The neuropeptide PACAP contributes to the glucagon response to insulin-induced hypoglycaemia in mice

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide in the autonomic nerves innervating the pancreatic islets and previous studies have shown that it stimulates insulin and glucagon secretion. It is known that autonomic nerve activation contributes to the glucagon response to hypoglycaemia. In the present study, we evaluated whether PACAP is involved in this glucagon response by examining the glucagon response to insulin-induced hypoglycaemia in mice genetically deleted of the specific PACAP receptor, the PAC1 receptor. We found that insulin (1 U kg-1 ip) reduced circulating glucose to a hypoglycaemic level of approximately 2.5 mmol L-1 in PAC1R-/- mice and their wild-type counterparts with no difference between the groups. However, the glucagon response to this hypoglycaemia was markedly impaired in the PAC1R-/- mice. Thus, after 120 min, plasma glucagon was 437 +/- 79 ng L-1 in wild-type mice vs. only 140 +/- 36 ng L-1 in PAC1R-/- mice (P=0.004). In contrast, the glucagon response to intravenously administered arginine (0.25 g kg-1) was the same in the two groups of mice. We conclude that PACAP through activation of PAC1 receptors contribute to the glucagon response to insulin-induced hypoglycaemia. Therefore, the glucagon response to hypoglycaemia is dependent not only on the classical neurotransmitters but also on the neuropeptide PACAP.     

Pituitary adenylate cyclase‐activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice

Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase‐activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9‐week‐old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293–1299, 2016. © 2016 Wiley Periodicals, Inc.     

Glucagon secretory response to hypoglycaemia,adrenaline and carbachol in streptozotocindiabetic rats

Glucagon response to insulin-induced hypoglycaemia is impaired 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 μ 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 ～ 45% in streptozotocindiabetic 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 impaired by ～ 58% in the diabetic rats (P= 0.028). We conclude that carbachol-stimulated glucagon secretion is impaired concomitantly with the impaired 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 insulininduced hypoglycaemia in diabetes.     

Pancreatic islet blood flow during euglycaemic, hyperinsulinaemic clamp in anaesthetized rats

Aims: Previous studies have demonstrated that pancreatic islet blood flow is crucially dependent on blood glucose concentration. Thus, hyperglycaemia increases and hypoglycaemia decreases islet blood perfusion, by a combination of nervous and metabolic signals. The aim of the present study was to evaluate if hyperinsulinaemia, without associated hypoglycaemia, affects islet blood flow. Methods: Thiobutabarbital‐anaesthetized Wistar–Furth rats were subjected to an euglycaemic, hyperinsulinaemic clamp, that is they were infused for 60 min with either saline, insulin (18 mU kg^?1 min^?1), glucose (27 mg kg^?1 min^?1) or both glucose and insulin. This was followed by islet blood flow measurements with a microsphere technique. Results: Animals receiving only glucose doubled their blood glucose and serum insulin concentrations, whereas rats receiving only insulin had blood glucose concentrations <2 mmol L^?1 and a 10‐fold increase in serum insulin concentrations. Animals given simultaneous glucose and insulin had normal blood glucose concentrations but a 10‐fold increase in serum insulin concentrations. Total pancreatic blood flow was unaffected in all animals. Islet blood flow was increased in hyperglycaemic and decreased in hypoglycaemic rats compared with control rats. Islet blood flow did not differ between clamped and control rats. Conclusions: Serum insulin concentration per se does not affect islet blood flow, whereas the ambient blood glucose concentration is of major importance in this context.     

PAC1 deficiency attenuates progression of atherosclerosis in ApoE deficient mice under cholesterol-enriched diet

The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is vasoactive and cytoprotective and exerts immunoregulatory functions throughout the nervous, neuroendocrine cardiovascular and immune systems in health and disease. PACAP mainly acts through PAC1 receptor signaling in neuronal communication, but the role of PAC1 in immune regulation of atherosclerosis is not known. Here, we generated PAC1^−/−/ApoE^−/− mice to test, whether PAC1^−/− influences plasma cholesterol-/triglyceride levels and/or atherogenesis in the brachiocephalic trunk (BT) seen in ApoE^−/− mice, under standard chow (SC) or cholesterol-enriched diet (CED). Furthermore, the effect of PAC1^−/−, on inflammatory, autophagy-, apoptosis- and necroptosis-relevant proteins in atherosclerotic plaques was determined. In plaques of PAC1^−/−/ApoE^−/− mice fed a SC, the immunoreactivity for apoptotic, autophagic, necroptotic and proinflammatory proteins was increased, however, proliferation was unaffected. Interestingly, without affecting hyperlipidemia, PAC1^−/− in ApoE^−/− mice remarkably reduced CED-induced lumen stenosis seen in ApoE^−/− mice. Thus, PAC1^−/− allows unchecked inflammation, necroptosis and decreased proliferation during SC, apparently priming the BT to develop reduced atheroma under subsequent CED. Remarkably, no differences in inflammation/necroptosis signatures in the atheroma under CED between PAC1^−/−/ApoE^−/− and ApoE^−/− mice were observed. These data indicate that selective PAC1 antagonists should offer potential as a novel class of atheroprotective therapeutics, especially during hypercholesterolemia.     

Reducing plasma free fatty acids by acipimox improves glucose tolerance in high‐fat fed mice

To study whether free fatty acids (FFAs) contribute to glucose intolerance in high‐fat fed mice, the derivative of nicotinic acid, acipimox, which inhibits lipolysis, was administered intraperitoneally (50 mg kg^?1) to C57BL/6J mice which had been on a high‐fat diet for 3 months. Four hours after administration of acipimox, plasma FFA levels were reduced to 0.46 ± 0.06 mmol L^?1 compared with 0.88 ± 0.10 mmol L^?1 in controls (P < 0.001). At this point, the glucose elimination rate after an intravenous glucose load (1 g kg^?1) was markedly improved. Thus, the elimination constant (K_G) for the glucose disposal between 1 and 50 min after the glucose challenge was increased from 0.54 ± 0.01% min^?1 in controls to 0.66 ± 0.01% min^?1 by acipimox (P < 0.001). In contrast, the acute insulin response to glucose (1–5 min) was not significantly different between the groups, although the area under the insulin for the entire 50‐min period after glucose administration was significantly reduced by acipimox from 32.1 ± 2.9 to 23.9 ± 1.2 nmol L^?1 × 50 min (P=0.036). This, however, was mainly because of lower insulin levels at 20 and 50 min because of the lowered glucose levels. In contrast, administration of acipimox to mice fed a normal diet did not affect plasma levels of FFA or the glucose elimination or insulin levels after the glucose load. It is concluded that reducing FFA levels by acipimox in glucose intolerant high‐fat fed mice improves glucose tolerance mainly by improving insulin sensitivity making the ambient islet function adequate, suggesting that increased FFA levels are of pathophysiological importance in this model of glucose intolerance.     

Nervous control of pancreatic endocrine secretion in pigs IV. The effect of somatostatin on the insulin and glucagon responses to electrical vagal stimulation and to intraarterial acetylcholine

We studied the effect of a primed i.v. infusion of somatostatin (0.5 μg×min^–1×kg^–1 on the glucose dependent insulin and glucagon responses to electrical stimulation of the vagus nerves or to i.a. acetylcholine in anesthetized pigs. Somatostatin completely abolished the insulin and glucagon responses to ongoing vagal stimulation; after 70 min somatostatin infusion the response to reiterated stimulation was profoundly inhibited. After termination of the somatostatin infusion, a considerable rebound secretion of insulin and glucagon was noted. By contrast, the endocrine response to acetylcholine persisted in spite of the somatostatin administration. Blood glucose increased slightly during somatostatin infusion. The results suggest that somatostatin inhibits the responses to vagal stimulation by interference with the neural transmission to the pancreatic islets rather than by inhibition of the islet cells themselves; acetylcholine may be involved in this neural transmission (acting on nicotinic receptors.     

Dose-response effects of free fatty acids on amino acid metabolism and ureagenesis

Aim: Free fatty acids (FFAs) are important fuels and have vital protein‐sparing effects, particularly during conditions of metabolic stress and fasting. However, it is uncertain whether these beneficial effects are evident throughout the physiological range or only occur at very high FFA concentrations. It is also unclear whether secondary alterations in hormone levels and ketogenesis play a role. We therefore aimed at describing dose–response relationships between amino acid metabolism and circulating FFA concentrations at clamped hormone levels. Methods: Eight healthy men were studied on four occasions (6 h basal, 2 h glucose clamp). Endogenous lipolysis was blocked with acipimox and Intralipid was infused at varying rates (0, 3, 6 or 12 μL kg^?1 min^?1) to obtain four different levels of circulating FFAs. Endogenous growth hormone, insulin and glucagon secretion was blocked by somatostatin (300 μg h^?1) and replaced exogenously. 15N‐phenylalanine, 2H4‐tyrosine and 13C‐urea were infused continuously to assess protein turnover and ureagenesis. Results: We obtained four distinct levels of FFA concentrations ranging from 0.03 to 2.1 mmol L^?1 and 3‐hydroxybutyrate concentrations from 10 to 360 μmol L^?1. Whole‐body phenylalanine turnover and phenylalanine‐to‐tyrosine degradation decreased with increasing FFA levels as did insulin‐stimulated forearm fluxes of phenylalanine. Phenylalanine, tyrosine and urea concentrations also decreased progressively, whereas urea turnover was unperturbed. Conclusion: Circulating FFAs decrease amino acid concentrations and inhibit whole‐body phenylalanine fluxes and phenylalanine‐to‐tyrosine conversion. Our data cover FFA concentrations from 0 to 2 mmol L^?1 and indicate that FFAs exert their protein conserving effects in the upper physiological range (>1.5 mmol L^?1).     

Mice deficient in pituitary adenylate cyclase activating polypeptide display increased sensitivity to renal oxidative stress in vitro

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide, showing widespread occurrence in the nervous system and also in peripheral organs. The neuroprotective effects of PACAP are well-established in different neuronal systems against noxious stimuli in vitro and in vivo. Recently, its general cytoprotective actions have been recognized, including renoprotective effects. However, the effect of endogenous PACAP in the kidneys is not known. The main aim of the present study was to investigate whether the lack of this endogenous neuropeptide influences survival of kidney cells against oxidative stress. First, we determined the presence of endogenous PACAP from mouse kidney homogenates by mass spectrometry and PACAP-like immunoreactivity by radioimmunoassay. Second, primary cultures were isolated from wild type and PACAP deficient mice and cell viability was assessed following oxidative stress induced by 0.5, 1.5 and 3 mM H₂O₂. Our mass spectrometry and radioimmunoassay results show that PACAP is endogenously present in the kidney. The main part of our study revealed that the sensitivity of cells from PACAP deficient mice was increased to oxidative stress: both after 2 or 4 h of exposure, cell viability was significantly reduced compared to that from control wild type mice. This increased sensitivity of kidneys from PACAP deficient mice could be counteracted by exogenously given PACAP38. These results show, for the first time, that endogenous PACAP protects against oxidative stress in the kidney, and that PACAP may act as a stress sensor in renal cells. These findings further support the general cytoprotective nature of this neuropeptide.     

Plasma levels of oxytocin after food deprivation and hypoglycaemia,and effects of 1–deamino-2–D-Tyr—(OEt)-4–Thr-8–Orn-oxytocin on blood glucose in rats

Björkstrand , E., Eriksson , M. & Uvnäs -Moberg , K. 1992. Plasma levels of oxytocin after food deprivation and hypoglycaemia, and effects of l-deamino-2–D-Tyr-(OEt)-l-Thr-8–Om-oxytocin on blood glucose in rats. Acta Physiol Scand 144 , 355–359. Received 1 March 1 991 , accepted 25 October 1991. ISSN 0001–6772. Department of Pharmacology, Karolinska Institute, Sweden. Oxytocin has been shown to influence insulin, glucagon and blood glucose levels in various experimental situations. The present study was performed in order to obtain support for a possible interaction of glucose and oxytocin under physiological conditions. We therefore studied whether or not short-term food deprivation (24 hours) affects basal oxytocin levels in male, female and lactating rats, since this is a situation when glucose is mobilized to prevent hypoglycaemia. Secondly, we studied whether oxytocin levels rise in a situation when blood glucose levels fall, i.e. following i.p. injection of insulin (20 U kg^-1). In order to explore the role of oxytocin more directly, we investigated whether i.p. injection of the oxytocin antagonist 1–deamino-2–D-Tyr-(OEt)-4–Thr-8–Orn-oxytocin affects blood glucose levels. Plasma levels of oxytocin, insulin and glucagon were measured with radioimmunoassay in samples obtained after decapitation. We found that oxytocin levels were significantly increased following short-term food deprivation in lactating rats. We also found that insulin-induced hypoglycaemia could elevate plasma levels of oxytocin in female and male rats. In addition, administration of an oxytocin antagonist cause a small, hut significant decrease in blood glucose levels after 30 min. These data imply that oxytocin may he one of several factors that take part in the control of blood glucose regulation.     

The role of the autonomic innervation in the control of glucagon release during hypoglycaemia in the calf

1. The extent to which the autonomic innervation to the pancreas is implicated in the control of glucagon release during hypoglycaemia has been investigated in calves 3-6 weeks after birth.2. A pronounced rise in plasma glucagon concentration occurred in normal conscious calves in response to hypoglycaemia following administration of insulin (0.1 u./kg). Prior treatment with atropine caused no significant change in the hypoglycaemic response to insulin in these animals but the rise in plasma glucagon concentration was delayed.3. Section of both splanchnic nerves produced no significant change in the tolerance of conscious calves to this small dose of insulin and the changes in plasma glucagon concentration in these animals were within the normal range.4. In contrast, the same dose of insulin produced severe hypoglycaemia, accompanied by convulsions, in atropinized calves with cut splanchnic nerves. In spite of the intensity of the hypoglycaemic stimulus the rise in plasma glucagon concentration was both delayed and diminished in these animals.5. Administration of atropine alone (0.2 mg/kg) to normal fasting calves produced a significant fall in the mean plasma concentrations of both glucose and glucagon (P < 0.01) within 30 min, without affecting that of insulin.6. A significant increase in plasma glucagon concentration also occurred in response to stimulation of the peripheral ends of the thoracic vagi in adrenalectomized calves with cut splanchnic nerves under barbiturate anaesthesia. A rise in mean plasma glucose concentration was also observed in these experiments and found to be significantly correlated with the glucagon response.7. It is concluded that changes in either sympathetic or parasympathetic efferent activity may modify plasma glucagon concentration in the conscious calf, but that only the latter mechanism is likely to be implicated in the response to changes in plasma glucose concentration within the physiological range.     

Effect of C-peptide administration on whole body glucose utilization in STZ-induced diabetic rats

Recent studies suggest that C-peptide stimulates glucose transport in isolated skeletal muscle. In order to determine the effect of C-peptide on whole body glucose utilization, streptozotocin (60 mg kg^-1) (STZ)-induced diabetic and normal rats were studied using the euglycaemic clamp procedure and continuous infusion of somatostatin (1.0 μg kg^-1 min^-1) in pentobarbital-anaesthetized rats. Plasma insulin levels during the 6.0- and 30.0-mU kg^-1 min^-1 insulin infusions rose to 70–90 μU mL^-1 and 500–700 μU mL^-1, respectively. Blood glucose concentrations were clamped at 7.5–7.9 mmol L^-1 in the diabetic rats and at basal levels or 7.7 mmol L^-1 in the non-diabetic (normal) rats. Biosynthetic human C-peptide (0.5 nmol kg^-1 min^-1) was infused in 12 diabetic and 11 normal rats, resulting in concentrations of 26–41 nmol L^-1. The metabolic clearance rate of glucose (MCR) for the diabetic rats receiving C-peptide (12.0±1.0 mL kg^-1 min^-1) was significantly (P<0.01) higher than that in the diabetic rats given saline (6.3±0.7 mL kg^-1 min^-1) or a randomly scrambled C-peptide (7.8±1.3 mL kg^-1 min^-1) at low-dose insulin infusion but not at the high-dose insulin infusion. In normal rats C-peptide did not significantly increase the MCR for glucose. These results thus demonstrate that C-peptide has the capacity to increase glucose utilization in STZ-induced diabetic rats.     

Induction of hypoglycaemia in Japanese encephalitis virus infection: the role of T lymphocytes

We report here development of hypoglycaemia in the convalescent phase of Japanese encephalitis virus (JEV) infection in mice by the induction of antigen-specific Ly1⁻2⁺ T cells in the spleen which mediate hypoglycaemia through the generation of soluble T cell hypoglycaemic factor (TCHF). The TCHF acted in a dose-dependent manner and was found to be trypsin-sensitive and thermolabile. It was purified on Superose-12 high performance liquid chromatography (HPLC) gel filtration column and purified protein migrated as a ~25-kD band on SDS–PAGE. The JEV-induced hypoglycaemia coincided with an increased circulating glucagon level, without any alterations in blood insulin and growth hormone concentrations. These effects were mimicked by TCHF. These results indicate that JEV-primed T lymphocytes mediate hypoglycaemia through the production of a soluble hypoglycaemic factor.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the brain

Recent progress in research on pituitary adenylate-activating polypeptide (PACAP) with a special emphasis on the brain is reviewed. PACAP is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide family. PACAP functions as a hypothalamic hormone, neurotransmitter, neuromodulator, and neurotrophic factor. Studies on the gene encoding the PACAP precursor and the specific PACAP receptor (PAC1-R) and its subtypes have provided information on the control of gene expression for PACAP, and the relationship between the receptor subtypes and the signal transduction pathways. The PAC1-R is a G protein-coupled receptor with seven transmembrane domains and belongs to the VIP receptor family. At least eight subtypes of PAC1-R result from alternate splicing. Each subtype is coupled to specific signaling pathways, and its expression is tissue or cell specific. PACAP stimulates the release of arginine vasopressin and increases cytosolic Ca2+ ([Ca2+]i). PACAP serves as a neurotransmitter and/or neuromodulator and the activation of the PAC1-R stimulates a cAMP-protein kinase A signal transduction pathway which in turn evokes the [Ca2+]i signaling system. More importantly, PACAP is a neurotrophic factor that may play an important role during the development of the brain. The PAC1-R is actively expressed in different neuroepithelia from early developmental stages and expressed in various brain regions during prenatal and postnatal development. In the adult brain, PACAP appears to function as a neuroprotective factor that attenuates the neuronal damage resulting from various insults.     

Nervous control of pancreatic endocrine secretion in pigs

The increases in the concentrations of insulin and pancreatic glucagon in portal venous and arterial plasma in response to electrical stimulation of the vagus nerves were studied in anesthetized splanchnicotomized young pigs. The responses were frequence dependent; threshold frequency was below 1 Hz and maximum response was reached at 8–12 Hz. With maximal stimulation responses of magnitudes comparable to the responses to maximal arginine (glucagon) and glucose stimulation (insulin) were observed. However, both the insulin and the glucagon response were critically dependent on the blood glucose concentration during the stimulation: the glucagon response was inversely correlated to blood glucose, whereas the insulin response was positively correlated to blood glucose at concentrations above 4.5 mmol · 1^-1. Below this glucose concentration there was no detectable insulin response and above 8.0 mmol ·^-1 no glucagon response to vagal stimulation. A stimulated secretion of glucagon as well as insulin was maintained for up to 30 min stimulation, but insulin secretion tended to decrease, whereas glucagon secretion tended to increase. Above blood glucose concentrations of 4 mmol · 1^-1, blood glucose concentrations increased slightly in response to vagal stimulation, whereas no change was noted during stimulations performed at lower blood glucose concentrations.     

Glucagon-like peptide-1 reduces hepatic glucose production indirectly through insulin and glucagon in humans

The effect of glucagon-like peptide-1 (GLP-1) on hepatic glucose production and peripheral glucose utilization was investigated with or without infusion of somatostatin to inhibit insulin and glucagon secretion in 13 healthy, non-diabetic women aged 59 years. After 120 min 3-³H-glucose infusion, GLP-1 was added (4.5 pmol kg^?1 bolus + 1.5 pmol kg^?1 min^?1). Without somatostatin (n = 6), GLP-1 decreased plasma glucose (from 4.8 ± 0.2 to 4.2 ± 0.3 mmol L^?1, P = 0.007). Insulin levels were increased (48 ± 3 vs. 243 ± 67 pmol L^?1, P = 0.032), as was the insulin to glucagon ratio (P = 0.044). The rate of glucose appearance (R_a) was decreased (P = 0.003) and the metabolic clearance rate of glucose (MCR) was increased during the GLP-1 infusion (P = 0.024 vs. saline). Also, the rate of glucose disappearance (R_d) was reduced during the GLP-1 infusion (P = 0.004). Since R_a was reduced more than R_d, the net glucose flow was negative, which reduced plasma glucose. Somatostatin infusion (500 μg h^?1, n = 7) abolished the effects of GLP-1 on plasma glucose, serum insulin, insulin to glucagon ratio, R_a, R_d, MCR and net glucose flow. The results suggest that GLP-1 reduces plasma glucose levels mainly by reducing hepatic glucose production and increasing the metabolic clearance rate of glucose through indirectly increasing the insulin to glucagon ratio in healthy subjects.     

The effects of long-term hyperinsulinaemia on insulin sensitivity in rats

The effects of long-term exposure (7 wk) to hyperinsulinaemia on insulin sensitivity were studied in female rats. The rats were made hyperinsulinaemic by implantation of osmotic minipumps that were changed once a week. Elevated adrenergic activity and secretion of glucocorticoids were controlled by another minipump with propranolol and adrenalectomy with corticosterone substitution, respectively. This resulted in hyperinsulinaemia and moderate hypoglycaemia, the latter probably counteracted by overeating and increased glucagon secretion, as indicated by increased body weight and lower liver glycogen contents, respectively. Euglycaemic, hyperinsulinaemic clamp measurements showed a significantly higher glucose disposal rate (P < 0.05) in the hyperinsulinaemic rats 18.8± 1.1 mg kg^-1 min^-1 compared with the control groups 14.6±0.4 and 15.4±0.9 mg kg^-1 min^-1. Insulin stimulation of 2-deoxyglucose as well as glycogen synthesis was measured in the extensor digitorum longus muscle, the red and white part of the gastrocnemius, the soleus muscle, the liver and in parametrial, retroperitoneal, and inguinal adipose tissue. No differences were found between the groups in the insulin response of the 2-deoxyglucose uptake. Glycogen synthesis was significantly elevated in all muscles in the insulin treated compared with the control rats but no differences were found in the liver. Capillary density was significantly elevated per unit muscle surface area in the soleus and extensor digitorum longus muscles of the insulin-exposed rats. These results suggest that long-term exposure to insulin is followed by increased insulin sensitivity, apparently localized to the insulin regulation of glycogen synthesis in muscles. Muscle capillary density is elevated in parallel.     

The role of nitric oxide in mediating pancreatic endocrine responses to insulin-induced hypoglycaemia in the conscious calf

The role of nitric oxide (NO) in mediating pancreatic endocrine responses to moderate hypoglycaemia has been investigated in conscious unrestrained calves. The synthesis of endogenous NO was inhibited by the administration of N-nitro-L-arginine methyl ester (L-NAME; 100 mg kg-1 I.A.), while sodium nitroprusside was infused continuously (2-4 microg min-1 kg-1 I.V.) to mimic the tonic production of NO. This effectively abolished the rise in plasma pancreatic polypeptide (PP) concentration during moderate hypoglycaemia (0.7 nmol kg-1 insulin I.V.) and significantly reduced the response to more intense hypoglycaemia (2.0 nmol kg-1 insulin I. V.). In contrast, the glucagon response was not significantly affected in either group, although consistently higher plasma glucagon values were obtained in response to the higher dose of insulin following the administration of L-NAME. It is concluded that, in the absence of L-NAME, production of NO contributes to the PP response, but not the glucagon response to hypoglycaemia in this species under physiological conditions.