Expression of the hypothalamic transcription factor Nhlh2 is dependent on energy availability

Abstract

Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 colocalises with the hypothalamic thyrotrophin-releasing hormone (TRH) neurones in the paraventricular nucleus (PVN) and pro-opiomelanocortin (POMC) neurones in the arcuate nucleus. We show that Nhlh2 expression is significantly reduced in response to 24-h food deprivation in the arcuate nucleus, PVN, lateral hypothalamus, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). Food intake for 2 h following deprivation stimulates Nhlh2 expression in the arcuate nucleus and the PVN, and leptin injection following deprivation results in increased Nhlh2 expression in the arcuate nucleus, PVN, lateral hypothalamus, VMH, and DMH. Hypothalamic Nhlh2 expression in response to leptin injection is maximal by 2 h. Following leptin injection, Nhlh2 mRNA colocalises in POMC neurones in the arcuate nucleus and TRH neurones in the PVN. Nhlh2 mRNA expression in POMC neurones in the arcuate nucleus and TRH neurones in the PVN is reduced with energy deprivation and is stimulated with food intake and leptin injection. Modulation of POMC expression in response to changes in energy availability is not affected in mice with a targeted deletion of Nhlh2. However, deletion of Nhlh2 does result in loss of normal TRH mRNA expression in mice exposed to food deprivation and leptin stimulation. These data implicate Nhlh2 as a regulatory target of the leptin-mediated energy availability network of the hypothalamus, and TRH as a putative downstream target of Nhlh2.

Fig. 1

Hypothalamic Nhlh2 expression changes with respect to energy status. Whole hypothalamic RNA was isolated from wild-type animals that were given (A,B) ad lib access to food (Ad lib), food deprived for 24 h (Deprived), or food deprived for 24 h followed by 2 h of ad lib food (Deprived + -Food), and given (C,D) ad lib access to food (Ad lib), food deprived for 24 h, given a phosphate buffered saline (PBS) injection and euthanised after 2 h (Deprived + PBS) or food deprived for 24 h, given a leptin injection and euthanised after 2 h (Deprived + Leptin). (A,C) Relative expression levels of Nhlh2 RNA via quantitative real-time reverse transcription polymerase chain reaction using primers to Nhlh2 normalised to β-actin and (B,D) corresponding serum leptin levels. The results are expressed as mean ± SEM (n = 5 animals per condition) (A–D). *P ≤ 0.05, **P ≤ 0.01. Horizontal lines indicate significance between food deprived and either food return or leptin injected groups. All other asterisks indicate significance compared to ad lib fed animals.

Fig. 2

Expression of Nhlh2 in the hypothalamus responds to food intake. A ³³P-labelled cRNA probe to Nhlh2 was used to label hypothalamic sections of brains from wild-type mice under different states of energy availability [Ad lib: A, arcuate nucleus; B, paraventricular nucleus (PVN)], energy deficit (24-h food deprivation, Deprived: C, arcuate nucleus; D, PVN) and energy availability following an energy deficit (24-h food deprivation followed by 2 h of ad lib access to food, Deprived + Food: E, arcuate nucleus; F, PVN) (× 10 magnification with × 40 magnification inset). Nhlh2 expression density in the arcuate nucleus (G) or PVN (H) displayed as total pixel density. The results are expressed as mean ± SEM (n = 6 animals per condition in both the arcuate nucleus and PVN) (A–H). **P ≤ 0.01. Horizontal lines indicate significance between food deprived and food return groups. All other asterisks indicate significance compared to ad lib fed animals.

Fig. 3

Expression of Nhlh2 in the hypothalamus responds to leptin injection. A ³³P-labelled cRNA probe to Nhlh2 was used to label hypothalamic sections of brains from wild-type mice under different states of energy availability [Ad lib: A, arcuate nucleus; B, paraventricular nucleus (PVN)], energy deficit [24-h food deprivation with phosphate buffered saline (PBS) injection, Deprived + PBS: C, arcuate nucleus; D, PVN] and energy availability following an energy deficit (24-h food deprivation, injection of leptin 2 h prior to sacrifice, Deprived + Leptin: E, arcuate nucleus; F, PVN) (× 10 magnification with × 40 magnification inset). Nhlh2 expression density in the arcuate nucleus (G) or PVN (H) displayed as total pixel density. The results are expressed as mean ± SEM [n = 6 animals per condition in the arcuate nucleus) (A,C,E,G) and n = 7 animals per condition in the PVN (B,D,F,H)]. **P ≤ 0.01. Horizontal lines indicate significance between food deprived and leptin injected groups. All other asterisks indicate significance compared to ad lib fed animals.

Fig. 4

Expression of Nhlh2 in other hypothalamic nuclei is responsive to leptin injection. A ³³P-labelled cRNA probe to Nhlh2 was used to label hypothalamic sections of brains from wild-type (WT) mice under different states of energy availability [Ad lib: A, dorsomedial hypothalamus (DMH); B, lateral hypothalamus; C, ventromedial hypothalamus (VMH); Deprived + phosphate buffered saline (PBS): D, DMH; E, lateral hypothalamus; F, VMH; Deprived + Leptin: G, DMH; H, lateral hypothalamus; I, VMH] (× 10 magnification with × 40 magnification, inset). (J–L) Nhlh2 expression density in DMH (J), lateral hypothalamus (K) and VMH (L) of WT mice under different states of energy availability displayed as pixel density. The results are expressed as mean ± SEM (n = 6 animals per condition in all three regions of the hypothalamus) (A–L). **P ≤ 0.01. Horizontal lines indicate significance between food deprived and leptin injected groups. All other asterisks indicate significance compared to ad lib fed animals.

Fig. 5

Increase in Nhlh2 mRNA expression is rapid following a leptin injection of deprived mice. (A) Nhlh2 mRNA expression at the indicated time points following leptin injection was quantified using quantitative real-time reverse transcription polymerase chain reaction (QPCR) and normalised to β-actin expression. (B) pro-opiomelanocortin (POMC) and (C) thyrotrophin-releasing hormone (TRH) mRNA expression at the indicated time points following leptin injection was quantified using QPCR and normalised to β-actin expression. (D) Serum leptin concentration levels were analysed via enzyme-linked immunosorbent assay at the indicated time points following leptin injection (n = 5 animals per time point) (A–D). *P ≤ 0.05, **P ≤ 0.01. Horizontal lines indicate significance between two time points. All other asterisks indicate significance compared to time point zero.

Fig. 6

Nhlh2 is coexpressed with pro-opiomelanocortin (POMC) neurones in the arcuate nucleus and thyrotrophin-releasing hormone (TRH) neurones in the paraventricular nucleus (PVN) under states of variable energy status. Dual label in situ hybridisation of hypothalamic sections of brains from wild-type mice were compared under states of energy availability (Ad lib: A, arcuate nucleus; E, PVN), energy deficit (Deprived: B, arcuate nucleus; F, PVN) and energy availability following an energy deficit (Deprived + Food return: C, arcuate nucleus; G, PVN; Deprived + Leptin: D, arcuate nucleus; H, PVN). Animals deprived given an injection of phosphate buffered saline (a control group for leptin injection) were measured, were identical to the deprived group and therefore not shown (× 10 magnification with × 40 magnification, inset). Silver grains (black) show Nhlh2 expression. Brown stain indicates POMC (A–D) or TRH (E–H) expression. (I,J) Nhlh2 expression density over POMC stained cells in the arcuate nucleus (I) and over TRH stained cells in the PVN (J) under different states of energy availability displayed as pixel density. The results are expressed as mean ± SEM (n = 6 animals under ad lib and deprived condition and n = 3 animals under food return and leptin injected conditions). **P ≤ 0.01. Horizontal lines indicate significance between deprived, food deprived and leptin injected groups. All other asterisks indicate significance compared to ad lib fed animals.

Fig. 7

Altered expression of thyrotrophin-releasing hormone (TRH) mRNA in mice with a targeted deletion of Nhlh2. Whole hypothalamic RNA was isolated from wild-type (black bars) and N2KO (grey bars) animals that were given ad lib access to food (Ad lib), food deprived for 24 h with a phosphate buffered saline (PBS) injection 2 h prior to euthanisation (Deprived + PBS), food deprived for 24 h with injection leptin given 2 h prior to euthanisation (Deprived + Leptin). Relative expression levels of pro-opiome-lanocortin (POMC) (A) or TRH (B) via QPCR using appropriate primers and normalised to β-actin mRNA expression (n = 3 animals per genotype and condition). ** P ≤ 0.01.