Reduced GLP-1 and insulin responses and glucose intolerance after gastric glucose in GRP receptor-deleted mice.

By applying a newly developed ELISA technique for determining biologically active intact glucagon-like peptide [GLP-1, GLP-1-(7-36)amide] in mouse, plasma baseline GLP-1 in normal NMRI mice was found to be normally distributed (4.5 +/- 0.3 pmol/l; n = 72). In anesthetized mice, gastric glucose (50 or 150 mg) increased plasma GLP-1 levels two- to threefold (P < 0.01). The simultaneous increase in plasma insulin correlated to the 10-min GLP-1 levels (r = 0.36, P < 0.001; n = 12). C57BL/6J mice deleted of the gastrin-releasing peptide (GRP) receptor by genetic targeting had impaired glucose tolerance (P = 0.030) and reduced early (10 min) insulin response (P = 0.044) to gastric glucose compared with wild-type controls. Also, the GLP-1 response to gastric glucose was significantly lower in the GRP receptor-deleted mice than in the controls (P = 0.045). In conclusion, this study has shown that 1) plasma levels of intact GLP-1 increase dose dependently on gastric glucose challenge in correlation with increased insulin levels in mice, and 2) intact GRP receptors are required for normal GLP-1 and insulin responses and glucose tolerance after gastric glucose in mice.

An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin.

Immunoassay technology is routinely used to measure concentrations of proteins and polypeptides in biological matrices. Increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharmacokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report describes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY355101 and LY396623, one and two amino acid changes relative to native human leptin, respectively). Rabbits were immunized with either LY355101 or LY396623. Antisera were screened to determine if any showed increased specificity for the analog relative to native human leptin. Antisera showing increased specificity for the leptin analog were then treated by immunoabsorption against native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays. which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 pharmacokinetics that were not confounded by the high levels of endogenous human leptin found in obese subjects. This technique has the potential for broad application in the development of assays capable of specifically measuring protein analogs without cross-reactivity to an endogenous substance.

Relationship between serum leptin immunoreactivity and body fat mass as estimated by use of a novel gas-phase Fourier transform infrared spectroscopy deuterium dilution method in cats.

OBJECTIVE: To validate a recently developed commercially available leptin radioimmunoassay (RIA) for use with feline serum and evaluate the relationship between serum leptin concentrations and body fat mass in domestic cats. ANIMALS: 19 sexually intact male specific-pathogen-free domestic cats that weighed 3.8 to 7.1 kg and were 1.1 to 3.5 years old. PROCEDURE: Specificity for feline leptin was evaluated by use of gel filtration chromatography and reverse-phase high-performance liquid chromatography fractionation of serum. Body fat mass was determined by use of the deuterium oxide (D2O) dilution method. Serum water D2O enrichment was measured by use of gas-phase Fourier transform infrared spectroscopy. RESULTS: Body fat mass and percentage body fat ranged from 0.3 to 2.3 kg and 7.5 to 34.9%, respectively. Serum leptin concentrations were lower in the unfed versus the fed state and ranged between 1.6 and 4.9 ng/ml human equivalent (HE); mean +/- SD value was 2.9 +/- 0.2 ng/ml HE. Leptin concentrations increased with increasing body fat mass and percentage of body fat. CONCLUSIONS: Leptin is in the serum of domestic cats in free (> 78%) and apparently bound forms. The relationship between body fat and serum leptin concentration was similar to that observed in humans and rodents and indicative of a lipostatic role for leptin in cats. Cats that have an overabundance of body fat appear to be less sensitive to the weight-normalizing action of leptin than cats of ideal body condition.

Cerebrospinal fluid and plasma leptin measurements: covariability with dopamine and cortisol in fasting humans.

Leptin (OB protein) is an important signal in the regulation of energy balance. Leptin levels correlate with adiposity, but also decrease acutely with caloric restriction and increase with refeeding. The brain is an established critical site of leptin function, yet little is known about leptin concentrations in the central nervous system relative to plasma levels, psychiatric diagnoses, and other endocrine parameters. Therefore, using a novel ultrasensitive leptin assay, we explored relationships of human plasma and cerebrospinal fluid (CSF) leptin levels to body mass index, smoking, posttraumatic stress disorder diagnosis, and levels of dopamine, monoamine metabolites, beta-lipotropin, glucocorticoid, and thyroid and cytokine hormones. A strong linear relation between CSF and plasma leptin levels in the am (r = 0.63; P < 0.002) and afternoon (r = 0.90; P < 0.0001) was revealed. CSF and plasma leptin concentrations decreased during a 12- to 20-h period of fasting. A strong association was found between plasma leptin and CSF dopamine levels (r = 0.74; P < 0.01) as well as between CSF leptin levels and urinary free cortisol (r = 0.73; P < 0.01). Both of these parameters covaried with leptin independently of adiposity, as estimated by body mass index. Implications for leptin transport, regulation, and its potential role in therapeutic strategies for obesity and diabetes are discussed.

Radioimmunoassay of rat leptin: sexual dimorphism reversed from humans.

Adipose tissue secretes leptin, which interacts with receptors in the hypothalamus. In rodent models of obesity, leptin increases metabolism and decreases food intake, which helps to maintain normal body composition. Accurate and precise methods to quantitate circulating leptin concentrations are needed for physiological studies. We developed an RIA to measure leptin in rat plasma, serum, or adipocyte culture fluids. The working range of the assay, defined by the detection limit and the highest calibrator, was 0.5-50 micrograms/L. Recovery of 1.6-11.6 micrograms/L leptin added to serum was 92-103%. The rat leptin RIA correlated well with a previously developed mouse RIA when rat plasma was assayed with both methods (r = 0.94), but the mouse leptin assay underestimated rat leptin in plasma. Within- and between-run CVs were 2.4% to 5.7%. Plasma leptin concentrations correlated directly with percentage of body fat, and correlation improved when the results were separated by gender (r = 0.796, P < 0.001 for males; r = 0.710, P < 0.001 for females). Leptin concentrations were generally higher in male rats than in females; plasma leptin increased 0.60 microgram/L for each percentage of increase in body fat for males but only 0.22 microgram/L for females. We conclude that rat serum/plasma leptin concentrations are accurately and precisely measured with this new RIA.

Regulation of plasma leptin in mice: influence of age, high-fat diet, and fasting.

Mechanisms regulating circulating leptin are incompletely understood. We developed a radioimmunoassay for mouse leptin to examine the influence of age, dietary fat content, and fasting on plasma concentrations of leptin in the background strain for the ob/ob mouse, the C57BL/6J mouse. Plasma leptin increased with age [5.3 +/- 0.6 ng/ml at 2 mo (n = 23) vs. 14.2 +/- 1.6 ng/ml at 11 mo (n = 15), P < 0.001]. Across all age groups (2-11 mo, n = 160), log plasma leptin correlated with body weight (r = 0.68, P < 0.0001), plasma insulin (r = 0.38, P < 0.001), and amount of intra-abdominal fat (r = 0.90, P < 0.001), as revealed by magnetic resonance imaging. Plasma leptin was increased by a high-fat diet (58% fat for 10 mo) and reduced by fasting for 48 h. The reduction of plasma leptin was correlated with the reduction of plasma insulin (r = 0.43, P = 0.012) but not with the initial body weight or the change in body weight. Moreover, the reduction in plasma leptin by fasting was impaired by high-fat diet. Thus plasma leptin in C57BL/6J mice 1) increases with age or a high-fat diet; 2) correlates with body weight, fat content, and plasma insulin; and 3) is reduced during fasting by an action inhibited by high-fat diet and related to changes of plasma insulin.

Sexual dimorphism in plasma leptin concentration.

Leptin, the obese (ob) gene product, is thought to be a lipostatic hormone that contributes to body weight regulation through modulating feeding behavior and/or energy expenditure. The determinants of plasma leptin concentration were evaluated in 267 subjects (106 with normal glucose tolerance, 102 with impaired glucose tolerance, and 59 with noninsulin-dependent diabetes). Fasting plasma leptin levels ranged from 1.8-79.6 ng/mL (geometric mean, 12.4), were higher in the obese subjects, and were not related to glucose tolerance. Women had approximately 40% higher leptin levels than men at any level of adiposity. After controlling for body fat, postmenopausal women had still higher leptin levels than men of similar age, and their levels were not different from those in younger women. Multiple regression analysis showed that adiposity, gender, and insulinemia were significant determinants of leptin concentration, explaining 42%, 28%, and 2% of its variance, respectively. Neither age nor the waist/hip ratio was significantly related to leptin concentration. Thus, our data indicate that gender is a major determinant of the plasma leptin concentration. This sex difference is not apparently explained by sex hormones or body fat distribution. Leptin's sexual dimorphism suggests that women may be resistant to its putative lipostatic actions and that it may have a reproductive function.

Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sustained weight loss.

Leptin, the product of the human homologue of the ob gene, which is defective in the obese (ob/ob) mouse, may be a humoral regulator of human adiposity. Plasma leptin concentrations were measured by RIA in 19 normal weight [body mass index (BMI) = 24.5 +/- 0.6 kg/m2] and 19 overweight to obese (BMI = 34.7 +/- 1.2 kg/m2) nondiabetic postmenopausal women on sequential controlled weight-maintaining diets containing 31%, 23%, and 14% of energy as fat, each for 4-6 weeks. Thereafter, the subjects ate a very low fat diet (< 15%) ad libitum; plasma leptin and insulin concentrations, BMI, percent body fat (%BF), and resting energy expenditure were determined after 6 and 8 months. Absolute and adiposity-corrected plasma leptin levels were higher in overweight/obese women (37.7 +/- 3.5 ng/mL; 1.01 +/- 0.07 ng.mL-1.%BF-1) than in normal weight women (16.9 +/- 2.2 ng/mL; 0.57 +/- 0.06 ng.mL-1.%BF-1, both P < 0.005 vs. obese), but were not different between the 31%, 23%, and 14% fat diets when body weight was stable. Plasma leptin was highly correlated with BMI (r = 0.81, P < 0.0001), %BF (r = 0.80, P < 0.0001), and fasting plasma insulin (r = 0.61, P < 0.0001). After 8 months on the ad libitum low fat diet, the women had lost an average of 6.9 +/- 1.0% of body mass (-2.0 +/- 0.3 kg/m2, P < 0.0001). In 15 subjects who lost more than 7% of body mass (-12.3 +/- 1.0%), plasma leptin concentrations decreased (-9.6 +/- 1.9 ng/mL, P < 0.0005), and the decrease of plasma leptin per change of adiposity (delta leptin/delta %BF) was greater in overweight/obese women (3.6 +/- 0.5) than in normal weight women (0.9 +/- 0.4, P < 0.01 vs. obese). In 18 other subjects who lost less than 7% of body mass (-2.7 +/- 0.6%), plasma leptin was unchanged (+1.4 +/- 1.4 ng/mL). Overall, the change of plasma leptin was significantly correlated with change of BMI (r = 0.43, P < 0.02), the change of %BF (r = 0.49, P < 0.005), the change of resting energy expenditure (r = 0.40, P < 0.02), and with the change of plasma insulin independently of changes of body adiposity (r = 0.45, P < 0.01). We conclude that plasma leptin concentrations are: 1) not affected by dietary fat content per se; 2) highly correlated with BMI, %BF, and plasma insulin in both overweight/obese and normal weight women; 3) decreased in parallel with plasma insulin after sustained weight loss; and 4) decreased more in overweight/obese than in normal weight women.

Des-(27-31)C-peptide. A novel secretory product of the rat pancreatic beta cell produced by truncation of proinsulin connecting peptide in secretory granules.

Insulin and connecting peptide (C-peptide) are produced in equimolar amounts during proinsulin conversion in the pancreatic beta cell secretory granule. To determine whether insulin and C-peptide are equally stable in beta cell granules (and thus secreted in equimolar amounts), neonatal and adult rat beta cells were pulse-chased, and radiolabeled insulin and C-peptide analyzed by high performance liquid chromatography. A novel truncated C-peptide was identified and shown by mass spectrometry to be des-(27-31)C-peptide (loss of 5 C-terminal amino acids). Des-(27-31)C-peptide is a major beta cell secretory product, accounting for 37.4 +/- 1.6% (neonatal) and 8.5 +/- 0.6% (adult) of total labeled C-peptide in secretory granules after 10 h of chase. Des-(27-31)C-peptide is also secreted in a glucose-sensitive manner from the perfused adult rat pancreas, accounting for approximately 10% of total C-peptide immunoreactivity secreted. Human C-peptide is also a substrate for truncation in granules. Thus, when human proinsulin was expressed (infection with recombinant adenovirus) in transformed (INS) rat beta cells, human des-(27-31)C-peptide was secreted along with the intact human peptide and both intact and truncated rat C-peptide. In addition to truncation, 33.1 +/- 1.2% of C-peptide in neonatal but not adult rat beta cell granules was further degraded. Such degradation was completely inhibited by ammonium chloride (known to neutralize intra-granular pH), whereas truncation was only partially inhibited by approximately 50%. In conclusion, a novel beta cell secretory product, des-(27-31)C-peptide, has been identified and should be considered as a potential bioactive peptide. Both truncation and degradation of C-peptide are responsible for non-equimolar secretion of insulin and C-peptide in rat beta cells.

Leptin concentrations in relation to overall adiposity and regional body fat distribution in Mexican Americans.

OBJECTIVE: Leptin, the product of the human OB gene is increased in obese individuals suggesting resistance to its effect. However, there is variability in leptin levels at each level of body mass index suggesting that genetic and environmental factors other than overall adiposity may regulate leptin concentrations. Moreover, the relation of leptin to various adipose depots may differ. Upper body (or central adiposity) is more metabolically active than peripheral adiposity. METHODS: We examined the relation of serum leptin levels to body fat distribution in 147 non-diabetic subjects from the San Antonio Heart Study. RESULTS: Leptin concentrations in men were significantly correlated with body mass index (BMI) (r = 0.741), waist-to-hip ratio (WHR) (r = 0.567), waist circumference (r = 0.840), hips circumference (r = 0.842) triceps skinfold (r = 0.520) and subscapular skinfold (r = 0.668) but not with subscapular to triceps skinfold (r = 0.185). Leptin concentrations in women were significantly correlated with BMI (r = 0.814), WHR (r = 0.377), waist circumference (r = 0.718), hips circumference (r = 0.779), subscapular skinfolds (r = 0.636) and triceps skinfolds (r = 0.587) but not with the ratio of subscapular to triceps skinfolds (r = 0.184) in women. CONCLUSIONS: Since the associations of leptin with body mass index (a surrogate for overall adiposity), waist circumference (a surrogate for upper body) and hips circumference (a surrogate for lower body adiposity) are similar, we conclude that leptin concentrations are associated with all adipose tissue depots and not disproportionately with upper body or central adiposity.

Pancreatic polypeptide administration improves abnormal glucose metabolism in patients with chronic pancreatitis.

Chronic pancreatitis (CP) is associated with lowered plasma levels and a blunted nutrient-induced release of pancreatic polypeptide (PP). To investigate the possible role of PP on glucose metabolism, we studied male patients with documented CP (n = 5) and obesity-matched control subjects (NL) (n = 6). Hepatic glucose production (HGP) and overall glucose disposal rates were determined by [3-3H]glucose infusion during a hyperinsulinemic-euglycemic clamp during three separate admissions. Basal rates of HGP were higher in CP patients. In response to an infusion of insulin (60 pmol.m-2.min-1), HGP fell 91 +/- 5% in NL subjects but only 68 +/- 8% in CP subjects (P < 0.05). One month later, the clamp was repeated during the final 2 h of an 8-h infusion of bovine PP (2 pmol.kg-1.min-1). HGP before the insulin infusion and its subsequent suppression (NL: 83 +/- 5%; CP: 86 +/- 15%) were nearly identical between groups. In follow-up studies 1 month after the PP infusion, HGP both basally and in response to insulin alone were similar to the first study. During oral glucose tolerance tests (OGTT) performed 18 h after the PP infusion, subjects with normal (n = 7) baseline OGTT responses showed no effect. All patients with diabetic (n = 3) or nondiagnostic (n = 1) OGTT responses, however, demonstrated lowered mean plasma glucose levels (approximately -2.3 mmol/L; range: -0.6 to -7.2 mmol/L). OGTTs repeated 1 month after the PP treatment showed a return to pretreatment responses. We conclude that chronic pancreatitis accompanied by PP deficiency is associated with partial hepatic resistance both in the basal state and in response to hyperinsulinemia. This impairment is reversed after iv PP administration. PP deficiency may therefore play a role in the development of pancreatogenic diabetes caused by pancreatic injury.

Interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals.

Transcapillary insulin transport has been considered a rate-limiting step of insulin action. However, direct measurement of interstitial insulin levels during physiologic levels of insulinemia have not been performed. We determined changes in interstitial insulin in eight healthy non-obese men and seven healthy obese men by microdialysis during a euglycemic-hyperinsulinemic clamp. Interstitial insulin was determined in the subcutaneous tissue of the abdomen and thigh. Steady-state insulin concentrations were reached approximately 10 minutes after the start of insulin infusion in the subcutaneous tissue of the abdomen and thigh and returned to basal levels approximately 10 minutes after the infusion was discontinued. There was no difference in the rapidity of change in interstitial insulin between obese and lean individuals at either site studied, irrespective of the pattern of fat distribution. The relative change in dialysate insulin concentration during the euglycemic clamp did not differ between obese and lean individuals at either site studied. It was also unaffected by the waist to hip ratio. The rapid change in interstitial insulin concentration could be of physiologic significance in determining the effects of changes in circulating insulin concentration. We conclude that transcapillary insulin transport in adipose tissue is unaffected by obesity and the pattern of fat distribution in healthy men. It is also concluded that when interstitial insulin is determined directly, transcapillary insulin transport is rapid and does not demonstrate a significant lag phase.

Leptin concentrations in diabetic and nondiabetic Mexican-Americans.

Leptin, the product of the OB gene, is increased in obese individuals, suggesting resistance to its effect. We questioned whether subjects with NIDDM have an altered regulation of serum leptin levels. We used a radioimmunoassay to measure serum leptin levels in three groups from the San Antonio Heart Study: 1) 50 Mexican-Americans with NIDDM; 2) 50 nondiabetic Mexican-Americans matched by age and sex to the diabetic Mexican-Americans; and 3) 50 nondiabetic Mexican-Americans matched by age, sex, and BMI to the diabetic Mexican-Americans. Leptin concentrations did not differ significantly by diabetic status. Leptin concentrations were significantly correlated with BMI in all groups (NIDDM women: r = 0.637; nondiabetic women: r = 0.772; NIDDM men: r = 0.849; and nondiabetic men: r = 0.686; all P < 0.001). Leptin levels were higher in women than in men regardless of diabetic status. We concluded that the leptin concentrations were not different in diabetic and nondiabetic subjects and that the association of leptin with obesity was similar in diabetic and nondiabetic subjects.

Radioimmunoassay of leptin in human plasma.

Recent studies suggest that leptin, the ob gene product absent in ob/ob mice, is a negative regulator of adiposity. We developed an RIA to measure human leptin in plasma or serum. The minimum detectable concentration by the assay is 0.5 microg/L leptin and the limit of linearity is 100 microg/L. Recovery of leptin added to serum was 99-104% over by the linear range of the assay. The RIA agreed reasonably well with rough quantification by Western blot (RIA = 0.90 blot + 3.7 microg/L, Sy/x = 10.9 microg/L). CVs within- and between-run ranged from 3.4% to 8.3% and from 3.6% to 6.2%, respectively. Variation in plasma leptin concentrations in specimens collected on consecutive mornings was large (CVs of 10.9% and 22.5%). After an overnight fast, leptin concentrations were similar to those 1-2 h after 1-2 meals. Plasma leptin concentrations in specimens from 83 lean and obese adults correlated directly with body mass index (BMI; kg/m2): r = 0.72, P <0.001. Correlations were significantly improved by separating results by gender (men r = 0.84, women r = 0.87; p <0.001). The increase in leptin concentrations with increasing BMI was greater in women than in men (slope 2.53 vs 0.97 microg/L per unit BMI, respectively). Leptin concentrations determined in lean subjects (BMI between 18 and 25) were higher in women (7.36 +/- 3.73 microg/L) than in men (3.84 +/- 1.79 microg/L) (P <0.001). Plasma leptin varied little with age and no significant difference was observed between whites and blacks. We conclude that: (a) plasma leptin concentrations are accurately and precisely measured by this new RIA; (b) leptin concentrations vary little due to short-term fasting, age, or race; but (c) plasma leptin concentrations are gender specific.

Effects of cholecystokinin and glucagon-like peptide 1 on the secretion of pancreatic polypeptide in mice.

The gut hormones, cholecystokinin (CCK) and truncated glucagon-like peptide 1 (GLP-1(7-36)amide or GLP-1) both stimulate insulin secretion and affect glucagon secretion in mice, but their effects on the secretion of other islet hormones have not been established in rodents. In the present study, we have examined the influence of the C-terminal octapeptide of CCK, CCK-8, and GLP-1 on the secretion of pancreatic polypeptide (PP) in the mouse by the use of a radioimmunoassay for rodent PP. Mice were injected intravenously with CCK-8 (doses in the range of 0.053-5.3 nmol/kg) or with GLP-1 (doses in the range of 1-32 nmol/kg) and blood was sampled at 2, 6 or 10 min after the injection. Controls were injected with saline. It was found that CCK-8 at 5.3 nmol/kg increases plasma levels of both PP and insulin when the sample was taken at 2 min, but not at 6 or 10 min, after injection. These effects were blocked by the selective CCKA-receptor antagonist, L-364,718 (2.4 micromol/kg). GPL-1 increased plasma insulin levels at 32 nmol/kg at 2 and 6 min after the injection, but plasma PP levels were unaltered. In conclusion, this study, using a newly developed radioimmunoassay for PP in rodents, shows that CCK-8 but not GPL-1 stimulated PP secretion in mice at dose levels where both peptides stimulate insulin secretion. Furthermore, PP secretion in response to CCK-8 showed a similarity with that of insulin in terms of dose- and time-response characteristics as well as sensitivity to CCKA-receptor antagonism.

Are specific serum insulin levels low in impaired glucose tolerance and type II diabetes?: measurement with a radioimmunoassay blind to proinsulin, in the population of Wadena, Minnesota.

It has been suggested that serum insulin levels in subjects with recently diagnosed type II diabetes have been overestimated, and that after correction for proinsulin, true insulin levels are depressed rather than elevated. We tested this possibility in a cross-sectional study of a population-based sample of 328 adults living in Wadena, a Minnesota community in which residents are of northern European background. Specificity of insulin measurements was provided by an antibody blind to proinsulin and its major metabolite. Oral glucose tolerance and liquid mixed-meal (Ensure-Plus) tests were performed on separate days. Mean insulin levels before and 90 minutes after the mixed meal were as follows. Among 302 randomly ascertained adults not previously known to have diabetes, both fasting and postmeal levels in subjects with impaired glucose tolerance (IGT) and newly identified type II diabetes were equal to or greater than levels in subjects with normal glucose tolerance (fasting: normal 52 pmol/L, IGT 78, new type II 87; postmeal: 317, 565, and 406, respectively). The fasting insulin to glucose ratio was significantly increased in IGT and new type II diabetes subjects. Among 26 established (previously known) type II diabetic subjects not taking insulin, fasting levels were elevated and postmeal levels were normal in absolute terms (75 and 328), but were normal or low with respect to plasma glucose. Relationships among the groups were not materially changed by adjustment for body mass index (BMI), sex, age, or blood pressure. There was marked overlap of individual insulin levels from group to group. In summary, randomly selected adults in Wadena with IGT or asymptomatic diabetes showed, on average, elevated insulin levels, but physician-diagnosed diabetes was associated with relative diminution of serum insulin. In this population, the current view of insulin resistance in "early" diabetes was supported by insulin-specific measurements.

Glucagon secretory response to hypoglycaemia, adrenaline and carbachol in streptozotocin-diabetic rats.

Glucagon response to insulin-induced hypoglycaemia is impared in diabetes, but the mechanism is not established. Pancreatic A cell hyporesponsiveness to adrenergic or cholinergic stimulation could contribute to the impairment. We therefore compared the plasma glucagon responses to intravenous infusion of adrenaline (1200 ng kg(-1) min(-1) for 20 min) or to intravenous injection of the cholinergic agonist carbachol (50 micrograms kg(-1)) in chloral hydrate-anaesthetized rats made diabetic with the use of streptozotocin (80 mg kg(-1) subcutaneously) 6 weeks before and in anaesthetized control rats. Insulin was infused intravenously to reduce plasma glucose levels to below 1.8 mmol L(-1). As expected, the plasma glucagon response was reduced by approximately 45% in streptozotocin-diabetic rats compared with controls (P = 0.045). During adrenaline infusion, plasma glucagon levels increased by 277 +/- 92 pg mL(-1) in controls (P = 0.009) and by 570 +/- 137 pg mL(-1) in the diabetic rats (P = 0.002). Thus, the plasma glucagon response to adrenaline was approximately doubled in the diabetic rats (P = 0.045). Following carbachol injection, plasma glucagon levels were raised by 1211 +/- 208 pg mL(-1) (P < 0.001) in controls but only by 555 +/- 242 pg mL(-1) in the diabetic rats (P = 0.049). Thus, the plasma glucagon response to carbachol was impared by approximately 58% in the diabetic rats (P = 0.028). We conclude that carbachol-stimulated glucagon secretion is impared concomitantly with the impared glucagon response to hypoglycaemia in streptozotocin-diabetic rats, whereas adrenaline-induced glucagon secretion is exaggerated. We suggest that a reduced pancreatic A cell responsiveness to cholinergic stimulation could contribute to the impairment of the glucagon response to insulin-induced hypoglycaemia in diabetes.

Parathyroid hormone decreases in vivo insulin effect on glucose utilization.

Hyperparathyroidism is associated with impaired glucose tolerance, and parathyroidectomy may improve carbohydrate homeostasis. It has been suggested that parathyroid hormone (PTH) suppresses insulin secretion but it is unclear whether it also interferes with the peripheral action of insulin. To evaluate in vivo effects of PTH on insulin-mediated glucose utilization, 15 male Sprague Dawley rats were continuously infused with rat PTH (1-34) using an Alzet miniosmotic pump at a rate of 0.03 nm/hour. Controls were infused with the vehicle alone. Following 5 days of PTH infusion, plasma calcium (Ca) levels were higher in the PTH-infused rats (12.3 +/- 0.2 versus 9.9 +/- 0.1 mg/dl, P < 0.01). On the 5th day, glucose (700 mg/kg) and insulin (0.175 U/kg) were given as a bolus infusion through the left femoral vein, blood samples were obtained from the right femoral vein, and plasma glucose and insulin were measured at basal (0 minutes) and at 2, 5, 10, and 20 minutes postinfusion. Basal, nonfasting glucose levels were higher (166 +/- 4 versus 155 +/- 4 mg/dL, P < 0.04) in the PTH-infused rats but their insulin levels were similar to those of controls (6.5 +/- 0.6 versus 5.6 +/- 0.5 ng/ml). Postinfusions and maximal (2 minutes) glucose and insulin levels were similar in both groups. However, although insulin levels were similar in both groups at all measured time points, glucose levels at 20 minutes were higher in the PTH-treated rats (205 +/- 13 versus 173 +/- 9; P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)