Hindbrain catecholamine regulation of ventromedial hypothalamic nucleus glycogen metabolism during acute versus recurring insulin-induced hypoglycemia in male versus female rat

Abstract

Ventromedial hypothalamic nucleus (VMN) glycogen metabolism affects local glucoregulatory signaling. The hindbrain metabolic-sensitive catecholamine (CA) neurotransmitter norepinephrine controls VMN glycogen phosphorylase (GP)-muscle (GPmm) and -brain (GPbb) type expression in male rats. Present studies addressed the premise that CA regulation of hypoglycemic patterns of VMN glycogen metabolic enzyme protein expression is sex-dimorphic, and that this signal is responsible for sex differences in acclimation of these profiles to recurrent insulin-induced hypoglycemia (RIIH). VMN tissue was acquired by micropunch-dissection from male and female rats pretreated by caudal fourth ventricular administration of the CA neurotoxin 6-hydroxydopamine (6OHDA) before single or serial insulin injection. 6-OHDA averted acute hypoglycemic inhibition of VMN glycogen synthase (GS) and augmentation of GPmm and GPbb protein expression in males, and prevented GPmm and -bb down-regulation in females. Males recovered from antecedent hypoglycemia (AH) exhibited neurotoxin-preventable diminution of baseline GS profiles, whereas acclimated GPmm and -bb expression in females occurred irrespective of pretreatment. RIIH did not alter VMN GS, GPmm, and GPbb expression in vehicle- or 6-OHDA-pretreated animals of either sex. VMN glycogen content was correspondingly unchanged or increased in males versus females following AH; 6-OHDA augmented glycogen mass in AH-exposed animals of both sexes. RIIH did not alter VMN glycogen accumulation in vehicle-pretreated rats of either sex, but diminished glycogen in neurotoxin-pretreated animals. AH suppresses baseline GS (CA-dependent) or GPmm/GPbb (CA-independent) expression in male and female rats, respectively, which corresponds with unaltered or augmented VMN glycogen content in those sexes. AH-associated loss of sex-distinctive CA-mediated enzyme protein sensitivity to hypoglycemia (male: GS, GPmm, GPbb; female: GPmm, Gpbb) may reflect, in part, VMN target desensitization to noradrenergic input.

Keywords: 6-hydroxydopamine; Glycogen phosphorylase; Glycogen synthase; Recurrent insulin-induced hypoglycemia; Sex differences; Ventromedial hypothalamic nucleus.

Fig. 1.

Experimental Design. Rats of each sex were randomly assigned to groups that were pretreated by neurotoxin (N) versus vehicle (V) injection into the caudal fourth ventricle on study days 1 and 2. On days 3–6, groups were injected subcutaneously with either insulin (I) or vehicle (V) to expose animals to single or serial hypoglycemia.

Fig. 2.

Effects of Caudal Fourth Ventricular (CV4) Administration of the Catecholamine Neurotoxin 6-Hydroxydopamine (6-OHDA) on Ventromedial Hypothalamic Nucleus (VMN) Glycogen Synthase (GS) Protein Expression during Single versus Serial Exposure to Insulin-Induced Hypoglycemia (IIH) in Male versus Female Rats. Groups of testes-intact and ovariectomized, estradiol-implanted female rats were pretreated by intra-CV4 administration on study days 1 and 2 of 6-OHDA (N) or vehicle (V; sterile apyrogenic water) prior to subcutaneous (sc) insulin (I; 10.0 U/kg bw) injection on one or four consecutive days (study days 3–6) according to the following treatment paradigm: sc vehicle (V; sterile diluent) injection on 3–6 (VVVV); sc V injection on study days 3–5, I treatment on day 6 (VVVI); sc I injection on days 3–5, V treatment on day 6 (IIIV); sc I injection on study days 3–6 (IIII). Data depict mean normalized GS protein optical density (O.D.) values + S.E.M. for micropunch-dissected VMN tissue acquired from groups of VVVV, VVVI, IIIV, and IIII male (Panel A) or female (Panel B) rats. In each Panel, groups given V or 6-OHDA pretreatment are depicted at left versus right, respectively. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 3.

Effects of Antecedent Hypoglycemia Exposure on Baseline and Hypoglycemic Patterns of VMN Glycogen Phosphorylase-Muscle Type (GPmm) Protein Expression in Male and Female Rats: Impact of Hindbrain 6-OHDA Pretreatment. Panels A (male rats) and B (female rats) depict mean normalized VMN GPmm O.D. values + S.E.M. for groups of rats pretreated with V (left-hand side) versus N (right-hand side) on days 1 and 2 prior to subcutaneous VVVV, VVVI, IIIV, or IIII injection regimens on days 3–6. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 4.

Hindbrain Neurotoxin Pretreatment Effects on VMN Glycogen Phosphorylase-Brain Type (GPbb) Protein Responses to Single versus Serial IIH Exposure in Male and Female Rats. Data depict mean normalized VMN GPbb O.D. values + S.E.M. for groups of testes-intact male (Panel A) or ovariectomized, estradiol-implanted female (Panel B) rats pretreated with V (left-hand side) versus N (right-hand side) prior to subcutaneous VVVV, VVVI, IIIV, or IIII injection regimens. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 5.

Effects of Acute or Recurring IIH on VMN Glycogen Content in Male versus Female Rats: Impact of 6-OHDA Pretreatment. Panels A (male rats) and B (female rats) depict mean VMN glycogen concentrations + S.E.M. for groups of rats pretreated with V (left-hand side) versus N (right-hand side) on days 1 and 2 prior to subcutaneous VVVV, VVVI, IIIV, or IIII injection regimens on days 3–6. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.