Lactate preserves neuronal metabolism and function following antecedent recurrent hypoglycemia

Abstract

Hypoglycemia occurs frequently during intensive insulin therapy in patients with both type 1 and type 2 diabetes and remains the single most important obstacle in achieving tight glycemic control. Using a rodent model of hypoglycemia, we demonstrated that exposure to antecedent recurrent hypoglycemia leads to adaptations of brain metabolism so that modest increments in circulating lactate allow the brain to function normally under acute hypoglycemic conditions. We characterized 3 major factors underlying this effect. First, we measured enhanced transport of lactate both into as well as out of the brain that resulted in only a small increase of its contribution to total brain oxidative capacity, suggesting that it was not the major fuel. Second, we observed a doubling of the glucose contribution to brain metabolism under hypoglycemic conditions that restored metabolic activity to levels otherwise only observed at euglycemia. Third, we determined that elevated lactate is critical for maintaining glucose metabolism under hypoglycemia, which preserves neuronal function. These unexpected findings suggest that while lactate uptake was enhanced, it is insufficient to support metabolism as an alternate substrate to replace glucose. Lactate is, however, able to modulate metabolic and neuronal activity, serving as a "metabolic regulator" instead.

Figures

Figure 1. Under hyperinsulinemic-hypoglycemic clamp conditions and [3-13C]-lactate infusion, animals preexposed to recurrent hypoglycemia show a markedly faster metabolite enrichment time course than controls.

Shown here are group averages for (A) plasma glucose levels, (B) glucose infusion rates, (C) plasma lactate concentrations during tracer infusion, (D) plasma [3-13C]-lactate enrichment, (E) brain [4-13C-]-glutamate enrichment time courses, and (F) brain [4-13C]-glutamine enrichment during tracer infusion (black squares represent control, white squares represent 3dRH; [3-13C]-lactate infusion begins at time t= 0 minutes; data reflect mean ± SEM of 6 animals per group).

Figure 2. Under hyperinsulinemic-euglycemic clamp conditions and [3-13C]-lactate infusion, control and 3dRH animals show similar metabolite enrichment time courses.

Shown here are group averages for (A) plasma glucose levels, (B) glucose infusion rates, (C) plasma lactate concentrations during [3-13C]-lactate infusion, (D) plasma 13C-lacate enrichment, (E) brain [4-13C]-glutamate enrichment time courses during [3-13C]-lactate infusion, and (F) brain [4-13C]-glutamine enrichment during [3-13C]-lactate infusion (black squares represent control, white squares represent 3dRH; [3-13C]-lactate infusion begins at timet = 0 minutes; data reflect mean ± SEM of 6 animals per group).

Figure 3. Metabolic model and in vivo glutamate enrichment time course together reveal that lactate is predominantly metabolized in neurons.

(A) Schematic representation of uptake and relevant metabolic fluxes for [3-13C]-lactate and glucose in astrocytes and neurons. (B) In vivo POCE difference spectrum showing the increase of 13C labeling in glutamate (Glu) and glutamine (Gln) C3 and C4 during infusion of [3-13C]-lactate. Glu4, glutamate labeled at C4; Gln4, glutamine labeled at C4; Glx3, sum of glutamate and glutamine labeled at C3; Lac3, lactate labeled at C3; Tmax, Michaelis-Menten constant for glucose transport kinetics; Vpc, pyruvate carboxylase flux; VxA and VxN, exchange rate from α-ketoglutarate (α-KG) to glutamate in astrocytes and neurons; VpdhA andVpdhN, pyruvate dehydrogenase flux in astrocytes and neurons; Vgln, glutamine synthesis rate; Veff, glutamine efflux, set to balance Vpc; Ac-CoA, acetyl-coenzyme A complex.

Figure 4. Metabolic fluxes during [3-13C]-lactate infusions in control and recurrently hypoglycemic animals under clamped euglycemia and hypoglycemia.

(A) Unidirectional lactate uptake from blood to brain (Vin). (B) Concentration-independent Vin of lactate from blood to brain, reflecting transport changes across the blood-brain barrier (Vin/[lac]pl). (C) Unidirectional lactate outflow from brain to blood (Vout). (D) Cerebral metabolic rate of lactate oxidation (CMRlac). (E) Cerebral metabolic rate of glucose oxidation [CMRglc(ox)]. (F) Maximal glucose transport capacity into the brain [Tmax(glc)]. (G) Neuronal TCA cycle flux (VtcaN). The data reflect mean ± SEM. One-way ANOVA was used to calculate statistical significance between all 4 groups. A post-hoc analysis for prespecified comparisons (CtrlEU vs. CtrlHYPO; 3dRHEU vs. 3dRHHYPO; CtrlHYPO vs. 3dRHHYPO) was then used to determine statistically significant difference after Bonferroni correction. Symbols indicate individual rats. *P≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 5. Glucose transporter expression levels in protein extracts from the brains of hypoglycemic CtrlHYPO and 3dRHHYPO animals that received [3-13C]-lactate infusions in the NMR experiments remain stable after exposure to recurrent hypoglycemia.

(A) Western blot of endothelial and glial GLUT1 isoforms together with β-actin loading control bands of control and 3dRH animals. (B) Western blot of GLUT3 together with β-actin loading control bands of control and 3dRH animals. (C) Gel band density ratios of GLUT1 and GLUT3 over β-actin loading controls. No statistically significant differences were detected between the ratios of control and 3dRH animals (Student’s t test). Data represent mean ± SEM. Symbols indicate individual rats.

Figure 6. Lactate normalizes electrophysiological responses under clamped hypoglycemia in 3dRH animals but not in control animals.

Representative traces of somatosensory cortical LFPs, MUA, and EEG responses to 3 Hz, 2 mA forepaw stimuli in control and 3dRH animals under different glycemic conditions: (A) CtrlEU; (B) CtrlHYPO; (C) CtrlHYPO with lactate infusion; (D) 3dRHEU; (E) 3dRHHYPO; (F) 3dRHHYPO with lactate infusion. (Note that the initial positive deflection in MUA traces reflect stimulus artifact.) Average of the root mean square LFP responses to forepaw stimuli in control (black bars) and 3dRH animals (white bars) under (G) EU clamp, (H) HYPO clamp, and (I) HYPO clamp plus lactate-infused conditions (6 recordings per animal; n = 5 per group; comparison by Student’s t test; *P≤ 0.05).

Figure 7. Enhanced lactate-stimulated CBF response under hypoglycemia in control and recurrently hypoglycemic animals.

Control (black bars) and 3dRH (white bars) cortical CBF changes (ΔCBF) in percentage of hypoglycemic baseline, as measured via a laser Doppler flow probe (n = 5 per group;P < 0.05 at each time point).