Antti Virkamäki

Chronic Hyperglycemia Impairs Endothelial Function and Insulin Sensitivity Via Different Mechanisms in Insulin-Dependent Diabetes Mellitus

BACKGROUNDnWe explored whether chronic hyperglycemia is associated with defects in endothelium-dependent vasodilatation in vivo and whether defects in the hemodynamic effects of insulin explain insulin resistance.nnnMETHODS AND RESULTSnVasodilator responses to brachial artery infusions of acetylcholine, sodium nitroprusside, and NG-monomethyl-L-arginine and, on another occasion, in vivo insulin sensitivity (euglycemic insulin clamp combined with the forearm catheterization technique) were determined in 18 patients with insulin-dependent diabetes mellitus (IDDM) and 9 normal subjects. At identical glucose and insulin levels, insulin stimulation of whole-body and forearm glucose uptake was 57% reduced in the IDDM patients compared with normal subjects (P < .001). The defect in forearm glucose uptake was attributable to a defect in glucose extraction (glucose AV difference, 1.1 +/- 0.2 versus 1.9 +/- 0.2 mmol/L, P < .001, IDDM versus normal subjects), not blood flow. Within the group of IDDM patients, hemoglobin A1c was inversely correlated with forearm blood flow during administration of acetylcholine (r = -.50, P < .02) but not sodium nitroprusside (r = .07). The ratio of endothelium-dependent to endothelium-independent blood flow was approximately 40% lower in patients with poor glycemic control than in normal subjects or patients with good or moderate glycemic control.nnnCONCLUSIONSnWe conclude that chronic hyperglycemia is associated with impaired endothelium-dependent vasodilatation in vivo and with a glucose extraction defect during insulin stimulation. These data imply that chronic hyperglycemia impairs vascular function and insulin action via distinct mechanisms. The defect in endothelium-dependent vasodilatation could contribute to the increased cardiovascular risk in diabetes.

Differential Effects of Oral and Transdermal Estrogen Replacement Therapy on Endothelial Function in Postmenopausal Women

BackgroundWe 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 ResultsEndothelial function was assessed from blood flow responses to intrabrachial artery infusions of endothelium-dependent (7.5 and 15 &mgr;g/min acetylcholine) and endothelium-independent (3 and 10 &mgr;g/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. ConclusionsWe conclude that oral but not transdermal estradiol induces potentially antiatherogenic changes in in vivo endothelium-dependent vasodilatation and lipid concentrations.

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).

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

Summary 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 μmol · 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. [Diabetologia (1996) 39: 1477–1482]

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 µmol · kg−1 · min−1) than when measured with 2-[1-14C]deoxyglucose (166 ± 10 µmol · kg−1 · 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 µmol · kg−1 · min−1) than when measured with [3-3H]glucose (72 ± 15 µmol · kg−1 · 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 µmol · kg−1 · 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 µmol · kg−1 dry · 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.

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.