Analysis of Western diet, palmitate and BMAL1 regulation of neuropeptide Y expression in the murine hypothalamus and BMAL1 knockout cell models.

Western diets that are high in saturated fat and sugar disrupt circadian rhythms, induce weight gain, and lead to metabolic diseases including obesity. However, the mechanistic link between altered circadian rhythms and energy homeostasis remains poorly understood. In C57BL/6J mice, consuming a Western diet for 16 weeks significantly reduced food intake (at zeitgeber 12-16), in association with decreases in hypothalamic expression of the orexigenic neuropeptides, neuropeptide Y (Npy) and agouti-related peptide (AgRP). To examine the acute effects of the most prevalent saturated fatty acid in a Western diet, palmitate, and the role of the core clock gene, Bmal1, in the regulation of hypothalamic feeding neuropeptides, we used heterogeneous and clonal BMAL1 knockout (KO) immortalized hypothalamic cell lines, expressing specific neuropeptides, derived from male (M) and female (F) mice. Both mHypoA-BMAL1-KO/F and mHypoA-BMAL1-KO/M cells demonstrated a loss of circadian rhythmicity in expression of the clock gene, Per2, as compared to wild-type (control) cultures. Loss of BMAL1 also altered the time-dependent expression of Npy and proopiomelanocortin, and disrupted AgRP rhythmicity. Furthermore, palmitate increased BMAL1 binding to the Npy promotor region, and palmitate treatment (50 µM for 24 h) stimulated Npy expression in a BMAL1-dependent manner in both heterogeneous and clonal NPY-expressing female-derived cell models. The results of this study demonstrate that circadian expression of Bmal1 serves as a mechanistic link between Western diet- and palmitate-induced disruptions of the normal rhythmic patterns in hypothalamic feeding-related neuropeptides.

Tumour necrosis factor α induces neuroinflammation and insulin resistance in immortalised hypothalamic neurones through independent pathways.

The links between obesity, inflammation and insulin resistance, which are all key characteristics of type 2 diabetes mellitus, are yet to be delineated in the brain. One of the key neuroinflammatory proteins detected in the hypothalamus with over-nutrition is tumour necrosis factor (TNF)α. Using immortalised embryonic rat and mouse hypothalamic cell lines (rHypoE-7 and mHypoE-46) that express orexigenic neuropeptide Y and agouti-related peptide, we investigated changes in insulin signalling and inflammatory gene marker mRNA expression after TNFα exposure. A quantitative polymerase chain reaction array of 84 inflammatory markers (cytokines, chemokines and receptors) demonstrated an increase in the expression of multiple genes encoding inflammatory markers upon exposure to 100 ng mL-1 TNFα for 4 hours. Furthermore, neurones pre-exposed to TNFα (50 ng mL-1 ) for 6 or 16 hours exhibited a significant reduction in phosphorylated Akt compared to control after insulin treatment, indicating the attenuation of insulin signalling. mRNA expression of insulin signalling-related genes was also decreased with exposure to TNFα. TNFα significantly increased mRNA expression of IκBα, Tnfrsf1a and IL6 at 4 and 24 hours, activating a pro-inflammatory state. An inhibitor study using an inhibitor of nuclear factor kappa B kinase subunit ß (IKK-ß) inhibitor, PS1145, demonstrated that TNFα-induced neuroinflammatory marker expression occurs through the IKK-ß/nuclear factor-kappa B pathway, whereas oleate, a monounsaturated fatty acid, had no effect on inflammatory markers. To test the efficacy of anti-inflammatory treatment to reverse insulin resistance, neurones were treated with TNFα and PS1145, which did not significantly restore the TNFα-induced changes in cellular insulin sensitivity, indicating that an alternative pathway may be involved. In conclusion, exposure to the inflammatory cytokine TNFα causes cellular insulin resistance and inflammation marker expression in the rHypoE-7 and mHypoE-46 neurones, consistent with effects seen with TNFα in peripheral tissues. It also mimics insulin- and palmitate-induced insulin resistance in hypothalamic neurones. The present study provides further evidence that altered central energy metabolism may be caused by obesity-induced cytokine expression.

Role of the saturated fatty acid palmitate in the interconnected hypothalamic control of energy homeostasis and biological rhythms.

The brain, specifically the hypothalamus, controls whole body energy and glucose homeostasis through neurons that synthesize specific neuropeptides, whereas hypothalamic dysfunction is linked directly to insulin resistance, obesity, and type 2 diabetes mellitus. Nutrient excess, through overconsumption of a Western or high-fat diet, exposes the hypothalamus to high levels of free fatty acids, which induces neuroinflammation, endoplasmic reticulum stress, and dysregulation of neuropeptide synthesis. Furthermore, exposure to a high-fat diet also disrupts normal circadian rhythms, and conversely, clock gene knockout models have symptoms of metabolic disorders. While whole brain/animal studies have provided phenotypic end points and important clues to the genes involved, there are still major gaps in our understanding of the intracellular pathways and neuron-specific components that ultimately control circadian rhythms and energy homeostasis. Because of its complexity and heterogeneous nature, containing a diverse mix cell types, it is difficult to dissect the critical hypothalamic components involved in these processes. Of significance, we have the capacity to study these individual components using an extensive collection of both embryonic- and adult-derived, immortalized hypothalamic neuronal cell lines from rodents. These defined neuronal cell lines have been used to examine the impact of nutrient excess, such as palmitate, on circadian rhythms and neuroendocrine signaling pathways, as well as changes in vital neuropeptides, leading to the development of neuronal inflammation; the role of proinflammatory molecules in this process; and ultimately, restoration of normal signaling, clock gene expression, and neuropeptide synthesis in disrupted states by beneficial anti-inflammatory compounds in defined hypothalamic neurons.