Astrocyte Glycogen Is a Major Source of Hypothalamic Lactate in Rats With Recurrent Hypoglycemia

Abstract

Lactate is an important metabolic substrate for sustaining brain energy requirements when glucose supplies are limited. Recurring exposure to hypoglycemia (RH) raises lactate levels in the ventromedial hypothalamus (VMH), which contributes to counterregulatory failure. However, the source of this lactate remains unclear. The current study investigates whether astrocytic glycogen serves as the major source of lactate in the VMH of RH rats. By decreasing the expression of a key lactate transporter in VMH astrocytes of RH rats, we reduced extracellular lactate concentrations, suggesting excess lactate was locally produced from astrocytes. To determine whether astrocytic glycogen serves as the major source of lactate, we chronically delivered either artificial extracellular fluid or 1,4-dideoxy-1,4-imino-d-arabinitol to inhibit glycogen turnover in the VMH of RH animals. Inhibiting glycogen turnover in RH animals prevented the rise in VMH lactate and the development of counterregulatory failure. Lastly, we noted that RH led to an increase in glycogen shunt activity in response to hypoglycemia and elevated glycogen phosphorylase activity in the hours following a bout of hypoglycemia. Our data suggest that dysregulation of astrocytic glycogen metabolism following RH may be responsible, at least in part, for the rise in VMH lactate levels.

Article highlights: Astrocytic glycogen serves as the major source of elevated lactate levels in the ventromedial hypothalamus (VMH) of animals exposed to recurring episodes of hypoglycemia. Antecedent hypoglycemia alters VMH glycogen turnover. Antecedent exposure to hypoglycemia enhances glycogen shunt activity in the VMH during subsequent bouts of hypoglycemia. In the immediate hours following a bout of hypoglycemia, sustained elevations in glycogen phosphorylase activity in the VMH of recurrently hypoglycemic animals contribute to sustained elevations in local lactate levels.

Figure 1

(A–G) Immunohistochemical staining demonstrating cellular specificity of the AAV vector for targeting astrocytes in VMH sections. A and E show labeling of the AAV’s GFP reporter gene in red. B and F show the labeling of GFAP, an astrocyte marker or NeuN, a neuronal marker, in green. C and G are the merged images showing colocalization of the GFP signal with GFAP on top and distinct labeling of the GFP reporter and neurons (NeuN) on the bottom, indicating the AAV vector specifically targets expression to astrocytes and not neurons. D is a magnified image of the area outlined in C that more clearly shows the colocalization. (H) Quantitative RT-PCR analysis showing the MCT1 siRNA-expressing AAV (MCT1 siRNA; n = 4) decreased the expression of MCT1 mRNA in the VMH by ∼35% (*P < 0.05) compared with controls receiving the AAV expressing the scrambled RNA (Scrambled; n = 4). (I) Quantitative RT-PCR showing an increase in MCT1 mRNA expression in RH rats (squares; N = 8) compared with controls (circles; N = 6) and normalization of MCT1 mRNA levels in the VMH of RH animals using the astrocyte targeting AAV (RH+MCT1 siRNA; triangles; N = 6). (J) Extracellular lactate concentrations in the VMH were elevated in RH animals (squares), and these levels were restored to normal following knockdown of MCT1 (RH + MCT1 siRNA; triangles). RH increased extracellular lactate concentrations (squares) compared with hypoglycemia-naïve control animals (circles). Reducing astrocytic MCT1 expression in the VMH of recurrently hypoglycemic rats (triangles) decreased extracellular lactate levels to normal. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 vs. Scrambled/Control and RH+MCT1 siRNA; 3V, 3rd ventricle.

Figure 2

(A) Plasma glucose concentrations of all treatment groups during the glucose clamp procedure. Plasma glucose concentrations were well matched between animals either receiving a saline infusion or undergoing the hyperinsulinemic-euglycemic clamp. Plasma glucose concentrations were matched for groups undergoing the hypoglycemic clamp as well. (B) Average glucose infusion rates (GIR) during the last 20 min of the clamp procedure for all treatment groups. (C) VMH glycogen levels following the clamping procedure. (D) Basal extracellular lactate concentrations in the VMH. (E) Peak plasma glucagon concentrations during the clamping procedure. (F) Peak plasma epinephrine concentrations during the clamping procedure. C-S: N = 6; C-E: N = 6; C-H: N = 8; RH: N = 8; RH+DAB: N = 8. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Immunoblot analysis of (A) GP and (B) GS protein levels in the VMH of hypoglycemia-naïve controls (Control; N = 8) and RH (N = 8) animals. (Top) Representative immunoblot images of GP, GS and B-actin bands. (Bottom) Densitometric analysis of the immunoblots standardized to the B-actin loading controls. (C) GP and (D) GS activity in the VMH of rats exposed to a single acute bout of hypoglycemia (AH; N = 8 per time point) or after the final bout of RH (N = 8 per time point). Baseline GP and GS activity levels in the VMH were determined in a group of hypoglycemia-naïve animals (N = 8) and are demarcated by the solid line, with SEM indicated by the dotted lines. The time points indicate the amount of time proceeding the last episode of hypoglycemia. (E) The ratio of GP to GS activity standardized to that of the baseline hypoglycemia-naïve animals. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. baseline.