Paraventricular nucleus Sim1 neuron ablation mediated obesity is resistant to high fat diet.

Single minded 1 (SIM1) is a transcription factor involved in brain patterning and control of energy balance. In humans, haploinsufficiency of SIM1 causes early-onset obesity. Mice deficient in the homologous gene, SIM1, also exhibit early onset obesity and increased sensitivity to a high fat diet. SIM1 is expressed in several areas of the brain implicated in control of energy balance including the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the medial amygdala and nucleus of the lateral olfactory tract. We have previously shown that mice with global Sim1 neuron ablation exhibit obesity with hyperphagia as the primary defect. The PVN has a critical role in feeding and in high-fat appetite, thus, we sought to determine the effect of Sim1 neuron ablation limited to the PVN. We achieved PVN-SIM1 limited ablation through stereotactic injection of diphtheria toxin into the PVN of Sim1Cre-iDTR mice. The specificity of this ablation was confirmed by immunohistochemistry and quantitative real time PCR of the PVN, supraoptic nucleus and the amygdala. Mice with PVN Sim1 neuron ablation, similar to mice with global Sim1 neuron ablation, exhibit early onset obesity with hyperphagia as the primary defect. However, PVN-Sim1 neuron ablated mice have a decreased response to fasting-induced hyperphagia. Consistent with this decrement, PVN-Sim1 neuron ablated mice have a decreased hyperphagic response to PVN injection of agouti-related peptide (AgRP). When PVN-Sim1 neuron ablated mice are placed on a high fat diet, surprisingly, their intake decreases and they actually lose weight. When allowed ad lib access to high fat diet and normal chow simultaneously, PVN-Sim1 neuron ablated mice exhibit overall decreased intake. That is, in PVN-Sim1 neuron ablated mice, access to fat suppresses overall appetite

Response of Sim1creiDTR and iDTR mice to high fat diet.

<p>(A) Food intake and (B) energy intake of Sim1creiDTR and iDTR mice. Mice were fed with chow for a week, switched to HF for a week, then back to chow(n=6 for each group). (C) Average daily body weight change of Sim1creiDTR and iDTR mice when fed with a chow diet or HF diet (n=6 for each group). (D)(E)Daily food intake (E) and energy intake (F) of Sim1creiDTR and iDTR mice fed with mixed diet of chow and HF (n=7 to 9 for each group). * p<0.05.</p

Immunofluorescence and quantitation of <i>SIM1</i> neurons in PVN and SON of iDTR and Sim1Cre mice.

<p>(A) PVN of iDTR mice shows robust expression of <i>SIM1</i>, (B) <i>SIM1</i> staining was dramatically decreased in PVN of Sim1creiDTR mice. Robust expression of <i>SIM1</i> was observed in SON of both iDTR (C) and Sim1creiDTR mice (D). (E) Quantitation of <i>SIM1</i> positive neurons in PVN and SON reveals similar numbers of <i>SIM1</i> positive neurons in SON of both iDTR and Sim1Cre mice but a significant decrease in SIM1 neurons of PVN of Sim1Cre mice relative to iDTR mice (n=3 for each group, * p<0.05). Scale bar: 40 µm for A, B; and 20 µm for C, D.</p

Body weight (A)(D), food intake (B)(E) and feeding efficiency of male and female Sim1creiDTR and iDTR mice.

<p>Body weight and food intake were measured weekly on a chow diet (n= 10 for male groups, n=9 for female groups, *p<0.05) (C) (F) feeding efficiency of male and female mice calculated as the ratio between weekly body weight change (g) and food intake (g) (n=8 for each group, * p<0.05).</p

Food intake after 12 hour fast, food intake after AgRP injection and metabolic rate after MC4R agonist injection.

<p>(A) (B) 4 hour and 24 hour intake of normal chow after fasting in Sim1creiDTR and iDTR mice after overnight 12 hour food deprivation (n≥4 for each group). (C)(D) 4 hour and 24 hour intake of normal chow in Sim1creiDTR and iDTR mice after injection of AgRP at 20 pmol into the PVN (n≥4 for each group). (E)(F) Oxygen consumption and metabolic rate of Sim1creiDTR and iDTR mice at 0min, 15 min, 30min, 60 min, and 120 min after injection of MC4R selective agonist into the PVN (n=3 for each group). Energy expenditure was measured in CLAMS cages during light cycle. *p< 0.05, **p<0.01, ***p<0.001. </p

Schema depicting Sim1 as global regulator of intake.

<p>In this model loss of Sim1 neurons in the PVN leads to increased Sim1 mRNA transcription in the amygdala and increased sensitivity of the amygdala to the satiety inducing effects of fat.</p

Real-time quantitative PCR comparing Sim1 mRNA, MC4R mRNA, and Galanin mRNA in PVN and Amygdala of Sim1creiDTR and iDTR mice, and comparing OXTmRNA in PVN of Sim1creiDTR and iDTR mice.

<p>Relative mRNA abundance of <i>SIM1</i> (A), MC4R (B), OXT (C), and GAL (F) expression in the PVN and <i>SIM1</i> (D), MC4R (E), and GAL (G) amygdala. (n=4 to 9 for each group, * p< 0.05, ** p<0.01, ***p<0.001) .</p