Blood glucose lowering and glucagonostatic effects of glucagon-like peptide I in insulin-deprived diabetic dogs.

To establish potential effects of glucagon-like peptide I (GLP-I) on blood glucose control in insulin-deficient states, GLP-I [GLP-I(7-36) amide; 10 pmol x kg(-1) x min(-1)] was infused intravenously in six fasting, canine C-peptide-negative, chronically diabetic dogs for 8 h. Blood samples were saved for the analysis of hormones, metabolites, and turnover rates of glucose (6-(3)H-glucose), alanine (U-(14)C-alanine), and urea ((15)N(2)-urea) starting 22 h after the last subcutaneous dose of exogenous insulin. Circulating plasma GLP-I levels rose under infusion from 2.9 +/- 0.8 to 41.4 +/- 10.1 pmol/l. This was efficient to significantly reduce the preexisting diabetic hyperglucagonemia. Since in the utilized model functioning pancreatic beta-cells are lacking, GLP-I had no insulinogenic effect. Compared with control experiments in the same animals receiving saline infusion, glycemia dropped from 20.8 +/- 1.9 to 16.2 +/- 1.0 mmol/l (P < 0.05). This was in parallel to the infusion of GLP-I and was most likely caused by a decrease of elevated glucose production since overall glucose turnover decreased with no alteration in glucose metabolic clearance. Alanine turnover was significantly reduced, obviously reflecting a decline in alanine production in relation to changed muscle glucose uptake under conditions of lower glycemia and overall glucose turnover. There was, however, neither an effect of GLP-I on alanine conversion into circulating glucose nor an effect on urea production rate, indicating unchanged gluconeogenesis from amino acid precursors. We conclude that the blood glucose-lowering effect of GLP-I in an animal model of insulinopenia was shown to be due to a reduction in hepatic glucose output, possibly secondary to reduction in glucagon concentrations leading to decreased glycogenolysis. Whether GLP-I might be therapeutically useful in clinical insulin-deficient diabetes needs to be verified.

In vitro stimulation by islet antigen facilitates the detectability of beta-cell reactive cells in diabetes-prone BB/OK rats.

It has been supposed that beta-cell destruction in man and animals is due to autoreactive T-cells. We used the [51Cr]-release assay to identify the presence of beta-cell reactive cells in the spleen of diabetes-prone BB/OK rats before and after diabetes manifestation as well as in long-term normoglycaemic rats with a reduced diabetes risk of 3%. Splenic mononuclear cells (MNCs) obtained from diabetes-resistant LEW.1W and the majority of long-term normoglycaemic BB/OK rats (86.4%) showed no reactivity to pancreatic islets in vitro. In contrast, beta-cell reactive cells were identified in dependence on age in 30.4-65.0% of 75-120 days old normoglycaemic rats and in relation to diabetes duration (1 and 20 days) in 75.0% and 16.0% of diabetic BB/OK rats. Islet antigen-specific stimulation of splenic MNCs, that showed no spontaneous islet-directed reactivity, resulted in a concentration-dependent activation of cytolytically reactive cells in BB/OK but not in LEW.1W rats. Splenic MNCs derived from all diabetic, from 82.4% of young normoglycaemic and from 46.2% of long-term normoglycaemic BB/OK rats developed an islet-directed reactivity in vitro. Phenotyping of MNCs showed a significant increase of activated IL2R+ T-lymphocytes in diabetic BB/OK rats, but without any correlation to their cytolytic potential in the [51Cr]-release assay. Despite this fact, IL2R+ cells enriched from the pool of MNCs mediated an enhanced [51Cr]-release from islets, indicating their relevance in the beta-cell destruction. These data suggest, that functional reactivity rather than phenotypic characterization of MNCs is useful to identify the existence of beta-cell reactive cells. Furthermore, for screening diabetes risk in young normoglycaemic BB/OK rats besides the detection of beta-cell reactive cells the occurrence of regulatory cells seems to be decisive.

Glucagon-like peptide-1 has no insulin-like effects in insulin-dependent diabetic dogs maintained normoglycemic and normoinsulinemic.

A pharmacological concentration of glucagon-like peptide-1 (GLP-1) in the insulin-deficient state clearly decreases the blood glucose level. Therefore, this study was designed to evaluate a putatively relevant effect of the gastrointestinal peptide as an adjuvant to insulin replacement therapy. GLP-1 (GLP-1(7-36) amide 10 pmol x kg(-1) x min(-1)) was infused intravenously over 8 hours in nine fasting, C-peptide-negative diabetic dogs. The animals were under normoglycemic control by glucose-controlled insulin infusion (GCII) during the night before and during GLP-1 administration. During the paired control tests, the animals received saline infusion instead of GLP-1. In addition to the insulin infusion rates required to maintain normoglycemia, hormones, metabolites, and the turnover rates for glucose (6-3H-glucose), alanine (U-14C-alanine), and urea (15N2-urea) were measured during the final 2 hours of GLP-1 administration. Circulating plasma GLP-1 levels increased from 3+/-1 to 17+/-7 pmol/L. There was no significant difference in the insulin infusion rate between the experimental and control groups (0.43+/-0.05 v. 0.40+/-0.05 mU x kg(-1) x h(-1), average over the entire interval). Glycemia was maintained at a practically identical level (4.9+/-0.3 v. 4.8+/-0.4 mmol/L). Also, the concentration of plasma insulin-which was not hyperinsulinemic--and pancreatic glucagon remained unaltered. We found no appreciable effect of GLP-1 on glucose production and metabolic clearance, alanine turnover and the formation of glucose from alanine (1.8+/-0.2 v. 1.4+/-0.2 micromol x kg(-1) x min(-1), or the urea production rate as a measure of overall amino acid catabolism (4.1+/-0.4 v. 4.1+/-0.4 micromol x kg(-1) x min(-1)). Thus, no conclusive adjuvant effect of GLP-1 was ascertained in insulin-treated diabetic dogs under normoglycemic control.

Estimation of urea production rate with [(15)n(2)]urea and [(13)c]urea to measure catabolic rates in diabetes mellitus.

Abstract For verifying catabolic states in insulin-dependent patients and dogs the method estimating urea production rates with (13)C and with doubly (15)N labeled urea, respectively, has been established. For a fast steady state of urea tracer dilution, a prime of 600 times the continuous infusion rate had to be injected. Urea was isolated from plasma samples by protein precipitation and cation exchange chromatography with a consecutive derivatization of the dried urea fraction (trimethylsilyl derivatives). The masses of the fragment ions m/z 189 ((14)N(14)N), 190 ((14)N(15)N) and 191 ((15)N(15)N) urea are monitored to estimate the [(15)N(2)]urea frequency in the overall body urea pool in mol percent excess (MPE). 1 to 15 ng of derivatized urea were measured efficiently. An excellent correlation between expected standard and measured MPE (r = 0.9977) was achieved from solutions containing 1 to 7% [(15)N(2)]urea. The interassay coefficient of variation amounted to < 10% for a [(15)N(2)]urea portion of ≥ 3%. Normoglycemic diabetic patients who were treated with insulin overnight showed significantly higher urea production compared to healthy controls (9.22 ± 2.07 vs. 5.4 ± 0.32 µmol·kg(-1) · min(-1); p < 0.05). Measurements in chronic diabetic dogs proved an increased rate of amino acid catabolism (+ 20% urea production) in systemic versus portal application of insulin in paired studies. This increased nitrogen load in diabetics may accelerate progression of diabetic nephropathy. - Thus, the established stable isotope technique may serve as a sensitive and useful indicator of amino acid catabolism in clinical and experimental research.

Estimation of urea production rate with [15N2]urea and [13C]urea to measure catabolic rates in diabetes mellitus.

For verifying catabolic states in insulin-dependent patients and dogs the method estimating urea production rates with 13C and with doubly 15N labeled urea, respectively, has been established. For a fast steady state of urea tracer dilution, a prime of 600 times the continuous infusion rate had to be injected. Urea was isolated from plasma samples by protein precipitation and cation exchange chromatography with a consecutive derivatization of the dried urea fraction (trimethylsilyl derivatives). The masses of the fragment ions m/z 189 (14N14N), 190 (14N15N) and 191 (15N15N) urea are monitored to estimate the [15N2] urea frequency in the overall body urea pool in mol percent excess (MPE). 1 to 15 ng of derivatized urea were measured efficiently. An excellent correlation between expected standard and measured MPE (r = 0.9977) was achieved from solutions containing 1 to 7% [15N2]urea. The interassay coefficient of variation amounted to < 10% for a [15N2]urea portion of > or = 3%. Normoglycemic diabetic patients who were treated with insulin overnight showed significantly higher urea production compared to healthy controls (9.22 +/- 2.07 vs. 5.4 +/- 0.32 mumol.kg-1.min-1; p < 0.05). Measurements in chronic diabetic dogs proved an increased rate of amino acid catabolism (+20% urea production) in systemic versus portal application of insulin in paired studies. This increased nitrogen load in diabetics may accelerate progression of diabetic nephropathy. Thus, the established stable isotope technique may serve as a sensitive and useful indicator of amino acid catabolism in clinical and experimental research.

Differences in protein and energy metabolism following portal versus systemic administration of insulin in diabetic dogs.

AIMS/HYPOTHESIS: In non-diabetic people, insulin levels in the liver are two-fold higher than those in the systemic circulation. In contrast, patients with type 1 diabetes have similar hepatic and systemic insulin levels because insulin is administered peripherally. The aim of this study was to compare the effects of systemic (SI) and pre-portal (PI) insulin administration on energy, glucose and protein metabolism in chronic insulin-dependent ketosis-prone diabetic dogs. MATERIALS AND METHODS: We applied glucose-controlled insulin infusion, indirect calorimetry and stable isotope and radioisotope techniques to measure energy, protein and glucose metabolism. We maintained near-normoglycaemia at identical levels under both study conditions for 20 h. RESULTS: SI was associated with lower oxygen consumption (130+/-13 vs 161+/-8 ml/min), CO(2) production (99+/-10 vs 130+/-8 ml/min), respiratory quotient (0.76+/-0.02 vs 0.81+/-0.01) and energy expenditure (870+/-90 vs 1089+/-60 kcal/24 h) (p<0.05 for all differences). PI increased the respiratory quotient from the insulin-deprived state, whereas SI did not. Glucose kinetics were similar for SI and PI, whereas leucine oxidation (36+/-4 vs 54+/-5 micromol kg(-1) min(-1)) and the fractional synthesis rates of liver tissue protein (0.68+/-0.6 vs 0.83+/-0.07%/h), albumin (0.55+/-0.06 vs 0.68+/-0.4%/h), and fibrinogen (1.73+/-0.23 vs 2.59+/-0.25%/h) were all lower during SI than PI (p<0.05). CONCLUSIONS/INTERPRETATION: The route of insulin administration did not alter glucose metabolism but did affect protein synthesis in the liver. The potential impact of this altered liver protein metabolism on chronic complications needs careful evaluation. A similar decrease in energy expenditure resulting from systemic insulin administration during tight glycaemic control is a potential cause of weight gain.

Impaired hormonal regulation of adenosine 3',5'-monophosphate release in adipose tissue from hyperglycemic sand rats in vitro.

Egyptian sand rats (Psammomys obesus) developed under laboratory holding conditions and in dependence of different food intake various states of hyperglycemia and hyperinsulinism. Associated with the development of this syndrome insulin resistance of muscle- and adipose tissue appears. The question arose as to whether hyperglycemia and hyperinsulinism generally leads to hormone resistance. Therefore, we selected sand rats with different degrees of hyperglycemia and various stages of hyperinsulinism. Two groups of sand rats were investigated: normoglycemic and hyperglycemic animals. In these sand rats the catecholamine action on cAMP production adipose tissue was studied in vitro. There was a significant reduction of hormone responsiveness in adipose tissue of the hyperglycemic group compared with the noradrenaline response of adipose tissue of the normoglycemic sand rats. In no case had insulin significantly inhibited the noradrenaline stimulated cAMP production. By means of adenosine deaminase studies the interference of the eventual release of adenosine with the hormone action on adipose tissue in vitro could be excluded. In the sand rats correlations between IRI levels in the decapitation blood and noradrenaline stimulated cAMP release associated with simultaneous occurrence of insulin- and catecholamine resistance in adipose tissue seem to indicate that hormone resistance could be a general phenomenon during the development of hyperinsulinism.

Impaired hormonal regulation of lipolysis and the interference with adenosine in adipose tissue from hyperglycemic sand rats in vitro.

Sand rats (Psammomys obesus) developed in response to different food intake various states of hyperglycemia and hyperinsulinism. 12 normo- and 10 hyperglycemic animals were selected by means of a weekly control of plasma glucose and plasma insulin over a period of 12 weeks after separation from the mother. During this time also the development of body weight gain was checked. In both groups of rats the hormonal regulation of glycerol release by incubated adipose tissue was investigated. In any case, the fat tissue from hyperglycemic sand rats showed a lower lipolytic responsiveness to noradrenaline stimulation than that of their normoglycemic controls. This correlates well with previous results in hyperglycemic sand rats in which the catecholamine-stimulated cAMP production was disturbed (Knospe and Köhler 1981). Degradation of released adenosine by addition of adenosine deaminase significantly enhanced the noradrenaline action on glycerol release in both groups of sand rats. Even though the noradrenaline-stimulated lipolytic activity of adipose tissue from normo- and hyperglycemic animals was enhanced in the presence of adenosine deaminase, the hormone resistance of adipose tissue from hyperglycemic sand rats was nevertheless not abolished. The theophylline-mediated adenosine receptor blockade gave further evidence that particularly endogenous adenosine released during incubation of adipose tissue from sand rats inhibited the noradrenaline action on lipolysis. The antilipolytic action of insulin on glycerol release is negligibly low in normoglycemic as well as hyperglycemic sand rats. The degradation of adenosine by adenosine deaminase failed to improve the insulin action. Adenosine addition completely blocked the stimulating effects of noradrenaline on glycerol release.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-mediated immune reactions against islets of Langerhans in diabetes-prone BB rats.

The intact pancreatic islet can be destroyed by antibody-dependent cell-mediated cytotoxicity (ADCC). T cell-mediated cytotoxicity (CMC) might additionally be important in the pathogenesis of IDDM. However, in vitro alterations of islets due to CMC have so far not been demonstrated. For the evaluation of the cytotoxic attack caused by ADCC and CMC against the islets in the development of IDDM we used a BB rat line with a high incidence of the disease (dp BB/OK). Cytotoxicity tests were carried out on autologous and on nonsyngeneic islets with mononuclear spleen cells and serum from non-diabetic BB rats of different ages. The cytotoxic action of the mononuclear cells was quantified by the specific 51Cr-release from the islets after pretreatment with serum. It was found that an anti-islet cytotoxicity appears with a peak incidence between 40 and 60 days of age. The frequency of cytotoxicity in vitro was related to the incidence of diabetes as normally observed in this rat line. Furthermore, it was shown that both autologous, allogeneic and xenogeneic islets can be destroyed by mononuclear spleen cells and serum of dp BB/OK rats.--The frequency and the strength of anti-islet cytotoxicity in vitro were higher in these dp BB/OK animals than in a control group of non-diabetes prone BB/PhiK rats. There was an association between cytotoxic 51Cr-release in the positive assays and a reduction in the hormone content of pancreatic islets.--This report provides evidence of cell-mediated immune damage of islets during the prediabetic state of BB rats suggesting that both CMC and ADCC are involved in islet cell killing.

Significance of adenosine for the hormone responsiveness of adenylate cyclase in adipose tissue of normoglycemic and hyperglycemic sand rats.

In young sand rats, bred in our colony, the metabolism was directed from the normoglycemic state by means of feeding conditions. The noradrenaline action on adenylate cyclase is impaired in hyperglycemic, hyperinsulinemic sand rats. Generally, addition of adenosine to the in vitro system eliminates the responsiveness of the adenylate cyclase to noradrenaline in adipose tissue. The presence of adenosine deaminase in the incubation medium abolished this inhibition effect of added adenosine. The interference of released adenosine with the hormone action in vitro was excluded by addition of adenosine deaminase to the incubation medium. In both groups of sand rats adenosine deaminase did not increase the noradrenaline effect on adenylate cyclase. Investigations were carried out as a part of the research project "Diabetes mellitus and diseases of fat metabolism". These results along with the others on the measured adenosine release exclude adenosine as a reason for the disturbed hormone action on adenylate cyclase in hyperglycemic, hyperinsulinemic sand rats.

A specific method of high sensitivity for the determination of adenosine in the incubation medium of fat tissue.

A suitable method for the measurement of adenosine in the incubation medium of fat tissue (200-500 mg) has been developed. The method is based on the specificity of the adenosine deaminase reaction and on the high sensitivity of a fluorescent method for adenine derivatives. The decrease of fluorescence in a sample after treatment with this enzyme is used for measuring adenosine in the range of 50-500 pmoles/tube. This method is highly specific and is not affected by other adenine derivatives present in the sample. Instead of acetic acid, perchloric acid was used in the fluorescent reaction, thus increasing the amount of adenosine dependent fluorescence. With this modification of the original fluorescent method, perchloric acid extracts can be used without further processing after deproteinization of the samples. Using this method, we could measure the adenosine release of fat pads of Wistar rats incubated in Krebs-Ringer-albumin buffer without concentration or purification procedures.

The response to insulin by adipose tissue of insulin-dependent diabetics in vitro.

Subcutaneous adipose tissue was removed by surgical biopsy from 10 normal subjects and 4 insulin-dependent diabetics under exact intracutaneous anaesthesia. The adipose tissue fragments or isolated fat cells were incubated with a tracer amount of labeled glucose. In the presence of insulin (62.5 muU/ml) the production of carbon dioxide from glucose by adipose tissue fragments of nonobese controls as well as diabetics increased up to 286 +/- 62% and 198 +/- 14.7%, respectively. Furthermore, triglyceride synthesis was raised to 257 +/- 30% and 267 +/- 34%, respectively. There were no differences in the sensitivity of adipose tissue to insulin of healthy volunteers and juvenile diabetics.

Relationship between body fat mass, carbohydrate tolerance and IRI response during glucose infusion in subjects with early diabetes.

We have studied the interrelationship of total body fat mass, carbohydrate tolerance and IRI response in 17 non-obese and obese subjects, who were suspected of having early diabetes. We carried out an i.v. glucose infusion test consisting of a priming injection of 0.33 g/kg followed by constant glucose infusion of 12 mg/kg/min in all persons. Total body fat mass was estimated by the tritium dilution method. There was a positive correlation of body fat mass, fasting glucose concentration and blood glucose concentration at 150 min as well as a strong correlation between body fat mass and BG area 60--120 min as parameters of carbohydrate tolerance in all subjects, i.e. the degree of carbohyrate intolerance was directly related to the quantity of total body fat mass. A similar correlation was found when the non-obese and obese groups were analyzed separately. In neither group did total body fat mass correlate with parameters of IRI response. In obese subjects with pathological carbohydrate tolerance, however, a positive correlation of basal IRI concentration and total body fat mass was found. Furthermore, a close relation between basal IRI level and parameters of carbohydrate tolerance could be demonstrated in obese subjects. The present study failed to demonstrate any correlation of parameters of carbohydrate tolerance and glucose-induced IRI response in either group. Thus, the significant relationship between body fat mass and degree of carbohydrate intolerance indicates that body fat mass plays an important role in the disturbance of blood glucose homeostasis in early diabetes with and without obesity.

Autologous mixed lymphocyte reaction in newly diagnosed type-1 diabetes.

The autologous mixed lymphocyte reaction (AMLR) represents activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Deteriorations in AMLR have been reported in many autoimmune diseases and in diseases with a derangement in T cell regulatory function. We have studied AMLR in 23 newly diagnosed Type-1 diabetic patients and 32 healthy subjects. T and non-T cells were purified by rosetting mononuclear cells with sheep erythrocytes and separating the rosetted T cells from the nonrosetted non-T cells by density gradient centrifugation. Purity of T-lymphocytes isolated was 90% as determined by indirect immunofluorescent analysis with monoclonal antibodies. Proliferation of lymphocytes was measured in response to phytohaemagglutinin and of concanavalin A in a lymphocyte transformation test. In the present study, a deficient AMLR is demonstrated in patients with newly diagnosed Type-1 diabetes. Our data provide evidence for an aberrant immune regulation at the time of diabetes manifestation. The deficient AMLR may represent the in-vitro expression of an in-vivo process against pancreatic cells.

Effect of lymphocytes and serum from probands before and after manifestation of type 1 (insulin-dependent) diabetes on rat islets in vitro.

In a two-year follow-up study neonatal rat islets have been shown to be affected in vitro by lymphocytes and complement-inactivated serum obtained from newly diagnosed Type 1 (insulin-dependent) diabetic patients and probands who are at high risk for developing the disease. The effect was measured by 51Cr-release of the islets treated with the proband's serum after a 6 h-incubation with lymphocytes of the same donor. Nineteen newly diagnosed diabetic patients, 23 persons at risk and 11 control probands were studied. There was no appreciable cytotoxic activity in the control probands (with one exception) and in 7 out of the 19 newly diagnosed diabetics. Five of the diabetes-susceptible probands developed diabetes mellitus during the investigation period. Anti-islet cytotoxicity of lymphocytes was found in these individuals at least 8 months before diagnosis of Type 1 diabetes. The cytotoxic effect disappeared at various time intervals after disease manifestation. Islet cytotoxicity was intermittently found with lymphocytes from further 13 probands at risk, sometimes for more than one year. Our data indicate that mononuclear cells from probands who are at high risk for developing Type 1 diabetes can exert cytotoxicity on xenogenic neonatal islets in the presence of their own serum.

Effect of antibody-mediated crosslinking of insulin on its bioactivity and receptor binding.

Precipitating anti-insulin antibodies or anti-insulin IgG/anti-IgG complexes bind insulin in a highly aggregated form and thus should preferably be capable of inducing receptor aggregation, which has recently been suggested to be a precondition for insulin bioactivity. We, therefore, studied the influence of antibody-mediated crosslinking of insulin on the glucose conversion into CO2 in rat fat cells and glycogen synthesis in rat liver cells. As far as possible receptor-bound insulin was measured in parallel. Insulin bound to antibodies with low insulin precipitating capacity had no or little bioactivity and receptor reactivity on fat cells. The bioactivity, however, could be restored in part by addition of a second antibody. On liver cells, second antibody-mediated crosslinking of insulin-antibody complexes resulted in an enhancement of receptor reactivity, and insulin bioactivity of such crosslinked immune complexes was demonstrable. An insulin-precipitating antiserum was shown to form insulin-antibody complexes with significant bioactivity in fat cells which correlated with their receptor binding. In each case antibodies had no or little effect in the absence of insulin. Our data suggest that insulin neutralization by antibodies can be compensated by crosslinking the insulin-antibody complexes or by formation of big complexes precipitating by themselves. This is probably due to the induction of receptor aggregation.

Investigations on isolated islets of Langerhans in vitro. XIV. Insulin secretion and insulin stores of cultivated islets from sand rats (Psammomys obesus): investigations of glucose-dose response.

Release of immunoreactive insulin activity (IRI) and biological insulin like activity (ILA) from collagenase isolated pancreatic islets of sand rats (Psammomys obesus) maintained on a vegetable diet were examined at 60 min and 24-48 h intervals under culture conditions at 1.0 and 15.6 mM glucose. The glucose-insulin dose response curves for sand rats after 60 min incubation were compared with those after 24 or 48 h of incubation. The pancreatic islets responded to 5mM glucose with a high insulin release especially under culture conditions. A drastic depletion of stored insulin in the islets cultivated for 2 days at 5 or 15 mM glucose is accompanied by a continuous diminution of the glucose-induced insulin release with the prolongation of cultivation up to one week.

Insulin sensitivity of non-obese asymptomatic diabetics in vivo in relation to insulin responsiveness of their adipose tissue in vitro.

In twenty-four non-obese male subjects a 2-hour glucose infusion test (12 mg/kg/min) with initial bolus injection (0.33 g/kg) was performed as a test of carbohydrate tolerance. Sixteen individuals had a normal and eight a pathological carbohydrate tolerance (asymptomatic diabetes). All subjects received a one-hour insulin infusion (two 30-minutes periods of 8 or 16 mU/kg MC-Actrapid). After two days subcutaneous adipose tissue was removed from the abdominal wall by needle biopsy for characterization of insulin-stimulated (1-14C) glucose incorporation into triglycerides. Under in vivo conditions insulin provoked a decrease of blood glucose concentrations by 31 +/- 3.9% and 11.6 +/- 2.2% as well as of plasma free fatty acids levels by 60 +/- 4.6% and 37 +/- 6.8% in normal persons and asymptomatic diabetics, respectively (p less than 0.01). In vitro the insulin-stimulated incorporation of labeled glucose into triglycerides of adipose tissue was diminished in asymptomatic diabetics. Thus, the results indicate that both the in vivo insulin responsiveness and the in vitro insulin sensitivity of adipose tissue are reduced in early stages of diabetes. The findings suggest that changes in insulin target tissues are equally important in the development of carbohydrate intolerance in non-obese subjects.

Insulin responsiveness of adipose tissue from normal weight subjects with early diabetes.

In normal weight subjects, classified by a 2-h glucose infusion test as having normal (11), borderline (3) or pathological (9) carbohydrate tolerance (CHT), subcutaneous adipose tissue was removed under intracutaneous anesthesia by surgical biopsy. The biological responsiveness of isolated adipocytes as well as adipose tissue fragments measured as incorportion of (1-14C) glucose into CO2 or triglycerides was studied in the absence or presence of different insulin concentrations. In persons with normal CHT the insulin-stimulated (62.5 microU/ml) glucose conversion to CO2 by adipocytes as well as fat pads increased significantly up to 156 +/- 14% and 285 +/- 30%, respectively. Insulin enhanced the glucose incorporation into triglycerides up to 154 +/- 20% (fat cells) and 258 +/- 30% (fat pads) in adipose tissue from subjects displaying a normal CHT. Rates of glucose oxidation and triglyceride synthesis was markedly reduced in adipose tissue obtained from patients with borderline or pathological CHT. A significant positive relationship was found between glucose oxiation to CO2 and triglyceride production of fat cells and fat pads (r = 0.964 and 0.783, respectively). There was no correlation with responsiveness of adipose tissue to insulin and insulin secretion during glucose infusion test. The results indicate that sensitivity to insulin of target cells might be important for the development of carbohydrate intolerance also in normal weight subjects.

[Insulin binding to isolated cells]. / Untersuchungen zur Insulinbindung an isolierten Zellen.

Using mono-125I-insulin the hormone receptor binding on cultivated rat hepatocytes cultivated rat fibroblasts and freshly isolated adipocytes of wistar rat and man were characterized. No specific insulin binding was obtained in the case of long time cultivated hepatocytes. The fibroblasts show a low specific insulin binding with an affinity constant of K = 0.75. 10(9)M-1 and a binding capacity of q = 4000 binding sites per cell, and also an insulin response measureable by the lactate production. In agreement with a strong stimulation effect of insulin on the conversion of glucose to CO2 und triglycerides the freshly isolated adipocytes bind insulin with high affinity and capacity in comparison with the fibroblasts. For the high affinity population of insulin receptors at the adipocytes of wistar rat we found K = 2.8 . 10(9) M-1 and q = 22000 binding sites per cell, whereas 20 per cent of saturation of the receptors cause 90 per cent of the maximal stimulation effect on the bio-conversion of glucose. This shift of the binding curve to higher concentrations may be important for the ability of insulin to regulate the carbohydrate metabolism.