Defective suppression by insulin of leucine-carbon appearance and oxidation in type 1, insulin-dependent diabetes mellitus. Evidence for insulin resistance involving glucose and amino acid metabolism.

To determine whether a resistance to insulin in type 1, insulin-dependent diabetes mellitus (IDDM) is extended to both glucose and amino acid metabolism, six normal subjects and five patients with IDDM, maintained in euglycemia with intravenous insulin administration, were infused with L-[4,5-3H]leucine (Leu) and [1-14C]alpha ketoisocaproate (KIC). Steady-state rates of leucine-carbon appearance derived from protein breakdown (Leu + KIC Ra) and KIC (approximately leucine) oxidation were determined at basal and during sequential euglycemic, hyperinsulinemic (approximately 40, approximately 90 and approximately 1,300 microU/ml) clamps. In the euglycemic postabsorptive diabetic patients, despite basal hyperinsulinemia (24 +/- 6 microU/ml vs. 9 +/- 1 microU/ml in normals, P less than 0.05), Leu + KIC Ra (2.90 +/- 0.18 mumol/kg X min), and KIC oxidation (0.22 +/- 0.03 mumol/kg X min) were similar to normal values (Leu + KIC Ra = 2.74 +/- 0.25 mumol/kg X min) (oxidation = 0.20 +/- 0.02 mumol/kg X min). During stepwise hyperinsulinemia, Leu + KIC Ra in normals decreased to 2.08 +/- 0.19, to 2.00 +/- 0.17, and to 1.81 +/- 0.16 mumol/kg X min, but only to 2.77 +/- 0.16, to 2.63 +/- 0.16, and to 2.39 +/- 0.08 mumol/kg X min in the diabetic patients (P less than 0.05 or less vs. normals at each clamp step). KIC oxidation decreased in normal subjects to a larger extent than in the diabetic subjects. Glucose disposal was reduced at all insulin levels in the patients. In summary, in IDDM: (a) Peripheral hyperinsulinemia is required to normalize both fasting leucine metabolism and blood glucose concentrations. (b) At euglycemic hyperinsulinemic clamps, lower glucose disposal rates and a defective suppression of leucine-carbon appearance and oxidation were observed. We conclude that in type 1 diabetes a resistance to the metabolic effects of insulin on both glucose and amino acid metabolism is present.

Reduced in vivo biological activity of in vitro glycosylated insulin.

We evaluated the in vivo biological activity of in vitro extensively glycosylated insulin (GI) with the euglycemic-hyperinsulinemic glucose-clamp technique in postabsorptive nondiabetic subjects. Insulin-mediated glucose disposal was approximately 30% lower (P less than .03) with GI (9.2 +/- 1.2 mg.kg-1.min-1, mean +/- SE) than with the nonglycosylated hormone (12.6 +/- 0.7 mg.kg-1.min-1) at comparable plasma insulin concentrations (approximately 90 microU/ml). Binding of GI to a specific receptor on circulating cells (erythrocytes and monocytes) was normal. We conclude that in vitro extensive glycosylation of insulin reduces its biological activity in vivo, as reflected by insulin-mediated glucose disposal, probably at a postreceptor level.

Metabolic control of kidney hemodynamics in normal and insulin-dependent diabetic subjects. Effects of acetoacetic, lactic, and acetic acids.

Diabetes mellitus is associated with important changes in renal hemodynamics. The purpose of this study was to determine whether an increase in blood concentration patterns of ketone bodies and lactic acid, organic acids often elevated in poorly controlled insulin-dependent diabetes mellitus (IDDM), could contribute to increase glomerular filtration rate (GFR) and renal plasma flow (RPF) regardless of changes in circulating levels of glucose and insulin. Six IDDM patients and six normal subjects were given a saline infusion (15 mumol.min-1.kg-1) for 2 h, an acetoacetic acid infusion (15 mumol.min-1.kg-1) for another 2 h, and then a saline infusion after an overnight fast during euglycemic insulin-glucose clamp. Acetoacetic acid infusion resulted in an increase of blood ketone bodies in the range of 0.7-1.5 mM from a basal value of 0.1-0.3 mM. GFR was 125 +/- 16 and 136 +/- 17 ml.min-1.1.73 m-2 in normal and IDDM subjects, respectively, during baseline saline infusion and 138 +/- 21 (P less than .01 vs. basal level) and 158 +/- 15 ml.min-1.1.73 m-2 (P less than .001 vs. basal level) during acetoacetic acid infusion. During the last saline infusion, renal hemodynamic patterns decreased again to baseline levels. Another six IDDM patients and six normal subjects were given saline, lactic acid, and saline infusions at the same rates of infusion after an overnight fast during euglycemic insulin-glucose clamp. Lactic acid concentration increased from approximately 0.5-0.8 to 1.0-1.5 mM in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Ketone body metabolism in NIDDM. Effect of sulfonylurea treatment.

We assessed the metabolism of the two KBs, AcAc and 3-BOH; the relationships between ketogenesis and FFA inflow rate; and the effect of chronic sulfonylurea treatment in mild NIDDM patients (plasma glucose less than 10 mM). We studied 10 nonobese NIDDM patients in a crossover, randomized, double-blind, placebo-controlled fashion. Each patient was studied 4 times: after a run-in period with placebo, after 3 mo of placebo treatment, after 3 mo of glibenclamide treatments, respectively, and after 3 mo of sulfonylurea treatment during an acute exogenous Intralipid infusion. Ten normal, nondiabetic subjects served as the control group. Glibenclamide treatment decreased plasma FFAs. When these substrates were exogenously increased, plasma FFAs were comparable with placebo and baseline concentrations. In NIDDM patients, baseline and placebo blood total KB concentration was significantly higher than in control subjects (216 +/- 22 and 244 +/- 25, respectively vs. 127 +/- 18 microM; P less than 0.01). Glibenclamide treatment significantly decreased total KBs to 177 +/- 19 microM (P less than 0.05). When FFAs were exogenously increased, total KBs were similar to the placebo and baseline period. In the baseline study, the AcAc/3-BOH ratio was 0.72 +/- 0.06 in control subjects, whereas in NIDDM patients, the ratio was 1.61 +/- 0.13 at baseline (P less than 0.001 vs. control subjects), 1.66 +/- 0.15 during placebo, 1.57 +/- 0.09 during glibenclamide (NS vs. baseline), and 1.51 +/- 0.23 during glibenclamide plus placebo FFAs. Both the AcAc interconversion rate to 3-BOH and the 3-BOH interconversion rate to AcAc were significantly lower in NIDDM patients than in control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin sensitivity, binding, and kinetics in pancreatogenic and type I diabetes.

Pancreatogenic diabetes (PD), secondary either to chronic calcific pancreatitis or to pancreatectomy, is characterized by higher frequency of hypoglycemic events during insulin therapy in comparison with type I insulin-dependent diabetes (IDD). Not only glucagon deficiency, but an enhanced peripheral tissue sensitivity to insulin could account for this metabolic behavior. We investigated several facets of insulin action, e.g., tissue sensitivity to insulin, insulin binding to red cells, and insulin kinetics in seven patients with PD in comparison with type I. Tissue sensitivity to insulin was evaluated by means of the glucose-insulin clamp technique as M/I x 100 ratio (mg . kg .-1 min-1/muU . ml-1), where M is the amount of glucose infused by Biostator GCIIS to clamp BG at basal level and I is the free insulin plateau concentration achieved by a primed-constant insulin infusion. At high BG 15 h after the last injection of regular insulin M/I x 100 was 7.79 (range 4.25-9.75) in PD and 4.20 (range 1.20-6.91) in D (P less than 0.05). At low and equal BG M/I x 100 was 8.55 (range 6.35-9.72) in PD and 3.42 (range 1.19-6.75) in D (P less than 0.01). The rate of endogenous glucose production was nearly totally suppressed in both groups of patients. Just before the two clamps, 125I-insulin specific binding to red cells was studied. The maximum specific binding was significantly higher in PD than in D at high BG (10.7 +/- 1.7 vs. 7.4 +/- 0.8/10(9) red cells) and at low and equal BG (12.4 +/- 1.2 vs. 6.8 +/- 0.8). Receptor concentration also was significantly higher in PD thant in D (P less than 0.02) while no significant differences were found in high affinity (Ke). Insulin kinetic data were analysed by using both "Model independent" (or noncompartmental) method and compartmental modeling. Patients with PD had significantly higher (P less than 0.05) plasma clearance of insulin.

Effect of metformin on insulin-stimulated glucose turnover and insulin binding to receptors in type II diabetes.

Euglycemic insulin glucose-clamp and insulin-binding studies on erythrocytes and monocytes were performed in seven type II (non-insulin-dependent) diabetic subjects before and after 4 wk of metformin treatment (850 mg 3 times/day) and in five obese subjects with normal glucose tolerance. Glucose turnover was also measured at basal insulin concentrations and during hyperinsulinemic euglycemic clamps. During euglycemic insulin-glucose clamps, diabetic subjects showed glucose disposal rates of 3.44 +/- 0.42 and 7.34 +/- 0.34 mg X kg-1 X min-1 (means +/- SD) before metformin at insulin infusion rates of 0.80 and 15.37 mU X kg-1 X min-1, respectively. With the same insulin infusion rates, glucose disposal was 4.94 +/- 0.55 (P less than .01) and 8.99 +/- 0.66 (P less than .01), respectively, after metformin treatment. Glucose disposal rates in normal obese subjects were 5.76 +/- 0.63 (P less than .01) and 10.92 +/- 1.11 (P less than .01) at 0.80 and 15.37 mU X kg-1 X min-1, respectively. Insulin maximum binding to erythrocytes in diabetics was 9.6 +/- 4.2 and 5.8 +/- 2.6 X 10(9) cells (means +/- SD) before and after metformin treatment, respectively (NS). Insulin maximum binding to monocytes in diabetics was 6.2 +/- 2.3 X 10(7) cells before and 5.0 +/- 1.6% after metformin. Hepatic glucose production was higher in the diabetic patients at basal insulin levels, but not at higher insulin concentrations, and was not significantly changed by drug treatment. Basal glucose and insulin concentrations decreased with metformin. Thus, metformin treatment improved glucose disposal rate without significant effect on insulin-binding capacity on circulating cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular free calcium abnormalities in fibroblasts from non-insulin-dependent diabetic patients with and without arterial hypertension.

As arterial hypertension is frequently associated with diabetes, it is possible that altered intracellular free calcium ([Ca2+]i) handling, as reported in non-insulin-dependent diabetic patients, is accounted for by abnormalities caused by hypertension rather than diabetes. Our aim was to investigate [Ca2+]i transients triggered by two extracellular agonists, bradykinin and angiotensin II, with or without chronic insulin exposure, in cultured skin fibroblasts from 10 normotensive and 10 hypertensive non-insulin-dependent patients, matched for age, body mass index, and metabolic control, with fibroblasts from 10 healthy control subjects. Long-term cultured fibroblasts were loaded with fura 2-AM for measurement of [Ca2+]i. Resting [Ca2+]i levels were similar in the three groups of subjects. [Ca2+]i spikes stimulated by angiotensin II (0.1 mumol/L) and bradykinin (1 mumol/L) were significantly greater in hypertensive non-insulin-dependent diabetic patients (216 +/- 43 and 374 +/- 39 nmol/L, respectively) than in normotensive patients (174 +/- 16 and 267 +/- 55 nmol/L) and control subjects (188 +/- 29 and 320 +/- 78 nmol/L). Also, ionomycin evoked a greater [Ca2+]i response in hypertensive than normotensive non-insulin-dependent diabetic patients and in control subjects. Chronic insulin exposure increased by 70% to 90% the [Ca2+]i response to both angiotensin II and bradykinin in control subjects and normotensive non-insulin-dependent diabetic patients but not in hypertensive patients. The presence of abnormalities in [Ca2+]i transients in fibroblasts from only hypertensive non-insulin-dependent diabetic patients supports the possibility that these defects are a feature of concomitant arterial hypertension rather than of diabetes or its disturbed metabolic milieu.

Type I diabetes is characterized by insulin resistance not only with regard to glucose, but also to lipid and amino acid metabolism.

Resistance to the metabolic effects of insulin has been reported with regard to glucose disposal in type I diabetic patients (IDDM) even when they were euglycemic. Our aim was to study glucose, lipid, and amino acid metabolism during glucose clamping at multiple levels of insulin in 10 normal (N) and 6 IDDM patients. Blood glucose was maintained constant (4.7 mmol/liter) at three insulin plateaus (160 min each) [42 +/- 6 (SD) 89 +/- 11, and 1255 +/- 185 microU/ml in N and 36 +/- 4, 80 +/- 13, and 1249 +/- 107 microU/liter in IDDM]. Mean glucose disposal was 34 +/- 11, 69 +/- 10, and 84 +/- 22 mumol kg-1 min-1 in N and 16 +/- 5, 40 +/- 18, and 65 +/- 27 in IDDM, respectively. Baseline concentrations of blood lactate, pyruvate, alanine, and branched chain amino acids were 560 +/- 130, 36 +/- 9, 212 +/- 44, and 451 +/- 19 mumol/liter, in N and 793 +/- 179 (P less than 0.05), 45 +/- 14, 195 +/- 50, and 439 +/- 33 in IDDM, respectively. The maximum percent change of lactate during the euglycemic clamp was +147 +/- 23% in N and +75 +/- 15% (P less than 0.05) in IDDM; that of branched chain amino acids was -61 +/- 5% in N and -48 +/- 7% (P less than 0.01) in IDDM. Baseline concentrations of glycerol, FFA, and adipate were 44 +/- 15, 449 +/- 152, and 8 - 8 mumol/liter in N and 39 +/- 14, 473 +/- 44, and 41 +/- 14 (P less than 0.01) in IDDM. The maximum percent change of glycerol during the euglycemic clamp was -50 +/- 8% in N and -16 +/- 8% (P less than 0.01) in IDDM, that of FFA -98 +/- 3% in N and -70 +/- 4% in IDDM (P less than 0.05). No significant differences were found between N and IDDM with regard to blood concentrations of ketone bodies, citrate, ketoglutarate, and hydroxymethylglutaryl coenzyme A both before and during the euglycemic clamp. The lactate percent increase was significantly correlated to glucose disposal rate (P less than 0.001). The lactate turnover rate increased during the euglycemic clamp and was lower in IDDM than in N. We conclude that during euglycemic-multiple insulin clamp studies the greater lactate increase suggests that the flux of glycolysis is higher in N than in IDDM, tricarboxylic acid concentrations are comparable in N and IDDM, and FFA, glycerol, and branched chain amino acid decreases were less in IDDM than in N, suggesting that IDDM patients are resistant to insulin with regard to lipid and protein metabolism. The higher adipate basal values demonstrate enhanced omega-oxidation in IDDM.

Porcine and human insulin absorption from subcutaneous tissues in normal and insulin-dependent diabetic subjects: a deconvolution-based approach.

The mechanisms of sc insulin absorption are not understood, and models for interpreting in vivo data cannot be developed without gross simplification. To overcome this difficulty we developed a new approach which makes use of deconvolution analysis and does not require any model of the sc tissue. In five normal subjects and seven insulin-dependent diabetic (IDDM) patients endogenous insulin secretion was suppressed by means of a hypoglycemic glucose clamp procedure (approximately 2.8 mmol/L) sustained by a continuous insulin infusion (approximately 4 pmol/min.kg). A bolus injection of insulin (5.4 nmol) was administered iv, and plasma insulin concentrations were measured frequently for 2 h to assess iv insulin kinetics. Insulin then was injected sc in the abdominal region, and plasma insulin concentrations were measured for 8 h. Each subject was studied twice, with porcine and semisynthetic human insulin (Actrapid, Novo). The rate of insulin absorption was reconstructed by deconvolution from the plasma concentrations and iv insulin kinetic data. Linearity of the iv insulin kinetics, essential for deconvolution analysis, was confirmed by a dose-response study in the range of the measured concentrations (150-1800 pmol/L). In most instances, a two-compartment model was adequate to describe the iv response. The mean plasma insulin clearance rates were 15.5 +/- 1.9 (+/- SD) mL/min.kg (porcine) and 17.2 +/- 6.0 (human) in normal subjects and 20.7 +/- 8.8 (porcine) and 20.9 +/- 9.1 (human) in the IDDM patients. The rate of appearance of human insulin from sc tissue was faster than that of porcine insulin in both normal and IDDM subjects, but no significant differences were found in bioavailability, which was 55 +/- 12% (+/- SD; porcine) and 61 +/- 34% (human) in the normal subjects, and 84 +/- 28% (porcine) and 86 +/- 23% (human) in the IDDM patients. The rate of absorption and bioavailability were higher in the IDDM patients than in the normal subjects, a difference possibly related to increased sc blood flow in the IDDM patients. No differences were found with regard to glucose requirement values, normalized to plasma insulin concentrations, in agreement with the finding that the bioavailability of the two insulin species was similar.

Insulin resistance in Cushing's syndrome.

It is well established that cortisol excess causes insulin resistance in man, but the mechanisms responsible for this insulin resistance are poorly understood. We studied five women with Cushing's syndrome with impaired oral glucose tolerance tests and seven normal subjects, plotting the shape of the insulin-induced disposal dose-response curve obtained by means of the euglycemic clamp procedure during four different plasma insulin plateaus at four infusion rates of 21, 73, 760, and 1200 mU/M2 . min. Glucose disposal (M = mg/M2 . min) was calculated as glucose amount infused to maintain euglycemia. In Cushing's syndrome the dose-response curve was shifted to the right in comparison with normal subjects, with a significantly lower M (337 +/- 35 vs. 657 +/- 76 P less than 0.01) during the highest insulin infusion rate [maximal glucose disposal (MGD)] without any significant difference in the levels of insulin half-maximally effective in the stimulation of glucose utilization. Neither erythrocyte nor monocyte maximum insulin receptor binding were different between the two populations. Four Cushing's syndrome patients were studied again after surgical treatment. A marked improvement of MGD was observed without any significant change in insulin-binding capacity. These results, particularly the marked decrease in MGD, a typical feature of postreceptor defects, indicate that cortisol-induced insulin resistance in man is due to an impairment of peripheral insulin action located beyond the hormone-receptor binding step.

Carbohydrate and lipid metabolism in cirrhosis. Evidence that hepatic uptake of gluconeogenic precursors and of free fatty acids depends on effective hepatic flow.

Splanchnic arteriovenous differences for several intermediary metabolites of carbohydrate and lipid metabolism were determined simultaneously with hepatic blood flow in seven normal subjects, eight patients with cirrhosis, and six patients with cirrhosis after surgical portosystemic shunt ( SPSS ) after an overnight fast. Arteriovenous differences in the legs were also determined together with flux measurement. The individual turnover rates of acetoacetate (AcAc) and 3 hydroxybutyrate (beta OHB) were also determined by means of isotopic techniques. Splanchnic gluconeogenic precursors and FFA uptakes were lower in cirrhotic patients with SPSS than in normal subjects (P less than 0.05 and P less than 0.01, respectively). Splanchnic triglyceride output was also lower in cirrhotic patients with SPSS than in normal subjects (P less than 0.01), whereas no significant differences were found for AcAc, beta OHB, and glucose release. In the group of cirrhotic patients without SPSS , those patients with negligible signs of portal systemic shunt and normal splanchnic blood flow had uptake of gluconeogenic precursors and of FFA normal or higher than that of normal subjects, whereas those patients with signs of spontaneous portal systemic shunt behaved like cirrhotic patients with SPSS . Alanine release from the leg was lower in both cirrhotic patient groups. Tracer determined hepatic output of AcAc and beta OHB was higher in cirrhotic patients with SPSS (P less than 0.05). Plasma clearance rates of AcAc and beta OHB were significantly elevated in both cirrhotic patient groups. Close agreement was found between tracer and catheterization techniques in the evaluation of ketone body production in cirrhotic patients with SPSS , whereas in cirrhotic patients without SPSS tracer determined hepatic output was slightly lower, possibly because of extrahepatic splanchnic tissue ketone body uptake. In conclusion, our data in patients with cirrhosis indicate that: 1) splanchnic uptake of gluconeogenic precursors and of FFA was related to the degree of portal systemic shunt, e.g. to the degree of effective hepatic blood flow; 2) liver triglyceride but not ketone body output was decreased by the impaired FFA (and glycerol) liver uptake; 3) the higher circulating levels of gluconeogenic precursors (except alanine) and of FFA appeared at least partially due to lower hepatic removal of these metabolites; and 4) peripheral use of ketone bodies was increased and alanine release from the leg reduced in patients with cirrhosis.

Hormonal and metabolic profiles in patients with alcohol-induced, mixed hypertriglyceridemia before and after abstinence from ethanol and before and after a lipid-lowering diet.

The diurnal variation of intermediary metabolites and hormones was determined, as 24-h profiles, in a group of subjects with mixed hypertriglyceridemia while consuming a diet with excess alcohol and caloric intake (Hyp-I) or after a hypotriglyceridemic diet (Hyp-II), and in normal controls. Alcohol was excluded from the hypotriglyceridemic diet and on the days of the study. Hyp-I subjects showed higher 24-h levels of plasma triglyceride, glucose, insulin, lactate, pyruvate, free fatty acid and glycerol. After the hypotriglyceridemic diet the levels of pyruvate, free-fatty acids and glycerol in plasma were normalized, while triglyceride, insulin and glucose concentrations were significantly reduced but remained still higher than in controls. The elevated lactate concentration in Hyp-I subjects were unaffected by the diet. In Hyp-I subjects free-fatty acids and glycerol levels were not suppressed following the meal, in contrast to controls. After the diet this defect in the suppression of endogenous lipolysis was only partially reversed in Hyp-II subjects. Plasma alanine, total ketone body and glucagon concentrations were unaffected. In conclusion, in mixed hypertriglyceridemia high lactate concentration and a defect in the suppression of endogenous lipolysis after a meal could represent a factor enhancing triglyceride production.

Effects of chronic alcohol intake on carbohydrate and lipid metabolism in subjects with type II (non-insulin-dependent) diabetes.

PURPOSE: To study the effects of chronic alcohol intake on carbohydrate and lipid metabolism in subjects with non-insulin-dependent (type II) diabetes (NIDDM). To also evaluate the effect of alcohol withdrawal on metabolic control. PATIENTS AND METHODS: The study group consisted of 46 alcohol-consuming patients with NIDDM (NIDDM-B group), 35 non-alcohol-consuming patients with NIDDM (NIDDM group), and 40 normal control subjects. All patients were admitted to the hospital. Carbohydrate and lipid metabolism was assessed in these individuals immediately on admission to the hospital and during the following days. RESULTS: In the NIDDM-B group, blood alcohol (ethyl alcohol) concentration was very low. However, chronic alcohol intake was associated with higher fasting and postprandial glucose concentrations and higher hemoglobin A1c. No significant differences were found in C-peptide levels. Moreover, higher concentrations of 3-hydroxybutyrate and free fatty acids were observed in the NIDDM-B group than in the NIDDM group. No differences were found in triglyceride concentrations, acid-base patterns, or electrolyte levels. The metabolic effects of alcohol completely waned after 3 days of complete withdrawal. CONCLUSION: Chronic alcohol intake causes deterioration in metabolic control of persons with NIDDM. The effects induced by alcohol are completely reversed after a few days of withdrawal. Strict metabolic assessment is necessary when alcohol is an important constituent of the diet.

Metabolic effects of moderate alcohol intake with meals in insulin-dependent diabetics controlled by artificial endocrine pancreas (AEP) and in normal subjects.

In order to evaluate the effects of moderate alcohol intake on intermediate metabolites, five normal subjects and five euglycemic insulin-dependent diabetics (IDDM) were administered two different isocaloric diets; in one diet 35% of the caloric intake consisted of red wine. The insulin-dependent diabetics were connected to an artificial endocrine pancreas (AEP), and glucose levels were continuously monitored. Blood lactate, pyruvate, acetoacetate (AcAc), 3-hydroxybutyrate (3-OHB), glycerol, free fatty acids (FFA), and alanine levels were measured over a 15-hour period from 9 AM to 12 PM. The results showed that alcohol intake did not significantly influence the glucose profiles in either group (111 +/- 4 mg/100 ml versus 110 +/- 4 mg/100 ml for IDDM; 72 +/- 2 mg/100 ml versus 82 +/- 3 mg/100 ml for controls, 15-hour mean +/- SEM), but in both groups it induced a marked increased in the levels of lactate (1.115 +/- 0.067 mM/liter with alcohol versus 0.706 +/- 0.031 mM/liter without alcohol for IDDM; 0.847 +/- 0.052 mM/liter with alcohol versus 0.666 +/- 0.035 mM/liter without alcohol for controls), in the lactate/pyruvate ratio (24.04 +/- 2.12 with alcohol versus 11.42 +/- 0.20 without alcohol for IDDM; 20.84 +/- 2.16 with alcohol versus 11.62 +/- 0.27 without alcohol for controls), in the levels of 3-OHB (0.081 +/- 0.007 mM/liter with alcohol versus 0.046 +/- 0.003 mM/liter without alcohol for IDDM; 0.067 +/- 0.007 mM/liter with alcohol versus 0.025 +/- 0.002 mM/liter without alcohol for controls) and in the 3-OHB/AcAc ratio (1.452 +/- 0.153 with alcohol versus 0.599 +/- 0.036 without alcohol for IDDM; 1.723 +/- 0.198 with alcohol versus 0.439 +/- 0.040 without alcohol for controls) because of a more reduced redox state. Alcohol intake during meals depressed alanine concentration, while glycerol levels showed a transient increase. Reduced blood FFA concentrations after alcohol intake were observed only in controls. This study demonstrates that moderate alcohol intake with meals also affects intermediate metabolites despite euglycemia. These effects were similar both in normal subjects and in IDDM, even if the harmful effects of alcohol may be enhanced by poor metabolic control in the latter.

The antiketogenic effect of alanine in normal man: evidence for an alanine-ketone body cycle.

The effect of alanine on ketone body levels, independent of hormonal changes, in normal man has been investigated. Five normal subjects were given somatostatin infusions (200 micrograms/hour) for 3 hr. After 1 hr alanine or isotonic saline was infused for 2 hr. With saline blood beta-hydroxybutyrate and acetoacetate levels rose steadily to a peak of 0.230 plus or minus 0.053 and 0.112 plus or minus 0.023 mmole/l respectively. With alanine beta-hydroxybutyrate and acetoacetate levels plateaued at 0.099 plus or minus 0.020 and 0.055 plus or minus 0.006 mmole/l respectively. Alanine levels reached nearly 1 mmole/l but a significant effect on ketone body levels was apparent at physiologic levels (less than 0.6 mmole/l). Plasma fatty acid and glycerol levels did not change significantly. Insulin C-peptide and glucagon levels were suppressed to a similar extent in both experiments. These results support the view that alanine suppresses ketogenesis in man by a direct hepatic effect independent of insulin and glucagon. It is suggested that this forms part of a negative feedback substrate cycle between alanine and ketone bodies.

The medium-term effect of natural or extractive dietary fibres on plasma amino acids and lipids in type 1 diabetics.

We evaluated the effect of a diet rich in natural (NF) or extractive fibres (guar gum) on 12 male IDD (insulin-dependent diabetes) out-patients. The treatment lasted for 2 months. During the first month the patients were on an isocaloric diet containing 30 g of fibres and then they were randomly subdivided into two groups. One group followed an isocaloric diet rich in fibres (70 g/day), the second group an isocaloric diet enriched by guar (9 g of guar added to 30 g of natural fibres/day). Reduced serum levels of HbA1c and several amino acids showed that metabolic control significantly improved under each dietary regimen.

No effects of high-fiber diets on metabolic control and insulin-sensitivity in type 1 diabetic subjects.

The metabolic effects of a three-month treatment with a high-fiber diet (15 grams of guar-gum added to a standard diet) were investigated in seven type 1 diabetic subjects, with a moderately poor metabolic control. HbA1c levels, daily insulin requirement, cholesterol, triglyceride, amino acid and intermediate metabolite concentrations were evaluated before and following the high fiber diet, both in the postabsorptive state at euglycemia and during a euglycemic, hyperinsulinemic, hyperaminoacidemic clamp. Insulin-mediated glucose utilization, an index of insulin-sensitivity, was also measured during the clamp. Following the diet, no differences in HbA1c levels (7.6 +/- 0.7%----7.3 +/- 0.6%), daily insulin requirement (50 +/- 5----51 +/- 3 U/d), triglyceride, amino acid and intermediary metabolite concentrations in the basal, euglycemic state, were observed. Only cholesterol concentrations decreased significantly (from 165 +/- 12 to 142 +/- 12 mg/dl, P less than 0.01) after the diet. During the clamp, the concentrations of all measured substrates were comparable before and after high fiber treatment. Insulin-mediated glucose disposal was also unchanged by guar-gum treatment. Patients' body weights were not modified by the diet. In conclusion, our study shows that a high fiber diet, obtained with the addition of 15 grams of guar-gum to a standard diet, is of no benefit to IDDM either as regards the metabolic control or insulin sensitivity. Only cholesterol levels were decreased. Therefore, the costs and benefits of these diets in the treatment of IDDM should be reconsidered.

Abnormal Na+/H+ antiport activity in cultured fibroblasts from NIDDM patients with hypertension and microalbuminuria.

An increased activity of Na+/H+ antiport has been reported in leukocytes and fibroblasts from insulin-dependent diabetic (IDDM) patients with nephropathy. To test whether a similar abnormality is present in fibroblasts from non-insulin-dependent diabetic (NIDDM) patients with microalbuminuria and hypertension, we examined intracellular pHi and Na+/H+ antiport activity, using the pH sensitive dye 2', 7'-bis (2-carboxyethyl-5(6)-carboxyfluorescein (BCECF), in cultured skin fibroblasts obtained from 34 NIDDM patients, divided into four groups based upon whether they had microalbuminuria or hypertension, or both: Group 1, nine NIDDM patients with microalbuminuria and hypertension. Group 2, nine NIDDM patients with hypertension and normal albumin excretion rate. Group 3, seven NIDDM patients with microalbuminuria and normal blood pressure. Group 4, nine NIDDM patients with normal blood pressure and normal albumin excretion rate. Nine normal subjects served as control group. Resting pHi was more alkaline in fibroblasts from Group 1 (7.22 +/- 0.03; p < 0.05), Group 2 (7.21 +/- 0.02; p < 0.05) and Group 3 (7.19 +/- 0.02, p = 0.17) than in Group 4 and normal subjects. This was due to higher Vmax values of Na+/H+ antiport activity in cultured fibroblasts from Group 1 (52.1 +/- 5.3 mmol H+/min; p < 0.05), Group 2 (57.7 +/- 8.3; p < 0.05) and Group 3 (60.6 +/- 7.4, p < 0.05) than those from Group 4 (31.2 +/- 3.6) or control subjects (31.3 +/- 3.5). The intracellular pH for half-maximal activation, Hill coefficient and buffering power capacity was similar in all the groups. These data suggest that in vitro phenotypic abnormalities of long-term cultured fibroblasts from NIDDM patients with microalbuminuria and/ or hypertension are likely to be, at least in part, independent of the degree of metabolic control in vivo and to be an intrinsic feature of these cells.

Enhanced effects of insulin and angiotensin II on intracellular pH and free cytosolic calcium in fibroblasts from microalbuminuric patients with non-insulin-dependent diabetes mellitus.

1. Whether an alteration in cell membrane cation transport after exposure to insulin and angiotensin II (two important growth promoters that have been shown to be involved in the pathogenesis of atherosclerosis and hypertension) is present in cells from non-insulin-dependent diabetes patients with microalbuminuria, a known risk factor for cardiovascular and renal disease, is unknown. We therefore examined intracellular pH and calcium changes after acute exposure to insulin and angiotensin II in cultured skin fibroblasts from eight non-insulin-dependent diabetes patients with and eight others without microalbuminuria and from a group of seven matched, normal control subjects. 2. Cultured fibroblasts were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester or fura 2-acetoxymethyl ester for continuous monitoring of intracellular pH and free calcium concentrations respectively. 3. In quiescent growth-arrested cells, both intracellular pH and free calcium concentrations were similar in the three groups of subjects. Acutely, insulin induced a gradual alkalinization in all groups of patients. The pH increase was significantly greater in non-insulin-dependent diabetes mellitus patients with microalbuminuria (delta pH +0.24 +/- 0.04 pH units) than in normoalbuminuric patients with non-insulin-dependent diabetes mellitus (0.08 +/- 0.02; P < 0.01) and normal control subjects (0.05 +/- 0.01; P < 0.01). Although the alkalinizing effect of angiotensin II was smaller than that obtained by insulin, intracellular pH increase after angiotensin addition was more pronounced in non-insulin-dependent diabetes mellitus patients with microalbuminuria (delta pH +0.14 +/- 0.04 pH units) than in those without (0.08 +/- 0.02; P < 0.01) and in normal control subjects (0.02 +/- 0.02; P < 0.01). That the increase in intracellular pH was mediated by the sodium-hydrogen antiport was demonstrated by its dependence on the presence of sodium in the medium and its inhibition by amiloride. Whereas insulin addition did not evoke any significant increase in intracellular free calcium levels in fibroblasts from the three groups studied, angiotensin II evoked a fast and transient rise in intracellular free calcium that was higher in fibroblasts from microalbuminuric patients with non-insulin-dependent diabetes mellitus than in cells from normoalbuminuric patients with non-insulin-dependent diabetes mellitus and control subjects. In the whole population of patients with non-insulin-dependent diabetes mellitus, the increase in intracellular pH after exposure to angiotensin II was positively correlated with intracellular free calcium increase (r = 0.53; P < 0.05), suggesting a possible role of intracellular free calcium levels in the activation of the sodium-hydrogen antiport. 4. In conclusion, we have described an association between increased agonist-induced responsiveness of sodium-hydrogen antiport activity and the presence of microalbuminuria in patients with non-insulin-dependent diabetes mellitus. This increased responsiveness, persisting in cultured fibroblasts after several passages in vitro, suggests that in vitro phenotypic characteristics of fibroblasts are likely to be genetically determined and to be, at least in part, independent of the degree of metabolic control in vivo.