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Activation of lipid-burning pathways in the fat-storing white adipose tissue (WAT) is a promising strategy to improve metabolic health and reduce obesity, insulin resistance, and type II diabetes. For unknown reasons, bilirubin levels are negatively associated with obesity and diabetes. Here, using mice and an array of approaches, including MRI to assess body composition, biochemical assays to measure bilirubin and fatty acids, MitoTracker-based mitochondrial analysis, immunofluorescence, and high-throughput coregulator analysis, we show that bilirubin functions as a molecular switch for the nuclear receptor transcription factor peroxisome proliferator–activated receptor α (PPARα). Bilirubin exerted its effects by recruiting and dissociating specific coregulators in WAT, driving the expression of PPARα target genes such as uncoupling protein 1 (Ucp1) and adrenoreceptor β 3 (Adrb3). We also found that bilirubin is a selective ligand for PPARα and does not affect the activities of the related proteins PPARγ and PPARδ. We further found that diet-induced obese mice with mild hyperbilirubinemia have reduced WAT size and an increased number of mitochondria, associated with a restructuring of PPARα-binding coregulators. We conclude that bilirubin strongly affects organismal body weight by reshaping the PPARα coregulator profile, remodeling WAT to improve metabolic function, and reducing fat accumulation.

Obesity-mediated metabolic syndrome remains the leading cause of death worldwide. Among many potential targets for pharmacological intervention, a promising strategy involves the heme oxygenase (HO) system, specifically its inducible form, HO-1. This review collects and updates much of the current knowledge relevant to pharmacology and clinical medicine concerning HO-1 in metabolic diseases and its effect on lipid metabolism. HO-1 has pleotropic effects that collectively reduce inflammation, while increasing vasodilation and insulin and leptin sensitivity. Recent reports indicate that HO-1 with its antioxidants via the effect of bilirubin increases formation of biologically active lipid metabolites such as epoxyeicosatrienoic acid (EET), omega-3 and other polyunsaturated fatty acids (PUFAs). Similarly, HO-1and bilirubin are potential therapeutic targets in the treatment of fat-induced liver diseases. HO-1-mediated upregulation of EET is capable not only of reversing endothelial dysfunction and hypertension, but also of reversing cardiac remodeling, a hallmark of the metabolic syndrome. This process involves browning of white fat tissue (i.e. formation of healthy adipocytes) and reduced lipotoxicity, which otherwise will be toxic to the heart. More importantly, this review examines the activity of EET in biological systems and a series of pathways that explain its mechanism of action and discusses how these might be exploited for potential therapeutic use. We also discuss the link between cardiac ectopic fat deposition and cardiac function in humans, which is similar to that described in obese mice and is regulated by HO-1-EET-PGC1α signaling, a potent negative regulator of the inflammatory adipokine NOV.

Bilirubin is a component of the heme catabolic pathway that is essential for liver function and has been shown to reduce hepatic fat accumulation. High plasma bilirubin levels are reflective of liver disease due to an injurious effect to hepatocytes. In a healthy liver, bilirubin is conjugated and excreted to the intestine and converted by microbes to urobilinoids, which is reduced to the predominant pigment in feces, stercobilin, or reabsorbed. The function of urobilinoids in the gut or their physiological relevance of reabsorption is not well understood. In this review, we discuss the relationship of hepatic bilirubin signaling to the intestinal microbiota and their regulation of the liver to gut axis, as well as their capacity to mediate these processes.

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3β (GSK3β) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3β phosphorylates serine 73 (Ser(P)73) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3β activity and Ser(P)73 of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKβ-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

Several large population studies have demonstrated a negative correlation between serum bilirubin levels and the development of obesity, hepatic steatosis, and cardiovascular disease. Despite the strong correlative data demonstrating the protective role of bilirubin, the mechanism by which bilirubin can protect against these pathologies remains unknown. Bilirubin has long been known as a powerful antioxidant and also has anti-inflammatory actions, each of which may contribute to the protection afforded by increased levels. We have recently described a novel function of bilirubin as a ligand for the peroxisome proliferator-activated receptor-alpha (PPARα), which we show specifically binds to the nuclear receptor. Bilirubin may function as a selective PPAR modulator (SPPARM) to control lipid accumulation and blood glucose. However, it is not known to what degree bilirubin activation of PPARα is responsible for the protection afforded to reduce hepatic steatosis. We hypothesize that bilirubin, acting as a novel SPPARM, increases hepatic fatty acid metabolism through a PPARα-dependent mechanism which reduces hepatic lipid accumulation and protects against hepatic steatosis and non-alcoholic fatty liver disease (NAFLD).

Recent studies have identified imidazole-dioxolane based compounds as novel heme oxygenase (HO) inhibitors. While these compounds have been demonstrated to be specific HO inhibitors in vitro, they have yet to be used to inhibit renal HO activity in vivo. The goal of this study was to determine the effectiveness of the imidazole-dioxolane HO-1 inhibitor, QC-13, in the inhibition of renal HO activity in vivo. HO-1 was induced in mice by treatment with cobalt protoporphyrin (CoPP). After 5 days, QC-13 was delivered either by continuous intrarenal medullary interstitial infusion (IRMI) into one kidney at several concentrations for 72 h or by two intraperitoneal injections over a 48-h period. IRMI infusion of QC-13 at a concentration of 25 microM resulted in a significant decrease in medullary but not cortical HO activity as compared to CoPP treated kidneys. IRMI infusion of QC-13 at a lower concentration (2.5 microM) had no effect on either medullary or cortical HO activity in CoPP treated mice. In contrast, administration of QC-13 at a higher concentration (250 microM) resulted in a significant decrease in both medullary and cortical HO activity in CoPP treated mice. Systemic administration of QC-13 resulted in significant decrease both renal cortical and medullary HO activity in CoPP treated mice. In contrast to classical porphyrin based HO inhibitors, IRMI infusion of QC-13 did not induce HO-1 protein levels as determined by Western blot analysis of medullary protein samples. Our results demonstrated that imidazole-dioxolane inhibitors are renal HO inhibitors in vivo and can inhibit HO activity independent of HO-1 induction. These inhibitors may be useful tools to elucidate the role of renal HO-1 in numerous physiologic and pathophysiologic conditions.

The objective of this study was to determine whether heme oxygenase-1 (HO-1) or heme metabolites exert cytoprotective effects on interleukin-18-mediated endothelial cell (EC) death. Treatment with interleukin (IL)-18 increased NF-kappaB activation and PTEN induction, suppressed Akt activation, and stimulated EC death. While ectopic expression of p65 enhanced PTEN transcription, adenoviral transduction of dnIkappaB-alpha, dnp65, or dnIKKbeta was inhibitory. Furthermore, IL-18 suppressed HO-1 mRNA expression via enhanced mRNA degradation. Overexpression of HO-1, treatment with HO-1 inducer hemin, or the CO donor cobalt (III) protoporphyrin IX all reversed IL-18-mediated NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Furthermore, hemin induced HO-1 expression, and HO-1 knockdown, HO-1 inhibition, or CO scavengers all reversed the prosurvival effects of hemin. In addition, the CO donors CORM-1 and CORM-3 and the heme metabolites biliverdin and bilirubin attenuated IL-18-induced EC death via a similar signaling pathway. IL-18 induced p38alpha MAPK activation, and suppressed p38beta isoform expression. While p38alpha knockdown attenuated, p38beta knockdown potentiated IL-18-mediated EC death. Hemin and HO-1 reversed IL-18-mediated p38alpha induction and restored p38beta levels. These results demonstrate that IL-18 suppresses HO-1 expression and induces EC death. HO-1 overexpression, HO-1 induction, or treatment with heme metabolites all reverse IL-18-mediated p38alpha MAPK and NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Thus, HO-1 inducers and CO donors may have the therapeutic potential to effectively block IL-18 signaling and reduce IL-18-dependent vascular injury and inflammation.

Radiocontrast agents are thought to induce acute kidney injury in part through increased production of reactive oxygen species and increased cellular apoptosis. In this study we determined whether heme oxygenase-1 could prevent or reduce radiocontrast-induced acute kidney injury and, if so, what were the mechanisms by which this can occur. Sodium iothalamate was administered to uninephrectomized, salt-depleted male Sabra rats to initiate acute kidney injury. Heme oxygenase-1 was induced with cobalt protoporphyrin or inhibited with stannous mesoporphyrin. Inhibition of heme oxygenase exacerbated kidney injury as measured by an increase in plasma creatinine and in superoxide production. Heme oxygenase-1 induction prevented the increase in plasma creatinine and in superoxide in both the cortex and medulla compared to untreated rats with acute kidney injury. This protective effect of heme oxygenase-1 was associated with increased anti-apoptotic proteins Bcl-2 and Bcl-xl and a decrease of pro-apoptotic caspase-3 and caspase-9 along with increased expression of inactive BAX. Our study suggests that increased levels of heme oxygenase-1 are protective against acute kidney injury due to radiocontrast exposure.

Moxonidine is a centrally-active imidazoline compound with preferential affinity for imidazoline receptors (IR) over alpha(2)-adrenoceptors (alpha(2)AR). Clinically, moxonidine has proven advantageous for treating hypertension over pure alpha(2)-adrenergic agonists (i.e., guanabenz) due to its lowered incidence of sedative side effects. The present experiments reveal divergent behavioral effects of low doses of moxonidine and guanabenz in C57Bl/6 mice in an exploratory arena. Low-dose moxonidine (0.05 mg kg(-1) i.p.) elicited an increase in novel object contacts (+36%) and more movement into central space (+56%; P

Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.

As more effort is made to identify genes responsible for hypertension in human populations and genetically hypertensive animal models, the need for experimental systems in which the functional significance of genes, gene variants, and quantitative trait loci (QTL) can be determined is becoming increasingly important. Over the past five years, transgenic and gene-targeting technology has been utilized to study the cardiovascular effects of over-expression or ablation of genes which have been considered candidates in the genetic basis of hypertension. This review focuses on the most recent major advances in this area, and how this technology aids in our understanding of the molecular mechanisms by which newly discovered genes or gene variants affect blood pressure in the whole organism. We also discuss the potential use of transgenic models in refining the location of a QTL, and discuss some of the limitations and potential pitfalls in the application of these tools to the field of hypertension research. The coupling of genetic manipulations afforded by transgenesis and gene targeting, along with advances in our ability to assess the cardiovascular phenotype in the mouse, provides us with a powerful system for examining the genes responsible for causing essential hypertension.