Jayme Borensztajn

Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas

Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium–derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.

Coactivators in PPAR-Regulated Gene Expression.

Peroxisome proliferator-activated receptor (PPAR)α, β (also known as δ), and γ function as sensors for fatty acids and fatty acid derivatives and control important metabolic pathways involved in the maintenance of energy balance. PPARs also regulate other diverse biological processes such as development, differentiation, inflammation, and neoplasia. In the nucleus, PPARs exist as heterodimers with retinoid X receptor-α bound to DNA with corepressor molecules. Upon ligand activation, PPARs undergo conformational changes that facilitate the dissociation of corepressor molecules and invoke a spatiotemporally orchestrated recruitment of transcription cofactors including coactivators and coactivator-associated proteins. While a given nuclear receptor regulates the expression of a prescribed set of target genes, coactivators are likely to influence the functioning of many regulators and thus affect the transcription of many genes. Evidence suggests that some of the coactivators such as PPAR-binding protein (PBP/PPARBP), thyroid hormone receptor-associated protein 220 (TRAP220), and mediator complex subunit 1 (MED1) may exert a broader influence on the functions of several nuclear receptors and their target genes. Investigations into the role of coactivators in the function of PPARs should strengthen our understanding of the complexities of metabolic diseases associated with energy metabolism.

Reversal of advanced atherosclerosis in Rhesus monkeys: Part 1. Light-microscopic studies

Abstract The regression of atherosclerotic lesions in Rhesus monkeys was evaluated by means of a low-fat, low-cholesterol diet with or without N-γ-phenylpropyl-N-benzyloxy acetamide (W-1372). Moderate to severe aortic and coronary atherosclerosis was induced by feeding 4 groups of male monkeys a high-fat, high-cholesterol diet for 18 months, after which the first group was autopsied for assessment of the lesions. During a subsequent 18-month regression period, the second group of animals was fed a low-fat, low-cholesterol diet with W-1372, and the third group the low-fat, low-cholesterol diet without W-1372. A pair of monkeys (the fourth “group”) was fed an atherogenic diet throughout the experiment. Serum cholesterol, which increased about 5-fold during the induction period, returned to baseline values or below in the 2 treated groups. Evidence of regression of lesions was obtained in both these groups, but was most noticeable in the monkeys fed the low-fat, low-cholesterol diet without W-1372. The aortas of the animals treated with the low-fat, low-cholesterol diet with or without W-1372 showed about two-thirds as many lesions which were on the average about half as severe as those in the animals killed at 18 months. The coronary artery lesions showed a similar contrast, with the treated groups having about one-third to one-half as many lesions which were about one-half to two-thirds as severe. In both locations the differences in frequency and severity of arterial lesions were statistically significant when the reference group killed at 18 months was compared with the group treated with the low-fat, low-cholesterol diet without W-1372.

Salmonella enterica Serovar Typhimurium Requires Nonsterol Precursors of the Cholesterol Biosynthetic Pathway for Intracellular Proliferation

ABSTRACT We have previously shown that Salmonella enterica serovar Typhimurium infection perturbs the host cholesterol biosynthetic pathway. Here we show that inhibiting the first step of this pathway (3-hydroxy-3-methylglutaryl coenzyme A reductase) reduces the growth of intracellular S. enterica serovar Typhimurium and has no effect on extracellular bacterial growth. Selectively inhibiting synthesis of downstream sterol components has no effect on infection, suggesting that the effect of statins on host nonsterol intermediates is detrimental to bacterial growth. Furthermore, statins also reduce bacterial proliferation in the S. enterica serovar Typhimurium mouse model. This suggests that blocking the production of nonsterol precursors in the host cell can be used to reduce infection.

Macrophage uptake of low-density lipoprotein bound to aggregated C-reactive protein: possible mechanism of foam-cell formation in atherosclerotic lesions.

Foam cells found in atherosclerotic lesions are believed to derive from macrophages that take up aggregated low-density lipoprotein (LDL) particles bound to the extracellular matrix of arterial walls. C-reactive protein (CRP) is an acute-phase protein found in atherosclerotic lesions, which when immobilized on a solid phase, can bind and cluster LDL particles in a calcium-dependent manner. In the present study, we examined whether CRP-bound aggregated LDL could be taken up by macrophages in culture. CRP molecules were aggregated in the presence of calcium and immobilized on the surface of polystyrene microtitre wells. Human LDL added to the wells bound to and aggregated on the immobilized CRP, also in a calcium-dependent manner. On incubation with macrophages, the immobilized CRP-bound LDL aggregates were readily taken up by the cells, as demonstrated by immunofluorescence microscopy, by the cellular accumulation of cholesterol and by the overexpression of adipophilin. Immunofluorescence microscopy and flow-cytometry analysis established that the uptake of the LDL-CRP complex was not mediated by the CRP receptor CD32. These observations with immobilized CRP and LDL, approximating the conditions that exist in the extracellular matrix of the arterial wall, thus suggest that CRP may contribute to the formation of foam cells in atherosclerotic lesions by causing the aggregation of LDL molecules that are then taken up by macrophages through a CD32-independent pathway.

Sustained activation of PPARα by endogenous ligands increases hepatic fatty acid oxidation and prevents obesity in ob/ob mice

Obesity, a major health concern, results from an imbalance between energy intake and expenditure. Leptin‐deficient ob/ob mice are paradigmatic of obesity, resulting from excess energy intake and storage. Mice lacking acyl‐CoA oxidase 1 (Acox1), the first enzyme of the peroxisomal fatty acid β‐oxidation system, are characterized by increased energy expenditure and a lean body phenotype caused by sustained activation of peroxisome proliferator‐activated receptor α (PPARα) by endogenous ligands in liver that remain unmetabolized in the absence of Acox1. We generated ob/ob mice deficient in Acox1 (Acox1‐/‐) to determine how the activation of PPARα by endogenous ligands might affect the obesity of ob/ob mice. In contrast to Acox1‐/‐ (14.3± 1.2 g at 6 mo) and the Acox1‐deficient (ob/ob) double‐mutant mice (23.8±4.6 g at 6 mo), the ob/ob mice are severely obese (54.3±3.2 g at 6 mo) and had significantly more (P<0.01) epididymal fat content. The resistance of Acox1‐/‐/ob/ob mice to obesity is due to increased PPARα‐mediated up‐regulation of genes involved in fatty acid oxidation in liver. Activation of PPARα in Acox1‐deficient ob/ob mice also reduces serum glucose and insulin (P<0.05) and improves glucose tolerance and insulin sensitivity. Further, PPARα activation reduces hepatic steatosis and increases hepatocellular regenerative response in Acox1‐/‐/ob/ob mice at a more accelerated pace than in mice lacking only Acox1. However, Acox1‐/‐/ob/ob mice manifest hepatic endoplasmic reticulum (ER) stress and also develop hepatocellular carcinomas (8 of 8 mice) similar to those observed in Acox1‐/‐ mice (10 of 10 mice), but unlike in ob/ob (0 of 14 mice) and OB/OB (0 of 6 mice) mice, suggesting that superimposed ER stress and PPARα activation contribute to carcinogenesis in a fatty liver. Finally, absence of Acox1 in ob/ob mice can impart resistance to high‐fat diet (60% fat)‐induced obesity, and their liver had significantly (P<0.01) more cell proliferation. These studies with Acox1‐/‐/ob/ob mice indicate that sustained activation of lipid‐sensing nuclear receptor PPARα attenuates obesity and restores glucose homeostasis by ameliorating insulin resistance but increases the risk for liver cancer development, in part related to excess energy combustion.—Huang, J., Jia, Y., Fu, T., Viswakarma, N., Bai, L., Sambasiva Rao, M., Zhu, Y., Borensztajn, J., Reddy, J. K. Sustained activation of PPARα by endogenous ligands increases hepatic fatty acid oxidation and prevents obesity in ob/ob mice. FASEB J. 26, 628–638 (2012). www.fasebj.org

Overexpression of SR-BI by adenoviral vector reverses the fibrateinduced hypercholesterolemia of apolipoprotein E-deficient mice.

The hypercholesterolemia characteristic of apolipoprotein (apoE)-deficient mice fed on a regular chow diet is caused by the abnormal accumulation of apoB-48-carrying remnants of chylomicrons and very low density lipoproteins in the plasma. Treatment of apoE-deficient mice with ciprofibrate or other peroxisome proliferator-activated receptor α agonists severely aggravates their hypercholesterolemia by interfering with one or more mechanisms of remnant removal from the circulation that do not require mediation by apoE (Fu, T., Kashireddy, P., and Borensztajn, J. (2003) Biochem. J. 373, 941–947). In the present investigation we report that ciprofibrate treatment causes the down-regulation of hepatic scavenger receptor class B, type I (SR-BI) protein expression in the livers of apoE-deficient mice. On cessation of the treatment SR-BI expression returns to its pretreatment levels, coinciding with a reversal of the hypercholesterolemia to base-line concentrations. Restoration of SR-BI expression in ciprofibrate-treated apoE-deficient mice by recombinant adenoviral gene transfer abolishes the ciprofibrate-induced over accumulation of apoB-48-carrying remnants in the plasma. We also report that remnants isolated from the plasma of ciprofibrate-treated apoE-deficient mice bind to murine SR-BI expressed in stably transfected cultured cells. These observations suggest that, in addition to its well established role as high density lipoprotein receptor, SR-BI can also function as a remnant receptor responsible for the clearance of remnants from the circulation of apoE-deficient mice.

The role of glucagon in the regulation of myocardial lipoprotein lipase activity.

Abstract The role of glucagon in regulating the lipoprotein lipase activities of rat heart and adipose tissue was examined. When starved rats were fed glucose, heart lipoprotein lipase activity decreased while that of adipose tissue increased. Glucagon administration to these animals at the time of glucose feeding prevented the decline in heart lipoprotein lipase activity, but had no effect on the adipose tissue enzyme. When glucagon was administered to fed rats, heart lipoprotein lipase activity increased to levels found in starved animals but there was no change in the adipose tissue enzyme. It is suggested that the reciprocal lipoprotein lipase activities in heart and adipose tissue of fed and starved animals may be regulated by the circulating plasma insulin and glucagon concentrations.

The peroxisome-proliferator-activated receptor alpha agonist ciprofibrate severely aggravates hypercholesterolaemia and accelerates the development of atherosclerosis in mice lacking apolipoprotein E.

Mice lacking apolipoprotein E (apoE) are characterized by severe hypercholesterolaemia, caused by an abnormal accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins (VLDL) in the plasma, and by the spontaneous development of atherosclerotic lesions. Ciprofibrate is a hypolipidaemic compound that acts primarily by enhancing the oxidation of fatty acids in the liver and, consequently, decreasing the production of hepatic VLDL. In the present study, homozygous apoE-deficient mice were fed with a normal chow diet, supplemented with ciprofibrate. We report that, as anticipated, ciprofibrate treatment (a) stimulated hepatic fatty acid oxidation, as indicated by an increase in the mRNA levels of peroxisomal fatty acyl-CoA oxidase (AOX) and peroxisomal bifunctional enzyme, and (b) decreased the hepatic secretion of VLDL into the plasma, as determined by treating the animals with Triton WR-1339. Paradoxically, the apoE-deficient mice developed a 3-4-fold increase in their plasma cholesterol levels. A similar effect was observed in apoE-deficient mice treated with other peroxisome-proliferator-activated receptor alpha agonists (fenofibrate, bezafibrate and WY14,643). By FPLC of the plasma and Western-blot analysis, we determined that the enhanced hypercholesterolaemia was due to an increased accumulation of apoB-48-carrying lipoprotein remnants in the plasma. Consistent with this finding, atherosclerotic lesions in animals treated with ciprofibrate for 90 days were considerably more advanced than in untreated animals. These results indicate that the ciprofibrate-induced accumulation of apoB-48-carrying remnants in apoE-deficient mice is caused by the inhibition of an as yet uncharacterized apoE-independent mechanism of removal of remnant from the circulation by the liver.

Transcription Coactivator Mediator Subunit Med1 is Required for the Development of Fatty Liver in the Mouse

Peroxisome proliferator‐activated receptor‐γ (PPARγ), a nuclear receptor, when overexpressed in liver stimulates the induction of adipocyte‐specific and lipogenesis‐related genes and causes hepatic steatosis. We report here that Mediator 1 (MED1; also known as PBP or TRAP220), a key subunit of the Mediator complex, is required for high‐fat diet–induced hepatic steatosis as well as PPARγ‐stimulated adipogenic hepatic steatosis. Mediator forms the bridge between transcriptional activators and RNA polymerase II. MED1 interacts with nuclear receptors such as PPARγ and other transcriptional activators. Liver‐specific MED1 knockout (MED1ΔLiv) mice, when fed a high‐fat (60% kcal fat) diet for up to 4 months failed to develop fatty liver. Similarly, MED1ΔLiv mice injected with adenovirus‐PPARγ (Ad/PPARγ) by tail vein also did not develop fatty liver, whereas mice with MED1 (MED1fl/fl) fed a high‐fat diet or injected with Ad/PPARγ developed severe hepatic steatosis. Gene expression profiling and northern blot analyses of Ad/PPARγ–injected mouse livers showed impaired induction in MED1ΔLiv mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin, and lipid droplet‐associated genes, including caveolin‐1, CideA, S3‐12, and others. These adipocyte‐specific and lipogenesis‐related genes are strongly induced in MED1fl/fl mouse liver in response to Ad/PPARγ. Re‐expression of MED1 using adenovirally‐driven MED1 (Ad/MED1) in MED1ΔLiv mouse liver restored PPARγ‐stimulated hepatic adipogenic response. These studies also demonstrate that disruption of genes encoding other coactivators such as SRC‐1, PRIC285, PRIP, and PIMT had no effect on hepatic adipogenesis induced by PPARγ overexpression. Conclusion: We conclude that transcription coactivator MED1 is required for high‐fat diet–induced and PPARγ‐stimulated fatty liver development, which suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis. (HEPATOLOGY 2011;)