Signal transducer and activator of transcription-3 is required in hypothalamic agouti-related protein/neuropeptide Y neurons for normal energy homeostasis

Abstract

Signal transducer and activator of transcription (Stat)-3 signals mediate many of the metabolic effects of the fat cell-derived hormone, leptin. In mice, brain-specific depletion of either the long form of the leptin receptor (Lepr) or Stat3 results in comparable obese phenotypes as does replacement of Lepr with an altered leptin receptor locus that codes for a Lepr unable to interact with Stat3. Among the multiple brain regions containing leptin-sensitive Stat3 sites, cells expressing feeding-related neuropeptides in the arcuate nucleus of the hypothalamus have received much of the focus. To determine the contribution to energy homeostasis of Stat3 expressed in agouti-related protein (Agrp)/neuropeptide Y (Npy) arcuate neurons, Stat3 was deleted specifically from these cells, and several metabolic indices were measured. It was found that deletion of Stat3 from Agrp/Npy neurons resulted in modest weight gain that was accounted for by increased adiposity. Agrp/Stat3-deficient mice also showed hyperleptinemia, and high-fat diet-induced hyperinsulinemia. Stat3 deletion in Agrp/Npy neurons also resulted in altered hypothalamic gene expression indicated by increased Npy mRNA and decreased induction of suppressor of cytokine signaling-3 in response to leptin. Agrp mRNA levels in the fed or fasted state were unaffected. Behaviorally, mice without Stat3 in Agrp/Npy neurons were mildly hyperphagic and hyporesponsive to leptin. We conclude that Stat3 in Agrp/Npy neurons is required for normal energy homeostasis, but Stat3 signaling in other brain areas also contributes to the regulation of energy homeostasis.

Figure 1

Loss of Stat3 from Agrp/Npy neurons resulted in increased adiposity. A, CON mice (n = 6–22) and DEL littermates (n = 5–21) were weighed weekly for 20 wk. Two-way ANOVA indicated statistically significant effects of both time and genotype (P < 0.001) and a significant interaction between these factors (P < 0.05). B, Body weights of 12-wk-old males (n = 5/group) and females (n = 8–11/group) showed DEL mice to weigh more (P < 0.001) than littermate controls. Twelve-week-old males (n = 5/group) were analyzed for body fat (C and D) and lean mass (E) as described in Materials and Methods. F, Weights of epididymal fat pads from 20-wk-old fed or fasted (48 h) mice (n = 4 mice/group). Statistical analysis by two-way ANOVA indicates significant effects of genotype (P < 0.01) and fasting (P < 0.05) but no interaction effect. Values are means ± sem. *, Statistical significance at P < 0.05; ***, P < 0.001 by two-tailed unpaired t test.

Figure 2

Hormones and glucose levels of 20-wk-old fed or fasted (48 h) CON and DEL littermates (n = 4–6/group). Values are means ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test between indicated comparisons. For all measures, a two-way ANOVA indicated a significant fasting effect with no interaction. Only leptin showed a significant (P < 0.001) effect of genotype.

Figure 3

Effect of 6-wk HFD on metabolic indices of CON and DEL littermates. Mice were 18 wk old and fasted for 24 h before they were killed (n = 5/group). A, Two-way ANOVA of mice switched to HFD at wk 12 indicated significant (P < 0.0001) effects of both age and genotype on body weight. Data for mice maintained on chow for 18 wk are from Fig. 2A and are presented here for comparison with mice switched to HFD at wk 12. B, Body weights for mice at 18 wk of age either maintained on chow (data from Fig 2A) or switched from chow to HFD at 12 wk of age. C–F, Additional metabolic indices of mice on HFD. Statistical analyses for B–F are by two-tailed, unpaired t tests: ***, P < 0.001; *, P < 0.05; values are means ± sem.

Figure 4

Fasting-induced rise in CFLIR was decreased in Agrp/Npy neurons in DEL mice. CFLIR by immunofluorescence in the arcuate nucleus in the fed state (A) and after 48 h fasting (B). Fasting-induced CFLIR (C) and green fluorescent cells expressing Egfp (D) in AgrpCre/ROSA26Egfp mice are shown. E, Colocalization of CFLIR and Egfp in AgrpCre/ROSA26Egfp mice. Fasting-induced CFLIR (F) and green fluorescent cells (G) expressing Egfp in PomcEgfp mice. H, CFLIR and Egfp in PomcEgfp mice. I, CFLIR by avidin biotin complex staining in the arcuate of 48-h fasted 8- to 12-wk-old mice was greater in CON mice (I–K) than DEL littermates (L–N). O and P, Surface plots of CFLIR by immunofluorescence after 48 h fasting in CON (O) and DEL littermates (P). Q, Means ± sem of summed FI units for fasting-induced CFLIR by immunofluorescence in arcuate nucleus of CON and DEL littermates (n = 4/group).

Figure 5

Effect of leptin on body weight (A and B) of 22-wk-old CON mice and DEL littermates (n = 5/group). Leptin was administered as described in the text. Two-way ANOVA of body weight from d 0 to d 19 indicated an effect of genotype (P < 0.0001). B, Body weight differences from d 0 were determined for the 4 d of leptin treatment and group differences tested by two-tailed unpaired t tests. C, Two-way ANOVA of food intake showed significant effects of both genotype (P < 0.0001) and time (P < 0.0001) with no interaction. Group differences in daily food intake were analyzed by two-tailed, unpaired t tests. Mice were killed after the last day of feeding measurements, and neuropeptide and Socs-3 transcripts from whole hypothalamus (D) were quantified. *, P < 0.05; **, P < 0.01. Values are means ± sem.

Figure 6

Effect of leptin on whole hypothalamic neuropeptide and Socs-3 mRNA in 24-wk-old CON and DEL littermates. Twenty-four-week-old CON (n = 9) and DEL (n = 9) mice had food removed on d 1. On the same day, mice from each group were injected with 5 mg/kg ip leptin (n = 5/group) or vehicle (n = 4/group) at 1000 and 1600 h. On the following day, the same mice were injected again with leptin (5 mg/kg ip) or vehicle at 0900 h and were killed 1 h later. *, P < 0.05; ***, P < 0.001. Values are means ± sem.