br Materials and Methods br Results br

Materials and Methods
Results
Discussion While circadian clocks throughout the body clearly play a role in regulating metabolic pathways and their homeostatic function, increasing evidence for diet-induced alterations in clock gene oscillations and behavioral rhythms suggests that nutrient metabolism may, in turn, feed back on the clock mechanism and modulate fundamental properties of circadian rhythms. HFD has been shown to alter circadian timekeeping function although its effects vary greatly among clocks in the brain and peripheral tissues. For example, HFD had no effect on core clock gene oscillations in the hypothalamus and only produced small increases in the free-running period of the SCN-regulated rhythm of locomotor activity (Kohsaka et al., 2007; Xu et al., 2014). Consistent with the effects of HFD on circadian clocks in the brain, palmitate alters clock gene expression and the amplitude of their rhythmic profiles in a hypothalamic cell line and this effect is not accompanied by a corresponding induction of inflammatory cytokines such as IL-6 (Fick et al., 2011; Greco et al., 2014). In contrast, HFD-induced dysregulation of circadian timekeeping is more robust in peripheral clocks; in HFD-fed mice, clock gene oscillations are distinguished by severe damping in both liver and fat tissue (Kohsaka et al., 2007) and by circadian period increases of up to 9h in adipose tissue and bone marrow-derived macrophages (Xu et al., 2014). Similar to the latter effect of HFD, prolonged treatment with palmitate induced large increases in the period of fibroblast Bmal1-dLuc rhythms, suggesting that this proinflammatory SFA is a key mediator of HFD-induced modulation of peripheral circadian clocks. Importantly, our findings indicate that the effects of HFD and its major constituent, palmitate, on circadian clock properties are not limited to changes in period and rhythm amplitude, but include differential phase shifts of clock gene oscillations in peripheral tissues. Previous studies have established that phase-shifting responses to HFD again differ among peripheral and central circadian clocks. In this regard, HFD treatment for 7days has been shown to shift Per2 oscillations, producing large advances in the liver and small delays in the spleen but no effect on the phase of SCN rhythms (Pendergast et al., 2013). In the present study, 4-hour exposure of peripheral cell types to palmitate similarly induced marked phase shifts of clock gene oscillations that varied in amplitude depending on the time of treatment. It is noteworthy that the time-dependent nature of palmitate-mediated phase shifts of the Bmal1 rhythm was cell-specific; the phase responses of undifferentiated fibroblasts to this proinflammatory SFA were maximal at hour 12 and negligible at hour 6 when palmitate-induced phase advances were at peak amplitude in differentiated adipocytes. Because the omega-3 fatty order Necrostatin 1 DHA had little or no phase-shifting effects on clock gene oscillations in both fibroblasts and differentiated adipocytes, these findings collectively suggest that SFAs may differentially reset the clock mechanism in some but not all cells within a given tissue, thereby altering the local coordination of circadian timekeeping among individual cellular clocks. How HFD and palmitate modulate circadian timekeeping is unknown. The present evidence for the close coincidence between the time-dependent variation in inflammatory and phase-shifting responses to palmitate raises the possibility that mutual interactions between peripheral circadian clocks and inflammatory signaling pathways may play a key role in the underlying mechanism. In both Bmal1-dLuc fibroblasts and differentiated adipocytes, the maxima and minima for palmitate-induced inflammatory signaling were contemporaneous with the equivalent variation in the amplitude of the phase shifts induced by this SFA. Thus, the signaling cascades through which HFD and SFAs induce inflammation in peripheral tissues may govern the modulatory effects of palmitate on circadian timekeeping. The induction of inflammatory signaling by palmitate and other SFAs is mediated through activation of NF-κB, ultimately leading to the secretion of proinflammatory cytokines in various peripheral tissues (Weigert et al., 2004; Ajuwon and Spurlock, 2005; Jové et al., 2006). In addition, the nutrient sensor, AMP-activated protein kinase (AMPK), is involved in coupling fatty acid metabolism to inflammatory responses. Palmitate and other SFAs decrease AMPK activity (Sun et al., 2008; Lindholm et al., 2013) and correspondingly their inductive effects on NF-κB activation and the expression of proinflammatory cytokines are repressed by AMPK activators (Yang et al., 2010; Green et al., 2011). The role of inflammatory signaling in SFA-mediated phase regulation of peripheral circadian clocks is directly supported by the present findings that the anti-inflammatory inhibitor of NF-ĸB signaling, cardamonin, and the AMPK activator, AICAR, suppress peak induction of both inflammatory and phase-shifting responses to palmitate at hour 12. Because DHA represses inflammation-induced NF-ĸB signaling and cytokine expression via activation of AMPK (Xue et al., 2012), the observed inhibition of palmitate-induced inflammation and phase shifts following pretreatment with this PUFA also underscores the importance of clock-gated inflammatory signaling in the mechanism by which palmitate alters circadian timekeeping. In conjunction with evidence for the circadian regulation of AMPK activity (Um et al., 2011) and inflammatory responses of the NF-ĸB pathway (Spengler et al., 2012) in different tissues, these observations indicate that mutual interactions between peripheral clocks and inflammatory signaling mediate the time-dependent variation in the extent to which SFA overload triggers inflammation and in turn, leads to the feedback modulation of circadian timekeeping.