Proinflammatory cytokines inhibit secretion in rat bile duct epithelium.

BACKGROUND AND AIMS: Cholestatic disorders often are associated with portal inflammation, but whether or how inflammation contributes to cholestasis is unknown. Thus we studied the effects of proinflammatory cytokines on bile duct epithelia secretory mechanisms. METHODS: Isolated bile duct units (IBDUs) were cultured with interleukin (IL)-6, interferon gamma, tumor necrosis factor (TNF)-alpha, and IL-1 alone or in combination. Ductular secretion was measured using video-optical planimetry. Bicarbonate and Cl(-) transport were assessed microfluorimetric measuring pH(i) (BCECF) and [Cl(-)](i) transients (MEQ). Expression of Cl(-)/HCO(3)(-) exchanger (AE-2), cystic fibrosis transmembrane conductance regulator (CFTR), and the secretin receptor (SR) were assessed by ribonuclease protection assay. Cellular cyclic adenosine monophosphate (cAMP) levels were studied by enzymatic immunoassay. Paracellular permeability was assessed using fluorescein-labeled dextrans (FD) in cholangiocyte monolayers (NRC-1). RESULTS: Although not effective when given alone, each combination of IL-6, interferon gamma, IL-1, and TNF-alpha inhibited secretion in IBDU. Cytokines inhibited cAMP formation, AE-2 activity, and cyclic AMP-dependent Cl(-) efflux, but not that induced by purinergic agonists. AE-2 gene expression was unaffected by proinflammatory cytokines, whereas CFTR and SR expression was increased. In addition, paracellular transit of FD across NRC-1 monolayers was increased. CONCLUSIONS: Inflammatory cytokines inhibit cAMP-dependent fluid secretion in cholangiocytes and impair the barrier functions of biliary epithelia. These changes may represent the molecular mechanisms by which inflammation leads to ductular cholestasis in vivo.

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.

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.

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.

Forearm ketone body metabolism in normal and in insulin-dependent diabetic patients.

In insulin deficiency, there is excessive arterial delivery of free fatty acid (FFA) to muscles where they are converted to acetoacetyl-CoA and acetyl-CoA. These intermediates may be metabolized further to acetoacetate and beta-hydroxybutyrate, which can be released into the venous circulation. When ketone body (KB) tracers are infused in vivo, they are diluted across muscle tissue. This dilution may be due to 1) KB newly formed within muscle (ketogenesis); 2) exchange of tracer between labeled and unlabeled acetyl-CoA and acetoacetyl-CoA, intermediates common to the metabolism of both FFA and KB (pseudoketogenesis). Thus this study assessed whether such label exchange could be detected across the human forearm and whether an increased delivery of FFA in insulin-sufficient controls provoked dilution of labeled KB tracer comparable to that observed in insulin-deficient diabetics. Five normal and five insulin-dependent diabetic (IDDM) subjects were infused with labeled [3,4-13C2]-acetoacetate. [13C]KB enrichments were lower in forearm vein than in the artery, and dilution of labeled KB was always higher than that which could be explained by arterial-venous differences of unlabeled KB. When arterial FFA concentrations in normals were raised (Intralipid+heparin) to values comparable to those of the diabetics, no additional increase in forearm arteriovenous dilution of labeled KB was observed. Neither in the basal state nor under conditions of increased plasma FFA were we able to detect venous appearance of KB labeled in the first and in the second carbon atoms, a necessary consequence of pseudoketogenesis.(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)

The effects of different plasma insulin concentrations on lipolytic and ketogenic responses to epinephrine in normal and type 1 (insulin-dependent) diabetic humans.

This study was performed to verify: (1) the ability of different insulin concentrations to restrict the lipolytic and ketogenic responses to exogenous epinephrine administration; (2) whether the ability of insulin to suppress the lipolytic and ketogenic responses during epinephrine administration is impaired in Type 1 (insulin-dependent) diabetic patients. Each subject was infused on separate occasions with insulin at rates of 0.2, 0.4, and 0.8 mU.kg-1.min-1 while normoglycaemic. To avoid indirect adrenergic effects on endocrine pancreas secretions, the so-called "islet clamp" technique was used. Rates of appearance of palmitic acid, acetoacetate, and 3-hydroxybutyrate were simultaneously measured with an infusion of 13C-labelled homologous tracers. After a baseline observation period epinephrine was exogenously administered at a rate of 16 ng.kg-1.min-1. At low insulin levels (20 microU/ml) the lipolytic response of comparable magnitude was detected in normal and Type 1 diabetic patients. However, the ketogenic response was significantly higher in Type 1 diabetic patients. During epinephrine administration, similar plasma glucose increments were observed in the two groups (from 4.74 +/- 0.53 to 7.16 +/- 0.77 mmol/l (p less than 0.05) in Type 1 diabetic patients and from 4.94 +/- 0.20 to 7.11 +/- 0.38 mmol/l (p less than 0.05) in normal subjects, respectively). At intermediate insulin levels (35 microU/ml) no significant differences were found between Type 1 diabetic patients and normal subjects, whereas plasma glucose levels rose from 4.98 +/- 0.30 to 6.27 +/- 0.66 mmol/l (p less than 0.05) in Type 1 diabetic patients, and from 5.05 +/- 0.13 to 6.61 +/- 0.22 mmol/l (p less than 0.05) in normal subjects. At high insulin levels (70 microU/ml) the lipolytic response was detectable only in Type 1 diabetic patients; the ketogenic response was reduced in both groups. During the third clamp, a significant rise in plasma glucose concentration during epinephrine infusion was observed in both groups. In conclusion this study shows that at low insulin levels Type 1 diabetic patients show an increased ketogenic response to epinephrine, despite their normal nonesterified fatty acid response. Instead, high insulin levels are able to restrict the ketogenic response to epinephrine in both normal and Type 1 diabetic subjects, although there is a still detectable lipolytic response in the latter. In the presence of plasma free insulin levels that completely restrict the ketogenic response in the same group, there is still a distinct glycaemic response. Plasma insulin levels in Type 1 diabetic patients are a major determinant of the metabolic response to epinephrine.

Effects of insulin and amino acid infusion on leucine and phenylalanine kinetics in type 1 diabetes.

To evaluate the anabolic effects of hyperinsulinemia and hyperaminoacidemia on amino acid (and protein) metabolism in type 1 (insulin-dependent) diabetes mellitus (IDDM), we studied leucine and phenylalanine kinetics in nine IDDM and seven control subjects, both at basal euglycemic conditions and during a euglycemic hyperinsulinemic clamp (approximately 60-80 microU/ml of plasma free insulin), combined with an intravenous infusion of amino acids (AA), which doubled plasma concentrations of most AA. In the basal state, euglycemia was maintained in IDDM subjects at the expense of a peripheral free insulin level (16 +/- 2 microU/ml) greater (P less than 0.05) than controls (9 +/- 1 microU/ml). Despite that, leucine rate of appearance (Ra), alpha-ketoisocaproate oxidation (approximating leucine-carbon oxidation), and nonoxidative leucine disposal, were greater (P less than 0.05) in IDDM than in control subjects. Phenylalanine Ra was slightly but not significantly greater in IDDM vs. control subjects. During the clamp, at comparable plasma free insulin and amino acid concentrations, oxidation was similar in the two groups, endogenous leucine and phenylalanine Ra remained significantly greater (P less than 0.05) in IDDM than in normal subjects, and leucine disposal tended also to be greater in IDDM subjects. Thus, in IDDM subjects maintained at euglycemia, endogenous Ra of essential amino acid(s) (index of endogenous proteolysis) is increased, both in the postabsorptive state and after hyperinsulinemia combined with hyperaminoacidemia, while leucine utilization for protein synthesis is not impaired.

Impaired response to angiotensin II in type 1 (insulin-dependent) diabetes mellitus. Role of prostaglandins and sodium-lithium countertransport activity.

The pathogenesis of diabetic nephropathy remains elusive. A role for renal prostaglandins in antagonizing the hormonal effects of renin-angiotensin II has been postulated as a putative factor leading to hyperfiltration in patients with Type 1 (insulin-dependent) diabetes mellitus. Our aim was to elucidate the effects of angiotensin II on kidney haemodynamics and on blood pressure in eight normal subjects, in nine normotensive, in nine hypertensive with normal sodium-lithium countertransport activity in erythrocytes, in seven hypertensive without and in eight hypertensive Type 1 diabetic patients with microalbuminuria and with high sodium-lithium countertransport activity in erythrocytes. Angiotensin II infusion (4 ng.kg-1.min-1 for 60 min) decreased the glomerular filtration rate to a greater extent in normal subjects (-20%), than in normotensive patients (-5% p less than 0.01), in hypertensive patients with normal sodium-lithium countertransport activity in erythrocytes (-8% p less than 0.01) in hypertensive patients with high sodium-lithium countertransport (-6% p less than 0.01) and in hypertensive microalbuminuric patients (-5% p less than 0.01) with Type 1 diabetes. The urinary excretion rate of vasodilatory prostaglandins was two-three fold higher in all patients than in normal subjects. Acute indomethacin treatment restored a normal response to angiotensin II infusion in normotensive patients, but did not change the renal haemodynamic response in normal subjects. With regard to hypertensive patients with and without microalbuminuria indomethacin treatment restored a normal response to angiotensin II in some but not all patients.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Partial recovery of insulin secretion and action after combined insulin-sulfonylurea treatment in type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral agents.

Metabolic control, insulin secretion and insulin action were evaluated in seven Type 2 (non-insulin-dependent) diabetic patients with secondary failure to oral antidiabetic agents before and after two months of combined therapy with supper-time insulin (Ultratard: 0.4 U/kg body weight/day) plus premeal glibenclamide (15 mg/day). Metabolic control was assessed by 24 h plasma glucose, NEFA, and substrate (lactate, alanine, glycerol, ketone bodies) profile. Insulin secretion was evaluated by glucagon stimulation of C-peptide secretion, hyperglycaemic clamp (+ 7 mmol/l) and 24 h free-insulin and C-peptide profiles. The repeat studies, after two months of combined therapy, were performed at least 72 h after supper-time insulin withdrawal. Combining insulin and sulfonylurea agents resulted in a reduction in fasting plasma glucose (12.9 +/- 7 vs 10.4 +/- 1.2 mmol/l; p less than 0.05) and hepatic glucose production (13.9 +/- 1.1 vs 11.1 +/- 1.1 mumol.kg-1.min-1; p less than 0.05). Mean 24 h plasma glucose was also lower (13.7 +/- 1.2 vs 11.1 +/- 1.4 mmol/l; p less than 0.05). Decrements in fasting plasma glucose and mean 24 h profile were correlated (r = 0.90; p less than 0.01). HbA1c also improved (11.8 +/- 0.8 vs 8.9 +/- 0.5%; p less than 0.05). Twenty-four hour profile for NEFA, glycerol, and ketone bodies was lower after treatment, while no difference occurred in the blood lactate and alanine profile. Insulin secretion in response to glucagon (C-peptide = +0.53 +/- 0.07 vs +0.43 +/- 0.07 pmol/ml) and hyperglycaemia (freeinsulin = 13.1 +/- 2.0 vs 12.3 +/- 2.2 mU/l) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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 bodies increase glomerular filtration rate in normal man and in patients with type 1 (insulin-dependent) diabetes mellitus.

The purpose of this study was to investigate whether the administration of acetoacetic and hydrochloric acids in a group of control and Type 1 (insulin-dependent) diabetic patients influenced renal haemodynamics. Renal plasma flow increased from 657 +/- 88 to 762 +/- 81 ml X min-1. 1.73 m-2 in diabetic patients (p less than 0.01) and from 590 +/- 71 to 691 +/- 135 in control subjects (p less than 0.01). Glomerular filtration rate increased from 135 +/- 9 to 180 +/- 8 ml X min-1. 1.73 m-2 in diabetic patients (p less than 0.001) and from 117 +/- 8 to 145 +/- 7 in control subjects (p less than 0.01). Similar effects on renal haemodynamics, even if less pronounced, were observed with low dose acetoacetic but not with hydrochloric acid infusion. Total protein, beta 2-microglobulin but not albumin excretion rates were increased by acetoacetic acid. We conclude that an acute increase in blood concentration of ketone bodies within the range found in diabetic patients with poor metabolic control increases renal plasma flow and glomerular filtration rate both in control subjects and diabetic patients and causes a tubular proteinuria.

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)

Dose-response curves of effects of insulin on leucine kinetics in humans.

To determine the effects of physiological and pharmacological insulin concentrations on leucine-carbon kinetics in vivo, eight postabsorptive normal volunteers were infused with L-[4,5-3H]leucine and alpha-[1-14C]ketoisocaproate (KIC). Insulin concentrations were sequentially raised from 8 +/- 1 to 43 +/- 6 and 101 +/- 14 and to 1,487 +/- 190 microU/ml, while maintaining euglycemia with adequate glucose infusions. At the end of each 140-min insulin-infusion period, steady-state estimates of leucine and KIC rates of appearance (Ra), KIC (approximately leucine-carbon) oxidation, nonoxidized leucine-carbon flux [an index of leucine incorporation into protein (Leu----P)], and leucine and KIC interconversion rates were obtained. After the three insulin infusions, leucine Ra decreased by a maximum of approximately 20%. KIC Ra decreased by a maximum of approximately 50%. The sum of leucine plus KIC Ra in the basal state was 2.59 +/- 0.24 mumol X kg-1 X min-1 and decreased by approximately 30% at the maximal insulin concentrations. KIC oxidation decreased by a maximum of approximately 65%. Leu----P did not increase after hyperinsulinemia. Interconversion rates were promptly and markedly suppressed by 50-70%. Leucine clearance increased by approximately 120%. We conclude that euglycemic hyperinsulinemia, at physiological and pharmacological concentrations, decreased leucine and KIC concentrations, leucine-carbon turnover and oxidation, and leucine and KIC interconversions in a dose-dependent manner in vivo.