A role for the forebrain in mediating time-of-day differences in glucocorticoid counterregulatory responses to hypoglycemia in rats.

The time of day influences the magnitude of ACTH and corticosterone responses to hypoglycemia. However, little is known about the mechanisms that impart these time-of-day differences on neuroendocrine CRH neurons in the hypothalamic paraventricular nucleus (PVH). Rats received 0-3 U/kg insulin (or 0.9% saline) to achieve a range of glucose nadir concentrations. Brains were processed to identify phosphorylated ERK1/2 (phospho-ERK1/2)-immunoreactive cells in the PVH and hindbrain and CRH heteronuclear RNA in the PVH. Hypoglycemia did not stimulate ACTH and corticosterone responses in animals unless a glucose concentration of approximately 3.15 mM or below was reached. Critically the glycemic thresholds required to stimulate ACTH and corticosterone release in the morning and night were indistinguishable. Yet glucose concentrations below the estimated glycemic threshold correlated with a greater increase in corticosterone, ACTH, and phospho-ERK1/2-immunoreactive neurons in the PVH at night, compared with morning. In these same animals, the number of phospho-ERK1/2-immunoreactive neurons in the medial part of the nucleus of the solitary tract was unchanged at both times of day. These data collectively support a model whereby changes in forebrain mechanisms alter the sensitivity of neuroendocrine CRH to the hypoglycemia-related information conveyed by ascending catecholaminergic afferents. Circadian clock-driven processes together with glucose-sensing elements in the forebrain would seem to be strong contenders for mediating these effects.

Activation of hindbrain neurons is mediated by portal-mesenteric vein glucosensors during slow-onset hypoglycemia.

Hypoglycemic detection at the portal-mesenteric vein (PMV) appears mediated by spinal afferents and is critical for the counter-regulatory response (CRR) to slow-onset, but not rapid-onset, hypoglycemia. Since rapid-onset hypoglycemia induces Fos protein expression in discrete brain regions, we hypothesized that denervation of the PMV or lesioning spinal afferents would suppress Fos expression in the dorsal medulla during slow-onset hypoglycemia, revealing a central nervous system reliance on PMV glucosensors. Rats undergoing PMV deafferentation via capsaicin, celiac-superior mesenteric ganglionectomy (CSMG), or total subdiaphragmatic vagotomy (TSV) were exposed to hyperinsulinemic-hypoglycemic clamps where glycemia was lowered slowly over 60-75 min. In response to hypoglycemia, control animals demonstrated a robust CRR along with marked Fos expression in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus. Fos expression was suppressed by 65-92% in capsaicin-treated animals, as was epinephrine (74%), norepinephrine (33%), and glucagon (47%). CSMG also suppressed Fos expression and CRR during slow-onset hypoglycemia, whereas TSV failed to impact either. In contrast, CSMG failed to impact upon Fos expression or the CRR during rapid-onset hypoglycemia. Peripheral glucosensory input from the PMV is therefore required for activation of hindbrain neurons and the full CRR during slow-onset hypoglycemia.

The locus for hypoglycemic detection shifts with the rate of fall in glycemia: the role of portal-superior mesenteric vein glucose sensing.

OBJECTIVE: To ascertain whether portal glucose sensing extends beyond the portal vein to the superior mesenteric vein and then test whether the role of portal-superior mesenteric glucose sensors varies with the rate of fall in glycemia. RESEARCH DESIGN AND METHODS: Chronically cannulated rats underwent afferent ablation of the portal vein (PV) or portal and superior mesenteric veins (PMV) or sham operation (control). One week later, animals underwent hyperinsulinemic-hypoglycemic clamps in which the hypoglycemic nadir, 2.48 +/- 0.06 mmol/l, was reached at a rate of decline in glucose of -0.09 or -0.21 mmol x l(-1) x min(-1) (PMV and control only). Additional PMV and control animals received an intravenous injection of the glucopenic agent 2-deoxyglucose. RESULTS; Inducing hypoglycemia slowly, at a rate of -0.09 mmol x l(-1) x min(-1), resulted in a 26-fold increase in epinephrine (23.39 +/- 0.62 nmol/l) and 12-fold increase in norepinephrine (11.42 +/- 0.92 nmol/l) for controls (P < 0.001). The epinephrine response to hypoglycemia was suppressed by 91% in PMV (2.09 +/- 0.07 nmol/l) vs. 61% in PV (9.05 +/- 1.59 nmol/l) (P < 0.001). The norepinephrine response to hypoglycemia was suppressed by 94 and 80% in PMV and PV, respectively, compared with that in controls. In contrast, when arterial glucose was lowered to 2.49 +/- 0.06 mmol/l within 20 min, no significant differences were observed in the catecholamine responses for PMV and controls over the first 45 min of hypoglycemia (20-65 min). Only at min 105 were catecholamines significantly lower for PMV vs. controls. Injection of 2-deoxyglucose induced a very rapid sympathoadrenal response with no significant differences between PMV and controls. CONCLUSIONS: The critical locus for hypoglycemic detection shifts away from the portal-mesenteric vein to some other loci (e.g., the brain) when hypoglycemia develops rapidly.

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.

Hypoglycemic detection at the portal vein is mediated by capsaicin-sensitive primary sensory neurons.

To elucidate the type of spinal afferent involved in hypoglycemic detection at the portal vein, we considered the potential role of capsaicin-sensitive primary sensory neurons. Specifically, we examined the effect of capsaicin-induced ablation of portal vein afferents on the sympathoadrenal response to hypoglycemia. Under anesthesia, the portal vein was isolated in rats and either capsaicin (CAP) or the vehicle (CON) solution applied topically. During the same surgery, the carotid artery (sampling) and jugular vein (infusion) were cannulated. One week later, all animals underwent a hyperinsulinemic hypoglycemic clamp, with glucose (variable) and insulin (25 mU x kg(-1) x min(-1)) infused via the jugular vein. Systemic hypoglycemia (2.76 +/- 0.05 mM) was induced by minute 75 and sustained until minute 105. By design, no significant differences were observed in arterial glucose or insulin concentrations between groups. When hypoglycemia was induced in CON, the plasma epinephrine concentration increased from 0.67 +/- 0.05 nM at basal to 36.15 +/- 2.32 nM by minute 105. Compared with CON, CAP animals demonstrated an 80% suppression in epinephrine levels by minute 105, 7.11 +/- 0.55 nM (P < 0.001). A similar response to hypoglycemia was observed for norepinephrine, with CAP values suppressed by 48% compared with CON. Immunohistochemical analysis of the portal vein revealed an 85% decrease in the number of calcitonin gene-related peptide-reactive nerve fibers following capsaicin-induced ablation. That the suppression in the sympathoadrenal response was comparable to our previous findings for total denervation of the portal vein indicates that hypoglycemic detection at the portal vein is mediated by capsaicin-sensitive primary sensory neurons.