Human resistin in chemotherapy-induced heart failure in humanized male mice and in women treated for breast cancer.

Resistin is a circulating mediator of insulin resistance mainly expressed in human monocytes and responsive to inflammatory stimuli. Recent clinical studies have connected elevated resistin levels with the development and severity of heart failure. To further our understanding of the role of human resistin in heart failure, we studied a humanized mouse model lacking murine resistin but transgenic for the human Retn gene (Hum-Retn mice), which exhibits basal and inflammation-stimulated resistin levels similar to humans. Specifically, we explored whether resistin underlies acute anthracycline-induced cardiotoxicity. Remarkably, doxorubicin (25mg/kg ip) led to a 4-fold induction of serum resistin levels in Hum-Retn mice. Moreover, doxorubicin-induced cardiotoxicity was greater in the Hum-Retn mice than in littermate controls not expressing human resistin (Retn(-/-)). Hum-Retn mice showed increased cardiac mRNA levels of inflammatory and cell adhesion genes compared with Retn(-/-) mice. Macrophages, but not cardiomyocytes, from Hum-Retn mice treated with doxorubicin in vitro showed dramatic induction of hRetn (human resistin) mRNA and protein expression. We also examined resistin levels in anthracycline-treated breast cancer patients with and without cardiotoxicity. Intriguingly, serum resistin levels in women undergoing anthracycline-containing chemotherapy increased significantly at 3 months and remained elevated at 6 months in those with subsequent cardiotoxicity. Further, elevation in resistin correlated with decline in ejection fraction in these women. These results suggest that elevated resistin is a biomarker of anthracycline-induced cardiotoxicity and may contribute in the development of heart failure via its direct effects on macrophages. These results further implicate resistin as a link between inflammation, metabolism, and heart disease.

Inverse regulation of inflammation and mitochondrial function in adipose tissue defines extreme insulin sensitivity in morbidly obese patients.

Obesity is associated with insulin resistance, a major risk factor for type 2 diabetes and cardiovascular disease. However, not all obese individuals are insulin resistant, which confounds our understanding of the mechanistic link between these conditions. We conducted transcriptome analyses on 835 obese subjects with mean BMI of 48.8, on which we have previously reported genetic associations of gene expression. Here, we selected ~320 nondiabetic (HbA(1c) <7.0) subjects and further stratified the cohort into insulin-resistant versus insulin-sensitive subgroups based on homeostasis model assessment-insulin resistance. An unsupervised informatics analysis revealed that immune response and inflammation-related genes were significantly downregulated in the omental adipose tissue of obese individuals with extreme insulin sensitivity and, to a much lesser extent, in subcutaneous adipose tissue. In contrast, genes related to ß-oxidation and the citric acid cycle were relatively overexpressed in adipose of insulin-sensitive patients. These observations were verified by querying an independent cohort of our published dataset of 37 subjects whose subcutaneous adipose tissue was sampled before and after treatment with thiazolidinediones. Whereas the immune response and inflammation pathway genes were downregulated by thiazolidinedione treatment, ß-oxidation and citric acid cycle genes were upregulated. This work highlights the critical role that omental adipose inflammatory pathways might play in the pathophysiology of insulin resistance, independent of body weight.

Resistin levels in lupus and associations with disease-specific measures, insulin resistance, and coronary calcification.

OBJECTIVE: To evaluate levels of resistin in female subjects with systemic lupus erythematosus (SLE) compared to age and race-matched controls and to determine the relationship between resistin and systemic inflammation, disease measures, and coronary artery calcification (CAC). METHODS: Resistin levels were measured on stored samples from 159 women with SLE and 70 controls as an extension of a previous cross-sectional study. Spearman correlations and multivariable regressions were used to examine whether resistin levels were associated with SLE, disease-specific and inflammatory markers, insulin resistance, and CAC. RESULTS: In a multivariable linear regression model, a diagnosis of SLE was significantly associated with higher resistin levels independent of age, race, renal function, body mass index (BMI), high-sensitivity CRP (hsCRP), hypertension, diabetes, and steroid use. In SLE, resistin levels correlated positively with Systemic Lupus International Collaborating Clinics Damage Index, glomerular filtration rate (GFR), hsCRP, erythrocyte sedimentation rate, homocysteine, and disease duration (all p < 0.03). Resistin level did not correlate with markers of insulin resistance or body adiposity, including homeostatic model assessment or BMI. Resistin levels were significantly elevated in SLE cases with CAC compared to cases without CAC (16.58 vs 13.10 ng/ml, respectively; p = 0.04). In multivariate logistic regression, the association was not present after adjustment for age, race, and GFR. CONCLUSION: SLE was independently associated with higher resistin levels. Among subjects with SLE, higher resistin level correlated positively with renal dysfunction, inflammatory markers, and disease damage but not with insulin resistance or BMI. SLE cases with CAC had higher resistin levels than cases without CAC; however, this relationship was dependent on other established risk factors.

Inflammatory induction of human resistin causes insulin resistance in endotoxemic mice.

OBJECTIVE: Although adipocyte-derived murine resistin links insulin resistance to obesity, the role of human resistin, predominantly expressed in mononuclear cells and induced by inflammatory signals, remains unclear. Given the mounting evidence that obesity and type 2 diabetes are inflammatory diseases, we sought to determine the relationship between inflammatory increases in human resistin and insulin resistance. RESEARCH DESIGN AND METHODS: To investigate the role of human resistin on glucose homeostasis in inflammatory states, we generated mice lacking murine resistin but transgenic for a bacterial artificial chromosome containing human resistin (BAC-Retn), whose expression was similar to that in humans. The metabolic and molecular phenotypes of BAC-Retn mice were assessed after acute and chronic endotoxemia (i.e., exposure to inflammatory lipopolysaccharide). RESULTS: We found that BAC-Retn mice have circulating resistin levels within the normal human range, and similar to humans, lipopolysaccharide markedly increased serum resistin levels. Acute endotoxemia caused hypoglycemia in mice lacking murine resistin, and this was attenuated in BAC-Retn mice. In addition, BAC-Retn mice developed severe hepatic insulin resistance under chronic endotoxemia, accompanied by increased inflammatory responses in liver and skeletal muscle. CONCLUSIONS: These results strongly support the role of human resistin in the development of insulin resistance in inflammation. Thus, human resistin may link insulin resistance to inflammatory diseases such as obesity, type 2 diabetes, and atherosclerosis.

Cell-specific determinants of peroxisome proliferator-activated receptor gamma function in adipocytes and macrophages.

The nuclear receptor peroxisome proliferator activator receptor gamma (PPARgamma) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARgamma is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPARgamma binding in macrophages occurs at genomic locations the same as or different from those in adipocytes. Here, utilizing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we demonstrate that PPARgamma cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycolate-elicited macrophages, PPARgamma colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPARgamma, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell specificity of PPARgamma function is regulated by cell-specific transcription factors, chromatin accessibility, and histone marks. Our data support the existence of an epigenomic hierarchy in which PPARgamma binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks, including histone acetylation.

Constitutive androstane receptor mediates the induction of drug metabolism in mouse models of type 1 diabetes.

UNLABELLED: Untreated type 1 diabetes increases hepatic drug metabolism in both human patients and rodent models. We used knockout mice to test the role of the nuclear xenobiotic receptors constitutive androstane receptor (CAR) and pregnane and xenobiotic receptor (PXR) in this process. Streptozotocin-induced diabetes resulted in increased expression of drug metabolizing cytochrome P450s and also increased the clearance of the cytochrome P450 substrate zoxazolamine. This induction was completely absent in Car(-/-) mice, but was not affected by the loss of PXR. Among the many effects of diabetes on the liver, we identified bile acid elevation and activated adenosine monophosphate-activated protein kinase as potential CAR-activating stimuli. Expression of the CAR coactivator peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha was also increased in mouse models of type 1 diabetes. CONCLUSION: The CAR-dependent induction of drug metabolism in newly diagnosed or poorly managed type 1 diabetes has the potential for significant impact on the efficacy or toxicity of therapeutic agents.

Endoplasmic reticulum stress regulates adipocyte resistin expression.

OBJECTIVE: Resistin is a secreted polypeptide that impairs glucose metabolism and, in rodents, is derived exclusively from adipocytes. In murine obesity, resistin circulates at elevated levels but its gene expression in adipose tissue is paradoxically reduced. The mechanism behind the downregulation of resistin mRNA is poorly understood. We investigated whether endoplasmic reticulum (ER) stress, which is characteristic of obese adipose tissue, regulates resistin expression in cultured mouse adipocytes. RESEARCH DESIGN AND METHODS: The effects of endoplasmic stress inducers on resistin mRNA and secreted protein levels were examined in differentiated 3T3-L1 adipocytes, focusing on the expression and genomic binding of transcriptional regulators of resistin. The association between downregulated resistin mRNA and induction of ER stress was also investigated in the adipose tissue of mice fed a high-fat diet. RESULTS: ER stress reduced resistin mRNA in 3T3-L1 adipocytes in a time- and dose-dependent manner. The effects of ER stress were transcriptional because of downregulation of CAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma transcriptional activators and upregulation of the transcriptional repressor CAAT/enhancer binding protein homologous protein-10 (CHOP10). Resistin protein was also substantially downregulated, showing a close correspondence with mRNA levels in 3T3-L1 adipocytes as well as in the fat pads of obese mice. CONCLUSIONS: ER stress is a potent regulator of resistin, suggesting that ER stress may underlie the local downregulation of resistin mRNA and protein in fat in murine obesity. The paradoxical increase in plasma may be because of various systemic abnormalities associated with obesity and insulin resistance.

Macrophage-derived human resistin exacerbates adipose tissue inflammation and insulin resistance in mice.

Resistin is an adipokine that contributes to insulin resistance in mice. In humans, however, studies investigating the link between resistin and metabolic disease are conflicting. Further complicating the matter, human resistin is produced mainly by macrophages rather than adipocytes. To address this important issue, we generated mice that lack adipocyte-derived mouse resistin but produce human resistin in a pattern similar to that found in humans, i.e., in macrophages (humanized resistin mice). When placed on a high-fat diet, the humanized resistin mice rapidly developed accelerated white adipose tissue (WAT) inflammation, leading to increased lipolysis and increased serum free fatty acids. Over time, these mice accumulated lipids, including diacylglycerols, in muscle. We found that this resulted in increased Pkcq pathway activity, leading to increased serine phosphorylation of Irs-1 and insulin resistance. Thus, although the site of resistin production differs between species, human resistin exacerbates WAT inflammation and contributes to insulin resistance.

Re-expression of GATA2 cooperates with peroxisome proliferator-activated receptor-gamma depletion to revert the adipocyte phenotype.

Nuclear peroxisome proliferator-activated receptor-gamma (PPARgamma) is required for adipocyte differentiation, but its role in mature adipocytes is less clear. Here, we report that knockdown of PPARgamma expression in 3T3-L1 adipocytes returned the expression of most adipocyte genes to preadipocyte levels. Consistently, down-regulated but not up-regulated genes showed strong enrichment of PPARgamma binding. Surprisingly, not all adipocyte genes were reversed, and the adipocyte morphology was maintained for an extended period after PPARgamma depletion. To explain this, we focused on transcriptional regulators whose adipogenic regulation was not reversed upon PPARgamma depletion. We identified GATA2, a transcription factor whose down-regulation early in adipogenesis is required for preadipocyte differentiation and whose levels remain low after PPARgamma knockdown. Forced expression of GATA2 in mature adipocytes complemented PPARgamma depletion and impaired adipocyte functionality with a more preadipocyte-like gene expression profile. Ectopic expression of GATA2 in adipose tissue in vivo had a similar effect on adipogenic gene expression. These results suggest that PPARgamma-independent down-regulation of GATA2 prevents reversion of mature adipocytes after PPARgamma depletion.

Retinol saturase promotes adipogenesis and is downregulated in obesity.

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor gamma (PPARgamma). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity. On the other hand, ectopic RetSat with an intact, but not a mutated, FAD/NAD dinucleotide-binding motif increased endogenous PPARgamma transcriptional activity and promoted adipogenesis. Indeed, RetSat was not required for adipogenesis when cells were provided with exogenous PPARgamma ligands. In adipose tissue, RetSat is expressed in adipocytes but is unexpectedly downregulated in obesity, most likely owing to infiltration of macrophages that we demonstrate to repress RetSat expression. Thiazolidinedione treatment reversed low RetSat expression in adipose tissue of obese mice. Thus, RetSat plays an important role in the biology of adipocytes, where it favors normal differentiation, yet is reduced in the obese state. RetSat is thus a novel target for therapeutic intervention in metabolic disease.

Loss of orphan receptor small heterodimer partner sensitizes mice to liver injury from obstructive cholestasis.

UNLABELLED: The orphan nuclear hormone receptor small heterodimer partner (SHP) regulates the expression of several genes involved in bile acid homeostasis in the liver. Because bile acid toxicity is a major source of liver injury in cholestatic disease, we explored the role of SHP in liver damage induced by common bile duct ligation (BDL). Shp(-/-) mice show increased sensitivity in this model of acute obstructive cholestasis, with greater numbers of bile infarcts and higher mortality than wild-type C57BL/6 mice. This increased sensitivity could not be accounted for by differences in expression of bile acid homeostatic genes 2 or 5 days after BDL. Instead, higher basal expression of such genes, including the key biosynthetic enzyme cholesterol 7alpha hydroxylase (Cyp7A1) and the bile salt export pump, is associated with both an increase in bile flow prior to BDL and an increase in acute liver damage at only 1.5 hours after BDL in Shp(-/-) mice, as shown by bile infarcts. At 3 hours, Cyp7A1 expression still remained elevated in Shp(-/-) with respect to wild-type mice, and the hepatic and serum bile acid levels and total hepatobiliary bile acid pool were significantly increased. The increased sensitivity of mice lacking SHP contrasts with the decreased sensitivity of mice lacking the farnesoid X receptor (FXR; nuclear receptor subfamily 1, group H, member 4) to BDL, which has been associated with decreased intraductal pressure and fewer bile infarcts. CONCLUSION: We propose that differences in acute responses to BDL, particularly the early formation of bile infarcts, are a primary determinant of the differences in longer term sensitivity of the Fxr(-/-) and Shp(-/-) mice to acute obstructive cholestasis.

Rev-erbalpha, a heme sensor that coordinates metabolic and circadian pathways.

The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. We show that heme binds reversibly to the orphan nuclear receptor Rev-erbalpha, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbalpha-mediated gene repression. Thus, Rev-erbalpha serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism.

Mechanisms of obesity-associated insulin resistance: many choices on the menu.

Obesity-associated insulin resistance is a major risk factor for type 2 diabetes and cardiovascular disease. In the past decade, a large number of endocrine, inflammatory, neural, and cell-intrinsic pathways have been shown to be dysregulated in obesity. Although it is possible that one of these factors plays a dominant role, many of these factors are interdependent, and it is likely that their dynamic interplay underlies the pathophysiology of insulin resistance. Understanding the biology of these systems will inform the search for interventions that specifically prevent or treat insulin resistance and its associated pathologies.

Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration.

Liver mass depends on one or more unidentified humoral signals that drive regeneration when liver functional capacity is diminished. Bile acids are important liver products, and their levels are tightly regulated. Here, we identify a role for nuclear receptor-dependent bile acid signaling in normal liver regeneration. Elevated bile acid levels accelerate regeneration, and decreased levels inhibit liver regrowth, as does the absence of the primary nuclear bile acid receptor FXR. We propose that FXR activation by increased bile acid flux is a signal of decreased functional capacity of the liver. FXR, and possibly other nuclear receptors, may promote homeostasis not only by regulating expression of appropriate metabolic target genes but also by driving homeotrophic liver growth.

Role of the constitutive androstane receptor in xenobiotic-induced thyroid hormone metabolism.

The induction of hepatic drug metabolizing enzymes alters not only the metabolism of the xenobiotic substances that induce them but also the metabolism of various endogenous hormones. The xenobiotic receptor constitutive androstane receptor (CAR) (NR1I3) mediates the well-studied induction of CYP2B genes and other drug-metabolizing enzymes by phenobarbital (PB), an antiepileptic drug that has been shown to alter thyroid hormone (TH) levels. Here we show that CAR is required for PB-mediated disruption of TH homeostasis and the induction of thyroid follicular cell proliferation. Treatment with PB or the more potent and more effective CAR ligand 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene resulted in universal induction of thyroid hormone glucuronidation and sulfation pathways in a CAR-dependent manner. This resulted in a decrease in serum T4 concentration and a concomitant increase in serum TSH levels. CAR activation also decreased serum T3 levels in mice in which T3 production was blocked. The increase in serum TSH levels resulted in the stimulation of thyroid-follicular cell proliferation. These results highlight the central role of the xenosensor CAR in drug-hormone interactions.

The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity.

A double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli. Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions. As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin. Comparisons of the single and double knockouts reveal specific roles for the two receptors. Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA). As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C. M., Nelson, M. C., Ong, E. S., Waxman, D. J., and Evans, R. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity. Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2. These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose. Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic. These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.

Alterations in the distribution and orexigenic effects of dexamethasone in CAR-null mice.

The constitutive androstane receptor (CAR, NR1I3) has emerged as an important regulator of drug metabolism. CAR responds to a wide spectrum of xenobiotics by inducing expression of cytochrome P450 (CYP) enzymes and a number of other proteins responsible for drug metabolism in the liver. The xenosensor function of CAR overlaps with that of the pregnane X receptor (PXR), another xenobiotic receptor that belongs to the nuclear hormone superfamily. We observed that injection of dexamethasone (Dex), a ligand for the glucocorticoid receptor (GR) and PXR but not CAR, results in an unexpected twofold increase in the stomach weight of CAR-null animals relative to wild-type animals. Here, we show that CAR knockout mice have elevated levels of Dex in the brain, resulting in a more rapid and robust increase in the hypothalamic expression of the GR-responsive target genes encoding neuropeptide Y (NPY) and neuropeptide Y receptor subtype 1 (NPY-R1). As expected, this is accompanied by a higher increase in the food intake of the CAR-null animals. The data described here highlight the complexity of the overlapping functions of CAR and PXR.

Induction of bilirubin clearance by the constitutive androstane receptor (CAR).

Bilirubin clearance is one of the numerous important functions of the liver. Defects in this process result in jaundice, which is particularly common in neonates. Elevated bilirubin levels can be decreased by treatment with phenobarbital. Because the nuclear hormone receptor constitutive androstane receptor (CAR) mediates hepatic effects of this xenobiotic inducer, we hypothesized that CAR could be a regulator of bilirubin clearance. Activation of the nuclear hormone receptor CAR increases hepatic expression of each of five components of the bilirubin-clearance pathway. This induction is absent in homozygous CAR null mice but is observed in mice expressing human CAR instead of mouse CAR. Pretreatment with xenobiotic inducers markedly increases the rate of clearance of an exogenous bilirubin load in wild-type but not CAR knockout animals. Bilirubin itself can also activate CAR, and mice lacking CAR are defective in clearing chronically elevated bilirubin levels. Unexpectedly, CAR expression is very low in livers of neonatal mice and humans. We conclude that CAR directs a protective response to elevated bilirubin levels and suggest that a functional deficit of CAR activity may contribute to neonatal jaundice.