Obesity is associated with many metabolic disorders, including type 2 diabetes, dyslipidemia, and cardiovascular disease. The hypothalamus is a critical brain site regulating body weight, energy balance, and modulating glucose homeostasis. Therefore, understanding molecular mechanisms responsible for body weight regulation and glucose homeostasis in the hypothalamus will provide strategies to develop pharmacological intervention to combat metabolic disorders. Collectively, our results provide additional evidence demonstrating a role for SF-1 neurons and related transcriptional programs to regulate energy expenditure. Our results also add FOXO1 (and gene programs regulated by FOXO1 in SF-1 neurons) to the growing list of factors regulating energy balance. We have found that FOXO1 in neurons in the VMH also regulates glucose homeostasis. FOXO1 ablation in VMH SF-1 neurons increases energy expenditure without changing food intake, resulting in a lean phenotype. Moreover, ablation of FOXO1 in SF-1 neurons results in mice that are resistant to HFD-induced obesity, which is due to increased energy expenditure. Mice lacking FOXO1 in SF-1 neurons lose more weight following leptin administration due to increased energy expenditure. At the other end of the energy balance spectrum, we have found that VMH-specific FOXO1 KO mice lose more weight during a fast, a response that is due to a failure to appropriately suppress energy expenditure.
We also identified the Sf1 gene itself as a putative target of FOXO1 in the VMH. Our results predict that suppression of SF-1 during a fast is due in part to direct binding of FOXO1 to the SF-1 promoter. Further, these data are consistent with a model in which falling levels of leptin and insulin during a fast result in less activation of PI3K activity and more nuclear accumulation of FOXO1 and lower levels of SF-1. This regulation of SF-1 is required for appropriate modulation of energy expenditure in the face of changing levels of energy availability. Thus, identification of key downstream transcriptional programs of SF-1 and FOXO1 in SF-1 neurons may provide insights into potential targets that affect energy expenditure without changing food intake.
This study proposes the involvement of the transcription factor FOXO1 in regulation of energy expenditure in the VMH. Upon activation by metabolic stimuli such as leptin and insulin in the VMH of the WT animals, FOXO1 will be phosphorylated by the activated PKB (pAkt) and excluded from the nucleus, ultimately leading actions of leptin and insulin. Since FOXO1 has been known as a negative regulator of the leptin/PI3K/pAkt signaling pathway, FOXO1 removal from the VMH may confer permanently increased sensitivity to leptin in the nucleus and may preferentially induce alternations in neuronal activity and regulation of downstream target genes. In addition, increased levels of plasma norepinephrine and elevated UCP1 levels in iBAT of Foxo1 KOSf1
animals suggest that inhibition of FOXO1 in the SF-1 neurons of the VMH leads to activation of the SNS. This is consistent with several previous studies that have shown that leptin activates the SNS in mammals through increased catecholamine output (25
). Moreover, leptin injections into the VMH induced Ucp1
mRNA levels through activation of the SNS (25
). These results support the notion that leptin action on the regulation of the SNS in the VMH may preferentially be mediated by PI3K/AKT/FOXO1 pathways rather than Jak/Stat3 pathways (24
). We attributed the increased energy expenditure to elevated UCP1 expression in iBAT of Foxo1 KOSf1
animals. However, we do expect that other organs or tissues, such as muscle and heart, will play critical roles in regulating energy metabolism in the Foxo1 KOSf1
mice, as they showed increased leptin sensitivity and glucose uptake. In this regard, more detailed approaches, including direct measurement of metabolic rates in individual organs, will be informative in future experiments.
Our current data are consistent with several additional lines of previously described evidence. First, mice lacking leptin receptors in VMH neurons display increases in body adiposity together with decreases in energy expenditure (12
). Second, removal of the catalytic PI3K subunit Pik3ca
(p110α), an upstream molecule of FOXO1, from VMH SF-1 neurons results in increased sensitivity to HFD, due mainly to impaired energy expenditure (20
). Third, VMH-specific SF-1 KO animals showed blunted energy expenditure in part due to impaired leptin action in the VMH (23
). Finally, diet-induced increases in activation of PI3K lead to resistance to HFD-induced obesity in the SF-1ΔIR
animals, without changing energy expenditure, suggesting that deletion of IR in the SF-1 neurons is primarily related to PIP3
channel modulation rather than the PI3K/pAkt/FOXO1 pathway (16
The hyperinsulinemic-euglycemic clamp studies demonstrated that Foxo1 KOSf1
animals have increased glucose uptake in heart and skeletal muscle, with comparable hepatic glucose production between genotypes. This is consistent with previous reports, in which the VMH is a critical site for the regulation of glucose uptake in peripheral tissues, including heart and skeletal muscle, primarily through the SNS (26
). Furthermore, several previous reports support our hypotheses that the deletion of FOXO1 in the VMH may lead to improved glucose homeostasis via changes in SNS signaling. For example, leptin injections into the VMH preferentially increased glucose uptake in skeletal muscle, heart, and brown adipose tissue (BAT), and this increased glucose uptake was impaired when the SNS was denervated (26
). In addition, leptin receptor mutant mice that cannot activate STAT3 pathway exhibited only a modest defect in glucose homeostasis, despite the fact that the mutant animal showed the severe hyperphagia and obesity, suggesting that the leptin effect on glucose/insulin sensitivity may be differentiated from STAT3 pathways (40
). Conversely, restoration of leptin receptors in the ARH and VMH of Kolesky rats (an animal model of mutated leptin receptors) using viral-mediated gene delivery improved glucose/insulin homeostasis. This effect was attenuated by applying PI3K inhibitors (24
). Finally, leptin administration activates the SNS, and PI3K signaling is required for this effect (45
). Collectively, these results indicate that leptin-mediated PI3K/pAkt/FOXO1 signaling in the VMH may play an important role, not only in mediation of sympathetic tone, but also in regulation of peripheral insulin action.
An intriguing finding of this study is that, whereas FOXO1 is involved in modulation of food intake through transcriptional regulation of NPY, AGRP, and POMC in the ARH, no clear difference in food intake was detected in Foxo1 KOSf1
). In addition, specific deletion of FOXO1 in ARH using POMC-Cre did not show any alternation in energy expenditure and peripheral glucose/insulin sensitivity (8
). In contrast, deletion of FOXO1 in the VMH altered energy expenditure and whole-body glucose metabolism, without significant changes in food intake. Elucidation of a potential direct regulatory loop between FOXO1 and SF-1 provides evidence that FOXO1 and SF-1 direct a broad spectrum of transcriptional targets in VMH neurons. In this regard, we clearly do not expect that the SF-1 is the only target of FOXO1 in the VMH, and elucidation of additional transcriptional targets of FOXO1 is of great interest.
In summary, our data suggest that FOXO1 signaling in the VMH plays key roles in regulation of leptin sensitivity, energy expenditure, peripheral insulin action, and glucose homeostasis. These results support the idea that the VMH is a crucial hypothalamic site of whole-body metabolism and suggest that transcriptional programs regulated by FOXO1 are key in regulating energy balance and glucose homeostasis.