Effects of recurrent hyperinsulinemia with and without hypoglycemia on counterregulation in diabetic rats

To understand the mechanisms whereby recurrent hypoglycemia increases the risk of subsequent hypoglycemia, it was necessary to differentiate the effects of recurrent hyperinsulinemia from those of hyperinsulinemic hypoglycemia. We examined basal and hypoglycemic endocrine function in normal rats, streptozotocin-diabetic controls, and diabetic rats exposed to 4 days of 2 episodes/day of hyperinsulinemic hypoglycemia (DH) or hyperinsulinemic hyperglycemia (DI). DH and DI rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. In diabetic controls, basal plasma ACTH tended to be increased, and plasma corticosterone, plasma somatostatin, and pancreatic prosomatostatin and proglucagon mRNA were increased (P < 0.05) vs. normal rats. These parameters were normalized in DH and DI rats. In diabetic controls, glucagon, epinephrine, norepinephrine, corticosterone, and peak glucose production responses to hypoglycemia were reduced (P < 0.05) vs. normal rats. In DI rats, epinephrine responses were normalized. Conversely, DH rats displayed marked further impairment of epinephrine and glucose production responses and increased peripheral insulin sensitivity (P < 0.05 vs. diabetic controls). Both insulin regimens partially normalized glucagon and fully normalized norepinephrine and corticosterone responses. In summary, recurrent hyperinsulinemia in diabetic rats normalized most pituitary-adrenal, sympathoadrenal, and pancreatic parameters. However, concurrent hypoglycemia further impaired epinephrine and glucose production responses and increased insulin sensitivity. We conclude that 1) recurrent hypoglycemia may increase the risk of subsequent hypoglycemia by increasing insulin sensitivity, and 2) epinephrine counterregulation is particularly sensitive to impairment by recurrent hypoglycemia.     

Portal vein hypoglycemia is essential for full induction of hypoglycemia-associated autonomic failure with slow-onset hypoglycemia

Antecedent hypoglycemia leads to impaired counterregulation and hypoglycemic unawareness. To ascertain whether antecedent portal vein hypoglycemia impairs portal vein glucose sensing, thereby inducing counterregulatory failure, we compared the effects of antecedent hypoglycemia, with and without normalization of portal vein glycemia, upon the counterregulatory response to subsequent hypoglycemia. Male Wistar rats were chronically cannulated in the carotid artery (sampling), jugular vein (glucose and insulin infusion), and mesenteric vein (glucose infusion). On day 1, the following three distinct antecedent protocols were employed: 1) HYPO-HYPO: systemic hypoglycemia (2.52 +/- 0.11 mM); 2) HYPO-EUG: systemic hypoglycemia (2.70 +/- 0.03 mM) with normalization of portal vein glycemia (portal vein glucose = 5.86 +/- 0.10 mM); and 3) EUG-EUG: systemic euglycemia (6.33 +/- 0.31 mM). On day 2, all groups underwent a hyperinsulinemic-hypoglycemic clamp in which the fall in glycemia was controlled so as to reach the nadir (2.34 +/- 0.04 mM) by minute 75. Counterregulatory hormone responses were measured at basal (-30 and 0) and during hypoglycemia (60-105 min). Compared with EUG-EUG, antecedent hypoglycemia (HYPO-HYPO) significantly blunted the peak epinephrine (10.44 +/- 1.35 vs. 15.75 +/- 1.33 nM: P = 0.01) and glucagon (341 +/- 16 vs. 597 +/- 82 pg/ml: P = 0.03) responses to next-day hypoglycemia. Normalization of portal glycemia during systemic hypoglycemia on day 1 (HYPO-EUG) prevented blunting of the peak epinephrine (15.59 +/- 1.43 vs. 15.75 +/- 1.33 nM: P = 0.94) and glucagon (523 +/- 169 vs. 597 +/- 82 pg/ml: P = 0.66) responses to day 2 hypoglycemia. Consistent with hormonal responses, the glucose infusion rate during day 2 hypoglycemia was substantially elevated in HYPO-HYPO (74 +/- 12 vs. 49 +/- 4 micromol x kg(-1) x min(-1); P = 0.03) but not HYPO-EUG (39 +/- 7 vs. 49 +/- 4 micromol x kg(-1) x min(-1): P = 0.36). Antecedent hypoglycemia local to the portal vein is required for the full induction of hypoglycemia-associated counterregulatory failure with slow-onset hypoglycemia.     

Effect of gastrointestinal hormones on choleresis from the isolated perfused rat liver

Using isolated perfused rat liver, the direct effect of secretin, glucagon, caerulein, insulin and somatostatin on choleresis was investigated. When the liver was perfused in the absence of sodium taurocholate, the bile volumes were: control, 0.33 +/- 0.01 (mean +/- S.E.M.) ml/10 g liver per 50 min; secretin 0.05 U/ml, 0.39 +/- 0.01 (P less than 0.01); glucagon 10(-10) M, 0.44 +/- 0.02 (P less than 0.01); caerulein 10(-8) M, 0.34 +/- 0.03 (n.s.); insulin 1 mU/ml, 0.35 +/- 0.02 (n.s.); glucagon plus somatostatin 10(-7) M, 0.46 +/- 0.03 (n.s. vs. glucagon alone), respectively. When 10(-5) M sodium taurocholate was present in the perfusate, the bile volumes were: control, 0.61 +/- 0.03; secretin, 0.63 +/- 0.01 (n.s.); glucagon, 0.70 +/- 0.01 (P less than 0.05); caerulein, 0.55 +/- 0.01 (n.s.); insulin, 0.62 +/- 0.04 (n.s.); somatostatin, 0.59 +/- 0.01 (n.s.); respectively. Glucagon increased glucose output and cyclic AMP in the effluent from the liver neither of which were suppressed by somatostatin. Secretin increased cyclic AMP but not glucose output. These results indicate that glucagon has the most potent action on bile acid-independent canalicular bile, that caerulein and insulin do not act on canalicular bile production directly and that somatostatin does not directly suppress canalicular bile production nor hepatic glucose output produced by glucagon in rats.     

Elevated endogenous cortisol reduces autonomic neuroendocrine and symptom responses to subsequent hypoglycemia

We tested the hypothesis that increased endogenous cortisol secretion reduces autonomic neuroendocrine and neurogenic symptom responses to subsequent hypoglycemia. Twelve healthy young adults were studied on two separate occasions, once after infusions of a pharmacological dose of alpha-(1-24)-ACTH (100 microg/h) from 0930 to 1200 and 1330 to 1600, which raised plasma cortisol levels to approximately 45 microg/dl on day 1, and once after saline infusions on day 1. Hyperinsulinemic (2.0 mU x kg(-1) x min(-1)) stepped hypoglycemic clamps (90, 75, 65, 55, and 45 mg/dl glucose steps) were performed on the morning of day 2 on both occasions. These markedly elevated antecedent endogenous cortisol levels reduced the adrenomedullary (P = 0.004, final plasma epinephrine levels of 489 +/-64 vs. 816 +/-113 pg/ml), sympathetic neural (P = 0.0022, final plasma norepinephrine levels of 244 +/-15 vs. 342 +/-22 pg/ml), parasympathetic neural (P = 0.0434, final plasma pancreatic polypeptide levels of 312 +/- 37 vs. 424 +/- 56 pg/ml), and neurogenic (autonomic) symptom (P = 0.0097, final symptom score of 7.1 +/-1.5 vs. 10.6 +/- 1.6) responses to subsequent hypoglycemia. Growth hormone, but not glucagon or cortisol, responses were also reduced. The findings that increased endogenous cortisol secretion reduces autonomic neuroendocrine and neurogenic symptom responses to subsequent hypoglycemia are potentially relevant to cortisol mediation of hypoglycemia-associated autonomic failure, and thus a vicious cycle of recurrent iatrogenic hypoglycemia, in people with diabetes mellitus.     

Insulin-independent effects of GLP-1 on canine liver glucose metabolism: duration of infusion and involvement of hepatoportal region

Whether glucagon-like peptide-1 (GLP-1) has insulin-independent effects on glucose disposal in vivo was assessed in conscious dogs by use of tracer and arteriovenous difference techniques. After a basal period, each experiment consisted of three periods (P1, P2, P3) during which somatostatin, glucagon, insulin, and glucose were infused. The control group (C) received saline in P1, P2, and P3, the PePe group received saline in P1 and GLP-1 (7.5 pmol.kg(-1).min(-1)) peripherally (Pe; iv) in P2 and P3, and the PePo group received saline in P1 and GLP-1 peripherally (iv) (P2) and then into the portal vein (Po; P3). Glucose and insulin concentrations increased to two- and fourfold basal, respectively, and glucagon remained basal. GLP-1 levels increased similarly in the PePe and PePo groups during P2 ( approximately 200 pM), whereas portal GLP-1 levels were significantly increased (3-fold) in PePo vs. PePe during P3. In all groups, net hepatic glucose uptake (NHGU) occurred during P1. During P2, NHGU increased slightly but not significantly in all groups. During P3, NHGU increased in PePe and PePo groups to a greater extent than in C, but no significant effect of the route of infusion of GLP-1 was demonstrated (16.61 +/- 2.91 and 14.67 +/- 2.09 vs. 4.22 +/- 1.57 micromol.kg(-1).min(-1), respectively). In conclusion: GLP-1 increased glucose disposal in the liver independently of insulin secretion; its full action required long-term infusion. The route of infusion did not modify the hepatic response.     

Glucagon secretion and autonomic signaling during hypoglycemia in late pregnancy

We examined net pancreatic norepinephrine (NE) spillover, pancreatic polypeptide (PP) release, and the decrement in C-peptide to identify factors involved in the blunted counterregulatory glucagon response in pregnancy. Conscious pregnant [pregnant hypoglycemic (Ph); 3rd trimester; n = 8] and nonpregnant [nonpregnant hypoglycemic (NPh); n = 6] dogs were studied during insulin-induced (approximately 12-fold basal insulin concentrations) hypoglycemia (plasma glucose 3.1 mM). Additional dogs were studied during hyperinsulinemic euglycemia [nonpregnant euglycemic (NPe), n = 4; pregnant euglycemic (Pe), n = 5; plasma glucose 6 mM]. Arterial glucagon concentrations declined similarly in NPe and Pe. Areas under the curve (AUCs) of the changes in glucagon and epinephrine were seven- and threefold greater in NPh than Ph (P < 0.05 between groups for both). Glucagon secretion fell below basal in NPe, Pe, and Ph but rose significantly in NPh. C-peptide declined 0.25 +/- 0.06, 0.12 +/- 0.11, 0.28 +/- 0.05, and 0.13 +/- 0.02 ng/ml in NPe, Pe, NPh, and Ph, respectively (P < 0.05, NPh vs. Ph). AUCs of NE spillover were 516 +/- 274, 265 +/- 303, 506 +/- 94, and -63 +/- 79 ng, respectively (P < 0.05, NPh vs. Ph). The AUC of PP release was approximately threefold greater in NPh than Ph (P < 0.05) but not different between euglycemic groups. The current evidence strongly suggests that the blunting of glucagon secretion during insulin-induced hypoglycemia in pregnancy is related to generalized impairment of a number of different signals, including parasympathetic and sympathoadrenal stimuli and altered sensing of circulating and/or intraislet insulin.     

Galanin and pancreastatin inhibit stimulated insulin secretion in the mouse: comparison of effects

The effects of the two intrapancreatic peptides galanin and pancreastatin on basal and stimulated insulin and glucagon secretion in the mouse were compared. It was found that at 2 min after intravenous injection of galanin or pancreastatin (4.0 nmol/kg), basal plasma glucagon and glucose levels were slightly elevated. Galanin was more potent than pancreastatin to elevate basal plasma glucagon levels: they increased from 60 +/- 15 to 145 +/- 19 pg/ml (p less than 0.01) after galanin compared to from 35 +/- 5 to 55 +/- 8 pg/ml (p less than 0.05) after pancreastatin. Plasma insulin levels were lowered by galanin (p less than 0.05), but not by pancreastatin. CCK-8 (6.3 nmol/kg) or terbutaline (3.6 mumol/kg) markedly increased the plasma insulin levels. Galanin (4.0 nmol/kg) completely abolished the insulin response to CCK-8 (p less than 0.001), but pancreastatin (4.0 nmol/kg) was without effect. Galanin inhibited the insulin response to terbutaline by approximately 60% (p less than 0.01), but pancreastatin inhibited the insulin response to terbutaline by approximately 35% only (p less than 0.05). CCK-8 and terbutaline did both elevate plasma glucagon levels by moderate potencies: neither pancreastatin nor galanin could affect these responses. Thus, in the mouse, galanin and pancreastatin both inhibit basal and stimulated insulin secretion, and stimulate basal glucagon secretion. Galanin is thereby more potent than pancreastatin. The study also showed that galanin potently inhibits insulin secretion stimulated by the octapeptide of cholecystokin and by the beta 2-adrenoceptor agonist terbutaline, and that pancreastatin inhibits terbutaline-induced insulin secretion.     

Pre-exposure to glucagon impairs glucose recovery after insulin-induced hypoglycemia in man

The effect of a two hour period of hypo- and hyperglucagonemia on a subsequent insulin-induced hypoglycemia was studied in nine healthy volunteers. Hypoglucagonemia was provoked by somatostatin (50 micrograms/h) and hyperglucagonemia by glucagon infusion (3.25 ng/kg/min) together with somatostatin, while saline alone was given as control. Hypoglycemia was induced by insulin infusion (2.4 U/h) for two hours. The hyperglycemic effect of glucagon was transient and similar nadir glucose levels were obtained in the three experiments. Preinfusion with glucagon impaired glucose recovery in spite of preserved secretion of epinephrine during restitution of blood glucose in this experiment. It is concluded, that a period of elevated glucagon levels deteriorates the restitution of blood glucose following hypoglycemia. Hyperglucagonemia, commonly apparent in poorly controlled diabetics, may therefore be of importance in explaining the impaired recovery of blood glucose seen in such patients after hypoglycemia.     

The selective serotonin reuptake inhibitor sertraline enhances counterregulatory responses to hypoglycemia

Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for patients with comorbid diabetes and depression. Clinical case studies in diabetic patients, however, suggest that SSRI therapy may exacerbate hypoglycemia. We hypothesized that SSRIs might increase the risk of hypoglycemia by impairing hormonal counterregulatory responses (CRR). We evaluated the effect of the SSRI sertraline on hormonal CRR to single or recurrent hypoglycemia in nondiabetic rats. Since there are time-dependent effects of SSRIs on serotonin neurotransmission that correspond with therapeutic action, we evaluated the effect of 6- or 20-day sertraline treatment on hypoglycemia CRR. We found that 6-day sertraline (SERT) treatment specifically enhanced the epinephrine response to a single bout of hypoglycemia vs. vehicle (VEH)-treated rats (t = 120: VEH, 2,573 +/- 448 vs. SERT, 4,202 +/- 545 pg/ml, P < 0.05). In response to recurrent hypoglycemia, VEH-treated rats exhibited the expected impairment in epinephrine secretion (t = 60: 678 +/- 73 pg/ml) vs. VEH-treated rats experiencing first-time hypoglycemia (t = 60: 2,081 +/- 436 pg/ml, P < 0.01). SERT treatment prevented the impaired epinephrine response in recurrent hypoglycemic rats (t = 60: 1,794 +/- 276 pgl/ml). In 20-day SERT-treated rats, epinephrine, norepinephrine, and glucagon CRR were all significantly elevated above VEH-treated controls in response to hypoglycemia. Similarly to 6-day SERT treatment, 20-day SERT treatment rescued the impaired epinephrine response in recurrent hypoglycemic rats. Our data demonstrate that neither 6- nor 20-day sertraline treatment impaired hormonal CRR to hypoglycemia in nondiabetic rats. Instead, sertraline treatment resulted in an enhancement of hypoglycemia CRR and prevented the impaired adrenomedullary response normally observed in recurrent hypoglycemic rats.     

Metabolic regulation of growth hormone by free fatty acids, somatostatin, and ghrelin in HIV-lipodystrophy

Human immunodeficiency virus (HIV)-lipodystrophy is a syndrome characterized by changes in fat distribution and insulin resistance. Prior studies suggest markedly reduced growth hormone (GH) levels in association with excess visceral adiposity among patients with HIV-lipodystrophy. We investigated mechanisms of altered GH secretion in a population of 13 male HIV-infected patients with evidence of fat redistribution, compared with 10 HIV-nonlipodystrophic patients and 11 male healthy controls similar in age and body mass index (BMI). Although similar in BMI, the lipodystrophic group was characterized by increased visceral adiposity, free fatty acids (FFA), and insulin and reduced extremity fat. We investigated ghrelin and the effects of acute lowering of FFA by acipimox on GH responses to growth hormone-releasing hormone (GHRH). We also investigated somatostatin tone, comparing GH response to combined GHRH and arginine vs. GHRH alone with a subtraction algorithm. Our data demonstrate an equivalent number of GH pulses (4.1 +/- 0.6, 4.7 +/- 0.8, and 4.5 +/- 0.3 pulses/12 h in the HIV-lipodystrophic, HIV-nonlipodystrophic, and healthy control groups, respectively, P > 0.05) but markedly reduced GH secretion pulse area (1.14 +/- 0.27 vs. 4.67 +/- 1.24 ng.ml(-1).min, P < 0.05, HIV-lipodystrophic vs. HIV-nonlipodystrophic; 1.14 +/- 0.27 vs. 3.18 +/- 0.92 ng.ml(-1).min, P < 0.05 HIV-lipodystrophic vs. control), GH pulse area, and GH pulse width in the HIV-lipodystrophy patients compared with the control groups. Reduced ghrelin (418 +/- 46 vs. 514 +/- 37 pg/ml, P < 0.05, HIV-lipodystrophic vs. HIV-nonlipodystrophic; 418 +/- 46 vs. 546 +/- 45 pg/ml, P < 0.05, HIV-lipodystrophic vs. control), impaired GH response to GHRH by excess FFA, and increased somatostatin tone contribute to reduced GH secretion in patients with HIV-lipodystrophy. These data provide novel insight into the metabolic regulation of GH secretion in subjects with HIV-lipodystrophy.     

Prolonged infusion of amino acids increases leucine oxidation in fetal sheep

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ～12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.     

Intraoperative protein sparing with glucose.

We examined the hypothesis that glucose infusion inhibits amino acid oxidation during colorectal surgery. We randomly allocated 14 patients to receive intravenous glucose at 2 mg x kg(-1) x min(-1) (glucose group) starting with the surgical incision or an equivalent amount of normal saline 0.9% (control group). The primary endpoint was whole body leucine oxidation; secondary endpoints were leucine rate of appearance and nonoxidative leucine disposal as determined by a stable isotope tracer technique (L-[1-(13)C]leucine). Circulating concentrations of glucose, lactate, insulin, glucagon, and cortisol were measured before and after 2 h of surgery. Leucine rate of appearance, an estimate of protein breakdown, and nonoxidative leucine disposal, an estimate of protein synthesis, decreased in both groups during surgery (P < 0.05). Leucine oxidation intraoperatively decreased from 13 +/- 3 to 4 +/- 3 micromol x kg(-1) x h(-1) in the glucose group (P < 0.05 vs. control group) whereas it remained unchanged in the control group. Hyperglycemia during surgery was more pronounced in patients receiving glucose (9.7 +/- 0.5 mmol/l, P < 0.05 vs. control group) than in patients receiving normal saline (7.1 +/- 1.0 mmol/l). The administration of glucose caused an increase in the circulating concentration of insulin (P < 0.05) resulting in a lower glucagon/insulin quotient than in the control group (P < 0.05). Intraoperative plasma cortisol concentrations increased in both groups (P < 0.05), whereas plasma concentrations of lactate and glucagon did not change. The provision of small amounts of glucose was associated with a decrease in amino acid oxidation during colorectal surgery.     

Superactive somatostatin analog decreases plasma glucose and glucagon levels in diabetic rats

The action of the new analog of somatostatin, D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160), on plasma glucagon and glucose levels was evaluated in streptozotocin-diabetic rats. The effect of this analog on the insulin-induced hypoglycemia in diabetic rats was also investigated in order to evaluate the risk of exacerbating hypoglycemia. Administration of analog RC-160, in a dose of 25 micrograms/kg b. wt. SC, inhibited plasma glucagon secretion and decreased plasma glucose levels. This effect also occurred when plasma glucagon and glucose levels were first elevated by arginine infusion, 1000 mg/kg/hr for 30 min. Subcutaneous injection of regular insulin, 15 U/kg b. wt., produced hypoglycemia with a progressive increase in glucagon levels. Analog RC-160 completely suppressed the hypoglycemia-induced glucagon release for up to 150 min after injection of the analog or insulin. A greater decrease in the plasma glucose level was observed in the group treated with insulin and the analog than in the group injected only with insulin. These results indicate that somatostatin analog RC-160 can produce a marked and prolonged inhibition of glucagon release and a decrease in the plasma glucose level in diabetic rats. This analog may be useful as an adjunct to insulin in the treatment of diabetic patients, although caution should be exercised, to prevent hypoglycemia when using somatostatin analogs together with insulin.     

Insulin and glucagon secretion in rats: effects of calcitonin gene-related peptide

Immunoreactive calcitonin gene-related peptide (CGRP) has been shown to occur in intrapancreatic nerves and islet somatostatin cells in the rat. Therefore, we investigated the effects of CGRP on insulin and glucagon secretion in the rat. CGRP was infused i.v. at one of 3 dose levels (4.3, 17 or 68 pmol/min). Infusion of CGRP alone was found to elevate basal plasma levels of both insulin and glucagon. In contrast, CGRP impaired the plasma insulin responses to both glucose (7 mg/min; P less than 0.001) and arginine (8.5 mg/min; P less than 0.001), and inhibited the arginine-induced increase in plasma glucagon concentrations (P less than 0.001). Since CGRP and somatostatin are colocalized within the D-cells, we also infused CGRP and somatostatin together at equimolar dose levels (17 pmol/min), with glucose (7 mg/min). By that, the increase in plasma insulin concentrations decreased more rapidly than during infusion of either peptide alone. Since alpha 2-adrenoceptor activation is known to inhibit glucose-stimulated insulin secretion, we also infused CGRP together with the specific alpha 2-adrenoceptor antagonist yohimbine (37 nmol/min). In that way, the plasma insulin-lowering effect of CGRP was prevented. We have shown in the rat: (1) that CGRP stimulates basal insulin and glucagon secretion; (2) that CGRP inhibits stimulated insulin and glucagon secretion; (3) that CGRP and somatostatin more rapidly induce a potent inhibitory action on glucose-stimulated insulin secretion when given together; and (4) that the alpha 2-adrenoceptor antagonist, yohimbine, counteracts the inhibitory action of CGRP on glucose-stimulated insulin secretion. We suggest that CGRP is of importance for the regulation of insulin and glucagon secretion in the rat. The mechanisms behind the islet effects of CGRP can not be established by the present results, though they apparently require intact alpha 2-adrenoceptors.     

Dissociation of hypothalamic noradrenergic activity and sympathoadrenal responses to recurrent hypoglycemia

This study evaluated whether attenuation of sympathoadrenal responses to recurrent hypoglycemia is mediated by diminished noradrenergic activity in the hypothalamus. Male Sprague-Dawley rats received either once daily insulin (1.0 units/kg) injections or an equal administration of saline for 3 days. Both groups received an administration of insulin on the fourth day, during which blood glucose and plasma catecholamines were determined, and extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH) or paraventricular hypothalamic nucleus (PVN) was monitored with microdialysis. The peak response of plasma epinephrine to insulin-induced hypoglycemia (nadir approximately 3.2 mmol/l) was significantly reduced during the fourth hypoglycemic episode (774 +/- 134 pg/ml) compared with the first episode (2,561 +/- 410 pg/ml, P < 0.001). Baseline levels of extracellular NE were elevated approximately 25% (P = 0.07) in the VMH and approximately 46% (P = 0.03) in the PVN after multiple hypoglycemic episodes. There was no difference in noradrenergic activity during the first or fourth hypoglycemic episode in either brain area. The reduced sympathoadrenal output after recurrent hypoglycemia is likely postsynaptic from hypothalamic NE release or is mediated via a collateral pathway.     

Hormonal studies in a patient with PP-producing tumors

Five years after the removal of pure pancreatic polypeptide (PP) producing tumors, concentrations of circulating levels of PP, insulin, glucagon, and growth hormone in the basal state, after insulin-induced hypoglycemia, and after a protein-rich meal were determined in a patient with previous truncal vagotomy and Billroth II gastrectomy. Basal plasma levels of PP ranged between 2180 and 2660 pg/ml suggesting persistence or recurrence of PP producing tumors. Concentrations of the other hormones were within normal values. After insulin injection (0.1 U/Kg) levels of PP and glucagon were not modified while those of GH rose from 3.2 to 22.6 ng/ml. After a protein meal (450 gms. of cooked ground beef meat) a sharp rise of plasma PP was observed to a peak of 11310 pg/ml at 10 min. Moreover, plasma levels of immunoreactive insulin also showed an equally prompt rise to a peak of 532 microU/ml while plasma glucagon rose simultaneously to 448 pg/ml. The cause of the abnormal PP, insulin and glucagon responses could not be ascertained but we postulate that they are derived from pancreatic tumors of mixed cell type.     

Impaired pancreatic polypeptide response to insulin hypoglycemia in obese subjects

We have studied the effect of insulin hypoglycemia on the secretion of pancreatic polypeptide (PP) in 14 obese subjects with normal glucose tolerance and in 6 normal controls. Infusion of insulin 0.1 U/kg/h in controls and 0.12 U/kg/h in the obese, for one hour, produced a progressive hypoglycemia, similar in both groups (nadir 2 mmol/l at 50 min). The secretion of PP was less in obese subjects than in controls (peak 116 mmol/l vs 184 pmol/l, P less than 0.01) (integrated secretion sigma delta PP 288 vs 472 pmol/l, P less than 0.01) and was also delayed in the obese subjects beginning at 50 min instead of 40 min. The secretion of glucagon and of C-peptide were not different in the two groups, but the integrated response of ACTH was higher in the obese (sigma delta ACTH 52 pmol/l vs 25 pmol/l, P less than 0.01). The secretory response of growth hormone (STH) was smaller in the obese group (peak 8.6 +/- 1.28 vs 21.4 +/- 6.4 ng/ml, P less than 0.01). The reduced secretion of PP in obese subjects could be due to impaired sensitivity to hypoglycemia of the central control mechanism for PP release. The similarity of the reductions in the secretion of both PP and STH support this hypothesis, although a reduction in the secretory capacity of pancreatic PP cells cannot be excluded.     

Interactions between oxytocin, glucagon and glucose in normal and streptozotocin-induced diabetic rats

Oxytocin has been suggested to have glucoregulatory functions in rats, man and other mammals. The hyperglycemic actions of oxytocin are believed to be mediated indirectly through changes in pancreatic function. The present study examined the interaction between glucose and oxytocin in normal and streptozotocin (STZ)-induced diabetic rats, under basal conditions and after injections of oxytocin. Plasma glucose and endogenous oxytocin levels were significantly correlated in cannulated lactating rats (r = 0.44, P less than 0.01). To test the hypothesis that oxytocin was acting to elevate plasma glucose, adult male rats were injected with 10 micrograms/kg oxytocin and killed 60 min later. Oxytocin increased plasma glucose from 6.1 +/- 0.1 to 6.8 +/- 0.2 mM (P less than 0.05), and glucagon from 179 +/- 12 to 259 +/- 32 pg/ml (P less than 0.01, n = 18). There was no significant effect of oxytocin on plasma insulin, although the levels were increased by 30%. A lower dose (1 microgram/kg) of oxytocin had no significant effect on plasma glucose or glucagon. To eliminate putative local inhibitory effects of insulin on glucagon secretion, male rats were made diabetic by i.p. injection of 100 mg/kg STZ, which increased glucose to greater than 18 mM and glucagon to 249 +/- 25 pg/ml (P less than 0.05). In these rats, 10 micrograms/kg oxytocin failed to further increase plasma glucose, but caused a much greater increase in glucagon (to 828 +/- 248 pg/ml) and also increased plasma ACTH. A specific oxytocin analog, Thr4,Gly7-oxytocin, mimicked the effect of oxytocin on glucagon secretion in diabetic rats. The lower dose of oxytocin also increased glucagon levels (to 1300 +/- 250 pg/ml), but the effect was not significant. A 3 h i.v. infusion of 1 nmol/kg per h oxytocin in conscious male rats significantly increased glucagon levels by 30 min in normal and STZ-rats; levels returned to baseline by 30 min after stopping the infusion. Plasma glucose increased in the normal, but not STZ-rats. The relative magnitude of the increase in glucagon was identical for normal and diabetic rats, but the absolute levels of glucagon during the infusion were twice as high in the diabetics. To test whether hypoglycemia could elevate plasma levels of oxytocin, male rats were injected i.p. with insulin and killed from 15-180 min later. Plasma glucose levels dropped to less than 2.5 mM by 15 min. Oxytocin levels increased by 150-200% at 30 min; however, the effect was not statistically significant.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of training on responses of beta-endorphin and other pituitary hormones to insulin-induced hypoglycemia

We studied whether the previously reported intensified beta-endorphin response to exercise after training might result from a training-induced general increase in anterior pituitary secretory capacity. Identical hypoglycemia was induced by insulin infusion in 7 untrained (VO2max 49 +/- 4 ml X (kg X min)-1, mean and SE) and 8 physically trained (VO2max 65 +/- 4 ml X (kg X min)-1) subjects. In response to hypoglycemia, levels of beta-endorphin and prolactin immunoreactivity in serum increased similarly in trained (from 41 +/- 2 pg X ml-1 and 6 +/- 1 pg X ml-1 before hypoglycemia to 103 +/- 11 pg X ml-1 and 43 +/- 9 pg X ml-1 during recovery, P less than 0.05) and untrained (from 35 +/- 7 pg X ml-1 and 7 +/- 2 pg X ml-1 to 113 +/- 18 pg X ml-1 and 31 +/- 8 pg X ml-1, P less than 0.05) subjects. Growth hormone (GH) was higher 90 min after glucose nadir in trained (61 +/- 13 mU X l-1) than in untrained (25 +/- 6 mU X l-1) subjects (P less than 0.05). Levels of thyrotropin (TSH) changed in neither of the groups. It is concluded that, in contrast to what has been formerly proposed, training does not result in a general increase in secretory capacity of the anterior pituitary gland. TSH responds to hypoglycemia neither in trained nor in untrained subjects. Finally, differences in beta-endorphin responses to exercise between trained and untrained subjects cannot be ascribed to differences in responsiveness to hypoglycemia.     

Counterregulatory deficits occur within 24 h of a single hypoglycemic episode in conscious, unrestrained, chronically cannulated mice

Hypoglycemia-induced counterregulatory failure is a dangerous complication of insulin use in diabetes mellitus. Controlled hypoglycemia studies in gene knockout models, which require the use of mice, would aid in identifying causes of defective counterregulation. Because stress can influence counterregulatory hormones and glucose homeostasis, we developed glucose clamps with remote blood sampling in conscious, unrestrained mice. Male C57BL/6 mice implanted with indwelling carotid artery and jugular vein catheters were subjected to 2 h of hyperinsulinemic glucose clamps 24 h apart, with a 6-h fast before each clamp. On day 1, blood glucose was maintained (euglycemia, 178 +/- 4 mg/dl) or decreased to 62 +/- 1 mg/dl (hypoglycemia) by insulin (20 mU x kg(-1) x min(-1)) and variable glucose infusion. Donor blood was continuously infused to replace blood sample volume. Baseline plasma epinephrine (32 +/- 8 pg/ml), corticosterone (16.1 +/- 1.8 microg/dl), and glucagon (35 +/- 3 pg/ml) were unchanged during euglycemia but increased significantly during hypoglycemia, with a glycemic threshold of approximately 80 mg/dl. On day 2, all mice underwent a hypoglycemic clamp (blood glucose, 64 +/- 1 mg/dl). Compared with mice that were euglycemic on day 1, previously hypoglycemic mice had significantly higher glucose requirements and significantly lower plasma glucagon and corticosterone (n = 6/group) on day 2. Epinephrine tended to decrease, although not significantly, in repeatedly hypoglycemic mice. Pre- and post-clamp insulin levels were similar between groups. We conclude that counterregulatory responses to acute and repeated hypoglycemia in unrestrained, chronically cannulated mice reproduce aspects of counterregulation in humans, and that repeated hypoglycemia in mice is a useful model of counterregulatory failure.