Kathleen K. Brown

The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16α-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7α-hydroxylase (Cyp7a1) and the Na+-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.

Peroxisome proliferator–activated receptor γ ligands inhibit development of atherosclerosis in LDL receptor–deficient mice

The peroxisome proliferator‐activated receptor γ (PPARγ) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARγ is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARγ-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor‐deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-α and gelatinase B, indicating both systemic and local actions of PPARγ. These findings suggest that PPARγ agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARγ ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities. J. Clin. Invest. 106:523‐531 (2000).

Comprehensive Messenger Ribonucleic Acid Profiling Reveals That Peroxisome Proliferator-Activated Receptor γ Activation Has Coordinate Effects on Gene Expression in Multiple Insulin-Sensitive Tissues

Peroxisome proliferator-activated receptor γ (PPARγ) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPARγ ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPARγ in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPARγ activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle...

Activation of the Nuclear Receptor Peroxisome Proliferator-activated Receptor γ Promotes Brown Adipocyte Differentiation

Brown adipose tissue (BAT) functions in non-shivering and diet-induced thermogenesis via its capacity for uncoupled mitochondrial respiration. BAT dysfunction in rodents is associated with severe defects in energy homeostasis, resulting in obesity and hyperglycemia. Here, we report that the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), a prostaglandin-activated transcription factor recently implicated as a central regulator of white adipose tissue differentiation, also regulates brown adipocyte function. PPARγ is abundantly expressed in both embryonic and adult BAT. Treatment of CD-1 rats with the PPARγ-selective ligand BRL49653, an anti-diabetic drug of the thiazolidinedione class, results in marked increases in the mass of interscapular BAT. In vitro, BRL49653 induces the terminal differentiation of the brown preadipocyte cell line HIB-1B as judged by both changes in cell morphology and expression of uncoupling protein and other adipocyte-specific mRNAs. These data demonstrate that PPARγ is a key regulatory factor in brown adipocytes and suggest that PPARγ functions not only in the storage of excess energy in white adipose tissue but also in its dissipation in BAT.

PPARγ agonists do not directly enhance basal or insulin-stimulated Na+ transport via the epithelial Na+ channel

Selective agonists of peroxisome proliferator-activated receptor gamma (PPARγ) are anti-diabetic drugs that enhance cellular responsiveness to insulin. However, in some patients, fluid retention, plasma volume expansion, and edema have been observed. It is well established that insulin regulates Na+ reabsorption via the epithelial sodium channel (ENaC) located in the distal tubule. Therefore, we hypothesized that these agonists may positively modulate insulin-stimulated ENaC activity leading to increased Na+ reabsorption and fluid retention. Using electrophysiological techniques, dose–response curves for insulin-mediated Na+ transport in the A6, M-1, and mpkCCDcl4 cell lines were performed. Each line demonstrated hormone efficacy within physiological concentration ranges and, therefore, can be used to monitor clinically relevant effects of pharmacological agents which may affect electrolyte transport. Immunodetection and quantitative PCR analyses showed that each cell line expresses viable and functional PPARγ receptors. Despite this finding, two PPARγ agonists, pioglitazone and GW7845 did not directly enhance basal or insulin-stimulated Na+ flux via ENaC, as shown by electrophysiological methodologies. These studies provide important results, which eliminate insulin-mediated ENaC activation as a candidate mechanism underlying the fluid retention observed with PPARγ agonist use.

Serum adiponectin as a biomarker for in vivo PPARgamma activation and PPARgamma agonist-induced efficacy on insulin sensitization/lipid lowering in rats

BackgroundPPARγ agonists ameliorate insulin resistance and dyslipidemia in type 2 diabetic patients. Adiponectin possesses insulin sensitizing properties, and predicts insulin sensitivity of both glucose and lipid metabolism. In diet-induced insulin resistant rats and ZDF rats, the current studies determined the correlation between PPARγ agonist-upregulated fatty acid binding protein(FABP3) mRNA in adipose tissue and PPARγ agonist-elevated serum adiponectin, and the correlation between PPARγ agonist-elevated serum adiponectin and PPARγ agonist-mediated efficacy in insulin sensitization and lipid lowering.ResultsParallel groups of SD rats were fed a high fat/sucrose (HF) diet for 4 weeks. These rats were orally treated for the later 2 weeks with vehicle, either PPARγ agonist GI262570 (0.2–100 mg/kg, Q.D.), or GW347845 (3 mg/kg, B.I.D). Rats on HF diet showed significant increases in postprandial serum triglycerides, free fatty acids (FFA), insulin, and area under curve (AUC) of serum insulin during an oral glucose tolerance test, but showed no change in serum glucose, adiponectin, and glucose AUC. Treatment with GI262570 dose-dependently upregulated adipose FABP3 mRNA, and increased serum adiponectin. There was a positive correlation between adipose FABP3 mRNA and serum adiponectin (r = 0.7350, p < 0.01). GI262570 dose-dependently decreased the diet-induced elevations in triglycerides, FFA, insulin, and insulin AUC. Treatment with GW347845 had similar effects on serum adiponectin and the diet-induced elevations. There were negative correlations for adiponectin versus triglycerides, FFA, insulin, and insulin AUC (For GI262570, r = -0.7486, -0.4581, -0.4379, and -0.3258 respectively, all p < 0.05. For GW347845, r = -0.6370, -0.6877, -0.5512, and -0.3812 respectively, all p < 0.05). In ZDF rats treated with PPARγ agonists pioglitazone (3–30 mg/kg, B.I.D.) or GW347845 (3 mg/kg, B.I.D.), there were also negative correlations for serum adiponectin versus glucose, triglycerides, FFA (for pioglitazone, r = -0.7005, -0.8603, and -0.9288 respectively; for GW347845, r = -0.9721, -0.8483, and -0.9453 respectively, all p < 0.01).ConclusionsThis study demonstrated that (a) PPARγ agonists improved insulin sensitivity and ameliorated dyslipidemia in HF fed rats and ZDF rats, which were correlated with serum adiponectin; (b) Serum adiponectin was positively correlated with adipose FABP3 mRNA in GI262570-treated rats. These data suggest that serum adiponectin can serve as a biomarker for both in vivo PPARγ activation and PPARγ agonist-induced efficacy on insulin resistance and dyslipidemia in rats.

PPARγ agonists inhibit vasopressin-mediated anion transport in the MDCK-C7 cell line

PPARgamma agonists are synthetic ligands for the peroxisome proliferator-activated receptor-gamma (PPARgamma). These agents have insulin-sensitizing properties but can cause fluid retention, thereby limiting their usefulness in patients at risk for cardiovascular disease. The side effect etiology is unknown, but the nature of presentation suggests modulation of renal salt and water homeostasis. In a well-characterized cell culture model of the principal cell type [Madin-Darby canine kidney (MDCK)-C7], PPARgamma agonists inhibit vasopressin-stimulated Cl(-) secretion with agonist dose-response relationships that mirror receptor transactivation profiles. Analyses of the components of the vasopressin-stimulated intracellular signaling pathway indicated no PPARgamma agonist-induced changes in basolateral membrane conductances, intracellular cAMP, protein kinase A, or total cellular adenine nucleotides. The PPARgamma agonist-induced decrease in anion secretion is the result of decreased mRNA of the final effector in the pathway, the apically located cystic fibrosis transmembrane regulator (CFTR). These data showing that CFTR is a target for PPARgamma agonists may provide new insights into the physiology of PPARgamma agonist-induced fluid retention.

Effects of a PPARgamma agonist, GI262570, on renal filtration fraction and nitric oxide level in conscious rats.

&NA; PPAR&ggr; agonists ameliorate insulin resistance and lower blood pressure. Volume expansion/edema has been observed in susceptible patients treated with these agents. Alterations of renal hemodynamics affect renal tubular reabsorption, and thus may contribute to volume expansion. This study seeks to determine whether volume expansion caused by a PPAR&ggr; agonist, GI262570, is related to changes in glomerular filtration rate, effective renal plasma flow, or renal filtration fraction. Chronically catheter‐implanted conscious rats were studied to determine the effects on glomerular filtration rate, effective renal plasma flow, and renal filtration fraction after 1, 4, and 10 days of GI262570 treatment (8 mg/kg, p.o., B.I.D.). Elevated adipose mRNA of PPAR&ggr; target genes confirmed PPAR&ggr; activation in GI262570‐treated rats. GI262570 treatment for 10 days decreased hematocrit, hemoglobin, and serum albumin (all P < 0.05), indicating volume expansion, but did not alter glomerular filtration rate, effective renal plasma flow, or renal filtration fraction. However, nitrate + nitrite was significantly higher in plasma and hind limb muscle of GI262570‐treated rats (both P < 0.05). This study demonstrated that treatment with PPAR&ggr; agonist GI262570 resulted in volume expansion and increased nitric oxide, but did not affect glomerular filtration rate, effective renal plasma flow, or renal filtration fraction, indicating PPAR&ggr; agonist‐induced volume expansion is not related to changes in renal filtration fraction, and increased nitric oxide may contribute to the PPAR&ggr; agonist‐induced blood‐pressure lowering.

589 Duodenojejunal Bypass Liner Increases Fasting and Postprandial Serum Levels of Bile Acids in Patients With Severe Obesity

information on anti-inflammatory or metabolic role of GPR120 at the level of intestinal epithelium is very limited. Further, GPR120 has been shown to be expressed in the intestine, however, its levels along the length of the intestine in various species have not been studied in detail. Aims: Our current studies, therefore, assessed the expression of GPR120 along the length of human, mouse and rat intestine. Further, we examined the anti-inflammatory effects and/or altered levels of the key aneroxigenic gut hormone glucagon-like peptide 1 (GLP1) in (i) model human intestinal epithelial Caco-2 and (ii) model mouse intestinal epithelial endocrine cell line STC-1 following GPR120 activation by synthetic (GW9508) or natural (ω-3 FA) agonists. Results: Both GPR120 mRNA and protein levels varied along the length of mouse and rat intestine in the order of proximal colon>cecum>distal colon>ileum>jejunum. Mucosal scrapings of different regions of organ donor human intestine also showed highest GPR120 protein level in the proximal colon. Cell-surface biotinylation and co-immunoprecipitation in Caco-2 cells, respectively, showed that GPR120 was internalized and bound to β-arrestin-2, a downstream signaling component of NF-κB activation, in response to 30 min exposure to GW9508 (50 μM) or eicosapentaenoic acid (EPA) (100 μM). These treatments also inhibited NF-κB reporter activity presumably via GPR120 binding to and sequestration of β-arrestin-2, inhibiting downstream signaling events. On the other hand, treatment of STC-1 cells with EPA or 10-trans,12-cis conjugated linoleic acid (100 μM, 6 hr) induced GLP1 intensity, as measured by immunofluorescence. However, in STC1 cells, GW9508/EPA treatments did not induce receptor internalization or sequestration of β-arrestin-2. Conclusion: Our studies for the first time demonstrate that agonist-stimulation of GPR120 in different cell types of the intestinal epithelium induces distinct signaling pathways to exert anti-inflammatory effects and modulate enteroendocrine function. Therefore, GPR120 appears to regulate diverse physiological processes at the level of intestinal epithelium to impact inflammatory and/or metabolic disorders. (Supported by NIH-NIDDK/ Bill & Melinda Gates Foundation).