetic subjects. Both the absolute and incremental increases in insulin-stimulated NOS activity were significantly greater in control vs. diabetic subjects at 240 min (Fig. 1Go). No significant change in NOS activity was observed between the baseline and 30-min insulin clamp muscle biopsies in either the control or diabetic groups (Fig. 1Go). Basal muscle NOS protein content was similar in diabetic and control groups, and there was no change in protein content in response to insulin in either group (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1. Basal and insulin-stimulated muscle NOS activity in control and type 2 diabetic patients. *, P < 0.01 diabetic subjects vs. controls; **, P < 0.05 diabetic subjects vs. controls; {dagger}, P < 0.001 240 min vs. basal in controls.

 
Basal and insulin-stimulated levels of VCAM and ICAM were markedly higher in diabetic vs. control subjects (P < 0.05) (Fig. 2Go). No change in plasma ICAM or VCAM levels was observed in either group during insulin stimulation (P = NS).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Plasma ICAM (top) and VCAM (bottom) concentrations in control (open bars) and type 2 diabetic subjects (solid bars) under basal conditions and during the euglycemic insulin clamp. *, P < 0.05 diabetic subjects vs. controls.

 
In control subjects, insulin-stimulated NOS activity correlated inversely with the fasting plasma insulin concentration (r = –0.58, P = 0.05) and positively with Rd (r = 0.71, P = 0.03). In diabetic subjects Rd correlated positively with NOS activity (r = 0.69, P < 0.02). In control and diabetic groups collectively, Rd correlated positively with insulin-stimulated NOS activity (r = 0.62, P = <0.02). In the control group, plasma VCAM and ICAM levels correlated inversely with basal NOS activity (r = –0.71, P = 0.03 and r = –0.65, P < 0.05, respectively). In diabetic subjects, the VCAM concentration correlated positively with HbA1c (r = 0.64, P = 0.04). In the diabetic group, plasma VCAM levels correlated positively with Rd (r = 0.65, P < 0.01).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Compared with age-, weight-, gender-, and ethnicity-matched healthy normal glucose-tolerant subjects, type 2 diabetic patients were severely insulin resistant and demonstrated markedly reduced basal and insulin-stimulated levels of NOS activity, even though the number of subjects studied was modest (n = 11) (Fig. 1Go). A strong positive relationship was observed between insulin-stimulated Rd (primarily reflects muscle) and muscle NOS activity. These results are consistent with in vivo (20, 32) and in vitro (21, 23, 24, 25, 26) rodent studies, which demonstrate an important role for NO in the stimulation of muscle glucose transport. Recently asymmetric dimethyl arginine, an endogenous competitive inhibitor of NOS, has been shown to be increased in T2DM subjects and correlate closely with the severity of insulin resistance and hyperinsulinemia (33). Collectively these results suggest that a common mechanism underlies the association between insulin-stimulated muscle glucose uptake and NO generation.

The relationship among insulin resistance, NOS activity, and NO generation in vascular tissue has been extensively studied. Insulin is a vasodilator and its vasodilatory effect is mediated by NO generation (17, 18). Insulin-resistant states, such as diabetes and obesity, are characterized by impaired insulin-mediated vasodilation (13, 16, 18), and the defect in insulin-stimulated vasodilation has been attributed to impaired NO generation by NOS (34, 35). In vascular endothelial cells, insulin stimulates NO generation in a dose-dependent manner by the phosphatidylinositol 3-kinase (PI-3 kinase)/protein kinase B pathway (15, 36). In the aorta and mesenteric artery of insulin-resistant rats, stimulation of the insulin receptor substrate (IRS)-1/PI-3 kinase pathway by insulin is severely impaired (37, 38). Because NOS activation by insulin in arteriolar muscle cells is dependent on an intact IRS-1/PI-3 pathway (15, 36), insulin resistance in this pathway (37, 38) would be expected to impair insulin-mediated vasodilation and result in endothelial dysfunction. In previous studies in type 2 diabetic subjects (30) and the normal glucose-tolerant insulin-resistant offspring of two diabetic parents (39), we have shown that the ability of insulin to activate the IRS-1/PI-3 kinase pathway in muscle is markedly impaired. This defect could explain the severe impairment in insulin-stimulated muscle NOS activity observed in the present study (Fig. 1Go). In both human muscle (30, 39) and rat arterial tissue (38) from type 2 diabetics, despite the severe defect in insulin-stimulated PI3-kinase activity, activation of the MAPK or mitogenic pathway by insulin is completely intact. Enhanced stimulation of the MAPK pathway results in vascular smooth muscle cell proliferation and migration and increased release of growth factors and inflammatory cytokines (TNF{alpha}, IL-6, monocyte chemotactic protein), which are instrumental in the development of atherosclerosis (38). Thus, intact MAPK pathway activity, in combination with reduced NO generation and endothelial dysfunction, may play a central role in the development of atherosclerosis in a variety of diverse phenotypes characterized by insulin resistance (1, 40).

In healthy insulin-sensitive, nondiabetic subjects, there was a small, statistically insignificant rise in NOS activity at 30 min, and by 240 min NOS activity was increased 2–2.5-fold (Fig. 1Go). Given the relatively short time (4 h) required for insulin to activate NOS, it is unlikely that augmented NOS transcription and translation can explain the increased NOS activity, and this is substantiated by the failure to observe any changes in NOS protein content during the 4-h study (data not shown). nNOS is the major isoform in muscle, but small amounts of eNOS and iNOS also are present (41). Because the polyclonal antibody used to quantitate NOS protein cross-reacts with all three NOS isoforms, it is possible that changes in the level of a minor isoform could occur undetectably. Although possible, we think it unlikely that a small increase in one specific NOS isoform could have occurred and accounted for the very large (2-fold) insulin-stimulated increase in NOS activity. Future studies employing antibodies that are specific for each of the three NOS isoforms are needed to examine this possibility. Although we cannot determine which NOS isoform is responsible for the defect in insulin-stimulated NOS activity in diabetic individuals, total NO generation would be expected to be deficient, and it is NO per se that is responsible for its metabolic and vasodilatory effects.

The most likely mechanism responsible for the insulin-stimulated increase in NOS activity in the control group involves serine phosphorylation of nNOS, the major isoform in muscle (41). Both nNOS and eNOS contain serine/threonine protein kinase phosphorylation sites at their C termini. Phosphorylation of human (or bovine) eNOS isoforms at serine 1177 (or 1179) causes a 2- to 4-fold increase in the rate of NO synthesis (42, 43, 44, 45). The homologous serine residue to S1177 in eNOS is S1412 in nNOS, and mutation of nNOS S1412 (46), like that of eNOS S1117 (47), to aspartate mimics the negative charge imparted by phosphorylation and, in the case of eNOS, leads to an increase in the rate of NO production. Insulin has been shown to increase serine phosphorylation of eNOS by a pathway involving Akt kinase (48).

In type 2 diabetics several potential mechanisms could explain the reduced ability of insulin to increase muscle NOS activity. Hyperglycemia inhibits phosphorylation at serine 1177 of eNOS (49) and interferes with insulin signaling via O-linked glycosylation of the metabolic branch point IRS/PI3-kinase/AKT (50). However, our diabetic patients were reasonably well controlled (HbA1c = 6.8%), and it is unknown whether this level of hyperglycemia in vivo is able to inhibit NOS activity. Reduced muscle concentration of L-arginine (43) or one of the essential cofactors, tetrahydrobiopterin (51), also could contribute to the defective stimulation of NOS by insulin in diabetics. However, deficiencies in these cofactors cannot explain the differences in NO production between the diabetic and control subjects in the present study because all required cofactors and L-arginine are provided in the assay mixture. Excessive accumulation of asymmetric dimethyl arginine in the muscle extract also is unlikely because this would have been diluted in the assay mixture.

The relationship between insulin resistance (Rd) and other known risk factors for atherosclerotic cardiovascular disease also was examined. Type 2 diabetic subjects were characterized by elevated levels of systolic and diastolic blood pressure, increased plasma triglyceride and FFA concentrations, and reduced plasma HDL cholesterol concentration. When the diabetic and control subjects were analyzed collectively, we observed an inverse correlation between Rd and systolic blood pressure, diastolic blood pressure, plasma triglyceride concentration, and plasma FFA concentration and a positive correlation between Rd and HDL concentration. Similar correlations have been reported in previous publications (52, 53). We also observed a significant increase in plasma ICAM and VCAM concentrations, established cardiovascular risk markers (54, 55), in diabetic subjects (Fig. 2Go), and an inverse correlation between increased plasma ICAM and VCAM levels and the reduced muscle NOS activity. Previous studies have shown that NO modulates leukocyte adhesion to the endothelium by regulating the production/release of ICAM and VCAM by endothelial and vascular smooth muscle cells (56). In our diabetic subjects, a strong correlation between VCAM and both HbA1c and severity of insulin resistance was found. These observations suggest that chronic hyperglycemia and impaired insulin action, perhaps by causing a reduction in NOS activity, lead to increased ICAM/VCAM production, contributing to the development of accelerated atherosclerosis.

In summary, insulin resistance in T2DM is not only a metabolic disorder characterized by decreased insulin mediated Rd but also a vascular disorder associated with impaired NOS activity and elevated circulating risk factors for atherosclerotic cardiovascular disease. Our results are consistent with the concept that reduced basal NOS activity and impaired insulin-stimulated NOS activity contribute to accelerated atherosclerosis, impaired endothelium-dependent vasodilatation, and hypertension in type 2 diabetic individuals.


   Acknowledgments
 
We thank all volunteers and the invaluable efforts of the General Clinical Research Center nursing staff who were involved in their care. We are thankful for the skilled nursing assistance of James King, John Kincade, Norma Diaz, and Tricia Wolff, who helped to perform the metabolic studies. Elva Gonzales and Lorrie Albarado provided expert secretarial support in preparing the manuscript. We also thank Richard Castillo, Cindy Munoz, Sheila Taylor, and Kathy Camp for their technical support in performing the many substrate, hormone, and isotopic assays.


   Footnotes
 
This work was supported by National Institutes of Health Grant NRSA (to S.R.K.), National Institute of Diabetes and Digestive and Kidney Diseases Grant AM 24092, a Veterans Affairs Merit Award (to R.A.D.), General Clinical Research Center Grant RR01346, National Institute of General Medical Sciences Grant GM52419, and National Heart, Lung, and Blood Institute Grant HL 30050 (to B.S.S.M.).

First Published Online November 23, 2004

1 S.R.K. and L.J.R. contributed equally to this work. Back

Abbreviations: eNOS, Endothelial NOS; FFA, free fatty acid; FPG, fasting plasma glucose; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; ICAM-1, intracellular adhesion molecule-1; iNOS, inducible NOS; IRS, insulin receptor substrate; NO, nitric oxide; NOS, NO synthase; nNOS, neuronal NOS; PI-3 kinase, phosphatidylinositol 3-kinase; Rd, insulin-stimulated glucose disposal; T2DM, type 2 diabetes mellitus; VCAM, vascular cellular adhesion molecule.

Received April 26, 2004.

Accepted November 14, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. DeFronzo RA, Ferrannini E 1991 Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and ASCVD. Diabetes Care 14:173–194 (Review)[Abstract]
  2. Haffner SM, Stern MP, Hazuda HP, Mitchell BD, Patterson JK 1990 Cardiovascular risk factors in confirmed prediabetic individuals: does the clock for coronary heart disease start ticking before the onset of clinical diabetes? JAMA 263:2893–2898[Abstract/Free Full Text]
  3. Hsueh WA, Quinones MJ, Creager MA 1997 Endothelium in insulin resistance and diabetes. Diabetes Rev 5:343–352
  4. Ross R 1993 The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809[CrossRef][Medline]
  5. Cannon R 1998 Role of nitric oxide in cardiovascular disease: focus on the endothelium. Clin Chem 44:1809–1819[Abstract/Free Full Text]
  6. Duplain H, Burcelin R, Sartori C, Cook S, Egli M, Lepori M, Vollenweider P, Pedrazzini T, Nicod P, Thoren B, Scherrer U 2001 Insulin resistance, hyperlipemia, and hypertension in mice lacking endothelial nitric oxide synthase. Circulation 104:342–345[Abstract/Free Full Text]
  7. Shankar R, Wu Y, Shen H, Zhu J, Baron A 2000 Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Diabetes 49:684–687[Abstract]
  8. Kapur S, Bedard S, Marcotte B, Cote CH, Marette A 1997 Expression of nitric oxide synthase in skeletal muscle. A novel role for nitric oxide as a modulator of insulin action. Diabetes 46:1691–1700[Abstract]
  9. Perreault M, Marette A 2001 Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Nature 7:1138–1143[CrossRef][Medline]
  10. Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA 1990 Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 323:22–27[Abstract]
  11. Gilligan DM, Guetta V, Panza JA, Garcia CE, Quyyumi AA, Cannon III RO 1994 Selective loss of microvascular endothelial function in human hypercholesterolemia. Circulation 90:35–41[Abstract/Free Full Text]
  12. Zeiher AM, Drexler H, Wollschlager H, Just H 1991 Modulation of coronary vasomotor tone in humans. Progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 83:391–401[Abstract/Free Full Text]
  13. Steinberg HO, Chaker H, Learning R, Johnson A, Brechtel G, Baron AD 1996 Obesity/insulin resistance is associated with endothelial dysfunction. J Clin Invest 97:2601–2610[Medline]
  14. Aljada A, Saadeh R, Assian E, Ghanim H, Dandona P 2000 Insulin inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide. J Clin Endocrinol Metab 85:2572–2575[Abstract/Free Full Text]
  15. Schnyder B, Pittet M, Durand J, Schnyder-Candrian S 2002 Rapid effects of glucose on the insulin signaling of endothelial NO generation and epithelial Na transport. Am J Physiol Endocrinol Metab 282:E87–E94
  16. Laakso M, Edelman SV, Brechtel G, Baron AD 1990 Decreased effect of insulin to stimulate skeletal muscle flow in obese men. J Clin Invest 85:1844–1852
  17. Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL 1991 Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans. J Clin Invest 87:2246–2252
  18. Steinberg JO, Brechtel G, Johnson A, Fineberg N, Baron AD 1994 Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent: a novel action of insulin to increase nitric oxide release. J Clin Invest 94:1172–1179
  19. DeFronzo RA 1997 Pathogenesis of type 2 diabetes: metabolic and molecular implications for identifying diabetes genes. Diabetes Rev 4:177–269
  20. Roy D, Perreault M, Marette A 1998 Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Am J Physiol Endocrinol Metab 274:E692–E699
  21. Young ME, Leighton B 1998 Evidence of altered sensitivity of the nitric oxide/cGMP signaling cascade in insulin-resistance skeletal muscle. Biochem J 329:73–79
  22. Young ME, Radda GK, Leighton B 1997 Nitric oxide stimulates glucose transport and metabolism in rat skeletal muscle in vitro. Biochem J 322:223–228
  23. Higaki Y, Hirschman MF, Fuji N, Goodyear LJ 2001 Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. Diabetes 50:241–247[Abstract/Free Full Text]
  24. Balon TW, Nadler JL 1997 Evidence that nitric oxide increases glucose transport in skeletal muscle. J Appl Physiol 82:359–363[Abstract/Free Full Text]
  25. Etgen GJ, Fryburg DA, Gibbs EM 1997 Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction and phosphatidylinositol-3-kinase-independent pathway. Diabetes 46:1915–1919[Abstract]
  26. Tanaka T, Nakatani K, Morioka K, Urakawa H, Maruyama N, Kitagawa N, Katsuki A, Araki-Sasaki R, Hori Y, Gabazza EC, Yano Y, Wada H, Nobori T, Sumida Y, Adachi Y 2003 Nitric oxide stimulates glucose transport through insulin-independent GLUT4 translocation in 3T3–L1 adipocytes. Eur J Endocrinol 49:61–67
  27. Meneilly GS, Elliot T, Battistimi B, Floras JS 2001 N(G)-monomethyl-L-arginine alters insulin mediated calf blood flow but not glucose disposal in the elderly. Metabolism 50:306–310[CrossRef][Medline]
  28. Zavaroni I, Platti PM, Monti LD, Gasparini P, Barilli LA, Massironi P, Ardigo D, Valsecchi G, Delsignore R, Reaven GM 2000 Plasma nitric oxide concentrations are elevated in insulin-resistant healthy subjects. Metabolism 49:959–961[CrossRef][Medline]
  29. DeFronzo RA 1999 Pharmacologic therapy for type 2 diabetes mellitus. Ann Intern Med 131:281–303[Abstract/Free Full Text]
  30. Cusi K, Maezono K, Osman A, Pendergrass M, Patti M, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ 2000 Insulin resistance differentially affects the PI3-kinase and MAP kinase-mediated signaling in human muscle. J Clin Invest 105:311–320[Medline]
  31. Towbin H, Staehelin J, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract/Free Full Text]
  32. Shiuchi T, Cui TX, Wu L, Nakagami H, Takeda-Matsubara Y, Iwai M, Horiuchi M 2002 ACE inhibitor improves insulin-resistance in diabetic mice mouse via bradykinin and nitric oxide. Hypertension 40:329–334[Abstract/Free Full Text]
  33. Stuhlinger MC, Abbasi F, Chu JW, Lamendola C, McLaughlin TL, Cooke JP, Reaven GM, Tsao PS 2002 Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA 287:1420–1426[Abstract/Free Full Text]
  34. Williams IL, Wheatcroft SB, Shah AM, Kearney MT 2002 Obesity, atherosclerosis and the vascular endothelium: mechanisms of reduced nitric oxide bioavailability in obese humans. Int J Obes 26:754–764
  35. Steinberg HO, Paradici G, Hook G, Crowder K, Cronin J, Baron AD 2000 Free fatty acids elevation impairs insulin-mediated vasodilation and nitric oxide production. Diabetes 49:1231–1238[Abstract]
  36. Zeng G, Quon MJ 1996 Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J Clin Invest 98:894–898[Medline]
  37. Jiang Z, Lin Y-W, Clermont A, Igarashi M, King G 1997 Direct demonstration of selective insulin resistance on PI 3 kinase pathway in vascular tissues of obese Zucker (fa/fa) rats. Diabetes 46:54A
  38. Hsueh WA, Law RF 1999 Insulin signaling in the arterial wall. Am J Cardiol 84:21J–24J
  39. Pratipanawatr W, Pratipanawatr T, Cusi K, Berria R, Adams JM, Jenkinson CP, Maezono K, DeFronzo RA, Mandarino LJ 2001 Skeletal muscle insulin resistance in normoglycemic subjects with strong family history of type 2 diabetes is associated with decreased insulin stimulation of insulin receptor substrate-1 tyrosine phosphorylation. Diabetes 50:2572–2578[Abstract/Free Full Text]
  40. Reaven GM 1988 Banting Lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607[Abstract]
  41. Groadanovic A 2001 NO message from muscle. Microsc Res Tech 55:148–153[CrossRef][Medline]
  42. Fulton D, Gratton J-P, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC 1999 Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399:597–601[CrossRef][Medline]
  43. Chen Z-P, Mitchelhill KI, Michell BJ, Stapleton D, Rodriquez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE 1999 AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443:285–289[CrossRef][Medline]
  44. Butt E, Bernhardt M, Smolenski A, Kotsonis P, Frohlich BG, Sickmann A, Meyer HE, Lohmann SM, Schmidt HW 2000 Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 275:5179–5187[Abstract/Free Full Text]
  45. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeihe AM 1999 Activation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399:601–605[CrossRef][Medline]
  46. Roman LJ, Martasek P, Miller RT, Harris DE, de La Garza MA, Shea TM, Kim JJ, Masters BS 2000 The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin. J Biol Chem 275:29225–29232[Abstract/Free Full Text]
  47. McCabe TJ, Fulton D, Roman LJ, Sessa WC 2000 Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem 275:6123–6128[Abstract/Free Full Text]
  48. Montagnani M, Chen H, Barr VA, Quon MJ 2001 Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser1179. J Biol Chem 276:30392–30398[Abstract/Free Full Text]
  49. Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee N 2001 Hyperglycemia inhibits endothelial nitric oxide synthase activity by post translational modification at the Akt site. J Clin Invest 108:1341–1348[CrossRef][Medline]
  50. Federici M, Menghini R, Mauriello A, Hribal ML, Ferrelli F, Lauro D, Sbraccia P, Spagnoli LG, Sesti G, Lauro R 2002 Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells. Circulation 106:466–472[Abstract/Free Full Text]
  51. Cosentino F, Luscher TF 1998 Tetrahydrobiopterin and endothelial function. Eur Heart J 19(Suppl G):G3–G8
  52. Abbasi F, Brown BW, Lamendola C, McLaughlin T, Reaven GM 2002 Relationship between obesity, insulin sensitivity and coronary artery disease risk. J Am Coll Cardiol 40:37–43
  53. Chen HS, Hwu CM, Kwok CF, Shih KC, Hsiao LC, Lee SH, Lin YT, Lin SH, Ho LT 2000 Insulin sensitivity in normotensive offspring of hypertensive parents. Horm Metab Res 32:110–114[Medline]
  54. Nakai K, Itoh C, Kawazoe K, Miura Y, Sotoyanagi H, Hotta K, Itoh T, Kamata J, Hiramori K 1995 Concentration of soluble vascular cell adhesion molecule-1 (VCAM-1) correlated with expression of VCAM-1 mRNA in the human atherosclerotic aorta. Coron Artery Dis 6:497–502[Medline]
  55. Haught WH, Mansour M, Rothlein R, Kishimoto TK, Mainolfi EA, Hendricks JB, Hendricks C, Mechta JL 1996 Alterations in circulating intercellular adhesion molecule-1 and L-selectin: further evidence for chronic inflammation in ischemic heart disease. Am Heart J 132:1–8[CrossRef][Medline]
  56. Kupatt C, Weber C, Wolf DA, Becker BF, Smith TW, Kelly RA 1997 Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: role of NF-{kappa}B. J Mol Cell Cardiol 29:2599–2609[CrossRef][Medline]



This article has been cited by other articles:


Home page
 DiabetesHome page
R. A. DeFronzo
From the Triumvirate to the Ominous Octet: A New Paradigm for the Treatment of Type 2 Diabetes Mellitus
Diabetes, April 1, 2009; 58(4): 773 - 795.
[Full Text] [PDF]

Home page
 DiabetesHome page
J. M. Haus, S. R. Kashyap, T. Kasumov, R. Zhang, K. R. Kelly, R. A. DeFronzo, and J. P. Kirwan
Plasma Ceramides Are Elevated in Obese Subjects With Type 2 Diabetes and Correlate With the Severity of Insulin Resistance
Diabetes, February 1, 2009; 58(2): 337 - 343.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. A. Lteif, A. D. Fulford, R. V. Considine, I. Gelfand, A. D. Baron, and K. J. Mather
Hyperinsulinemia fails to augment ET-1 action in the skeletal muscle vascular bed in vivo in humans
Am J Physiol Endocrinol Metab, December 1, 2008; 295(6): E1510 - E1517.
[Abstract] [Full Text] [PDF]

Home page
 Diabetes CareHome page
S. R. Kashyap, A. Lara, R. Zhang, Y. M. Park, and R. A. DeFronzo
Insulin Reduces Plasma Arginase Activity in Type 2 Diabetic Patients
Diabetes Care, January 1, 2008; 31(1): 134 - 139.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
P. Tessari, A. Coracina, L. Puricelli, M. Vettore, A. Cosma, R. Millioni, D. Cecchet, A. Avogaro, A. Tiengo, and E. Kiwanuka
Acute effect of insulin on nitric oxide synthesis in humans: a precursor-product isotopic study
Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E776 - E782.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
R. Muniyappa, M. Montagnani, K. K. Koh, and M. J. Quon
Cardiovascular Actions of Insulin
Endocr. Rev., August 1, 2007; 28(5): 463 - 491.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Q.-J. Zhang, Q.-X. Li, H.-F. Zhang, K.-R. Zhang, W.-Y. Guo, H.-C. Wang, Z. Zhou, H.-P. Cheng, J. Ren, and F. Gao
Swim training sensitizes myocardial response to insulin: Role of Akt-dependent eNOS activation
Cardiovasc Res, July 15, 2007; 75(2): 369 - 380.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
J. E. Ayala, D. P. Bracy, B. M. Julien, J. N. Rottman, P. T. Fueger, and D. H. Wasserman
Chronic Treatment With Sildenafil Improves Energy Balance and Insulin Action in High Fat-Fed Conscious Mice
Diabetes, April 1, 2007; 56(4): 1025 - 1033.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. P. Mauldin, S. Srinivasan, A. Mulya, A. Gebre, J. S. Parks, A. Daugherty, and C. C. Hedrick
Reduction in ABCG1 in Type 2 Diabetic Mice Increases Macrophage Foam Cell Formation
J. Biol. Chem., July 28, 2006; 281(30): 21216 - 21224.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kashyap, S. R.
Right arrow Articles by DeFronzo, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kashyap, S. R.
Right arrow Articles by DeFronzo, R. A.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Diabetes
Related Collections
Right arrow Diabetes and Insulin

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals