Naloxone, but not valsartan, preserves responses to hypoglycemia after antecedent hypoglycemia: role of metabolic reprogramming in counterregulatory failure

Abstract

Objective: Hypoglycemia-associated autonomic failure (HAAF) constitutes one of the main clinical obstacles to optimum treatment of type 1 diabetes. Neurons in the ventromedial hypothalamus are thought to mediate counterregulatory responses to hypoglycemia. We have previously hypothesized that hypoglycemia-induced hypothalamic angiotensin might contribute to HAAF, suggesting that the angiotensin blocker valsartan might prevent HAAF. On the other hand, clinical studies have demonstrated that the opioid receptor blocker naloxone ameliorates HAAF. The goal of this study was to generate novel hypothalamic markers of hypoglycemia and use them to assess mechanisms mediating HAAF and its reversal.

Research design and methods: Quantitative PCR was used to validate a novel panel of hypothalamic genes regulated by hypoglycemia. Mice were exposed to one or five episodes of insulin-induced hypoglycemia, with or without concurrent exposure to valsartan or naloxone. Corticosterone, glucagon, epinephrine, and hypothalamic gene expression were assessed after the final episode of hypoglycemia.

Results: A subset of hypothalamic genes regulated acutely by hypoglycemia failed to respond after repetitive hypoglycemia. Responsiveness of a subset of these genes was preserved by naloxone but not valsartan. Notably, hypothalamic expression of four genes, including pyruvate dehydrogenase kinase 4 and glycerol 3-phosphate dehydrogenase 1, was acutely induced by a single episode of hypoglycemia, but not after antecedent hypoglycemia; naloxone treatment prevented this failure. Similarly, carnitine palmitoyltransferase-1 was inhibited after repetitive hypoglycemia, and this inhibition was prevented by naloxone. Repetitive hypoglycemia also caused a loss of hypoglycemia-induced elevation of glucocorticoid secretion, a failure prevented by naloxone but not valsartan.

Conclusions: Based on these observations we speculate that acute hypoglycemia induces reprogramming of hypothalamic metabolism away from glycolysis toward β-oxidation, HAAF is associated with a reversal of this reprogramming, and naloxone preserves some responses to hypoglycemia by preventing this reversal.

FIG. 1.

Blood glucose concentration throughout the study for all experimental groups: Eu, 1XH, 5XH, Eu-V, 1XH-V, 5XH-V, 5XH-N. The x-axis indicates the day and time points of the study (with insulin or saline injected at time 0, and for the 5XH-N group, naloxone was injected 15 min prior). Data are means ± SE (n = 10 for all groups).

FIG. 2.

Counterregulatory hormones in mice exposed to antecedent hypoglycemia and naloxone (2 mg/kg) or valsartan (40 mg/kg/day). Trunk blood was collected at the time the animals were killed, 4 h after final insulin or saline injection. Hormone levels were measured by ELISA in the same groups as described in Fig. 1. Data are means ± SE (n = 10 for all groups). *P < 0.05 as compared with the euglycemic group (Dunnett test).

FIG. 3.

Real-time qPCR data for murine hypothalamic genes that do not correlate with counterregulatory failure or its reversal by naloxone or valsartan. Relative expression levels of (A) Hif3a, (B) S3-12, and (C) GLUT1 were assessed using custom PCR arrays in the same groups described in Fig. 1. Animals were killed 4 h after the final insulin or saline injection. Data for each gene were normalized to a panel of housekeeping transcripts and expressed as fold change compared with the saline-injected (euglycemic) group. Data are means ± SE (n = 6 for all groups). *P < 0.05 as compared with the euglycemic group (Dunnett test).

FIG. 4.

Real-time qPCR data for murine hypothalamic genes that fail to respond after antecedent hypoglycemia, and the failure was prevented by naloxone, but not valsartan. Relative expression levels of (A) Pdk4, (B) Gpd1, (C) Angptl4, and (D) Cdkn1a were assessed using custom PCR arrays from SABiosciences in the same groups described in Fig. 1. Animals were killed 4 h after the final insulin or saline injection. Data for each gene were normalized to a panel of housekeeping transcripts and expressed as fold change compared with the saline-injected (euglycemic) group. Data are means ± SE (n = 6 for all groups). *P < 0.05 as compared with the euglycemic group (Dunnett test).

FIG. 5.

Real-time qPCR data for murine hypothalamic genes whose regulation by hypoglycemia were not impaired by antecedent hypoglycemia but were prevented by naloxone, though not by valsartan. Relative expression levels of (A) Gpd2 (glycerol 3-phosphate dehydrogenase 2, mitochondrial), (B) Cxcl14, and (C) Sox17 were assessed using custom PCR arrays from SABiosciences in the same groups described in Fig. 1. Animals were killed 4 h after the final insulin or saline injection. Data for each gene were normalized to a panel of housekeeping transcripts and expressed as fold change compared with the saline-injected (euglycemic) group. Data are means ± SE (n = 6 for all groups). *P < 0.05 as compared with the euglycemic group (Dunnett test).

FIG. 6.

Hypothalamic expression of Cpt1a was significantly regulated by repetitive hypoglycemia but not acute hypoglycemia, and this inhibition was prevented by naloxone. Relative expression level of Cpt1a (carnitine palmitoyltransferease 1a, liver) was assessed with custom PCR arrays from SABiosciences in the same groups described in Fig. 1. Animals were killed 4 h after the final insulin or saline injection. Data for each gene were normalized to a panel of housekeeping transcripts and expressed as fold change compared with the saline-injected (euglycemic) group. Data are means ± SE (n = 6 for all groups). *P < 0.05 as compared with the euglycemic group (Dunnett test).

FIG. 7.

Hypothalamic expression of genes whose induction by acute hypoglycemia was prevented by antecedent hypoglycemia but not maintained by exposure to naloxone. Relative expression levels of (A) Ucp2 (uncoupling protein 2) and (B) Pnpla2 (patatin-like phospholipase domain containing 2) were assessed with custom PCR arrays from SABiosciences in the same groups described in Fig. 1. Animals were killed 4 h after the final insulin or saline injection. Data for each gene were normalized to a panel of housekeeping transcripts and expressed as fold change compared with the saline-injected (euglycemic) group. Data are means ± SE (n = 6 for all groups). *P < 0.05 as compared with the euglycemic group (Dunnett test).