Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis

Abstract

Blood glucose levels are tightly controlled, a process thought to be orchestrated primarily by peripheral mechanisms (insulin secretion by β cells, and insulin action on muscle, fat, and liver). The brain also plays an important, albeit less well-defined role. Subsets of neurons in the brain are excited by glucose; in many cases this involves ATP-mediated closure of K(ATP) channels. To understand the relevance of this, we are manipulating glucose sensing within glucose-excited neurons. In the present study, we demonstrate that glucose excitation of MCH-expressing neurons in the lateral hypothalamus is mediated by K(ATP) channels and is negatively regulated by UCP2 (a mitochondrial protein that reduces ATP production), and that glucose sensing by MCH neurons plays an important role in regulating glucose homeostasis. Combined, the glucose-excited neurons are likely to play key, previously unexpected roles in regulating blood glucose.

Figure 1. MCH neurons are glucose excited and express Sur1-containing K_ATP channels

(A) Structure of Mch-Cre BAC transgene. (B) Double immunohistochemistry for tdTomato (red) and MCH peptide (green) in the lateral hypothalamus of Mch-Cre/lox-tdTomato mice. (C-D) Representative traces recorded in the whole-cell patch clamp mode from GFP-positive neurons of Mch-Cre/Z/EG mice. (C) Spontaneously firing MCH neuron that responded to glucose. (D) Silent MCH neuron that responded to glucose with depolarizing current injection (20pA for 3 sec with 20sec interval). (E) Effects of glucose (5mM → 1mM → 5mM) on membrane potential of “tolbutamide-responding” GFP-positive neurons of Mch-Cre/Z/EG mice. The averaged membrane potential of the last three minutes of the indicated condition from each recording was used for calculation (n=9, mean ± SEM).

Figure 2. Expression of mutant Kir6.2 in MCH neurons blocks glucose sensing

(A-B) Effects of ATP on K_ATP channel current. Recordings were performed on inside-out patches derived from GFP-positive (i.e. MCH) neurons. (A) Representative macroscopic current traces recorded at -60 mV from patches derived from i) WT (Mch-Cre/Z/EG) and ii) Mch-Cre/mutKir6.2/Z/EG mice. Concentrations of ATP are indicated above the corresponding traces. (B) Summarized effects of ATP on K_ATP channel current. Data was normalized to the maximal current recorded in the absence of ATP (mean ± SEM, n=5-7). (C) Effects of glucose on MCH neurons within brain slices. Representative traces recorded in the whole-cell patch clamp mode from GFP-positive neurons derived from i) WT (Mch-Cre/Z/EG) and ii)Mch-Cre/mutKir6.2/Z/EG mice. (D) Effects of glucose on membrane potential (mean ± SEM, values above each point represent the number of neurons assessed). (E) Effects of mutant Kir6.2 on glucose tolerance in intact mice. Representative glucose tolerance curves of 8-week-old male mice (mean ± SEM, n=7-10 per genotype, 2 g/Kg glucose i.p.). Asterisk, P<0.05 (unpaired t Tests), compared with wildtype littermates at a given time point.

Figure 3. Deleting UCP2 in MCH neurons augments glucose sensing

(A) Double immunohistochemistry / in situ hybridization for MCH peptide (DAB, brown stain) and Ucp2 mRNA (³⁵S-labelled cRNA probe, silver grains) in wildtype mice. (B) Effects of UCP2 deletion on glucose sensing by MCH neurons. Dose response curve of glucose-stimulated depolarization of GFP-positive neurons from WT (Mch-Cre/Z/EG) and Mch-Cre/Ucp2^lox/lox/Z/EG mice (mean ± SEM values above each point represent the number of neurons assessed, two-way ANOVA test, p_interaction=0.95, p_colume=0.02, p_row=0.001). (C) Blood glucose levels of ad lib fed and overnight fasted, 8-week old male mice (mean ± SEM, n=8-10 per genotype). Asterisk, P<0.05; Two asterisks, P<0.01, compared with wildtype littermates (unpaired t Tests). (D) Representative glucose tolerance curves of 8-week-old male mice (mean ± SEM, n=8-10 per genotype, 2 g/Kg glucose i.p.). Asterisk, P<0.05; Two asterisks, P<0.01, compared with wildtype at a given time point (unpaired t Tests). (E) Insulin levels during glucose tolerance test. (mean ± SEM, n=8-10 per genotype, 2 g/Kg glucose i.p.).

Figure 4. Mutant K_ATP Channels Block the Effects of UCP2-deficiency in MCH Neurons

(A) Blood glucose levels of ad lib fed 8-week-old male mice (mean ± SEM, n=7-8 per genotype). Asterisk, P<0.05; Two asterisks, P<0.01, compared with littermates (one-way ANOVA assay). (B) Representative glucose tolerance curves of 8-week-old male mice (mean ± SEM, n=7-8 per genotype, 2 g/kg glucose i.p.). (C) GTT data from (B) expressed as Area Under the Curve. Asterisk, P<0.05; Two asterisks, P<0.01, Three asterisks, P<0.001, compared with littermates (one-way ANOVA assay).