Microglial activation as a mediator of hypothalamic leptin resistance: a target for bilirubin?
One of the phenomena that promote weight gain as people grow older is the development of hypothalamic leptin resistance.21 The hormone leptin is produced primarily in adipocytes, and its plasma levels rise as body fat mass increases. Leptin functions to counteract inappropriate weight gain by acting on leptin-responsive neurons in the hypothalamus to suppress appetite while also boosting metabolic rate via sympathetic activation.22–24 Of particular interest in this regard are leptin-responsive neurons in the arcuate nucleus of the mediobasal hypothalamus (MBH); the MBH has a poorly developed blood–brain barrier, and hence hormones, free fatty acids and other plasma components have ready access to it.25 Leptin-responsive neurons in the arcuate nucleus boost anorexic signalling by increasing neuronal release of pro-opiomelanocortin, while suppressing release of the orexigenic hormones neuropeptide Y and agouti-related peptide within this nucleus. The physiological importance of this mechanism, at least in mice, is confirmed by the fact that genetic strains of mice which are incapable of making either leptin (ob/ob) or functional leptin receptors (db/db) overeat and become obese and diabetic.26 27
Unfortunately, efforts to develop injectable leptin as an antiobesity drug have not been successful, as overweight subjects are resistant to its suppressive impact on appetite. Studies in rodents with diet-induced obesity suggest that this phenomenon reflects a loss of leptin responsiveness that is specific to the arcuate nucleus.21 28–30 Activated leptin receptors trigger JAK2-mediated phosphorylation of STAT3, which then migrates as a homodimer to the nucleus to modulate gene transcription. In lean chow-fed rodents, a leptin injection rapidly boosts pSTAT3 levels in the arcuate nucleus and suppresses feeding; this response is substantially blunted in obese rodents. In contrast, leptin is able to raise pSTAT3 levels in other leptin-responsive regions of the brain in obese rodents.31
Although the molecular biology underlying hypothalamic leptin resistance in obesity is still somewhat obscure, studies focusing on high-fat/high-sugar diet-induced obesity in rodents have yielded some intriguing findings. In particular, activation and proliferation of microglia in the MBH are noted in rodents with diet-induced obesity.32–34 The microglial activation noted in this situation appears to be mediated primarily by saturated fatty acids interacting with toll-like receptor-4 (TLR4) expressed by microglia.32 35 36 (Plasma-derived fetuin-A forms a trimeric complex with fatty acids and TLR4, catalysing this interaction.37–39) Hence, TLR4 antagonists—but not TLR2 antagonists—prevent microglial activation and development of leptin resistance in rats fed a fatty diet.35 Microglial proliferation is also noted in this circumstance, and measures which prevent microglial proliferation have likewise been found to prevent development of leptin resistance in rodents.32 33 How activated microglia act to impair leptin responsiveness in the arcuate nucleus is still unclear.
A key role for saturated fatty acids in driving leptin resistance might help to explain why risk for obesity is lower in those who habitually consume plant-based or ‘Mediterranean’ diets in which saturated fats constitute a relatively low percentage of total fatty acids.40–47 Risk for type 2 diabetes has been found to be markedly lower in individuals who follow a plant-based diet.48 Increased hepatic production of fibroblast growth factor 21 may also contribute to the obesity prevention associated with plant-based diets of modest protein content.49–51
Activation of microglia via TLR4—as with lipopolysaccharides—has been shown to entail activation of Nox2-dependent NADPH oxidase.52–54 Moreover, this activation is required for production of toxic oxidants such as peroxynitrite, and increased production of proinflammatory cytokines and prostanoids. Hence, it is straightforward to propose that bilirubin may have the ability to downregulate microglial activation by diminishing NADPH oxidase activation.55 In light of the foregoing discussion, a corollary of this is that elevated bilirubin—whether derived from plasma or from local haem oxygenase activity—may oppose the evolution of leptin resistance by inhibiting the activation (and likely proliferation) of microglia in the MBH. The ability of the HO-1 inducer haemin to alleviate hyperleptinaemia—a marker for leptin resistance—in fat-fed rats appears consistent with this possibility.56
With respect to bilirubin and microglia, it should be noted that, when unconjugated bilirubin exceeds its solubility limit (70 nM), it can disrupt membranes and promote microglial activation.57 58 This explains the neural damage and microglial activation associated with perinatal bilirubin encephalopathy, which can occur in newborns whose livers have limited capacity to conjugate bilirubin at a time when the blood–brain barrier is poorly formed. Analogously, bilirubin neurotoxicity is seen in Crigler-Najjar syndrome, in which mutations of the UGT1A1 render it non-functional, and plasma bilirubin levels are roughly an order of magnitude higher than those seen in GS.59 The concentrations of unconjugated bilirubin which result from haem oxygenase induction appear to be below its solubility limit, as such induction tends to suppress microglial activation and provide neuroprotection in rodent or cell culture models.60 In endothelial cells, bilirubin’s antioxidant activity has been found to be half-maximal at 11 nM; hence, bilirubin can function physiologically as an important intracellular antioxidant in concentrations far below its solubility limit.
One of the cytokines whose expression by microglia is contingent on Nox2 activity is tumour necrosis factor-alpha (TNFα).52 53 TNFα, via Nuclear factor-kappa beta (NF-kappaB) activation, provokes increased hypothalamic expression of phosphotyrosine phosphatase-1A (PTP1B), which functions as an antagonist of leptin signalling by reversing activating tyrosine phosphorylation of JAK2.61–64 Hypothalamic PTP1B activity increases in response to high-fat diets in rodents, and neuronal PTP1B knockout mice fail to develop leptin resistance and obesity when fed such diets; a similar effect is seen when hypothalamic PTP1B activity is inhibited with antisense oligonucleotides.61 65 66 Hence, the TNFα produced by microglia—and possibly other cytokines capable of inducing PTP1B in neurons—likely contributes to leptin resistance by boosting PTP1B expression.
Additionally, there is evidence that hypothalamic TNFα can oppose leptin resistance by additional mechanisms, likely including increased expression of suppressor of cytokine signalling-3 (SOCS-3).62 This protein, via an inhibitory interaction with JAK2, blocks all known signalling pathways activated by the leptin receptor. It is elevated in the hypothalamus of fat-fed rodents, and mice that are heterozygous for SOCS-3 gene deletion are resistant to diet-induced obesity.67 68 SOCS-3 is induced at the transcriptional level by leptin, and thus provides feedback regulation of leptin activity.69 TNFα can increase its expression by boosting the half-life of its mRNA, thereby amplifying the efficacy of this negative feedback mechanism.70 In obese mice whose leptin is clamped at a lower level similar to that of lean mice, an injection of leptin causes a normal rise in arcuate pSTAT3; this might reflect the fact that their baseline level of SOCS-3 in leptin-responsive arcuate neurons is relatively low.71
If we presume, not unreasonably, that hypothalamic leptin resistance tends to evolve and worsen gradually over a lifetime—possibly reflecting proliferation of activated microglia in the arcuate nucleus—then the fact that body composition is only slightly modified in young subjects with GS relative to controls may simply reflect the fact that bilirubin cannot influence leptin activity until leptin resistance begins to develop.