Over-Expression of Leptin Receptors in Hypothalamic POMC Neurons Increases Susceptibility to Diet-Induced Obesity

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

Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/−) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR−/−) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR−/− rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR−/− males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR−/− animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p