Degeneration and loss of orexin neurons are observed under several neuropathological conditions including narcolepsy, but the detailed mechanisms of these changes are yet unclear. A notable feature of orexin-A peptide is the presence of two intramolecular disulfide bonds located closely each other, which may render this neuropeptide prone to misfolding. We have recently demonstrated that nitric oxide (NO)-mediated inactivation of protein disulfide isomerase leads to formation of orexin-immunopositice intracellular aggregates and subsequent neuron loss. Moreover, we found that sleep deprivation promoted NO production by neuronal NO synthase in the hypothalamus, which resulted in specific pathological changes in orexin neurons (J Neurosci 33: 12557-12568, 2013). Here we investigated whether lifestyle-related factors other than sleep deprivation could also trigger NO-mediated neuropathological events in orexin neurons. Male C57BL/6 mice were fed with high fat diet for 8 weeks from 4 weeks old, and then, their hypothalami were examined by immunohistochemical procedures and Western blot. The lateral hypothalamus of mice fed with high fat diet contained fewer orexin-A-immunopositive neurons as compared to that of mice fed with normal diet. In addition, orexin-A-immunopositive intracellular aggregates were observed in a subset of hypothalamic neurons in mice with high fat diet. In comparison, melanin-concentrating hormone-immunopositive neurons in the lateral hypothalamus did not display abnormal immunohistochemical appearance even when mice were fed with high fat diet. Importantly, high fat diet increased expression of inducible NO synthase (iNOS) in the hypothalamus. Moreover, in iNOS-deficient mice, high fat diet did not induce formation of orexin-A-immunopositive intracellular aggregates or a decrease in the number of orexin-A-immunopositive neurons. These results suggest that, in contrast to the case with sleep deprivation, iNOS plays a dominant role in high fat diet-induced pathological changes in orexin neurons.