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.     

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.     

PVN activation is suppressed by repeated hypoglycemia but not antecedent corticosterone in the rat

The mechanism(s) underlying hypoglycemia-associated autonomic failure (HAAF) are unknown. To test the hypothesis that the activation of brain regions involved in the counterregulatory response to hypoglycemia is blunted with HAAF, rats were studied in a 2-day protocol. Neuroendocrine responses and brain activation (c-Fos immunoreactivity) were measured during day 2 insulin-induced hypoglycemia (0.5 U insulin x 100 g body x wt(-1) x h(-1) iv for 2 h) after day 1 hypoglycemia (Hypo-Hypo) or vehicle. Hypo-Hypo animals demonstrated HAAF with blunted epinephrine, glucagon, and corticosterone (Cort) responses and decreased activation of the medial hypothalamus [the paraventricular (PVN), dorsomedial (DMH), and arcuate (Arc) nuclei]. To evaluate whether increases in day 1 Cort were responsible for the decreased hypothalamic activation, Cort was infused intracerebroventricularly (72 microg) on day 1 and the response to day 2 hypoglycemia was measured. Intracerebroventricular Cort infusion failed to alter the neuroendocrine response to day 2 hypoglycemia, despite elevating both central nervous system and peripheral Cort levels. However, day 1 Cort blunted responses in two of the same hypothalamic regions as Hypo-Hypo (the DMH and Arc) but not in the PVN. These results suggest that decreased activation of the PVN may be important in the development of HAAF and that antecedent exposure to elevated levels of Cort is not always sufficient to produce HAAF.     

Effects of oral carbohydrate on autonomic nervous system counterregulatory responses during hyperinsulinemic hypoglycemia and euglycemia

The effects of oral carbohydrate on modulating counterregulatory responses in humans remain undecided. This study's specific aim was to determine the effects of oral carbohydrate on autonomic nervous system (ANS) and neuroendocrine responses during hyperinsulinemic hypoglycemia and euglycemia. Nineteen healthy volunteers were studied during paired, single blind experiments. Nine subjects underwent two-step glucose clamps consisting of 60 min of euglycemia (5.0 mmol/l) followed by either 15 g of oral carbohydrate (cal) as orange juice or a noncaloric control (nocal) and subsequent 90 min of clamped hypoglycemia (2.9 mmol/l). Ten other subjects underwent two randomized 150-min hyperinsulinemic-euglycemic clamps with cal or nocal control administered at 60 min. Oral carbohydrate initially blunted (P < 0.05) epinephrine, norepinephrine, cortisol, glucagon, pancreatic polypeptide, muscle sympathetic nerve activity (MSNA), symptom, and systolic blood pressure responses during hypoglycemia. However, by the end of 90 min of hypoglycemia, plasma epinephrine and norepinephrine responses had rebounded and were increased (P < 0.05) compared with control. MSNA and cortisol levels remained suppressed during hypoglycemia (P < 0.05) after cal, whereas pancreatic polypeptide, glucagon, symptom, and blood pressure responses increased similar to control following initial suppression. Oral carbohydrate had no effects on neuroendocrine or ANS responses during hyperinsulinemic euglycemia. These results demonstrate that oral carbohydrate can have differential effects on the time course of ANS and neuroendocrine responses during hypoglycemia. We conclude that gastro-splanchnic-portal sensing of an amount of carbohydrate recommended for use in clinical practice for correction of hypoglycemia can have widespread and significant effects on central nervous system mediated counterregulatory responses in healthy humans.     

Glucocorticoid-deficient corticotropin-releasing hormone knockout mice maintain glucose requirements but not autonomic responses during repeated hypoglycemia

Glucocorticoids have been implicated in hypoglycemia-induced autonomic failure but also contribute to normal counterregulation. To determine the influence of normal and hypoglycemia-induced levels of glucocorticoids on counterregulatory responses to acute and repeated hypoglycemia, we compared plasma catecholamines, corticosterone, glucagon, and glucose requirements in male wild-type (WT) and glucocorticoid-deficient, corticotropin-releasing hormone knockout (CRH KO) mice. Conscious, chronically cannulated, unrestrained WT and CRH KO mice underwent a euglycemic (Prior Eu) or hypoglycemic clamp (Prior Hypo) on day 1 followed by a hypoglycemic clamp on day 2 (blood glucose both days, 65 +/- 1 mg/dl). Baseline epinephrine and glucagon were similar, and norepinephrine was elevated, in CRH KO vs. WT mice. CRH KO corticosterone was almost undetectable (<1.5 microg/dl) and unresponsive to hypoglycemia. CRH KO glucose requirements were significantly higher during day 1 hypoglycemia despite epinephrine and glucagon responses that were comparable to or greater than those in WT. Hyperinsulinemic euglycemia did not increase hormones or glucose requirements above baseline. On day 2, Prior Hypo WT had significantly higher glucose requirements and significantly lower corticosterone and glucagon responses. Prior Hypo and Prior Eu CRH KO mice had similar day 2 glucose requirements. However, Prior Hypo CRH KO mice had significantly lower day 2 epinephrine and norepinephrine vs. Prior Eu CRH KO and tended to have lower glucagon than on day 1. We conclude that glucocorticoid insufficiency in CRH KO mice correlates with 1) impaired counterregulation during acute hypoglycemia and 2) complex effects after repeated hypoglycemia, neither preventing decreased hormone responses nor worsening glucose requirements.     

Inactivation of the DMH selectively inhibits the ACTH and corticosterone responses to hypoglycemia

We have previously reported that repeated bouts of insulin-induced hypoglycemia (IIH) in the rat result in blunted activation of the paraventricular, arcuate, and dorsomedial hypothalamic (DMH) nuclei. Because DMH activation has been implicated in the sympathoadrenal and hypothalamic-pituitary-adrenal (HPA) responses to stressors, we hypothesized that its blunted activation may play a role in the impaired counterregulatory response that is also observed with repeated bouts of IIH. In the present study, we evaluated the role of normal DMH activation in the counterregulatory response to a single bout of IIH. Local infusion of lidocaine (n = 8) to inactivate the DMH during a 2-h bout of IIH resulted in a significant overall decrease of the ACTH response and a delay of onset of the corticosterone response compared with vehicle-infused controls (n = 9). We observed suppression of the ACTH response at time (t) = 90 and 120 min (50 +/- 12 and 63 +/- 6%, respectively, of control levels) and early suppression of the corticosterone response at t = 30 min (59 +/- 13% of the control level). The epinephrine, norepinephrine, and glucagon responses were not altered by DMH inactivation. Our finding suggests that DMH inactivation may play a specific role in decreasing the HPA axis response after repeated bouts of IIH.     

Enhanced activation of RVLM-projecting PVN neurons in rats with chronic heart failure

Previous studies have indicated that there is increased activation of the paraventricular nucleus (PVN) in rats with chronic heart failure (CHF); however, it is not clear if the preautonomic neurons within the PVN are specifically overactive. Also, it is not known if these neurons have altered responses to baroreceptor or osmotic challenges. Experiments were conducted in rats with CHF (6-8 wk after coronary artery ligation). Spontaneously active neurons were recorded in the PVN, of which 36% were antidromically activated from the rostral ventrolateral medulla (RVLM). The baseline discharge rate in RVLM-projecting PVN (PVN-RVLM) neurons from CHF rats was significantly greater than in sham-operated (sham) rats (6.0 ± 0.6 vs. 2.6 ± 0.3 spikes/s, P < 0.05). Picoinjection of the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphonovaleric acid significantly decreased the basal discharge of PVN-RVLM neurons by 80% in CHF rats compared with 37% in sham rats. Fifty-two percent of spontaneously active PVN-RVLM neurons responded to changes in the mean arterial pressure (MAP). The changes in discharge rate in PVN-RVLM neurons after a reduction in MAP (+52 ± 7% vs. +184 ± 61%) or an increase in MAP (-42 ± 8% vs. -71 ± 6%) were significantly attenuated in rats with CHF compared with sham rats. Most PVN-RVLM neurons (63%), including all barosensitive PVN-RVLM neurons, were excited by an internal carotid artery injection of hypertonic NaCl (2.1 osmol/l), whereas a smaller number (7%) were inhibited. The increase in discharge rate in PVN-RVLM neurons to hypertonic stimulation was significantly enhanced in rats with CHF compared with sham rats (134 ± 15% vs. 92 ± 13%). Taken together, these data suggest that PVN-RVLM neurons are more active under basal conditions and this overactivation is mediated by an enhanced glutamatergic tone in rats with CHF. Furthermore, this enhanced activation of PVN-RVLM neurons may contribute to the altered responses to baroreceptor and osmotic challenges observed during CHF.     

Exercise training normalizes enhanced glutamate-mediated sympathetic activation from the PVN in heart failure

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the mechanisms are not known. We hypothesized that ExT would normalize the augmented glutamatergic mechanisms mediated by N-methyl-d-aspartic acid (NMDA) receptors within the paraventricular nucleus (PVN) that occur with HF. Four groups of rats were used: 1) sham-operated (Sham) sedentary (Sed), 2) Sham ExT, 3) HF Sed, and 4) HF ExT. HF was induced by left coronary artery ligation, and ExT consisted of 3 wk of treadmill running. In alpha-chloralose-urethane-anesthetized rats, the increase in renal sympathetic nerve activity in response to the highest dose of NMDA (200 pmol) injected into the PVN in the HF Sed group was approximately twice that of the Sham Sed group. In the HF ExT group the response was not different from the Sham Sed and Sham ExT groups. Relative NMDA NR1 receptor subunit mRNA expression was 63% higher in the HF Sed group compared with the Sham Sed group but in the HF ExT group was not different from the Sham Sed and Sham ExT groups. NR1 receptor subunit protein expression was increased 87% in the HF Sed group compared with the Sham Sed group but in the HF ExT group was not significantly different from the Sham Sed and Sham ExT groups. Thus one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of glutamatergic mechanisms within the PVN.     

Leg vasoconstriction during head-up tilt in patients with autonomic failure is not abolished

Maintaining blood pressure during orthostatic challenges is primarily achieved by baroreceptor-mediated activation of the sympathetic nervous system, which can be divided into preganglionic and postganglionic parts. Despite their preganglionic autonomic failure, spinal cord-injured individuals demonstrate a preserved peripheral vasoconstriction during orthostatic challenges. Whether this also applies to patients with postganglionic autonomic failure is unknown. Therefore, we assessed leg vasoconstriction during 60° head-up tilt in five patients with pure autonomic failure (PAF) and two patients with autonomic failure due to dopamine-β-hydroxylase (DBH) deficiency. Ten healthy subjects served as controls. Leg blood flow was measured using duplex ultrasound in the right superficial femoral artery. Leg vascular resistance was calculated as the arterial-venous pressure gradient divided by blood flow. DBH-deficient patients were tested off and on the norepinephrine pro-drug l-threo-dihydroxyphenylserine (l-DOPS). During 60° head-up tilt, leg vascular resistance increased significantly in PAF patients [0.40 ± 0.38 (+30%) mmHg·ml(-1)·min(-1)]. The increase in leg vascular resistance was not significantly different from controls [0.88 ± 1.04 (+72%) mmHg·ml(-1)·min(-1)]. In DBH-deficient patients, leg vascular resistance increased by 0.49 ± 0.01 (+153%) and 1.52 ± 1.47 (+234%) mmHg·ml(-1)·min(-1) off and on l-DOPS, respectively. Despite the increase in leg vascular resistance, orthostatic hypotension was present in PAF and DBH-deficient patients. Our results demonstrate that leg vasoconstriction during orthostatic challenges in patients with PAF or DBH deficiency is not abolished. This indicates that the sympathetic nervous system is not the sole or pivotal mechanism inducing leg vasoconstriction during orthostatic challenges. Additional vasoconstrictor mechanisms may compensate for the loss in sympathetic nervous system control.     

Water deprivation increases Fos immunoreactivity in PVN autonomic neurons with projections to the spinal cord and rostral ventrolateral medulla

The present study sought to determine whether water deprivation increases Fos immunoreactivity, a neuronal marker related to synaptic activation, in sympathetic-regulatory neurons of the hypothalamic paraventricular nucleus (PVN). Fluorogold (4%, 50 nl) and cholera toxin subunit B (0.25%, 20-30 nl) were microinjected into the spinal cord (T1-T3) and rostral ventrolateral medulla (RVLM), respectively. Rats were then deprived of water but not food for 48 h. Water deprivation significantly increased the number of Fos-positive nuclei throughout the dorsal, ventrolateral, and lateral parvocellular divisions of the PVN (water deprived, 215 +/- 23 cells; control, 45 +/- 7 cells, P < 0.01). Moreover, a significantly greater number of Fos-positive nuclei were localized in spinally projecting (11 +/- 3 vs. 2 +/- 1 cells, P < 0.025) and RVLM-projecting (45 +/- 7 vs. 7 +/- 1 cells, P < 0.025) neurons of the PVN in water-deprived vs. control rats, respectively. The majority of these double-labeled neurons was found in the ventrolateral and lateral parvocellular divisions of the ipsilateral PVN. Interestingly, a significantly greater percentage of RVLM-projecting PVN neurons were Fos positive compared with spinally projecting PVN neurons in the ventrolateral (25.8 +/- 0.7 vs. 8.0 +/- 1.5%, respectively, P < 0.01) and lateral (23.4 +/- 2.1 vs. 5.0 +/- 0.9%, respectively, P > 0.01) parvocellular divisions. In addition, we analyzed spinally projecting neurons of the RVLM and found a significantly greater percentage were Fos positive in water-deprived rats than in control rats (26 +/- 3 vs. 3 +/- 1%, respectively; P < 0.001). Collectively, the present findings indicate that water deprivation evokes a distinct cellular response in sympathetic-regulatory neurons of the PVN and RVLM.     

Decreased response of epinephrine and norepinephrine to insulin-induced hypoglycemia in diabetic autonomic neuropathy

The responses of epinephrine, norepinephrine and other counter-regulatory hormones to insulin-induced hypoglycemia were investigated in 5 diabetics who showed signs of autonomic neuropathy, in 7 age-matched diabetics without autonomic neuropathy and in 7 healthy subjects. The presence of autonomic neuropathy was evaluated by decreased beat-to-beat variation in heat rates during hyperventilation or orthostatic hypotension. Catecholamines were determined by a totally automated plasma catecholamine analyzing system using a two-column system of high performance liquid chromatography. Plasma epinephrine and norepinephrine responses to hypoglycemia in diabetics with autonomic neuropathy were significantly lower than those in diabetics without autonomic neuropathy. Plasma glucagon response in diabetics was apparently attenuated compared to normal controls and there was no significant difference in glucagon response between the two patient groups. Other counter-regulatory hormone responses did not differ among the three groups. The data demonstrate that the responses of plasma epinephrine and norepinephrine to insulin-induced hypoglycemia are impaired in diabetics with autonomic neuropathy.     

Blunted nitric oxide-mediated inhibition of renal nerve discharge within PVN of rats with heart failure

We have demonstrated a decreased neuronal nitric oxide (NO) synthase (nNOS) message in the hypothalamus of rats with heart failure (HF). Subsequently, we have demonstrated that NADPH diaphorase (a commonly used marker for nNOS activity) positive neurons are decreased in paraventricular nucleus (PVN) of rats with coronary artery ligation model of HF. The goal of the present study was to examine the influence of endogenous NO within the PVN on renal sympathetic nerve discharge (RSND) during HF. In alpha-chloralose- and urethane-anesthetized rats, an inhibitor of NO synthase, N(G)-monomethyl-L-arginine (L-NMMA) microinjected into the PVN (50, 100, and 200 pmol in 50-200 nl) produced a dose-dependent increase in RSND, blood pressure, and heart rate in control and HF rats. These responses were attenuated in rats with HF compared with control rats. On the other hand, the NO agonist, sodium nitroprusside, microinjected in PVN produced a dose-dependent decrease in RSND and blood pressure in control and HF rats. These responses were less in rats with HF compared with control rats. These data suggest that the endogenous NO-mediated effect within the PVN of HF rats is less potent in suppressing RSND compared with control rats. These data support the conclusion that the NO system within the PVN involved in controlling autonomic outflow is altered during HF and may contribute to the elevated levels of renal sympathoexcitation commonly observed in HF.     

Reduced endogenous GABA-mediated inhibition in the PVN on renal nerve discharge in rats with heart failure

One characteristic of heart failure (HF) is increased sympathetic activation. The paraventricular nucleus (PVN) of the hypothalamus (involved in control of sympathetic outflow) has been shown to have increased neuronal activation during HF. This study examined the influence of endogenous GABA input (inhibitory in nature) into the PVN on renal sympathetic nerve discharge (RSND), arterial blood pressure (BP), and heart rate (HR) in rats with HF induced by coronary artery ligation. In alpha-chloralose- and urethane-anesthetized rats, microinjection of bicuculline (a GABA antagonist) into the PVN produced a dose-dependent increase in RSND, BP, and HR in both sham-operated control and HF rats. Bicuculline attenuated the increase in RSND and BP in HF rats compared with control rats. Alternatively, microinjection of the GABA agonist muscimol produced a dose-dependent decrease in RSND, BP, and HR in both control and HF rats. Muscimol was also less effective in decreasing RSND, BP, and HR in HF rats than in control rats. These results suggest that endogenous GABA-mediated input into the PVN of rats with HF is less effective in suppressing RSND and BP compared with control rats. This is partly due to the post-release actions of GABA, possibly caused by altered function of post-synaptic GABA receptors in the PVN of rats with HF. Reduced GABA-mediated inhibition in the PVN may contribute to increased sympathetic outflow, which is commonly observed during HF.     

Intrinsic autonomic innervation of the stomach during aging

In the senescence the cholinergic network of the basal and interglandular lamina propria and of the muscularis mucosae and submucosa of the gastric wall undergoes small modifications, while the adrenergic innervation decreases severely in the muscularis mucosae and in the interglandular tissue. Having regard to this adrenergic failure we note histochemically a rupture of the cholinergic/adrenergic balance in favour of the cholinergic innervation. These modifications disturb the gastrointestinal motility but their influence on the secretory reflex has to be demonstrated.     

Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy is a disorder of glucose homeostasis and is characterized by unregulated insulin secretion and profound hypoglycemia. Loss-of-function mutations in the second nucleotide-binding fold of the sulfonylurea receptor, a subunit of the pancreatic-islet beta-cell ATP-dependent potassium channel, has been demonstrated to be causative for persistent hyperinsulinemic hypoglycemia of infancy. We now describe three additional mutations in the first nucleotide-binding fold of the sulfonylurea-receptor gene. One point mutation disrupts the highly conserved Walker A motif of the first nucleotide-binding-fold region. The other two mutations occur in noncoding sequences required for RNA processing and are predicted to disrupt the normal splicing pathway of the sulfonylurea-receptor mRNA precursor. These data suggest that both nucleotide-binding-fold regions of the sulfonylurea receptor are required for normal regulation of beta-cell ATP-dependent potassium channel activity and insulin secretion.