Hypothalamic DNA 5-hydroxymethylation levels are altered by diet-induced weight gain during the development of obesity in a sex-specific manner.

Obesity is a major health concern that is associated with altered gene transcription in the hypothalamus. However, the mechanisms controlling this gene expression dysregulation remain largely unknown. DNA 5-hydroxymethylation (5-hmC) is a potent transcriptional activator that is expressed at 10 times higher levels in the brain than the periphery. Despite this, no study has examined if DNA 5-hmC is altered in the brain following exposure to obesogenic diets or contributes to abnormal weight gain over time. Here, we used a rodent diet-induced obesity model in combination with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic DNA 5-hmC in abnormal weight gain in male and female rats. We found that males, but not females, have decreased levels of DNA 5-hmC in the hypothalamus following exposure to a high fat diet, which directly correlate with increased body weight. Short-term exposure to a high fat diet, which does not result in significant weight gain, resulted in decreased hypothalamic DNA 5-hmC levels, suggesting these changes occur prior to obesity development. Moreover, decreases in DNA 5-hmC persist even after the high fat diet is removed, though the extent of this is diet-dependent. Importantly, CRISPR-dCas9-mediated upregulation of DNA 5-hmC enzymes in the male, but not female, ventromedial nucleus of the hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. These results suggest that hypothalamic DNA 5-hmC is an important sex-specific regulator of abnormal weight gain following exposure to high fat diets.