Functional role of suppressor of cytokine signaling 3 upregulation in hypothalamic leptin resistance and long-term energy homeostasis

Abstract

Objective: Hypothalamic leptin resistance is found in most common forms of obesity, such as diet-induced obesity, and is associated with increased expression of suppressor of cytokine signaling 3 (Socs3) in the hypothalamus of diet-induced obese animals. This study aims to determine the functional consequence of Socs3 upregulation on leptin signaling and obesity, and to investigate whether Socs3 upregulation affects energy balance in a cell type-specific way.

Research design and methods: We generated transgenic mice overexpressing Socs3 in either proopiomelanocortin (POMC) or leptin receptor-expressing neurons, at levels similar to what is observed in diet-induced obesity.

Results: Upregulation of Socs3 in POMC neurons leads to impairment of STAT3 and mammalian target of rapamycin (mTOR)-S6K-S6 signaling, with subsequent leptin resistance, obesity, and glucose intolerance. Unexpectedly, Socs3 upregulation in leptin receptor neurons results in increased expression of STAT3 protein in mutant hypothalami, but does not lead to obesity.

Conclusions: Our study establishes that Socs3 upregulation alone in POMC neurons is sufficient to cause leptin resistance and obesity. Socs3 upregulation impairs both STAT3 and mTOR signaling before the onset of obesity. The lack of obesity in mice with upregulated Socs3 in leptin receptor neurons suggests that Socs3's effect on energy balance could be cell type specific. Our study indicates that POMC neurons are important mediators of Socs3's effect on leptin resistance and obesity, but that other cell types or alteration of other signaling regulators could contribute to the development of obesity.

FIG. 1.

Generation and characterization of transgenic mice that express Cre-activatable Socs3. A: Transgenic construct to overexpress Socs3 in a Cre-activatable way. In cells in which Cre is absent, Socs3 is not expressed because of the upstream stop cassette, which consists of four copies of polyadenylation signal (pA). In cells in which Cre is expressed, the stop cassette is removed and Socs3 expression is driven by the CMV promoter. Nomenclature of mice used in this study is listed. B: Transgenic mice carrying the Tg.Socs3-OE allele did not show a difference in body weight (Tg.Socs3-OE/+) compared with wild-type (+/+) controls (controls n = 6–30, Tg.Socs3-OE n = 5–20). C: Transgenic Socs3 expression is Cre dependent. Tg.Socs3-OE mice were crossed with mice carrying Nestin-Cre. Semiquantitative real-time RT-PCR showing differential expression of transgenic Socs3 (Tg.Socs3) in the hypothalamus where Nestin-Cre is expressed, and in liver where Nestin-Cre is not expressed. Primers were specific to the Socs3 transgene. Hyp, hypothalamus; Liv, liver. D: Socs3 mRNA expression was analyzed in 0.5-week-old control and Nestin-Socs3-OE mutant mice (controls n = 12, mutants n = 6). E: Hypothalamic Socs3 mRNA expression was analyzed in 0.5- and 8-week-old wild-type C57BL6/J mice (n = 6–8). All mice were fed a chow diet. *P < 0.05 by Student t test.

FIG. 2.

Socs3 overexpression in POMC neurons leads to increased body weight and adiposity, decreased resting energy expenditure, and resistance to leptin's anorexigenic effects. A: Body weight of POMC-Socs3-OE on chow diet at indicated ages. Mutants became significantly heavier at 15 weeks of age (controls n = 9–13, mutants n = 7–10). B: Analysis of lean mass and fat mass by DEXA of POMC-Socs3-OE at 8 and 25 weeks of age (controls n = 10, mutants n = 8). (C) Plasma leptin levels under fed conditions for 25-week-old POMC-Socs3-OE mice were significantly higher (controls n = 9, mutants n = 8). D: A 24-h food intake was measured in 8-week-old controls and POMC-Socs3-OE mutant mice (controls n = 11, mutants n = 7). E and F: Oxygen consumption was measured in 8-week-old mice, and the values were normalized to lean body mass of each mouse. Daytime: 1100–1700; nighttime: 1900–0700. A representative trace is shown in F. G: The 9-week-old POMC-Socs3-OE and control mice were injected intraperitoneally with saline twice daily for 3 consecutive days and leptin (2.5 mg/kg) twice on the fourth day. A 24-h food intake after leptin treatment was reported as a ratio of food intake after saline treatment (controls n = 11, mutants n = 7). Data represent mean ± SEM. *P < 0.05, **P < 0.01, between controls and mutants as determined by 2-way ANOVA using litters and genotypes as variables. ns, nonsignificant.

FIG. 3.

Leptin-induced STAT3 signaling is reduced in POMC neurons of POMC-Socs3-OE mice. A: The 6-week-old control and POMC-Socs3-OE mice were injected with either saline or leptin (3 mg/kg), and perfused 45 min later. Double immunofluorescence analysis was carried out to examine pSTAT3 in the POMC neurons using antibodies against Tyr705-STAT3 (green) and ACTH (red). B: Number of pSTAT3-positive cells showed that leptin-induced pSTAT3 was reduced in the POMC but not other arcuate neurons in the mutant mice. C: Percentage of POMC neurons that are pSTAT3 positive upon saline or leptin treatment in control and mutant mice. n = 3–5 sections per mouse from 3–5 mice per group between bregma −1.22 and −1.58. Data represent mean ± SEM. *P < 0.05, between controls and mutants as determined by Student t test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

mTOR-S6 signaling is impaired in POMC neurons of POMC-Socs3-OE mice. A: The 6-week-old control and POMC-Socs3-OE mice were injected with either saline or leptin (3 mg/kg), and perfused 45 min later. Double immunofluorescence analysis was carried out to examine phosphorylation of S6 (pS6) in POMC neurons using antibodies against Ser235/236-S6 (green) and ACTH (red). B: Quantification of POMC and pS6-positive cells showed that pS6 was specifically reduced in POMC neurons but not in other cells in the arcuate nucleus of mutant mice. C: Percentage of POMC neurons that is positive for pS6 after either saline or leptin treatment. n = 3–5 sections per mouse from 3–5 mice per group between bregma −1.22 and −1.58. Data represent mean ± SEM. *P < 0.05, **P < 0.01 for comparisons shown as determined by Student t test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Upregulation of Socs3 in leptin receptor neurons does not result in obesity but rather in a subtle lean phenotype. A: Body weight of LepRb-Socs3-OE on chow diet at indicated ages. Mutants became slightly leaner at 7 weeks of age (controls n = 16–44, mutants n = 10–27). B: Length of LepRb-Socs3-OE at 5 weeks of age (controls n = 14, mutants n = 8) and 12 weeks of age (controls n = 12, mutants n = 7) was identical. C: Analysis of lean mass and fat mass by DEXA at 10 weeks of age (controls n = 12, mutants n = 8). D: The 12-week-old LepRb-Socs3-OE mutants showed significantly decreased interscapular, retroperitoneal, and total white adipose tissue on a chow diet (controls n = 11, mutants n = 8). E: Plasma leptin levels under fed conditions for LepRb-Socs3-OE mice at 12 weeks of age (controls n = 9, mutants n = 5). F: 24-h food intake of LepRb-Socs3-OE mice at 6–8 weeks of age was reduced (controls n = 6, mutants n = 6). G: The 6- to 8-week-old LepRb-Socs3-OE and control mice were injected intraperitoneally with saline twice daily for 3 consecutive days and leptin (2.5 mg/kg) twice on the fourth day. 24-h food intake after leptin treatment was reported as a ratio of food intake after saline treatment (controls n = 6, mutants n = 6). Data represent mean ± SEM. WAT, white adipose tissue. *P < 0.05, between controls and mutants as determined by 2-way ANOVA.

FIG. 6.

Upregulation of Socs3 in leptin receptor neurons does not result in obesity when fed a high-fat diet. A: Control and LepRb-Socs3-OE mice were placed on a high-fat diet (60% kcal from fat; Research Diet D12492) at 5 weeks of age for a total of 16 weeks (controls n = 16–19, mutants n = 9–10). B: Body length (nose to anus) of control and mutants was identical after 16 weeks on high-fat diet (controls n = 11, mutants n = 6). C: Body composition analysis by DEXA after 16 weeks on high-fat diet (controls n = 14, mutants n = 8). D: Weight of individual fat pads after 16 weeks on high-fat diet (controls n = 13, mutants n = 9). Data represent mean ± SEM. WAT, white adipose tissue.

FIG. 7.

Steady-state levels of total STAT3 and phosphorylated STAT3 are increased in LepRb-Socs3-OE mice. A: The 6-week-old control and LepRb-Socs3-OE mice were injected with either saline or leptin (3 mg/kg), and perfused 45 min later. Double immunofluorescence analysis was carried out to examine pSTAT3 in different areas of hypothalamus and in POMC neurons. ARC, arcuate nucleus; VMH, ventromedial hypothalamus; DMH, dorsomedial hypothalamus; PH, posterior hypothalamic area. Representatives of leptin-injected mice are shown to illustrate different regions of the hypothalamus. B: Quantification of pSTAT3-positive cells in different areas of the hypothalamus and in POMC neurons. n = 3–5 sections per mouse from 3–4 mice per group between bregma −1.94 and −2.30. C: LepRb-Socs3-OE and control mice were fasted overnight and injected with leptin (3 mg/kg). Mice were killed 1 h later; hypothalamic protein extract was prepared. Western blot analysis was performed with equal amount of total protein input, and pSTAT3 was examined. The same blots were stripped and blotted with an antibody to detect total STAT3 protein levels. Sal, saline; Lep, leptin. D: Free-fed LepRb-Socs3-OE and control mice were killed, and hypothalamic protein extracts were prepared. Western blot analysis was performed to examine total STAT3 expression levels. The same blot was stripped and subsequently blotted with GAPDH. *P < 0.05, between controls and mutants as determined by Student t test.

FIG. 8.

Effects of Socs3 upregulation on glucose homeostasis. A and B: POMC-Socs3-OE and control mice at 8 weeks (A) or 25 weeks (B) of age were fasted for 6 h and injected with 1.5 g/kg glucose. Glucose levels were measured from tail blood using a glucometer. Area under the curve (AUC) was presented. Control n = 32 (A), n = 6 (B); mutants n = 19 (A), n = 5 (B). C and D: LepRb-Socs3-OE and control mice at 5 (C) or 9–11 (D) weeks of age were fasted for 6 h, and injected with 1.5 g/kg glucose. Glucose levels were measured from tail blood using a glucometer. AUC was presented. Control n = 9–11, mutants n = 7–10. Data represent mean ± SEM. *P < 0.05 between controls and mutants. Analysis was performed by Student t test.