Switching from NPH insulin to once-daily insulin detemir in basal-bolus-treated patients with diabetes mellitus: data from the European cohort of the PREDICTIVE study.

BACKGROUND: The PREDICTIVE study is a multinational observational study designed to follow up patients with diabetes who started insulin detemir (IDet) in routine care. Recruitment started in June 2004 and is ongoing in some countries. METHODS: We report 12-week follow-up data for patients with type 1 (T1D) or type 2 diabetes (T2D) in the European cohort who, as part of basal-bolus therapy, switched from once- (qd) or twice-daily (bid) neutral protamine Hagedorn insulin (NPH) to qd IDet. End-points - evaluated from patients' records and diaries - were incidence of serious adverse drug reactions, glycaemic parameters, hypoglycaemia and weight change. RESULTS: A total of 3637 patients were included, n = 1500 T1D [mean age 40.9 years, body mass index (BMI) 25.0 kg/m(2), glycosylated haemoglobin (HbA(1c)) 7.9%] and n = 2137 T2D (mean age 60.5 years, BMI 31.9 kg/m(2), HbA(1c) 8.0%). IDet was well tolerated. Lower overall, major and nocturnal rates of hypoglycaemia were observed in T1D and T2D patients switching from NPH to IDet (overall, T1D: 38.2-18.56 episodes/patient year, p < 0.001; T2D: 13.8-3.3 [corrected] episodes/patient year, p < 0.001). Switching from bid NPH to qd IDet resulted in significant 12-week reductions in HbA(1c) (T1D: -0.40%; T2D: -0.56%; both p < 0.001). Switching from qd NPH to qd IDet, resulted in HbA(1c) reductions of: T1D -0.52%; T2D -0.56%; both p < 0.001. Fasting blood glucose levels were also significantly reduced in patients with T1D or T2D. Overall mean weight changes were: T1D: 0.0 kg, T2D: -0.2 kg after 12 weeks. CONCLUSION: In routine care, patients with T1D or T2D may be switched from NPH to IDet qd as part of a basal-bolus regimen.

Intramyocellular lipid is associated with resistance to in vivo insulin actions on glucose uptake, antilipolysis, and early insulin signaling pathways in human skeletal muscle.

To examine whether and how intramyocellular lipid (IMCL) content contributes to interindividual variation in insulin action, we studied 20 healthy men with no family history of type 2 diabetes. IMCL was measured as the resonance of intramyocellular CH(2) protons in lipids/resonance of CH(3) protons of total creatine (IMCL/Cr(T)), using proton magnetic resonance spectroscopy in vastus lateralis muscle. Whole-body insulin sensitivity was measured using a 120-min euglycemic-hyperinsulinemic (insulin infusion rate 40 mU/m(2). min) clamp. Muscle biopsies of the vastus lateralis muscle were taken before and 30 min after initiation of the insulin infusion to assess insulin signaling. The subjects were divided into groups with high IMCL (HiIMCL; 9.5 +/- 0.9 IMCL/Cr(T), n = 10) and low IMCL (LoIMCL; 3.0 +/- 0.5 IMCL/Cr(T), n = 10), the cut point being median IMCL (6.1 IMCL/Cr(T)). The groups were comparable with respect to age (43 +/- 3 vs. 40 +/- 3 years, NS, HiIMCL versus LoIMCL), BMI (26 +/- 1 vs. 26 +/- 1 kg/m(2), NS), and maximal oxygen consumption (33 +/- 2 vs. 36 +/- 3 ml. kg(-1). min(-1), NS). Whole-body insulin-stimulated glucose uptake was lower in the HiIMCL group (3.0 +/- 0.4 mg. kg(-1). min(-1)) than the LoIMCL group (5.1 +/- 0.5 mg. kg(-1). min(-1), P < 0.05). Serum free fatty acid concentrations were comparable basally, but during hyperinsulinemia, they were 35% higher in the HiIMCL group than the LoIMCL group (P < 0.01). Study of insulin signaling indicated that insulin-induced tyrosine phosphorylation of the insulin receptor (IR) was blunted in HiIMCL compared with LoIMCL (57 vs. 142% above basal, P < 0.05), while protein expression of the IR was unaltered. IR substrate-1-associated phosphatidylinositol (PI) 3-kinase activation by insulin was also lower in the HiIMCL group than in the LoIMCL group (49 +/- 23 vs. 84 +/- 27% above basal, P < 0.05 between HiIMCL and LoIMCL). In conclusion, IMCL accumulation is associated with whole-body insulin resistance and with defective insulin signaling in skeletal muscle independent of body weight and physical fitness.

Effects of oral and transdermal estrogen replacement therapy on markers of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in postmenopausal women.

We compared the effects of oral estradiol (2 mg), transdermal estradiol (50 microg), and placebo on measures of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in 27 postmenopausal women at baseline and after 2 and 12 weeks of treatment. Oral and transdermal estradiol induced similar increases in serum free estradiol concentrations. Oral therapy increased the plasma concentrations of factor VII antigen (FVIIag) and activated factor VII (FVIIa), and the plasma concentration of the prothrombin activation marker prothrombin fragment 1+2 (F1+2). Oral but not transdermal estradiol therapy significantly lowered plasma plasminogen activator inhibitor-1 (PAI-1) antigen and tissue-type plasminogen activator (tPA) antigen concentrations and PAI-1 activity, and increased D-dimer concentrations, suggesting increased fibrinolysis. The concentration of soluble E-selectin decreased and serum C-reactive protein (CRP) increased significantly in the oral but not in the transdermal or placebo groups. In the oral but not in the transdermal or placebo estradiol groups low-density-lipoprotein (LDL) cholesterol, apolipoprotein B and lipoprotein (a) concentrations decreased while high-density-lipoprotein (HDL) cholesterol, apolipoprotein AI and apolipoprotein All concentrations increased significantly. LDL particle size remained unchanged. In summary, oral estradiol increased markers of fibrinolytic activity, decreased serum soluble E-selectin levels and induced potentially antiatherogenic changes in lipids and lipoproteins. In contrast to these beneficial effects, oral estradiol changed markers of coagulation towards hypercoagulability, and increased serum CRP concentrations. Transdermal estradiol or placebo had no effects on any of these parameters. These data demonstrate that oral estradiol does not have uniformly beneficial effects on cardiovascular risk markers and that the oral route of estradiol administration rather than the circulating free estradiol concentration is critical for any changes to be observed.

A model to explore the interaction between muscle insulin resistance and beta-cell dysfunction in the development of type 2 diabetes.

Type 2 diabetes is a polygenic disease characterized by defects in both insulin secretion and insulin action. We have previously reported that isolated insulin resistance in muscle by a tissue-specific insulin receptor knockout (MIRKO mouse) is not sufficient to alter glucose homeostasis, whereas beta-cell-specific insulin receptor knockout (betaIRKO) mice manifest severe progressive glucose intolerance due to loss of glucose-stimulated acute-phase insulin release. To explore the interaction between insulin resistance in muscle and altered insulin secretion, we created a double tissue-specific insulin receptor knockout in these tissues. Surprisingly, betaIRKO-MIRKO mice show an improvement rather than a deterioration of glucose tolerance when compared to betaIRKO mice. This is due to improved glucose-stimulated acute insulin release and redistribution of substrates with increased glucose uptake in adipose tissue and liver in vivo, without a significant decrease in muscle glucose uptake. Thus, insulin resistance in muscle leads to improved glucose-stimulated first-phase insulin secretion from beta-cells and shunting of substrates to nonmuscle tissues, collectively leading to improved glucose tolerance. These data suggest that muscle, either via changes in substrate availability or by acting as an endocrine tissue, communicates with and regulates insulin sensitivity in other tissues.

Differential effects of oral and transdermal estrogen replacement therapy on endothelial function in postmenopausal women.

BACKGROUND: We determined whether the vascular effects of estradiol depend on the route of administration by comparing the effects of oral estradiol and transdermal placebo, transdermal estradiol and oral placebo, and transdermal placebo and oral placebo on in vivo endothelial function in 27 postmenopausal women. METHODS AND RESULTS: Endothelial function was assessed from blood flow responses to intrabrachial artery infusions of endothelium-dependent (7.5 and 15 microgram/min acetylcholine) and endothelium-independent (3 and 10 microgram/min of sodium nitroprusside) vasodilators at 0, 2, and 12 weeks. In the oral estradiol group, the increase in flow above basal during infusion of the low dose of acetylcholine at 0, 2, and 12 weeks averaged 6.0+/-0.8, 6.9+/-0.8, and 11.3+/-1.2 (P<0.01 versus 0 and 2 weeks) mL. dL(-1). min(-1) at 0, 2, and 12 weeks. The percentage increases versus 0 weeks averaged 21+/-14% at 2 and 120+/-34% at 12 weeks. During the high-dose acetylcholine infusion, the increase in flow above basal averaged 8.6+/-1.3, 10.2+/-1.5, and 15.1+/-1.8 (P<0.05 versus 0 weeks) mL. dL(-1). min(-1), respectively. The percentage increases versus 0 weeks averaged 22+/-10% at 2 weeks and 119+/-46% at 12 weeks. In the oral estradiol group, endothelium-independent vasodilatation also improved significantly, but less markedly than endothelium-dependent responses. In the transdermal and placebo groups, all vascular responses remained unchanged. Oral but not transdermal estradiol also induced significant decreases in LDL cholesterol and Lp(a) concentrations and an increase in HDL cholesterol within 2 weeks. CONCLUSIONS: We conclude that oral but not transdermal estradiol induces potentially antiatherogenic changes in in vivo endothelium-dependent vasodilatation and lipid concentrations.

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance.

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.

Effect of estrogen replacement therapy on insulin sensitivity of glucose metabolism and preresistance and resistance vessel function in healthy postmenopausal women.

In the present study, we hypothesized that estradiol, via its ability to vasodilate in an endothelium-dependent manner, might enhance vascular effects of insulin. Basal and insulin-stimulated peripheral blood flow and resistance, arterial stiffness, and glucose metabolism were determined in 27 healthy postmenopausal women before and after 12 weeks of treatment with either transdermal or oral estradiol or corresponding placebo preparations. Whole body insulin sensitivity was determined using the euglycemic insulin clamp technique (rate of continuous insulin infusion 1 mU/kg.min), forearm blood flow with a strain-gauge plethysmography, and arterial stiffness using pulse wave analysis. Estradiol therapy increased basal peripheral blood flow (1.5 +/- 0.1 vs. 1.9 +/- 0.1 mL/dL.min, 0 vs. 12 weeks; P: < 0.01), decreased peripheral vascular resistance (65 +/- 3 vs. 52 +/- 3 mm Hg/mL/dL.min, respectively; P: < 0.01), and diastolic blood pressure (78 +/- 2 vs. 75 +/- 2 mm Hg, respectively; P: < 0.05) but had no effect on large artery stiffness. Infusion of insulin did not acutely alter peripheral blood flow but diminished large artery stiffness significantly both before and after the 12-week period of estradiol therapy. No measure of acute insulin action (glucose metabolism, blood flow, or large artery stiffness) was altered by estradiol or placebo treatment. These data demonstrate that insulin and estradiol have distinct hemodynamic effects. Physiological doses of estradiol increase peripheral blood flow but have no effects on large artery stiffness, whereas physiological concentrations of insulin acutely decrease stiffness without changing peripheral blood flow. Putative vasculoprotection by estradiol is, thus, not mediated via alterations in arterial stiffness or insulin sensitivity.

Autoantibodies against oxidized LDL and endothelium-dependent vasodilation in insulin-dependent diabetes mellitus.

We determined whether autoantibodies against oxidized LDL are increased in patients with IDDM, and if so, whether they are associated with endothelial dysfunction in vivo. Autoantibodies against oxidized LDL (ratio of antibodies against oxidized vs. native LDL, oxLDLab) were determined in 38 patients with IDDM (HbA(1c) 8.4+/-0.2%), who were clinically free of macrovascular disease, and 33 healthy normolipidemic subjects (HbA(1c) 5.1+/-0.1%, P<0.001 vs. IDDM). The groups had comparable serum total-, LDL- (2. 9+/-0.1 vs. 2.8+/-0.1 mmol/l, IDDM vs. controls), and HDL-cholesterol concentrations. OxLDLab were 1.5-fold higher in the IDDM patients (1.8+/-0.1) than in the normal subjects (1.2+/-0.1, P<0.001). OxLDLab were correlated with age in normal subjects, but not with age, duration of disease, LDL-cholesterol, HbA(1c) or degree of microvascular complications in patients with IDDM. To determine whether oxLDLab are associated with endothelial dysfunction in vivo, blood flow responses to intrabrachial infusions of acetylcholine, sodium nitroprusside and L-NMMA were determined in 23 of the patients with IDDM (age 33+/-1 years, body mass index 24. 3+/-0.6 kg/m(2), HbA(1c) 8.5+/-0.3%) and in the 33 matched normal males. OxLDLab were 41% increased in IDDM (1.7+/-0.2 vs. 1.2+/-0.1, P<0.01). Within the group of IDDM patients, HbA(1c) but not oxLDLab or LDL-cholesterol, was inversely correlated with the forearm blood flow response to acetylcholine (r=-0.51, P<0.02), an endothelium-dependent vasodilator, but not to sodium nitroprusside (r=0.06, NS). These data demonstrate that oxLDLab concentrations are increased in patients with IDDM, but show that chronic hyperglycemia rather than oxLDLab, is associated with impaired endothelium-dependent vasodilation in these patients.

Glutamine: fructose-6-phosphate amidotransferase activity and gene expression are regulated in a tissue-specific fashion in pregnant rats.

We examined whether regulation of glutamine: fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme of the hexosamine pathway, is tissue specific and if so whether such regulation occurs at the level of gene expression. We compared GFA activity and expression and levels of UDP-hexosamines and UDP-hexoses between insulin-sensitive (liver and muscle) tissues and a glucose-sensitive (placenta) tissue from 19 day pregnant streptozotocin diabetic and non-diabetic rats. In pregnant non-diabetic rats GFA activities averaged (1521+/-75 pmol/mg protein x min) in the placenta, 895+/-74 in the liver and 81+/-11 in muscle (p<0.001 between each tissue). In the diabetic rats, GFA activities were approximately 50% decreased both in the liver (340+/-42 pmol/mg protein x min, p<0.05 vs control rats) and in skeletal muscle (46+/-3, p<0.05) compared to control rats. In the placenta, GFA activities were identical between diabetic (1519+/-112 pmol/mg protein x min) and non-diabetic (1521+/-75) animals. In the liver, the reduction in GFA activity could be attributed to a significant decrease in GFA mRNA concentrations, while GFA mRNA concentrations were similar in the placenta between diabetic and non-diabetic animals. UDP-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine pathway, was significantly reduced in the liver and in skeletal muscle but similar in the placenta between diabetic and non-diabetic rats. In summary, GFA activity and expression and the concentration of UDP-GlcNAc are decreased in the liver but unaltered in the placenta, although GFA activity is almost 2-fold higher in this tissue than in the liver. These data provide the first evidence for tissue specific regulation of GFA and for its regulation at the level of gene expression.

Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle.

To explore potential cellular mechanisms by which activation of the hexosamine pathway induces insulin resistance, we have evaluated insulin signaling in conscious fasted rats infused for 2-6 h with saline, insulin (18 mU x kg(-1) x min(-1)), or insulin and glucosamine (30 micromol x kg(-1) x min(-1)) under euglycemic conditions. Glucosamine infusion increased muscle UDP-N-acetylglucosamine concentrations 3.9- and 4.3-fold over saline- or insulin-infused animals, respectively (P < 0.001). Glucosamine induced significant insulin resistance to glucose uptake both at the level of the whole body and in rectus abdominis muscle, and it blunted the insulin-induced increase in muscle glycogen content. At a cellular level, these metabolic effects were paralleled by inhibition of postreceptor insulin signaling critical for glucose transport and glycogen storage, including a 45% reduction in insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation (P = 0.02), a 44% decrease in IRS-1 association with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase (P = 0.03), a 34% reduction in IRS-1-associated PI 3-kinase activity (P = 0.03), and a 51% reduction in insulin-stimulated glycogen synthase activity (P = 0.03). These alterations in postreceptor insulin signaling were time-dependent and paralleled closely the progressive inhibition of systemic glucose disposal from 2 to 6 h of glucosamine infusion. We also demonstrated that glucosamine infusion results in O-linked N-acetylglucosamine modification of IRS-1 and IRS-2. These data indicate that activation of the hexosamine pathway may directly modulate early postreceptor insulin signal transduction, perhaps via posttranslation modification of IRS proteins, and thus contribute to the insulin resistance induced by chronic hyperglycemia.

Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart.

Glucosamine infusion induces insulin resistance in vivo, but the effect of glucosamine on intracellular metabolites of the hexosamine pathway, especially glucosamine-6-phosphate (GlcN6P) is unknown. Because of the structural similarity of glucose-6-phosphate (G-6-P) and GlcN6P, we hypothesized that accumulation of this metabolite might alter the activities of enzymes such as glycogen synthase and hexokinase. We infused glucosamine (30 micromol x kg(-1) x min(-1)) to induce insulin resistance in rats during a euglycemic-hyperinsulinemic clamp. Glucosamine induced whole-body insulin resistance, which was apparent after 90 min and continued progressively for 360 min. Despite inducing severe whole-body insulin resistance and decrease in glycogen synthase fractional activity in rectus abdominis muscle (69+/-3 vs. 83+/-1%, P<0.01) and heart (7+/-1 vs. 32+/-4%, P<0.001), glucosamine did not change the glycogen content in rectus and even increased it in the heart (209+/-13 vs. 117+/-9 mmol/kg dry wt, P<0.001). Glucosamine increased tissue concentrations of UDP-GlcNAc 4.4- and 4.6-fold in rectus abdominis and heart, respectively. However, GlcN6P concentrations increased 500- and 700-fold in glucosamine-infused animals in rectus abdominis (590+/-80 vs. 1.2+/-0.1 micromol/kg wet wt, P<0.001) and heart (7,703+/-993 vs. 11.2+/-2.3 micromol/kg wet wt, P<0.001). To assess the possible significance of GlcN6P accumulation, we measured the effect of GlcN6P on glycogen synthase and hexokinase activity in vitro. At the GlcN6P concentrations measured in rectus abdominis and heart in vivo, glycogen synthase was activated by 21 and 542%, while similar concentrations inhibited hexokinase activity by 5 and 46%, respectively. This study demonstrates that infusion of glucosamine during a euglycemic-hyperinsulinemic clamp results in marked accumulation of intracellular GlcN6P. The GlcN6P concentrations in the heart and rectus abdominis muscle reach levels sufficient to cause allosteric activation of glycogen synthase and inhibition of hexokinase.

Insulin and glucosamine infusions increase O-linked N-acetyl-glucosamine in skeletal muscle proteins in vivo.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of serine/threonine residues of nuclear and cytoplasmic proteins. We determined whether insulin or coinfusion of glucosamine (GlcN) with insulin alters O-GlcNAc of skeletal muscle proteins. Three groups of conscious fasted rats received 6-hour infusions of either saline (BAS), insulin 18 mU/kg.min and saline (INS), or insulin and GlcN 30 micromol/kg.min (GLCN) during maintenance of normoglycemia. At 6 hours, the concentrations of muscle UDP-GlcNAc, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), glycogen, and N and O-linked GlcNAc (galactosyltransferase labeling followed by beta elimination) were measured in freeze-clamped abdominis muscle. Insulin increased whole-body glucose uptake from 49 +/- 5 to 239 +/- 8 micromol/kg.min (P < .001) and glycogen in abdominis muscle from 138 +/- 11 to 370 +/- 26 mmol/kg dry weight (P < .001). Insulin increased the amount of cytosolic N - and O-linked GlcNAc by 56% from 362 +/- 30 to 564 +/- 45 dpm/microg protein . 100 min (P < .02), and O-GlcNAc from 221 +/- 16 to 339 +/- 27 dpm/microg . 100 min (P < .02). Glycogen content was positively correlated with the amount of total (r = .90, P < .005) and O-linked GlcNAc in insulin-infused animals. Coinfusion of GlcN with insulin increased muscle UDP-GlcNAc about fourfold (100 +/- 6 nmol/g) compared with insulin (27 +/- 1, P < .001) or saline (25 +/- 1, P < .001) infusion. GlcN also decreased glucose uptake over 6 hours by 30% to 168 +/- 8 micromol/kg . min (P < .001 for GLCN v INS) and muscle glycogen to 292 +/- 24 mmol/kg dry weight (P < .05 for GLCN v INS). Both total (635 +/- 60 dpm/microg . 100 min, P < .002) and O-linked GlcNAc (375 +/- 36 dpm/microg . 100 min, P < .002) in the cytosol were significantly higher in GLCN rats (635 +/- 60 dpm/microg) versus BAS rats (P < .002). As in INS rats, muscle glycogen and O-GlcNAc were positively correlated in GLCN rats (r = .54, P < .05). Variation in total and O-linked GlcNAc in GLCN rats was due both to GlcN (P < .02) and to variation in the glycogen content (P < .005).

Incorporation of [3-3H]glucose and 2-[1-14C]deoxyglucose into glycogen in heart and skeletal muscle in vivo: implications for the quantitation of tissue glucose uptake.

2-deoxyglucose has been widely used to quantitate tissue glucose uptake in vivo, assuming that 2-deoxyglucose is transported and phosphorylated but not further metabolized. We examined the validity of this assumption by infusing [3-3H]glucose and 2-[1-14C]deoxyglucose in a similar primed continuous fashion to chronically catheterized, freely moving rats during normoglycemic hyperinsulinemic conditions. The rates of 2-deoxyglucose uptake were determined from the accumulation of 2-[1-14C]deoxyglucose-6-phosphate and 2-[1-14C]deoxyglucose-6-phosphate combined with the rate of the incorporation of 2-[1-14C]deoxyglucose into glycogen in rectus abdominis muscle and the heart. When the rates of glycogen synthesis during the 2-h hyperinsulinemic period from the two tracers were compared in rectus abdominis muscle, the rate of glycogen synthesis was twofold higher when measured with [3-3H]glucose (337 +/- 14 micromol x kg(-1) x min(-1)) than when measured with 2-[1-14C]deoxyglucose (166 +/- 10 micromol x kg(-1) x min(-1), P < 0.001). In the heart, the rate of glycogen synthesis was twofold higher when measured with 2-[1-14C]deoxyglucose (141 +/- 20 micromol x kg(-1) x min(-1)) than when measured with [3-3H]glucose (72 +/- 15 micromol x kg(-1) x min(-1), P < 0.001). The rate of 2-deoxyglucose uptake was 29% underestimated in rectus abdominis muscle, when counts found in glycogen were not included in glucose uptake calculations (398 +/- 25 vs. 564 +/- 25 micromol x kg(-1) x min(-1), P < 0.001). In the heart, glucose uptake was underestimated by 7% if glycogen counts were not taken into account (1,786 +/- 278 vs. 1,926 +/- 291 micromol x kg(-1) dry x min(-1), P < 0.05). The fraction of [3-3H]glucose incorporated into glycogen of total glucose metabolism (calculated from 2-deoxyglucose conversion to 2-deoxyglucose-6-phosphate and glycogen) was 0.6 (337/564) in rectus abdominis muscle and 0.037 (72/1,926) in the heart. We conclude that 2-deoxyglucose is incorporated into glycogen in the heart and in skeletal muscle in vivo under normoglycemic hyperinsulinemic conditions in the rat. Failure to consider the incorporation of 2-deoxyglucose into glycogen will underestimate the rate of tissue glucose uptake. To avoid such problems, the amount of 2-deoxyglucose incorporated into glycogen should be quantitated in subsequent studies.

Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance in multiple insulin sensitive tissues.

We determined the effect of infusion of glucosamine (GlcN), which bypasses the rate limiting reaction in the hexosamine pathway, on insulin-stimulated rates of glucose uptake and glycogen synthesis in vivo in rat tissues varying with respect to their glutamine:fructose-6-phosphate amidotransferase (GFA) activity. Three groups of conscious fasted rats received 6-h infusions of either saline (BAS), insulin (18 mU/kg x min) and saline (INS), or insulin and GlcN (30 micromol/ kg x min, GLCN). [3-(3)H]glucose was infused to trace whole body glucose kinetics and glycogen synthesis, and rates of tissue glucose uptake were determined using a bolus injection of [1-(14)C]2-deoxyglucose at 315 min. GlcN decreased insulin-stimulated glucose uptake (315-360 min) by 49% (P < 0.001) at the level of the whole body, and by 31-53% (P < 0.05 or less) in the heart, epididymal fat, submandibular gland and in soleus, abdominis and gastrocnemius muscles. GlcN completely abolished glycogen synthesis in the liver. GlcN decreased insulin-stimulated glucose uptake similarly in the submandibular gland (1.3 +/- 0.2 vs. 2.0 +/- 0.3 nmol/mg protein x min, GLCN vs. INS, P < 0.05) and gastrocnemius muscle (1.4 +/- 0.3 vs. 3.1 +/- 0.5 nmol/mg protein x min), although the activity of the hexosamine pathway, as judged from basal GFA activity, was 10-fold higher in the submandibular gland (286 +/- 35 pmol/mg protein x min) than in gastrocnemius muscle (27 +/- 3 pmol/mg protein x min, P < 0.001). These data raise the possibility that overactivity of the hexosamine pathway may contribute to glucose toxicity not only in skeletal muscle but also in other insulin sensitive tissues. They also imply that the magnitude of insulin resistance induced between tissues is determined by factors other than GFA.

Hyperreactivity to nitrovasodilators in forearm vasculature is related to autonomic dysfunction in insulin-dependent diabetes mellitus.

BACKGROUND: The link between diabetes and vascular disease is poorly understood. Data regarding endothelial function in vivo in patients with insulin-dependent diabetes mellitus (IDDM) have been inconsistent with in vitro studies demonstrating hyperglycemia-induced impairments in endothelium-dependent vasodilation. METHODS AND RESULTS: We determined whether alterations in neural control of the vascular tone might contribute to blood flow responses to intrabrachial infusions of acetylcholine (ACh), sodium nitroprusside (SNP), and L-N-monomethyl-arginine (L-NMMA) in 22 men with IDDM (12 with normoalbuminuria. HbA1c = 8.6 +/- 0.3%; 10 with macroalbuminuria, HbA1c = 8.6 +/- 0.3%) and 11 matched normal men. Autonomic function was assessed from reflex vasoconstriction to cold, the blood pressure response to standing and hand grip, and heart rate variation, including spectral analysis, during controlled breathing, and the Valsalva maneuver. IDDM with macroalbuminuria exhibited hyperresponsiveness to both ACh and SNP compared with the patients with normoalbuminuria or normal subjects. Reflex sympathetic vasoconstriction to cold was severely impaired in the IDDM patients with macroalbuminuria (-19 +/- 6%) compared with normoalbuminuric patients (-39 +/- 5%, P < .05) and normal subjects (-54 +/- 7%, P < .001). The macroalbuminuric patients also had evidence of autonomic dysfunction during controlled and deep breathing tests and during the Valsalva maneuver. Within the group of IDDM patients, neither the urinary albumin excretion rate nor other parameters such as HbA1c or serum cholesterol correlated with forearm blood flow during the vasoactive drug infusions. There were, however, significant inverse correlations between several measures of both sympathetic and parasympathetic autonomic functions and vascular hyperresponsiveness to SNP and ACh. For example, the Valsalva ratio was inversely correlated with the increase in blood flow in response to infusion of 3 (r = -.74, P < .001) and 10 (r = -.73, P < .001) micrograms/min SNP and 7.5 (r = -.73, P < .001) and 15 (r = -.75, P < .001) micrograms/min ACh. CONCLUSIONS: These data are consistent with idea that altered neurotransmission is an important determinant of vascular reactivity of diabetic blood vessels to nitrovasodilators in vivo.

UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues.

Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using 3H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2-4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14-36-fold higher in submandibular gland and 5-18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 +/- 0.3 nmol/mg.min, which was not different from that in normal subjects (3.3 +/- 0.4 nmol/mg.min). A 180-min intravenous insulin infusion (40 mU/m2.min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions.

Dissociation between insulin sensitivity of glucose uptake and endothelial function in normal subjects.

Insulin increases limb blood flow in a time- and dose-dependent manner. This effect can be blocked by inhibiting nitric oxide synthesis. These data raise the possibility that insulin resistance is associated with endothelial dysfunction. To examine whether endothelial function and insulin sensitivity are interrelated we quantitated in vivo insulin-stimulated rates of whole body and forearm glucose uptake at a physiological insulin concentration (euglycaemic hyperinsulinaemic clamp, 1 mU.kg-1.min-1 insulin infusion for 2 h) and on another occasion, in vivo endothelial function (blood flow response to intrabrachial infusions of sodium nitroprusside, acetylcholine, and N-monomethyl-L-arginine) in 30 normal male subjects. Subjects were divided into an insulin-resistant (IR) and an insulin-sensitive (IS) group based on the median rate of whole body glucose uptake (31 +/- 2 vs 48 +/- 1 mumol.kg-1.min-1, p < 0.001). The IR and IS groups were matched for age, but the IR group had a slightly higher body mass index, percentage of body fat and blood pressure compared to the IS group. The IR group also had diminished insulin-stimulated glucose extraction (p < 0.05) compared to the IS group, while basal and insulin-stimulated forearm blood flow rates were identical. There was no difference between the IR and IS groups in the forearm blood flow response to endothelium-dependent (acetylcholine and N-monomethyl-L-arginine) or -independent (sodium nitroprusside) vasoactive drugs. In conclusion, the ability of insulin to stimulate glucose uptake at physiological insulin concentrations and endothelium-dependent vasodilatation are distinct phenomena and do not necessarily coexist.