Effect of glucagon and glomerulopressin on the renal function of the dog.

Glucagon was infused through the porta or through the left renal artery in dogs. Another group of dogs were infused with glomerulopressin through the left renal artery. It was observed that glucagon when infused through the portal vein enhanced the glomerulopressin production and the glomerular filtration rate (GFR). When glucagon was infused intrarenally it did not alter GRF but it had a direct tubular action decreasing sodium reabsorption in the proximal tubule. Glomerulopressin infused intrarenally increased GRF and potassium excretion. The results suggest that the increase in GFR was due to increase in glomerulopressin activity. There are three reasons for this statement: a) GRF increased when glomerulopressin activity was high, but not when there was a low activity, 5) intrarenally infused glomerulopressin produced a very significant change in the GFR of the infused kidney, while the GRF of the contralateral kidney remained unchanged and c) intrarenally administered glucagon had no effect on GFR.     

Response of extrapancreatic immunoreactive glucagon to intraluminal nutrients in pancreatectomized dogs

To investigate the response of extrapancreatic glucagon to intraluminal stimuli, nutrients were administered to normal and pancreatectomized dogs through a stomach tube in a fully conscious state after an overnight fast. Plasma immunoreactive glucagon was determined with antisera specific and nonspecific to glucagon and was designated as IRG and total IRG, respectively. Oral glucose load elicited a decrease in plasma IRG and a remarkable rise of plasma total IRG in a group of 6 pancreatectomized dogs, as in the control dogs. When arginine was given, both plasma IRG and total IRG significantly increased in a group of 5 pancreatectomized dogs, while only total IRG rose significantly in the normal control dogs. Butter load did not reveal any changes in plasma IRG and total IRG in a group of 5 pancreatectomized dogs, whereas only total IRG increased in the normal control dogs. It is concluded that extrapancreatic glucagon responds to intraluminal administration of nutrients, as pancreatic glucagon does. In addition, gut glucagon-like immunoreactivity increased following glucose or arginine ingestion in pancreatectomized dogs. Furthermore, the failure in response of plasma IRG and total IRG to butter load in pancreatectomized animals suggests that its intraluminal hydrolysis is important in the secretion of extrapancreatic immunoreactive glucagon.     

Kaluresis independent K-homeostasis: glucagon and B receptor blockade in pancreatectomized dogs

Seventy minutes post pancreatectomy, in dogs that are K loaded - made abruptly hyperkalemic and "life threatened" - by infusion with 2 mEgKC1/kg-/hr until prelethal ECG changes of hyperkalemic cardiotoxicity appear, a kaluresis independent K homeostatic mechanism transfers about 2/3 of administered K to intracellular fluid. Treatment of K loaded pancreatectomized dogs with glucagon or a B receptor blockading dosage of propranolol does not alter the proportion transferred, but treatment with glucagon and propranolol reduces it. It appears that in pancx dogs there is a reciprocal relation between insulin and B receptor mediated K transfer and that glucagon is involved in activity of the kaluresis independent K homeostatic mechanism.     

Oxytocin increases extrapancreatic glucagon secretion and glucose production in pancreatectomized dogs

Infusion of oxytocin into normal dogs increases plasma levels of insulin and glucagon and glucose production and uptake. To determine whether infused oxytocin also increases glucagon secretion from extrapancreatic sites, pancreatectomized dogs, off insulin for 18 hr, were infused with oxytocin and plasma glucagon, and glucose production and uptake were measured using the [6-3H]glucose primer-infusion technique. The diabetic dogs, in the control period, had elevated plasma glucose and glucagon levels, an increased rate of glucose production, and a relative decrease in glucose uptake (decreased clearance). Infusion of oxytocin (500 microU/kg/min) caused a rise in plasma glucagon and glucose levels, increased glucose production, and further decreased glucose clearance. It is concluded that oxytocin can stimulate secretion of extrapancreatic glucagon, which contributes to the increased glucose production.     

Physiological relationships between growth hormone level, glycemia and metabolic control in dogs

This study was undertaken to explore the physiological relationships between fasting glycemia, antecedent glycemic control and fasting growth hormone levels in pancreatectomized dogs. In contrast to other studies, we used continuous intravenous infusions of insulin in an attempt not only to normalize fasting plasma glycemia but also to eliminate the characteristic fluctuations of diabetes usually encountered in the postprandial and postabsorptive periods. For comparison, a similar group of healthy animals served as normal controls. In the healthy dogs, fasting growth hormone (GH) levels were stable and well within normal limits for this species, demonstrating an overall mean +/- SD of 2.50 +/- 0.46 ng/ml. In the pancreatectomized group as a whole, the fasting GH levels were significantly elevated (4.63 +/- 2.42 ng/ml, P less than 0.01) and significantly (P less than 0.001) more variable than in the controls. Multiple regression and analysis of variance confirmed the expected significant positive correlation between fasting GH and fasting plasma glucose levels, but also elucidated a heretofore unknown direct relationship between fasting GH levels and the preceding instability of glycemic control.     

The effect of insulin-induced hypoglycaemia on the secretion of glucagon and hepatic glucose production in pancreatectomized dogs

The concentration of plasma glucose in insulin deprived pancreatectomized dogs was decreased from the basal 385 +/- 44 to 65 +/- 12 mg/dL by the infusion of 7 mU X kg-1 X min-1 insulin. During the infusion, the plasma concentration of immunoreactive glucagon (IRG) did not change and hepatic glucose production was decreased. This is in contrast to earlier findings in alloxan diabetic dogs in which plasma IRG decreased in hypoglycaemia. The hypothesis is put forward that, in contrast to pancreatic alpha cells in which the effect of insulin prevails, neither insulin nor a decrease in the ambient concentration of glucose exerts any effect on the secretion of glucagon from extrapancreatic alpha cells.     

Effect of glomerulopressin on ovarian blood flow in dogs.

The present study showed that glomerulopressin decreased ovarian blood flow in normal dogs and that this effect could be inhibited by treatment with indomethacin. In addition diabetic dogs had a high plasma level of glomerulopressin and a low ovarian blood flow that was shown to increase after treatment with indomethacin. This suggests that the low ovarian blood flow in diabetis might be due to a prostaglandin synthesizing effect of glomerulopressin.     

Enhanced glomerulopressin production and glomerular filtration rate by amino acid infusion in normal humans

Amino acid infusion induces a rise in glomerular filtration rate (GFR) in normal subjects, but the mechanism is as yet unknown. Glomerulopressin infused into the renal arteries of rats and dogs increases GFR. The aim of this study was to ascertain whether amino acid infusion raised glomerulopressin production and GFR. Accordingly, before renal arteriovenography, in 11 potential kidney donors, the caval catheter was introduced into the right hepatic vein and 60-ml blood samples were collected at the beginning and end of each experiment; six patients received amino acid infusion and five a saline infusion. Glomerulopressin in ultrafiltrates from hepatic vein plasma was measured by toad bioassay and GFR determined with diethylenetriamine pentaacetic acid-Tc99. The amino acid-infused group showed significant glomerulopressin activity in ultrafiltrates, as well as a significant GFR increase, whereas in the control group no glomerulopressin activity was observed, and there was no change in GFR. These findings suggest that intravenous amino acid infusion stimulates glomerulopressin production, which may in turn induce an increase in GFR.     

Response of gastric inhibitory polypeptide (GIP) to oral administration of nutrients in normal and pancreatectomized dogs

In order to clarify the response of plasma gastric inhibitory polypeptide (GIP) to various nutrients and to investigate the relationship between the pancreas and GIP secretion, an experimental study was performed using normal and pancreatectomized dogs. Oral administration of glucose (2 g/kg) or butter (2 g/kg) resulted in an increase of plasma GIP in five normal dogs. In contrast, oral administration of arginine (1 g/kg) did not produce any discernible changes in plasma GIP in normal dogs. In a group of nine pancreatectomized dogs, the fasting level of plasma GIP did not differ from that of the control group. Furthermore, glucose ingestion in the pancreatectomized group resulted in the same pattern and the same degree of change in plasma GIP as it did in the normal controls. In contrast, plasma GIP did not change at all following fat loading in the pancreatectomized group. However, butter with pancreatic enzymes elicited a significant rise of plasma GIP in the pancreatectomized dogs. The present study indicates that plasma GIP increases following oral administration of glucose or fat but not arginine. Furthermore, it is demonstrated that GIP secretion following fat ingestion occurs only after fat digestion by pancreatic enzymes. In addition, the findings observed in the present study do not support the existence of feedback effect of insulin on GIP secretion.     

Induction of glomerulopressin production by cyclic AMP

Isolated rat livers were perfused with gassed Krebs-Ringer-Bicarbonate and different doses of theophylline and dibutyryl cyclic AMP were added to the perfusing solution. The perfusates were ultrafiltrated through Diaflo UM-05 membranes. The glomerulopressin activity of the ultrafiltrates were assayed in the tonic tension contraction (TTC) of isolated stomach fundus from rats. As glomerulopressin is known to be a glucuronide, it was inactivated with beta-glucuronidase to confirm that the effect on the stomach fundus was due to the glomerulopressin and not to another substance. It was observed that doses of theophylline between 2 x 10(-3) M and 2 x 10(-5) M enhanced glomerulopressin production. However, there was no relationship between dose of theophylline and the response, and a dose of theophylline 2 x 10(-6) M has no activity. The perfusion with dibutyryl cyclic AMP at 5 x 10(-8) M increased the amount of glomerulopressin produced by the liver. This was a log-dose response of glomerulopressin production to dibutyryl cyclic AMP between 5 x 10(-8) M and 5 x 10(-4) M. Theophylline (2 x 10(-6) M) potentiated the activity of cyclic AMP (5 x 10(-8) M). These results support the view that cyclic AMP is intracellular mediator of the hepatic production of glomerulopressin.     

Effect of bombesin upon plasma somatostatin-like immunoreactivity, insulin and glucagon in normal and chemically sympathectomized dogs

The present study was designed to determine the effects of intravenously infused bombesin (10 ng/kg/min) upon basal and postprandial plasma somatostatin-like immunoreactivity (SLI), glucagon, insulin and triglyceride levels in normal (n = 12) and chemically sympathectomized (n = 11) dogs. Basal plasma SLI, glucagon and insulin levels rose significantly during the infusion of bombesin in the normal dogs, and this was not altered by chemical sympathectomy. Bombesin infusion enhanced the postprandial SLI response, while attenuating the postprandial glucagon response by 50% and the insulin response in the early postprandial phase of the meal. Sympathectomy did not significantly alter the basal levels of these polypeptides, but augmented the postprandial plasma SLI response during the first 90 min, and reduced the postprandial glucagon response during the infusion of bombesin. The postprandial insulin response was not affected by sympathectomy. In both normal and chemically sympathectomized dogs the rise in postprandial triglyceride levels was attenuated by bombesin infusion.     

Minimal requirement of the remnant pancreas for protein and DNA synthesis of cultured hepatocytes prepared from pancreatectomized rats

The incorporation of 3H-thymidine and 3H-leucine into the hepatocytes was studied, using cultured hepatocytes prepared from normal and pancreatectomized rats. (1) In the cultured hepatocytes prepared from 80% pancreatectomized rats, the incorporation of 3H-thymidine and 3H-leucine into hepatocytes remained unchanged compared with those of sham-operated controls. In contrast, in those from totally pancreatectomized rats, the incorporation of 3H-thymidine and 3H-leucine decreased to approximately 67% and 37% respectively of sham-operated controls. However, those returned to near normal in the cultured hepatocytes from totally pancreatectomized rats treated by 0.8 IU/kg of insulin. (2) The addition of insulin (10(-4) M) to the culture medium stimulated the incorporation of 3H-thymidine into cultured hepatocytes prepared from normal rats to 148% of controls. The insulin-stimulated incorporation was inhibited by the addition of glucagon to the culture medium. The combined addition of insulin and glucagon did not synergistically act on DNA synthesis. It is suggested that the portal blood insulin in the presence of more than 20% of the pancreas is imperative for maintaining spontaneous regeneration.     

Important role of glucagon during exercise in diabetic dogs

To define the role of immunoreactive glucagon (IRG) during exercise in diabetes, 12 insulin-deprived alloxan-diabetic (A-D) dogs were run for 90 min (100 m/min, 12 degrees) with or without somatostatin (St 0.5 microgram . kg-1 . min-1). Compared with normal dogs, A-D dogs were characterized by similar hepatic glucose production (Ra), lower glucose metabolic clearance, and higher plasma glucose and free fatty acid levels during rest and exercise. In A-D dogs IRG was greater at rest and exhibited a threefold greater exercise increment than controls, whereas immunoreactive insulin (IRI) was reduced by 68% at rest but had similar values to controls during exercise. Basal norepinephrine, epinephrine, cortisol, and lactate levels were similar in normal and A-D dogs. However, exercise increments in norepinephrine, cortisol, and lactate were higher in A-D dogs. When St was infused during exercise in the A-D dogs, IRG was suppressed by 432 +/- 146 pg/ml below basal and far below the exercise response in A-D controls (delta = 645 +/- 153 pg/ml). IRI was reduced by 1.8 +/- 0.2 microU/ml with St. With IRG suppression the increase in Ra seen in exercising A-D controls (delta = 4.8 +/- 1.6 mg . kg-1 . min-1) was virtually abolished, and glycemia fell by 104 to 133 +/- 37 mg/dl. Owing to this decrease in glycemia, the increase in glucose disappearance was attenuated. Despite the large fall in glucose during IRG suppression, counterregulatory increases were not excessive compared with A-D controls. In fact, as glucose levels approached euglycemia, the increments in norepinephrine and cortisol were reduced to levels similar to those seen in normal exercising dogs. In conclusion, IRG suppression during exercise in A-D dogs almost completely obviated the increase in Ra, resulting in a large decrease in plasma glucose. Despite this large fall in glucose, there was no excess counterregulation, since glucose concentrations never reached the hypoglycemic range.     

Isolated dog coronary arteries response to glomerulopressin

Coronary arteries were excised from pentobarbital anesthetized normal dogs. The isolated coronary arteries were placed in an oxigenated KRB bathing solution maintained at 37 degrees C and pH 7.4 changes in the basal tone were measured. The addition of glomerulopressin to the bathing solution produced a decrease of 26 +/- 1.5 mg of the basal tone. This decrease was prolonged for more than 40 minutes. Several inhibitors of prostaglandins synthesis were added to the bath such as corticosterone (2 X 10(-5) M), indomethacin (6 X 10(-6) M), acetylsalicylic acid (1.8 X 10(-4) M) and tranylcypromine (4 X 10(-4) M). All these inhibitors blocked the action of glomerulopressin. We conclude that glomerulopressin relaxes the coronary arteries and that this relaxation may be mediated through the novobiosynthesis of prostaglandins.     

Role of insulin and glucagon in oxytocin effects on glucose metabolism.

Oxytocin (OT) infusion in normal dogs increases plasma insulin and glucagon levels and increases rates of glucose production and uptake. The purpose of this study was to determine whether the effects of OT on glucose metabolism were direct or indirect. The studies were carried out in normal, unanesthetized dogs in which OT infusion was superimposed on infusion of either somatostatin, which suppresses insulin and glucagon secretion, or clonidine, which suppresses insulin secretion only. Infusion of 0.2 microgram/kg/min of somatostatin suppressed basal levels of plasma insulin and glucagon and inhibited the OT-induced rise of these hormones by about 60-80% of that seen with OT alone. The rates of glucose production and uptake by tissues, measured with [6-3H] glucose, were significantly lower than those seen with OT alone, and the rise in glucose clearance was completely inhibited. Clonidine (30 micrograms/kg, sc), given along with an insulin infusion to replace basal levels of insulin, completely prevented the OT-induced rise in plasma insulin and markedly reduced the glucose uptake seen with OT alone, but did not reduce the usual increase in plasma glucose and glucagon levels or glucose production. To determine whether the OT-induced rise in plasma insulin was in response to the concomitant increase in plasma glucose, similar plasma glucose levels were established in normal dogs by a continuous infusion of glucose and an OT infusion was superimposed. OT did not raise plasma glucose levels further, but plasma insulin levels were increased, indicating that OT can stimulate insulin secretion independently of the plasma glucose changes. Studies by others have shown that the addition of OT to pancreatic islets or intact pancreas can stimulate insulin and glucagon secretion, indicating a direct effect. Our studies agree with that and suggest that in vivo, OT raises plasma insulin levels, at least in part, through a direct action on the pancreas. These studies also show that OT increases glucose production by increasing glucagon secretion and, in addition, a direct effect of OT on glucose production is likely. The OT-induced increase in glucose uptake is mediated largely by increased insulin secretion.     

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.     

Exenatide can reduce glucose independent of islet hormones or gastric emptying

Exenatide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic used in the treatment of type 2 diabetes. There is increasing evidence that GLP-1 can influence glycemia not only via pancreatic (insulinotropic and glucagon suppression) and gastric-emptying effects, but also via an independent mechanism mediated by portal vein receptors. The aim of our study was to investigate whether exenatide has an islet- and gastric-independent glycemia-reducing effect, similar to GLP-1. First, we administered mixed meals, with or without exenatide (20 microg sc) to dogs. Second, to determine whether exenatide-induced reduction in glycemia is independent of slower gastric emptying, in the same animals we infused glucose intraportally (to simulate meal test glucose appearance) with exenatide, exenatide + the intraportal GLP-1 receptor antagonist exendin-(9-39), or saline. Exenatide markedly decreased postprandial glucose: net 0- to 135-min area under the curve = +526 +/- 315 and -536 +/- 197 mg.dl(-1).min(-1) with saline and exenatide, respectively (P < 0.05). Importantly, the decrease in plasma glucose occurred without a corresponding increase in postprandial insulin but was accompanied by delayed gastric emptying and lower glucagon. Significantly lower glycemia was induced by intraportal glucose infusion with exenatide than with saline (92 +/- 1 vs. 97 +/- 1 mg/dl, P < 0.001) in the absence of hyperinsulinemia or glucagon suppression. The exenatide-induced lower glycemia was partly reversed by intraportal exendin-(9-39): 95 +/- 3 and 92 +/- 3 mg/dl with exenatide + antagonist and exenatide, respectively (P < 0.01). Our results suggest that, similar to GLP-1, exenatide lowers glycemia via a novel mechanism independent of islet hormones and slowing of gastric emptying. We hypothesize that receptors in the portal vein, via a neural mechanism, increase glucose clearance independent of islet hormones.     

Insulin dependence of the actions of growth hormone and somatostatin on splanchnic biogenic amines of the dog

It is known from studies previously conducted in this laboratory that an iv injection of ovine growth hormone (GH, 100 micrograms/kg BW) or an equimolar amount of somatostatin (SRIF, 7.5 micrograms/kg BW), given to normal conscious dogs into a saphenous vein, leads to a significant increase in hepatic portal plasma serotonin and a simultaneous decrease in the concentrations of dopamine, norepinephrine and epinephrine. The changes take place within 12 minutes after the injection and are observed only in the portal circulation. The purpose of the present experiment was to investigate whether or not similar results could be obtained in diabetic animals. Mongrel dogs were rendered diabetic by surgical pancreatectomy and fitted with an indwelling hepatic portal catheter. Radioenzymatic methods were employed for quantitative measurements of plasma free serotonin and catecholamines. No response was noted when the same type of experiments as those conducted in normal dogs were now carried out in trained, fully conscious totally pancreatectomized dogs deprived of exogenous insulin supply. When the same animals were given an injection into a peripheral vein of 50 mU/kg BW regular crystalline insulin (a small dose that affected neither plasma glucose nor biogenic amine levels) 10 minutes prior to the administration of the other hormones, the usual response to both GH and SRIF was restored, i.e. the data were comparable to those of normal dogs. It is concluded that the GH/SRIF effect on gut biogenic amines is insulin dependent.     

Urinary excretion of cGMP in response to atrial natriuretic peptide in dogs with acute pancreatitis

Previous studies have shown that when atrial natriuretic peptide (ANF) is given to anaesthetized dogs with hypovolemic acute pancreatitis, it will produce a diuresis and natriuresis but will not elevate the glomerular filtration rate (GFR). When the same dose of peptide is given to dogs equally hypovolemic (hemorrhage) but without pancreatitis, a brisk increment in GFR occurs. GFR will, however, rise in dogs with pancreatitis in response to other peptides, such as glucagon. In these studies we assessed cGMP excretion as a marker for ANF effect in both normal anaesthetized dogs and dogs with acute experimental pancreatitis. In each group, urinary output and sodium excretion increased significantly, but GFR rose only in the control group. Urinary excretion of cGMP rose equally and dramatically in both control and experimental animals. We conclude that GFR is prevented from rising in dogs with experimental pancreatitis following ANF, but this effect does not depend on depressed cGMP generation.     

Glycemic control in mice with targeted disruption of the glucagon receptor gene.

The action of glucagon in the liver is mediated by G-coupled receptors. To examine the role of glucagon in glucose homeostasis, we have generated mice in which the glucagon receptor was inactivated (GR(-/-) mice). Blood glucose levels were somewhat reduced in GR(-/-) mice relative to wild type, in both the fed and fasted state. Plasma insulin levels were not significantly affected. There was no significant effect on fasting plasma cholesterol or triglyceride levels associated with deletion of the glucagon receptor. Glucose tolerance, as assessed by an oral glucose tolerance test, improved. Plasma glucagon levels were strikingly elevated in both fed and fasted animals. Despite a total absence of glucagon receptors, these animals maintained near-normal glycemia and normal lipidemia, in the presence of circulating glucagon concentrations that were elevated by two orders of magnitude.