Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance

Abstract

Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions.

Keywords: POMC; PPARγ; neuron; rosiglitazone.

Fig. 1.

Validation of transgenic models. A: schematic representation of the inducible transgene expression system. B: PPARγ Western blot of total cellular protein from HEK293 cells transfected with empty vector (Con), CAG-PPARγ-P467L, or CAG-PPARγ-WT in the presence or absence of Cre-recombinase. C: expression of transgenic PPARγ mRNA and aP2 mRNA in 3T3-L1 preadipocytes transfected with Cre-recombinase activated constructs expressing either empty vector (control), PPARγ-P467L, or PPARγ-WT (*P < 0.05, n = 4 per group). D: expression of PPARγ in the liver of CAG-PPARγ-P467L transgenic and control littermate mice in response to intravenous injection of AdCre or AdeGFP. Shown is a representative blot of many experiments performed in independent lines of mice. E: expression of tdTomato (red) and PPARγ (green) in neurons (nuclei stained by DAPI, blue) along the needle tract (white dashed arrow) in the cerebral cortex of a CAG-PPARγ-WT transgenic mouse injected with AdCre. Similar results were obtained in CAG-PPARγ-P467L transgenic mice (data not shown). F: expression of PPARγ in the brain of Nes^CRE/PPARγ-P467L transgenic and control littermate mice. The presence of the transgene and Nestin-Cre is indicated. Each lane represents brain protein isolated from an individual mouse (n = 8).

Fig. 2.

Transgene expression in POMC^Cre/PPAR-P467L mice. A: immunofluorescence detection of PPARγ (green) and endogenous fluorescent detection of tdTomato (red) on coronal sections at the level of the mediobasal hypothalamus. Arrows indicate cells coexpressing PPARγ and tdTomato. B: POMC neurons immuno-labeled with antibody against ACTH compared with cells expressing tdTomato.

Fig. 3.

Body weight, composition, and glucose during 25 wk of 60% high-fat diet. Body weight in male (A) and female (B) POMC^Cre/PPARγ-P467L (n = 11 male, n = 15 female) mice compared with littermate controls (n = 15 males, n = 22 females). Body composition is shown in male (C) and female (D) POMC^Cre/PPARγ-P467L (n = 11 male, n = 8 female) and control (n = 15 male, n = 12 female) mice. Fasting glucose is shown in male (E) and female (F) POMC^Cre/PPARγ-P467L (n = 11 male, n = 12 female) and control (n = 15 male, n = 17 female) mice. Data are expressed as means ± SE.

Fig. 4.

Body weight and composition during 25 wk of 10% fat, isocaloric-match diet. Body weight response to low-fat isocaloric control diet in male (A) and female (C) POMC^Cre/PPARγ-P467L (n = 10 male, n = 7 female) and control (n = 10 male, n = 13 female) mice. B and D: body composition in male POMC^Cre/PPARγ-P467L (n = 10) and control (n = 10) mice as measured by nuclear magnetic resonance. Data are expressed as means ± SE. *P < 0.0001; **P < 0.05.

Fig. 5.

Leptin sensitivity. Change in body weight (BW, A and C) or food intake (B and D) in response to vehicle or leptin (1.0 mg/kg) administration in male POMC^Cre/PPARγ-P467L (n = 9) and control (n = 10) mice fed 10% fat diet (A and B) or male POMC^Cre/PPARγ-P467L (n = 11) and control (n = 15) mice fed 60% high-fat diet (C and D). Data are expressed as means ± SE. *P < 0.01 vs vehicle.

Fig. 6.

Body weight during 25 wk of 60% high-fat diet in POMC^Cre/PPARγ-WT mice. Body weight in male (A) and female (B) POMC^Cre/PPARγ-WT (n = 9 male, n = 9 female) and control (n = 16 male, n = 14 female) mice during 60% high-fat diet. Data are expressed as means ± SE.

Fig. 7.

Body weight response to rosiglitazone. A: the effect of rosiglitazone administration (28 mg/kg ip for 5 days) on body weight in lean POMC^Cre/PPARγ-WT (n = 8) and control (n = 11) female mice exposed to 45% high-fat diet. B: the effect of rosiglitazone administration (28 mg/kg ip for 5 days) on body weight in lean POMC^Cre/PPARγ-P467L (n = 7) and control (n = 7) female mice exposed to 45% high-fat diet. Data are expressed as means ± SE. *P < 0.01.