Hypothalamic ventromedial COUP-TFII protects against hypoglycemia-associated autonomic failure

Abstract

The nuclear receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) is an important coordinator of glucose homeostasis through its function in different organs such as the endocrine pancreas, adipose tissue, skeletal muscle, and liver. Recently we have demonstrated that COUP-TFII expression in the hypothalamus is restricted to a subpopulation of neurons expressing the steroidogenic factor 1 transcription factor, known to play a crucial role in glucose homeostasis. To understand the functional significance of COUP-TFII expression in the steroidogenic factor 1 neurons, we generated hypothalamic ventromedial nucleus-specific COUP-TFII KO mice using the cyclization recombination/locus of X-overP1 technology. The heterozygous mutant mice display insulin hypersensitivity and a leaner phenotype associated with increased energy expenditure and similar food intake. These mutant mice also present a defective counterregulation to hypoglycemia with altered glucagon secretion. Moreover, the mutant mice are more likely to develop hypoglycemia-associated autonomic failure in response to recurrent hypoglycemic or glucopenic events. Therefore, COUP-TFII expression levels in the ventromedial nucleus are keys in the ability to resist the onset of hypoglycemia-associated autonomic failure.

Fig. 1.

SF1–COUP-TFII mice blood parameters. (A) Blood glucose, plasma insulin, and glucagon concentrations in 5-mo-old fed or fasted male mice (18 h fast). White, (SF1)^CRE+/− mice; gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM. *P ≤ 0.05 (n = 12). (B) Glucose tolerance test (GTT) and insulin secretion during GTT on 3-mo-old mice. AUC, area under curve of plasma insulin concentrations. Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 9). *P ≤ 0.05; **P ≤ 0.01. (C) Insulin tolerance test (ITT) on 3-mo-old mice. Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). *P ≤ 0.05.

Fig. 2.

SF1–COUP-TFII mice have imbalanced energy homeostasis. (A) Ponderal growth over a period of 10 mo. Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 32). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. (B) Body composition of 3- or 6-mo-old male mice. Gray, COUP-TFII fl/wt male mice; black, (SF1)^CRE+/− COUP-TFII fl/wt male mice. Data are expressed as the mean ± SEM (n = 5). *P ≤ 0.05; **P ≤ 0.01 (C) Daily food intake and food intake during a 4 h refeeding after 18 h fast. White, (SF1)^CRE+/− mice; gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). (D) Time course of resting metabolic rate (RMR), respiratory quotient (RQ), and cost of activity from 12 h before to 7 h after ingestion of a test meal (t = 0). Gray line, COUP-TFII fl/wt mice; black line, (SF1)^CRE+/− COUP-TFII fl/wt mice. (E) Autoradiogram of a Western blot analysis of UCP3 and porine protein expressions in mitochondria isolated from skeletal muscles.

Fig. 3.

Decreased COUP-TFII expression in the VMN leads to an increased expression of POMC mRNA and increased insulin signaling. (A) POMC and agouti-related protein (AgRP) mRNA expression levels in the hypothalamus of fed SF1–COUP-TFII mice and their controls. White, (SF1)^CRE+/− mice; gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). *P ≤ 0.05. (B) Autoradiogram of a Western blot analysis of the STAT3, phosphorylated STAT3 proteins, FOXO1, and phosphorylated FOXO1 in the hypothalamus of 5-mo-old male mice. (C) EMSA with nuclear extracts of SF1–COUP-TFII or COUP-TFII fl/wt mice VMN. (Left) FOXO1 binding activity in absence (–) or presence (+) of a competing anti-FOXO1 antibody. (Right) CCAAT binding activity as internal control of nuclear extracts’ integrity.

Fig. 4.

SF1–COUP-TFII mice have a defective CRR to hypoglycemia. (A, Upper) ITT in 5-mo-old male mice (Left). Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). *P ≤ 0.05. (Lower) Glucagon, corticosterone, and growth hormone concentrations at time 0 and 60 min. White box, (SF1)^CRE+/− mice; gray box, COUP-TFII fl/wt; black box, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). (B, Upper) Blood glucose concentrations following a 2-DG injection (Left). Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). (Lower) Food intake over a 2 h period following a single 2-DG injection and glucagon secretion at time 60 min of the 2-DG injection. White box, (SF1)^CRE+/− mice; gray box, COUP-TFII fl/wt; black box, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 6). *P ≤ 0.05; **P ≤ 0.01.

Fig. 5.

SF1–COUP-TFII mice are more prone to HAAF. (A) Recurrent ITT, 5-mo-old male mice were subjected to a weekly insulin tolerance test. (Left) First insulin injection; (Right) third insulin injection. Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 10). *P ≤ 0.05; **P ≤ 0.01. (B) Blood glucose concentrations following recurrent 2-DG injections on 5-mo-old male mice. (Left) First injection; (Right) third injection. Light gray, (SF1)^CRE+/− mice; dark gray, COUP-TFII fl/wt; black, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 6). *P ≤ 0.05. (C) 2 h food intake following three 2-DG injection. White box, (SF1)^CRE+/− mice; gray box, COUP-TFII fl/wt; black box, (SF1)^CRE+/− COUP-TFII fl/wt mice. Data are expressed as the mean ± SEM (n = 8). (D) Autoradiogram of a Western blot analysis of the AMPKα and phosphorylated AMPKα in the hypothalamus of 5-mo-old male mice after four recurrent 2-DG injections.