We demonstrate for what we believe to be the first time that PKC-θ is expressed in the hypothalamus, is colocalized with neuronal populations previously shown to be critical for nutrient sensing and body weight regulation, and is responsive to palmitic acid, which induces translocation and activation of PKC-θ, resulting in inhibition of PI3K signaling. Furthermore, we provide evidence to suggest that in the brain, palmitic and not oleic acid, induces translocation of PKC-θ. Taken together, these findings suggest that diets high in palmitic acid confer central insulin resistance through PKC-θ in a mechanism similar to that which has been described in peripheral tissues.
The prevalence of obesity continues to rise at an alarming rate. There is increasing support for a role of elevated circulating fatty acids as a critical factor to explain the connection between obesity and insulin resistance (41
). An acute increase in plasma fatty acids by lipid infusion causes insulin resistance in animals (43
) as well as in healthy and diabetic humans (44
), potentially by altering insulin signaling downstream of PI3K (28
). Recently, we extended these findings to include fatty acid inhibition of insulin signaling in hypothalamic circuits that control energy homeostasis (10
). Here, we provide a potential mechanism by which diets high in palmitic acid interfere with hypothalamic insulin and leptin signaling through membrane translocation and activation of hypothalamic PKC-θ.
We further demonstrate that this phenomenon is specific for palmitic acid and does not occur following exposure to oleic acid. More than other fatty acids, palmitic acid is implicated in fatty acid–induced insulin resistance (48
) in the periphery and recently in the CNS (10
). Picinato et al. demonstrated that rats fed a diet enriched with olive oil for 6 weeks (10% of energy, 90% oleate acid) have improved glucose tolerance and insulin secretion compared with rats fed a diet high in animal fat (10% of energy, 30% palmitate acid, 50% oleate acid) diet (50
). Importantly, here we provide data to suggest that the relative ratios
of the fatty acids may be critical. Direct infusions or gavages of palmitic but not oleic acid induced arcuate membrane translocation of hypothalamic PKC-θ and impaired tyrosine phosphorylation of IRS proteins by increasing serine phosphorylation. Moreover, the specificity of palmitic acid is demonstrated by the data showing that oleic acid has no effect on the translocation of PKC-θ, consistent with maintenance of insulin signaling in animals maintained on a diet high in oleic acid. Therefore, these data suggest that the brain is responsive to changes in fatty acids and one mechanism by which the fatty acids impair insulin and leptin signaling and hepatic glucose production is through fatty acid mediated activation and translocation of PKC-θ in the hypothalamus.
Insulin rapidly lowers blood glucose by promoting glucose uptake and suppressing glucose production by both direct actions on the liver (51
) and by insulin signaling in extrahepatic tissues that in turn inhibit glucose production via neural and/or humoral mediators (2
). Inhibition of arcuate insulin action via antibodies, antisense oligonucleotides, or blockade of signaling intermediates (PI3K) leads to an attenuation of glucose production by circulating insulin (52
). Thus, insulin action in the hypothalamus is required for the full inhibitory effect of systemic insulin on glucose production and requires an intact insulin signaling cascade, involving the activation of the IR, IRS, and PI3K (2
). The arcuate has been repeatedly implicated as the crucial site for regulating peripheral glucose homeostasis (53
). Despite the fact in the arcuate insulin and leptin receptors are found on NPY/agouti-related protein (NPY/AgRP) and POMC/CART neurons (55
), there is no change in hepatic glucose production in mice lacking IRs in POMC neurons (56
). However, mice lacking IRs only on NPY/AgRP neurons fail to fully suppress hepatic glucose production (56
). Hence, it appears that suppression of hepatic glucose production by CNS insulin is mediated primarily by its effects on the NPY/AgRP neurons. Here, we confirm the findings of Dewing et al. that PKC-θ is expressed in neurons in the arcuate (37
) and extend those findings to also demonstrate PKC-θ is colocalized with AgRP/NPY neurons and its translocation and activity is modulated by fatty acids. Additionally, insulin effects in the CNS are mediated by activation of K+
ATP channels in arcuate neurons through insulin-induced activation of PI3K, resulting in subsequent membrane hyperpolarization. Our data suggest that palmitic acid exposure induces translocation of PKC-θ exclusively in arcuate NPY/AgRP neurons, which would lead to prevention of the activation of K+
ATP channels, reduced hyperpolarization, and eventually disinhibition of hepatic glucose production.
It has previously been demonstrated that PKC isoforms influence CNS regulation of hepatic glucose production (2
). Recently, Lam and colleagues (22
) reported activation of hypothalamic PKC-δ, via direct delivery of the PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG), suppressed hepatic glucose production. These authors concluded hypothalamic PKC-δ activation is sufficient and necessary to lower glucose production (22
). Where these findings might seem contradictory to our findings, we note that they addressed the role of PKC-δ, whereas we provide data on the effects of PKC-θ. It is well established that different PKC isoforms can and do exert opposing actions on physiological processes. Specifically in peripheral tissues, PKC-δ and -θ exert opposing actions on insulin signaling. Thus, both isoforms, localized to the arcuate nucleus, may operate under different mechanisms that coordinate the effects of central insulin (22
Importantly, fatty acids induce insulin resistance through accumulation of intracellular triglyceride and DAG, which induces recruitment and translocation of PKCs. Here, we demonstrate in the CNS, significant (P < 0.05) increases in DAG levels following exposure to the HFS diet and following the 3-day gavage of palmitic acid relative to the oleic acid and control diets/emulsions. Our findings provide further evidence that in the CNS elevated levels of DAG resulted in reduced insulin-stimulated IRS-1 tyrosine phosphorylation, increased serine 307 phosphorylation of IRS-1, and reduced PI3K activity, collectively consistent with fatty acid–mediated reductions in insulin signaling.
In a seminal paper, Shulman and colleagues (15
) demonstrated that mice lacking PKC-θ are resistant to fatty acid–induced insulin resistance. Importantly, they found that exposure to Intralipid (a mixture of fatty acids) increases translocation of PKC-θ from the cytosol to the cell membrane in skeletal muscle and consequently caused insulin resistance. However, a more recent paper reported the opposite effects (57
). We believe that these apparently contradictory findings may be explained by differences in the route, type, and duration of fatty acid administration. Here, we demonstrate that short-term infusions of fatty acids via gastric gavage, or directly into the CNS, were sufficient to induce translocation of PKC-θ and produce central insulin resistance. Interestingly, in the Shulman paper (15
), there was no modulation of glucose production during the clamp after lipid infusion between the PKC-θ knockout and WT mice. Here, we report that direct infusion of fatty acids into the CNS induced translocation of PKC-θ, reductions in insulin signaling, and modulation of hepatic glucose production. Our data suggest that activation and movement of this isozyme in the CNS is necessary and sufficient for HFS diet–induced hypothalamic insulin resistance and dysregulation of glucose homeostasis. The difference between our findings and those of Gao and colleagues (57
) may be due to the direct infusion of the fatty acids into the CNS as well as our isolation of the specific type of fatty acids important for dysregulation of glucose homeostasis.
Attention has been focused on understanding the role of high-fat diets in mediating insulin resistance and obesity. Importantly, in our experiments, we demonstrate that a short exposure to fatty acids resulted in significant (P
< 0.05) reductions in insulin signaling in the CNS. However, in light of recent data from Shulman et al. (58
), the liver also appears to become insulin resistant following 3-day exposure to fatty acids. Therefore, we can not conclude that the influence of the fatty acids is solely on the CNS. Many recent reports have linked insulin resistance with other disorders of the CNS, including Alzheimer disease and mild cognitive impairment (59
). However, perhaps because of their relative ubiquity in the CNS, investigation into the role of specific CNS PKCs in body weight regulation or CNS insulin signaling was previously unexplored. We demonstrate for the first time to our knowledge that short-term exposure to palmitic acid induced membrane translocation and activation of PKC-θ and that this was associated with reductions in hypothalamic insulin signaling. Finally, we demonstrate CNS fatty acid–induced translocation of PKC-θ is critical for mediating insulin resistance and obesity. Our data show that site-specific knockdown of PKC-θ in the arcuate attenuated diet-induced obesity and improved peripheral glucose homeostasis and insulin signaling in the arcuate nucleus. These data suggest that this specific PKC isozyme may represent an important yet unexplored mechanistic link between peripheral metabolic disease and impaired CNS insulin signaling.