Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.     

Insulin therapy in burn patients does not contribute to hepatic triglyceride production.

Lipid kinetics were studied in six severely burned patients who were treated with a high dose of exogenous insulin plus glucose to promote protein metabolism. The patients were 20+/-2-yr-old (SD) with 63+/-8% total body surface area burned. They were studied in a randomized order (a) in the fed state on the seventh day of a control period (C) of continuous high-carbohydrate enteral feeding alone, and (b) on the seventh day of enteral feeding plus exogenous insulin (200 pmol/h = 28 U/h) with extra glucose given as needed to avoid hypoglycemia (I+G). Despite a glucose delivery rate approximately 100% in excess of energy requirements, the following lipid parameters were unchanged: (a) total hepatic VLDL triglyceride (TG) secretion rate (0.165+/-0.138 [C] vs. 0.154+/- 0.138 mmol/kg . d-1 [I+G]), (b) plasma TG concentration (1.58+/-0.66 [C] vs. 1. 36+/-0.41 mmol/liter [I+G]), and (c) plasma VLDL TG concentration (0. 68+/-0.79 [C] vs. 0.67+/- 0.63 mmol/liter [I+G]). Instead, the high-carbohydrate delivery in conjunction with insulin therapy increased the proportion of de novo-synthesized palmitate in VLDL TG from 13+/-5% (C) to 34+/-14% (I+G), with a corresponding decreased amount of palmitate from lipolysis. In association with the doubling of the secretion rate of de novo-synthesized fatty acid (FA) in VLDL TG during insulin therapy (P > 0.5), the relative amount of palmitate and stearate increased from 35+/-5 to 44+/-8% and 4+/-1 to 7+/-2%, respectively, in VLDL TG, while the relative concentration of oleate and linoleate decreased from 43+/-5 to 37+/-6% and 8+/-4% to 2+/-2%, respectively. A 15-fold increase in plasma insulin concentration did not change the rate of release of FA into plasma (8.22+/-2.86 [C] vs. 8.72+/-6.68 mmol/kg.d-1 [I+G]. The peripheral release of FA represents a far greater potential for hepatic lipid accumulation in burn patients than the endogenous hepatic fat synthesis, even during excessive carbohydrate intake in conjunction with insulin therapy.     

Insulin-like growth factor-I improves glucose and lipid metabolism in type 2 diabetes mellitus.

Hyperglycemia, hyperinsulinemia, and insulin resistance cause vascular disease in type 2 diabetes mellitus. Dietary treatment alone often fails and oral drugs or insulin enhance hyperinsulinemia. In previous studies, an intravenous bolus of recombinant human insulin-like growth factor-I (rhIGF-I) caused normoglycemia in insulin-resistant diabetics whereas rhIGF-I infusions lowered insulin and lipid levels in healthy humans, suggesting that rhIGF-I is effective in insulin-resistant states. Thus, eight type 2 diabetics on a diet received on five treatment days subcutaneous rhIGF-I (2 x 120 micrograms/kg) after five control days. Fasting and postprandial glucose, insulin, C-peptide, proinsulin, glucagon, triglyceride, insulin-like growth factor-I and -II, and growth hormone levels were determined. RhIGF-I administration increased total IGF-I serum levels 5.3-fold above control. During the control period mean (+/- SD) fasting glucose, insulin, C-peptide, and total triglyceride levels were 11.0 +/- 4.3 mmol/liter, 108 +/- 50 pmol/liter, 793 +/- 250 pmol/liter, and 3.1 +/- 2.7 mmol/liter, respectively, and decreased during treatment to a nadir of 6.6 +/- 2.5 mmol/liter, 47 +/- 18 pmol/liter, 311 +/- 165 pmol/liter, and 1.6 +/- 0.8 mmol/liter (P < 0.01), respectively. Postprandial areas under the glucose, insulin, and C-peptide curve decreased to 77 +/- 13 (P < 0.02), 52 +/- 11, and 60 +/- 9% (P < 0.01) of control, respectively. RhIGF-I decreased the proinsulin/insulin ratio whereas glucagon levels remained unchanged. The magnitude of the effects of rhIGF-I correlated with the respective control levels. Since rhIGF-I appears to improve insulin sensitivity directly and/or indirectly, it may become an interesting tool in type 2 diabetes and other states associated with insulin resistance.     

Intracellular lactate- and pyruvate-interconversion rates are increased in muscle tissue of non-insulin-dependent diabetic individuals.

The contribution of muscle tissues of non-insulin-dependent diabetes mellitus (NIDDM) patients to blood lactate appearance remains undefined. To gain insight on intracellular pyruvate/lactate metabolism, the postabsorptive forearm metabolism of glucose, lactate, FFA, and ketone bodies (KB) was assessed in seven obese non-insulin-dependent diabetic patients (BMI = 28.0 +/- 0.5 kg/m2) and seven control individuals (BMI = 24.8 +/- 0.5 kg/m2) by using arteriovenous balance across forearm tissues along with continuous infusion of [3-13C1]-lactate and indirect calorimetry. Fasting plasma concentrations of glucose (10.0 +/- 0.3 vs. 4.7 +/- 0.2 mmol/liter), insulin (68 +/- 5 vs. 43 +/- 6 pmol/liter), FFA (0.57 +/- 0.02 vs. 0.51 +/- 0.02 mmol/liter), and blood levels of lactate (1.05 +/- 0.04 vs. 0.60 +/- 0.06 mmol/liter), and KB (0.48 +/- 0.04 vs. 0.29 +/- 0.02 mmol/liter) were higher in NIDDM patients (P < 0.01). Forearm glucose uptake was similar in the two groups (10.3 +/- 1.4 vs. 9.6 +/ 1.1 micromol/min/liter of forearm tissue), while KB uptake was twice as much in NIDDM patients as compared to control subjects. Lactate balance was only slightly increased in NIDDM patients (5.6 +/- 1.4 vs. 3.3 +/- 1.0 micromol/min/liter; P = NS). A two-compartment model of lactate and pyruvate kinetics in the forearm tissue was used to dissect out the rates of lactate to pyruvate and pyruvate to lactate interconversions. In spite of minor differences in the lactate balance, a fourfold increase in both lactate- (44.8 +/- 9.0 vs. 12.6 +/- 4.6 micromol/min/liter) and pyruvate-(50.4 +/- 9.8 vs. 16.0 +/- 5.0 micromol/min/liter) interconversion rates (both P < 0.01) were found. Whole body lactate turnover, assessed by using the classic isotope dilution principle, was higher in NIDDM individuals (46 +/- 9 vs. 21 +/- 3 micromol/min/kg; P < 0.01). Insights into the physiological meaning of this parameter were obtained by using a whole body noncompartmental model of lactate/pyruvate kinetics which provides a lower and upper bound for total lactate and pyruvate turnover (NIDDM = 46 +/- 9 vs. 108 +/- 31; controls = 21 +/- 3 - 50 +/-13 micromol/min/kg). In conclusion, in the postabsorptive state, despite a trivial lactate release by muscle, lactate- and pyruvate-interconversion rates are greatly enhanced in NIDDM patients, possibly due to concomitant impairment in the oxidative pathway of glucose metabolism. This finding strongly suggest a major disturbance in intracellular lactate/pyruvate metabolism in NIDDM.     

Release of somatostatin-like immunoreactivity from the perfused canine thyroid. Selective stimulatory effect of calcium ions.

It is well accepted that the C cells of the thyroid contain somatostatin, but the role in local endocrine function has not yet been firmly established in this organ, and it has not been proved that thyroidal somatostatin is released into the circulation. We have measured the contents of somatostatin-like immunoreactivity in the effluent of canine thyroid glands perfused without recirculation with a synthetic buffer medium. During basal conditions a definite release was consistently found in the order of 10 pg/ml corresponding to 12 pg/min. The somatostatin-like immunoreactivity was studied in dilution experiments and by gel-filtration chromatography, and found to have properties identical to those of synthetic cyclic somatostatin, which was also recovered quantitatively when added to sampling tubes. Various compounds were infused in concentrations that are highly active in pancreas perfusion experiments. 14-min infusion of arginine, 5 and 11.5 mmol/liter; isoproterenol, 10 and 23.7 nmol/liter and 68.7 mumol/liter; acetylcholine, 5 mumol/liter, carbamylcholine, 10 and 100 mumol/liter; glucagon, 1 and 30 nmol/liter; and porcine calcitonin, 1 and 100 ng/ml did not affect the basal release of somatostatin-like immunoreactivity significantly. Neither did an increase from the control level of 4 mmol/liter glucose of 10 or 20 mmol/liter, nor an increase in the control level of 4.4 mmol/liter K+ to 7.5 or 14.4 mmol/liter. Each of these compounds were tested in three or four dogs. The effect of an increase in Ca++ from the control level of 1.5 mmol/liter to 2.25, 3.0, and 4.5 mmol/liter was tested in random order in five thyroid lobes. All three doses elicited an immediate increase in effluent somatostatin-like immunoreactivity. In most experiments the response was biphasic with an early spike, followed by a stable level that was maintained during prolonged Ca++ infusion. The secretory response was not diminished through a series of repeated short pulses of calcium infusion. The response to 3.0 mmol/liter Ca++ (control period 8.4 +/- 1.5, test period 337 +/- 110 pg/ml, mean +/- SE) and 4.5 mmol/liter Ca++ (control period 9.5 +/- 1.4, test period 386 +/- 125) were significantly higher than 2.25 mmol/liter Ca++ (control period 7.2 +/- 1.0 test period 140 +/- 39), while there was no significant difference between responses to the two high doses. Infusion of salmon calcitonin, 10 ng/ml and 1 microgram/ml; or porcine calcitonin, 1 microgram/ml during calcium stimulation (2.25 mmol/liter of Ca++) did not induce alterations in the release of somatostatin-like immunoreactivity. The results demonstrate that thyroidal somatostatin is mobilizable, and it appears to be selectively sensitive to calcium stimulation, indicating a possible role in calcitonin release control.     

Nitric oxide, lipid peroxides, and uric acid levels in pre-eclampsia and eclampsia

The aim was to study the role of nitric oxide (NO), lipid peroxides (LPX), and uric acid in pre-eclampsia and eclampsia. Plasma levels of NO metabolites (nitrite+nitrate), malonyldialdehyde (MDA), and uric acid and erythrocyte MDA levels were compared between normal pregnant, pre-eclamptic, and eclamptic pregnant women in third trimester. Student's t-test was used for statistical evaluation. Plasma NO metabolites levels were higher in eclamptic group (35.7 +/- 16.5 micromol/liter, p < 0.05) but not in pre-eclamptic group (22.1 +/- 10.8 micromol/liter) than control group (18.8 +/- 6.9 micromol/liter). Plasma MDA and uric acid concentrations were higher in preeclamptic (4.4 +/- 1.7 nmol/ml, p < 0.05; 0.45 +/- 0.11 mmol/liter, p < 0.05, respectively) and eclamptic (5.8 +/- 1.9 nmol/ml, p < 0.05; 0.47 +/- 0.12 mmol/liter, p < 0.05) groups compared with control group (3.0 +/- 1.3 nmol/ml; 0.35 +/- 0.06 mmol/liter). Erythrocytes MDA concentrations were higher only in eclamptic group (174.4 +/- 62 nmol/gHb, p < 0.05) than control group (139.2 +/- 49.5 nmol/gHb). These results suggest that NO, LPX, and uric acid are important factors in the pathogenesis of pre-eclampsia and eclampsia, and that NO production and LPX are directly related to the severity of disease.     

Serum calcium ion activity. Some aspects on methodological differences and intraindividual variation.

Sera were analyzed for calcium ion activity on the Orion 99-20 and SS-20 systems. Samples from 84 healthy subjects were analyzed on both systems and, because of a change of electrode composition, 74 of them were reanalyzed after this change. Additional 89 were analyzed on only SS-20. The distribution of serum calcium ion activity was close to Gaussian. The difference between the 99-20 and SS-20 readings was 0.048+/-0.017 mmol/liter (range -0.01 to 0.08) before the electrode change and -0.085+/-0.027 mmol/liter (range -0.14 to 0.02) after. The reference ranges were 1.02--1.23 mmol/liter for 99-20 and 0.93--1.20 mmol/liter for SS-20 before the electrode change. The following characteristics may contribute to differences in the readings: higher temperature, higher flow rate, new calcium and reference electrodes, new liquid junction construction and a new pumping procedure. For 74 subjects the determination of serum calcium ion activity (99-20 system) was performed with a five year interval and the difference between the first and second measurements was -0.0046+/-0.0168 mmol/liter (mean +/-SD), ranging from -0.04 to 0.03 mmol/liter. This corresponded to an intraindividual variation of 1.5%. The corresponding variations for serum total calcium and corrected calcium were 4.8% and 3.3% respectively.     

Glucose-free fatty acid cycle operates in human heart and skeletal muscle in vivo.

Positron emission tomography permits noninvasive measurement of regional glucose uptake in vivo in humans. We employed this technique to determine the effect of FFA on glucose uptake in leg, arm, and heart muscles. Six normal men were studied twice under euglycemic hyperinsulinemic (serum insulin approximately 500 pmol/liter) conditions, once during elevation of serum FFA by infusions of heparin and Intralipid (serum FFA 2.0 +/- 0.4 mmol/liter), and once during infusion of saline (serum FFA 0.1 +/- 0.01 mmol/liter). Regional glucose uptake rates were measured using positron emission tomography-derived 18F-fluoro-2-deoxy-D-glucose kinetics and the three-compartment model described by Sokoloff (Sokoloff, L., M. Reivich, C. Kennedy, M. C. Des Rosiers, C. S. Patlak, K. D. Pettigrew, O. Sakurada, and M. Shinohara. 1977. J. Neurochem. 28: 897-916). Elevation of plasma FFA decreased whole body glucose uptake by 31 +/- 2% (1,960 +/- 130 vs. 2,860 +/- 250 mumol/min, P less than 0.01, FFA vs. saline study). This decrease was due to inhibition of glucose uptake in the heart by 30 +/- 8% (150 +/- 33 vs. 200 +/- 28 mumol/min, P less than 0.02), and in skeletal muscles; both when measured in femoral (1,594 +/- 261 vs. 2,272 +/- 328 mumol/min, 25 +/- 13%) and arm muscles (1,617 +/- 411 to 2,305 +/- 517 mumol/min, P less than 0.02, 31 +/- 6%). Whole body glucose uptake correlated with glucose uptake in femoral (r = 0.75, P less than 0.005), and arm muscles (r = 0.69, P less than 0.05) but not with glucose uptake in the heart (r = 0.04, NS). These data demonstrate that the glucose-FFA cycle operates in vivo in both heart and skeletal muscles in humans.     

In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway.

To study the role of apoC1 in lipoprotein metabolism, we have generated transgenic mice expressing the human APOC1 gene. On a sucrose-rich diet, male transgenic mice with high APOC1 expression in the liver showed elevated levels of serum cholesterol and triglyceride compared with control mice (5.7+/-0.7 and 3.3+/-2.1 vs. 2.7+/-0.1 and 0.4+/-0.1 mmol/liter, respectively). These elevated levels were mainly confined to the VLDL fraction. Female APOC1 transgenic mice showed less pronounced elevated serum lipid levels. In vivo VLDL turnover studies revealed that, in hyperlipidemic APOC1 transgenic mice, VLDL particles are cleared less efficiently from the circulation as compared with control mice. No differences were observed in the hepatic production and extrahepatic lipolysis of VLDL-triglyceride. Also, VLDL isolated from control and APOC1 transgenic mice were found to be equally good substrates for bovine lipoprotein lipase in vitro. These data indicate that the hyperlipidemia in APOC1 transgenic mice results primarily from impaired hepatic VLDL particle clearance, rather than a defect in the hydrolysis of VLDL-triglyceride. To investigate which hepatic receptor is involved in the apoC1-mediated inhibition of VLDL clearance, APOC1 transgenic mice were bred with an LDL receptor-deficient (LDLR(-/-)) background. In addition, control, LDLR(-/-), and LDLR(-/-)/APOC1 mice were transfected with adenovirus carrying the gene for the receptor-associated protein (Ad-RAP). Both serum cholesterol and triglyceride levels were strongly elevated in LDLR(-/-)/APOC1 mice compared with LDLR(-/-) mice (52+/-19 and 36+/-19 vs. 8.4+/-0.9 and 0.5+/-0.2 mmol/liter, respectively), indicating that apoC1 inhibits the alternative VLDL clearance pathway via the remnant receptor. Transfection of LDLR(-/-) mice with Ad-RAP strongly increased serum cholesterol and triglyceride levels, but to a lesser extent than those found in LDLR(-/-)/APOC1 mice (39+/-8 and 17+/-8 vs. 52+/-19 and 36+/-19 mmol/liter, respectively). However, in LDLR(-/-)/APOC1 mice the transfection with Ad-RAP did not further increase serum cholesterol and triglyceride levels (52+/-19 and 36+/-19 vs. 60+/-10 and 38+/-7 mmol/liter, respectively). From these studies we conclude that, in the absence of the LDLR, apoC1 inhibits the hepatic uptake of VLDL via a RAP-sensitive pathway.     

Role of blood flow in regulating insulin-stimulated glucose uptake in humans. Studies using bradykinin, [15O]water, and [18F]fluoro-deoxy-glucose and positron emission tomography.

Defects in insulin stimulation of blood flow have been used suggested to contribute to insulin resistance. To directly test whether glucose uptake can be altered by changing blood flow, we infused bradykinin (27 microgram over 100 min), an endothelium-dependent vasodilator, into the femoral artery of 12 normal subjects (age 25+/-1 yr, body mass index 22+/-1 kg/m2) after an overnight fast (n = 5) and during normoglycemic hyperinsulinemic (n = 7) conditions (serum insulin 465+/-11 pmol/liter, 0-100 min). Blood flow was measured simultaneously in both femoral regions using [15O]-labeled water ([15O]H2O) and positron emission tomography (PET), before and during (50 min) the bradykinin infusion. Glucose uptake was measured immediately after the blood flow measurement simultaneously in both femoral regions using [18F]-fluoro-deoxy-glucose ([18F]FDG) and PET. During hyperinsulinemia, muscle blood flow was 58% higher in the bradykinin-infused (38+/-9 ml/kg muscle x min) than in the control leg (24+/-5, P<0.01). Femoral muscle glucose uptake was identical in both legs (60.6+/-9.5 vs. 58.7+/-9.0 micromol/kg x min, bradykinin-infused vs control leg, NS). Glucose extraction by skeletal muscle was 44% higher in the control (2.6+/-0.2 mmol/liter) than the bradykinin-infused leg (1.8+/-0.2 mmol/liter, P<0.01). When bradykinin was infused in the basal state, flow was 98% higher in the bradykinin-infused (58+/-12 ml/kg muscle x min) than the control leg (28+/-6 ml/kg muscle x min, P<0.01) but rates of muscle glucose uptake were identical in both legs (10.1+/-0.9 vs. 10.6+/-0.8 micromol/kg x min). We conclude that bradykinin increases skeletal muscle blood flow but not muscle glucose uptake in vivo. These data provide direct evidence against the hypothesis that blood flow is an independent regulator of insulin-stimulated glucose uptake in humans.     

Angiotensin II depolarizes podocytes in the intact glomerulus of the Rat.

The aim of this study was to examine the effects of angiotensin II (Ang II) on cellular functions of rat podocytes (pod) in the intact freshly isolated glomerulus and in culture. Membrane voltage (Vm) and ion currents of pod were examined with the patch clamp technique in fast whole cell and whole cell nystatin configuration. Vm of pod was -38+/-1 mV (n = 86). Ang II led to a concentration-dependent depolarization of pod with an ED50 of 10(-8) mol/liter. In the presence of Ang II (10(-7) mol/liter, n = 20), pod depolarized by 7+/-1 mV. In an extracellular solution with a reduced Cl- concentration of 32 mmol/liter, the effect of Ang II on Vm was significantly increased to 14+/-4 mV (n = 8). The depolarization induced by Ang II was neither inhibited in an extracellular Na+-free solution nor in a solution with a reduced extracellular Ca2+ (down to 1 micromol/liter). Like Ang II, the calcium ionophore A23187 (10(-5) mol/liter, n = 9) depolarized pod by 10+/-2 mV, whereas forskolin (10(-5) mol/liter), 8-(4-chlorophenylthio)-cAMP and N2,2''-o-dibutyryl-cGMP (both 5 x 10(-4) mol/liter) did not alter Vm of pod. The angiotensin 1 receptor antagonist losartan (10(-7) mol/liter) completely inhibited the Ang II-induced (10(-7) mol/liter) depolarization (n = 5). Like pod in the glomerulus, pod in short term culture depolarized in response to Ang II (10(-8) mol/liter, n = 5). Our results suggest that Ang II depolarizes podocytes directly by opening a Cl- conductance. The activation of this ion conductance is mediated by an AT1 receptor and may be regulated by the intracellular Ca2+ activity.     

Antioxidant activity of a botanical extract preparation of Ilex paraguariensis: prevention of DNA double-strand breaks in Saccharomyces cerevisiae and human low-density lipoprotein oxidation

We analyzed the antioxidant properties of Ilex paraguariensis infusion (Ip) popularly known as mate (m?'tā), by using two experimental models: the induction of DNA double-strand breaks (DSB) by hydrogen peroxide (H(2)O(2)) and lethality in Saccharomyces cerevisiae, as well as peroxide and lipoxygenase-induced human low-density lipoprotein (LDL) oxidation. Diploid yeast cells were exposed to different concentrations of H(2)O(2) (5-10 mmol/L) in the absence or presence of Ip infusion (10(-1) v/v) or alpha-tocopherol (10(-2) mol/L). Both mate infusion and alpha-tocopherol significantly decreased the dose dependent DSB number, and the lethality induced by H(2)O(2). Peroxynitrite and lipoxygenase-induced human LDL oxidation are inhibited by Ip extracts in a potent, dose-dependent fashion. Dilutions of 5 x 10(-3) v/v provide 50% +/- 10% inhibition. Finally, Ip extracts are potent direct quenchers of the free radical 1,1-diphenyl-2-picrylhydrazyl. Dilutions of 2 x 10(-2) v/v produced quenching of more than 30%, which was comparable to that obtained with 0.5-1 mmol/L alpha-tocopherol or the quercetin aglycone, respectively. For comparison, total polyphenol content of Ip, green, and black tea (Camelia sinensis) were 6.5 +/- 0.8; 1.8 +/- 0.5; and 1.13 +/- 0.3 mmol of quercetin equivalents per liter, respectively. Their respective free radical quenching activities at dilutions of 1 x 10(-1) v/v were 75% +/- 5%; 35% +/- 5%; and 2% +/- 5%. Ip is thus a rich source of polyphenols and has antioxidant properties comparable to those of green tea which merit further in vivo intervention and cross-sectional studies.     

Enhancement of the anabolic effects of growth hormone and insulin-like growth factor I by use of both agents simultaneously.

The use of growth hormone (GH) as an anabolic agent is limited by its tendency to cause hyperglycemia and by its inability to reverse nitrogen wasting in some catabolic conditions. In a previous study comparing the anabolic actions of GH and IGF-I (insulin-like growth factor I), we observed that intravenous infusions of IGF-I (12 micrograms/kg ideal body wt [IBW]/h) attenuated nitrogen wasting to a degree comparable to GH given subcutaneously at a standard dose of 0.05 mg/kg IBW per d. IGF-I, however, had a tendency to cause hypoglycemia. In the present study, we treated seven calorically restricted (20 kcal/kg IBW per d) normal volunteers with a combination of GH and IGF-I (using the same doses as in the previous study) and compared its effects on anabolism and carbohydrate metabolism to treatment with IGF-I alone. The GH/IGF-I combination caused significantly greater nitrogen retention (262 +/- 43 mmol/d, mean +/- SD) compared to IGF-I alone (108 +/- 29 mmol/d; P < 0.001). GH/IGF-I treatment resulted in substantial urinary potassium conservation (34 +/- 3 mmol/d, mean +/- SE; P < 0.001), suggesting that most protein accretion occurred in muscle and connective tissue. GH attenuated the hypoglycemia induced by IGF-I as indicated by fewer hypoglycemic episodes and higher capillary blood glucose concentrations on GH/IGF-I (4.3 +/- 1.0 mmol/liter, mean +/- SD) compared to IGF-I alone (3.8 +/- 0.8 mmol/liter; P < 0.001). IGF-I caused a marked decline in C-peptide (1,165 +/- 341 pmol/liter; mean +/- SD) compared to the GH/IGF-I combination (2,280 +/- 612 pmol/liter; P < 0.001), suggesting maintenance of normal carbohydrate metabolism with the latter regimen. GH/IGF-I produced higher serum IGF-I concentrations (1,854 +/- 708 micrograms/liter; mean +/- SD) compared to IGF-I only treatment (1,092 +/- 503 micrograms/liter; P < 0.001). This observation was associated with increased concentrations of IGF binding protein 3 and acid-labile subunit on GH/IGF-I treatment and decreased concentrations on IGF-I alone. These results suggest that the combination of GH and IGF-I treatment is substantially more anabolic than either IGF-I or GH alone. GH/IGF-I treatment also attenuates the hypoglycemia caused by IGF-I alone. GH/IGF-I treatment could have important applications in diseases associated with catabolism.     

Detection of inhibitors in reduced nicotinamide adenine dinucleotide by kinetic methods.

Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.     

Anion transport as related to hemoglobin A1c in erythrocytes of diabetic children

We determined chloride and bicarbonate transport [Jcl and Jbic, nmol/(cm2.s)] under physiological conditions (Cl- 110 and HCO3- 25 mmol per liter, respectively, pH 7.4, 38 degrees C) across the erythrocyte membrane in blood samples from 12 diabetic (Jcl 26.1, SD +/- 3.7, n = 24; Jbic 7.6, SD +/- 0.9, n = 19) and 10 non-diabetic children (Jcl 30.6, SD +/- 4.6, n = 16; Jbic 7.3, SD +/- 1.0, n = 20) with mean hemoglobin A1c values of 11.08% (SD +/- 2.45%) and 5.36% (SD +/- 0.25%), respectively. The concentration of HbA1c, which also reflects the degree of glycation of the membrane proteins, differed significantly (P greater than 0.001) between the two groups, whereas there was no significant variation (P greater than 0.1) in Jcl and Jbic. We conclude that glycation of the integral transport protein in the erythrocyte membrane, capnophorin (also called "band 3"), which mediates a tightly coupled anion exchange, does not change the capacity of the transport system under physiological conditions. Thus the rate-limiting step of the exploitation of the CO2 transport capacity of the blood is not impaired in diabetics and consequently does not endanger the compensatory hyperventilation after ketoacidosis.     

Interaction between free fatty acids and insulin in the acute control of very low density lipoprotein production in humans.

Changes in VLDL triglyceride and VLDL apo B production were determined semiquantitatively in healthy young men by examining the effect of altering plasma insulin and/or FFA levels on the change in the slopes of the specific activity of VLDL [3H]triglyceride glycerol or the 131I-VLDL apo B versus time curves. In one study (n = 8) insulin was infused for 5 h using the euglycemic hyperinsulinemic clamp technique. Plasma FFA levels declined by approximately 80% (0.52 +/- 0.01 to 0.11 +/- 0.02 mmol/liter), VLDL triglyceride production decreased by 66.7 +/- 4.2% (P = 0.0001) and VLDL apo B production decreased by 51.7 +/- 10.6% (P = 0.003). In a second study (n = 8) heparin and Intralipid (Baxter Corp., Toronto, Canada) were infused with insulin to prevent the insulin-mediated fall in plasma FFA levels. Plasma FFA increased approximately twofold (0.43 +/- 0.05 to 0.82 + 0.13 mmol/liter), VLDL triglyceride production decreased to a lesser extent than with insulin alone (P = 0.006) (-31.8 +/- 9.5%, decrease from baseline P = 0.03) and VLDL apo B production did not decrease significantly (-6.3 +/- 13.6%, P = NS). In a third study (n = 8) when heparin and Intralipid were infused without insulin, FFA levels rose approximately twofold (0.53 +/- 0.04 to 0.85 +/- 0.1 mmol/liter), VLDL triglyceride production increased by 180.1 +/- 45.7% (P = 0.008) and VLDL apo B production increased by 94.2 +/- 28.7% (P = 0.05). We confirm our previous observation that acute hyperinsulinemia suppresses VLDL triglyceride and VLDL apo B production in healthy humans. In addition, we have demonstrated that elevation of plasma FFA levels acutely stimulates VLDL production in vivo in healthy young males. Elevating plasma FFA during hyperinsulinemia attenuates but does not completely abolish the suppressive effect of insulin on VLDL production, at least with respect to VLDL triglycerides. Therefore, in normal individuals the acute inhibition of VLDL production by insulin in vivo is only partly due to the suppression of plasma FFA, and may also be due to an FFA-independent process.     

Evaluation of the fructosamine test in obesity: consequences for the assessment of past glycemic control in diabetes

The effect of obesity in diabetic and nondiabetic states on serum fructosamine levels, as measured by the nitro blue tetrazolium reduction method, was investigated. In 26 nondiabetic obese subjects, the mean (SD) fructosamine (1.78 +/- 0.16 mmol/L) and protein corrected fructosamine concentrations (25.7 +/- 2.5 mumol/g) were significantly lower than in nondiabetic lean control subjects (2.06 +/- 0.18 mmol/L and 30.5 +/- 2.5 mumol/g, respectively; p less than 0.01). Hemoglobin A1C, blood glucose and serum protein concentrations were normal in obese subjects. Interference from hypertriglyceridemia, hemolysis, or drugs was excluded. In diabetic subjects, fructosamine correlated with hemoglobin A1C, but the least-squares regression lines were different in 16 nonobese and in 19 obese patients, so that for the same hemoglobin A1C value, fructosamine level was 16% lower in obese compared to nonobese diabetic subjects. In vitro studies showed a significant decrease in 14C-glucose incorporation in serum proteins of obese nondiabetic subjects compared to control subjects. Similarly, the rate of formation of fructosamine in sera of obese nondiabetic subjects incubated with 12 mmol/L and 30 mmol/L glucose concentrations was slower than in sera of control subjects. In conclusion, fructosamine is underestimated in obesity, both in diabetic and nondiabetic patients, and its validity as an index of glycemic control may be impaired in obese subjects. This decrease is due to an alteration in the glycation process itself.     

Multiple-organ pathology in septic shock in patients with hemoblastoses

A prospective study of 20 patients with hemoblastosis and septic shock (SS) was carried out by invasive monitoring of the central hemodynamics and oxygen transport, evaluation of biochemical and coagulological parameters, and assessment of the severity of clinical condition by the APACHE II and SOFA scores. Septic shock was effectively treated in 12 patients, 5 of them were discharged from the department (group 1) and 7 died in intensive care wards from various complications (group 2). Eight patients died during the first 2 days from SS resistant to therapy (group 3). Group 2 patients were in need of a longer inotropic support than group 1 patients (5.8 +/- 1.6 vs. 2.7 +/- 0.8 days, p < 0.01). The deficit of bases was more expressed in groups 2 and 3 in comparison with group 1 (-11.3 +/- 3 and -2.7 +/- 9.1 mmol/liter vs. 1.4 +/- 4.4 mmol/liter) and left ventricular stroke index (LVSI) and oxygen delivery were lower. LVSI and base deficit were in linear correlation (rho = 0.4, p < 0.05). XIIa-dependent fibrinolysis was suppressed in all patients, which was more pronounced in group 3 in comparison with groups 1 and 2 (135 +/- 47.4 vs. 103 +/- 27 and 88.3 +/- 42.3). According to SOFA score, the severity of cardiovascular disorders during day 1 of SS was the same in all groups, while starting from day 2 it decreased in patients who survived. Acute respiratory failure was lower in group 1 only on day 1 according to SOFA. More pronounced (according to SOFA) hepatorenal failure was observed in group 2 in comparison with other patients. Organ involvement in hemoblastosis was detected at autopsy in 8 out of 13 cases. Hence, the need in prolonged cardiovascular support of SS patients is associated with development of polyorgan involvement. Fibrinolysis suppression is a frequent early manifestation of hemostasis disorders. Specific neoplastic organ involvement was observed in 61.5% patients with hemoblastosis who died from SS and its complications.     

In vivo inhibition of glucagon secretion by paracrine beta cell activity in man.

The close anatomical relationships betaeen pancreatic alpha and beta cells makes possible their interaction at a local (paracrine) level. To demonstrate this in vivo, we have compared the acute glucagon response to intravenous arginine in the basal state and after beta cell suppression by infusions of insulin. The plasma glucose concentration was maintained by the glucose clamp technique. In six normal weight nondiabetics, infusion of insulin at 0.2 mU/kg per min (rate 1) raised the mean +/- SEM plasma insulin levels from 10 +/- 3 to 32 +/- 4 mU/liter and at 1 mU/kg per min (rate 2) raised plasma insulin to 84 +/- 8 mU/liter. This resulted in beta cell suppression, as shown by a diminution in the acute insulin response (incremental area under the insulin response curve, 0-10 min): basal = 283 +/- 61, 199 +/- 66 (rate 1) and 143 +/- 48 mU/liter per 10 min (rate 2) and a fall in prestimulus C-peptide from 1.05 +/- 0.17 to 0.66 +/- 0.15 and to 0.44 +/- 0.15 mM/liter (all P less than 0.01). This beta cell suppression was associated with increased glucagon responses to arginine: 573 +/- 75 (basal), 829 +/- 114 (rate 1), and 994 +/- 136 ng/liter per 10 min (rate 2) and increased peak glucagon responses 181 +/- 11 (basal), 214 +/- 16 (rate 1), and 259 +/- 29 ng/liter (rate 2) (all P less than 0.01). In all subjects, there was a proportional change between the rise in he acute glucagon response to arginine and the fall in the prearginine C-peptide level. To demonstrate that augmented glucagon response was due to betw cell suppression, and not to the metabolic effect of infused insulin, similar studies were performed in C-peptide-negative-diabetics. Their acute glucagon response to arginine was inhibited by the insulin infusion: 701 +/- 112 (basal), 427 +/- 33 (rate 1), and 293 +/- 67 ng/liter per 10 min (rate 2) as was their peak glucagon response: 268 +/- 69, 170 +/- 36, and 115 +/- 33 ng/liter (all P less than 0.01). Thus, hyperinsulinemia, within the physiological range achieved by insulin infusion, inhibits beta cell secretion which, via a paracrine mechanism, potentiates glucagon secretion.     

Demonstration of active potassium transport in the mammalian colon.

The mechanism responsible for K transport in the mammalian colon is controversial. Experiments were performed to determine whether K secretion involves active as well as passive driving forces in controls and in animals with a marked increase in K secretion. In these experiments a steady-state solution was established in proximal and distal colon of both control rats and animals fed a K-enriched diet during in vivo luminal perfusion, to compare the observed luminal [K] with predicted equilibrium [K] when net water and electrolyte movement approached zero. Transmural potential difference was measured simultaneously. A difference between the predicted equilibrium and observed luminal [K] under this condition indicates active transport. In controls the observed [K] of 20 mmol/liter in proximal colon markedly exceeded the predicted value of 6.2 +/- 0.3, mean +/- SE, indicating active secretion. In contrast, the observed [K] in distal colon of 5 mmol/liter was less than predicted (10.0 +/- 1.0), suggesting active absorption. In K-loaded animals active K secretion was demonstrable and increase above control in both segments of colon. In proximal colon the observed [K] rose to 40 mmol/liter, compared to a predicted value of 7.2 +/- 0.3, whereas in distal colon the observed [K] was 50 mmol/liter vs. a predicted value of 7.0 +/- 0.8. These studies suggest active K secretion in proximal, but not in distal colon of control animals. Further, these data suggest that the increase in the capacity for K secretion that occurs in response to chronic K loading involves stimulation of an active mechanism in both proximal and distal colon.