Over-expression of leptin receptors in hypothalamic POMC neurons increases susceptibility to diet-induced obesity

Abstract

Diet-induced obesity (DIO) in rodents is characterized by impaired activation of signal-transducer and activator of transcription 3 (STAT3) by leptin receptors (LepRb) within the hypothalamic arcuate nucleus. This signaling defect likely plays an important role in development of DIO. However, the neuro-chemical identity of the leptin-STAT3 resistant arcuate neurons has not been determined and the underlying mechanisms responsible for development of cellular leptin resistance remain unclear. To investigate this, we first measured arcuate gene expression of known key signaling components of the LepRb signaling pathway and tested whether specifically the critical arcuate pro-opiomelanocortin (POMC) neurons are resistant to LepRb-STAT3 signaling in mice given a high-fat-diet (HFD) compared to mice provided a low-fat control diet (LFD). We found that leptin-dependent STAT3 phosphorylation was decreased within POMC neurons of HFD mice. In addition, Leprb mRNA and suppressor of cytokine signaling 3 (Socs3) mRNA were elevated in the arcuate of HFD mice. To investigate whether increased LepRb expression per se in POMC neurons can influence development of DIO and Socs3 expression, we created mice that over-express LepRb selectively in POMC neurons (POMC-LepRb). No differences in body weight, fat mass or food intake were found between LFD POMC-LepRb mice and LFD controls. Surprisingly, body weight, fat mass and caloric intake of HFD POMC-LepRb mice was markedly higher than HFD control mice. In addition, arcuate Socs3 mRNA was increased in HFD POMC-LepRb mice compared to HFD controls. These data show that specifically POMC neurons of DIO mice are resistant to STAT3 activation by leptin, indicating that those cells might play a role in development of DIO. Furthermore, over-expression of LepRb selectively in POMC neurons increases susceptibility to the development of DIO. We propose a model where over-reactivity of the leptin-LepRb signaling system in arcuate neurons may play causal a role in development of diet-induced obesity.

Figure 1. Diet-induced obesity in C57Bl/6J mice.

A–C. Shown are body-weight curves of C57Bl/6J mice given HF or LF diets for 3, 7 and 11 weeks, respectively. D–F. Shown is cumulative food intake after 3, 7 and 11 weeks of diets, respectively. * = p<0.05; ** = p<0.01; # = p<0.001. N = 23–25 animals per group. Data are means +/− SEM.

Figure 2. Arcuate nucleus gene-expression in LFD and HFD C57Bl/6J mice.

Shown are real-time RT-PCR results from arcuate tissues isolated from LFD and HFD C57Bl/6J mice (Fig. 1). Data for HFD are represented as % change from the LFD control group from each time-point (i.e. 3, 7 or 11 weeks of diets). The average expression level in LFD is normalized to 0% at each time-point. * = p<0.05; ** = p<0.01. N = 22–24 mice per group. Data are means +/− SEM.

Figure 3. Arcuate neuronal leptin-sensitivity in LFD and HFD C57Bl/6J mice.

C57Bl/6J mice were given diets for 10 weeks and injected with leptin (0.6 mg/kg, 30 minutes). A. Shown are representative microphoto images of P-STAT3 immunohistochemistry in the ARC of leptin-treated LFD and HFD mice. Matched representative coronal sections from the rostral, medial and caudal mediobasal hypothalamus are presented. B. Number of P-STAT3-immunoreactive (IR) ARC neurons in LFD and HFD mice. N = 5 sections per animal, and N = 3 mice per group. Counts are from one hemisphere in each section. C. Shown is quantification of the combined immunoreactivity (P-STAT3 IR) of all ARC P-STAT3 positive neurons. N = 5 sections per animal, and N = 3 mice per group. Analyses are done on one hemisphere from each section. * = p<0.05. Arc = hypothalamic ARC nucleus. Data are means +/− SEM.

Figure 4. POMC polypeptide expression in LFD and HFD C57Bl/6J mice.

A. Shown are matched representative images of POMC neurons as detected in situ hybridization. Matched coronal sections from the rostral, medial and caudal mediobasal hypothalamus of a LFD mouse are presented. B. Shown are matched representative images POMC neurons as detected by POMC immunohistochemistry in the mediobasal hypothalamus of LFD and HFD mice. Matched coronal sections from the rostral, medial and caudal mediobasal hypothalamus are presented. C57Bl/6J mice were given diets for 10 weeks and injected with leptin (0.6 mg/kg) 30 minutes before sacrifice. C. Number of POMC-immunoreactive neurons per LFD and HFD mouse. N = 2 mice per group. D. Shown is quantification of POMC immunoreactivity per POMC neuron. N = 2 mice per group. Tmt = Mammillothalamic tract; fx = fornix; DMH = Dorsomedial hypothalamic nucleus; VMH = Ventromedial hypothalamic nucleus. Data are means +/− SD.

Figure 5. Leptin-dependent STAT3 phosphorylation in POMC neurons of LFD and HFD C57Bl/6J mice.

A. Shown are representative images of double immunohistochemistry for P-STAT3 (DAB) and β-Endorphin (green fluorescence) in the mediobasal hypothalamus of leptin-treated LFD and HFD mice. C57Bl/6J mice were given diets for 16 weeks and injected with leptin 30 minutes before sacrifice. B. Percentage of P-STAT3 immunoreactive POMC (β-Endorphin) neurons per hypothalamus. One hemisphere of 9–11 matched hypothalamic brain sections was analyzed in each animal. C. Shown is the total P-STAT3 immunoreactivity (P-STAT3 IR) in all POMC neurons. For B. and C. P-STAT3 was counted and quantified in 258±24 POMC neurons from LFD mice (N = 3) and in 289±25 POMC neurons from HFD mice (N = 4). VMH = Ventromedial hypothalamic nucleus; DMH = Dorsomedial hypothalamic nucleus. * = p<0.05. Data are means +/− SEM.

Figure 6. Body weight and caloric intake of POMC-LepRb mice on HFD and LFD.

A. Body-weight curves of C57Bl/6J control mice and POMC-LepRb (C57Bl/6J) mice given HFD or LFD diets. B. Final average body weights after 19 weeks on diets. C. Cumulative food intake during the 19 weeks on diets. * = p<0.05; ** = p<0.01; # = p<0.001; ns = Not significant. All mice are littermates. N = 8–9 POMC-LepRb and N = 20–21 control animals per group. Data are means +/− SEM.

Figure 7. Body composition of POMC-LepRb mice on HFD and LFD.

A. Total lean mass. B. Total adipose mass. C. Perigonadal adipose mass. D. Perirenal fat mass. All animals are littermates. * = p<0.05; ** = p<0.01. ns = Not significant. N = 6–7 POMC-LepRb and N = 15–16 control animals per group. Data are means +/− SEM.

Figure 8. Serum glucose in POMC-LepRb mice on HFD and LFD.

A. Ad libitum fed glucose levels. B. Overnight fasted glucose levels. Fed glucose was measured at 12 weeks and fasted levels at 17 weeks on diets. ns = Not significant. N = 8–9 POMC-LepRb and N = 18–19 control mice per group. Data are means +/− SEM.

Figure 9. Arcuate mRNA and hypothalamic neuropeptide levels in HFD POMC-LepRb mice.

A–C. Arcuate SOCS3 mRNA, PTP1B mRNA and TC-PTP mRNA in POMC-LepRb and control mice after 19 weeks of HFD. Each mRNA was normalized to cyclophilin mRNA in the same samples. N = 6 POMC-LepRb and N = 11 control mice. D-F. Whole hypothalamic α-MSH, NPY and AgRP neuropeptide levels in POMC-LepRb and control mice after 19 weeks of HF diet. α-MSH: N = 6 POMC-LepRb and N = 13 control mice. NPY and AgRP: N = 3 POMC-LepRb and N = 10 control mice. ** = p<0.01. Data are means +/− SEM.

Figure 10. Leptin-sensitivity in POMC-LepRb mice on HFD and LFD.

A. Acute food intake after leptin (5 mg/kg, i.p.) or vehicle (PBS) in POMC-LepRb and control mice on LFD. B. Body weight change (relative to preinjection weight) after leptin administration in POMC-LepRb and control mice on LFD. C. Food intake after leptin in POMC-LepRb and control mice after 5 weeks on HFD. B. Body weight change after leptin in POMC-LepRb and control mice after 5 weeks on HFD. N = 4 POMC-LepRb and N = 5 control mice per group. * = p<0.05; ** = p<0.01; ns = not significant. Data are means +/− SEM.

Figure 11. Melanocortin (MTII)-sensitivity in POMC-LepRb mice on HFD and LFD.

A. Acute food intake after MTII (5 mg/kg, i.p.) or vehicle in POMC-LepRb and control mice on LFD. B. Body weight change (relative to preinjection weight) after MTII administration in POMC-LepRb and control mice on LFD. C. Food intake after MTII in POMC-LepRb and control mice after 5 weeks on HFD. B. Body weight change after MTII in POMC-LepRb and control mice after 5 weeks on HFD. N = 4 POMC-LepRb and N = 5 control mice per group. * = p<0.05; ** = p<0.01; # = p<0.001. Data are means +/− SEM.