Accelerated diabetic nephropathy in mice lacking the peroxisome proliferator-activated receptor alpha

Peroxisome proliferator-activated receptor (PPAR)alpha, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at determining the role of PPARalpha in the pathogenesis of diabetic nephropathy using PPARalpha-knockout mice and cultured murine mesangial cells. Diabetes was induced using a low-dose streptozotocin protocol in 8-week-old male 129 SvJ PPARalpha-knockout and wild-type mice. Diabetic PPARalpha-knockout and wild-type mice developed elevated fasting blood glucose (P < 0.001) and HbA1c levels (P < 0.001). Renal functional and histopathological changes in diabetic and nondiabetic PPARalpha-knockout and wild-type mice were evaluated after 16 weeks of hyperglycemia. PPARalpha immunostaining of the cortical tubules of diabetic wild-type mice was elevated by hyperglycemia. In diabetic PPARalpha-knockout mice, renal disease with accompanying albuminuria, glomerular sclerosis, and mesangial area expansion was more severe than in diabetic wild-type mice (P < 0.05) and was accompanied by increased levels of serum free fatty acids and triglycerides (P < 0.01). Furthermore, they exhibited increased renal immunostaining for type IV collagen and osteopontin, which was associated with increased macrophage infiltration and glomerular apoptosis. There were no significant differences in these indexes of renal disease between nondiabetic PPARalpha-knockout and wild-type mice and diabetic PPARalpha wild-type mice. In vitro studies demonstrated that high glucose levels markedly increased the expression of type IV collagen, transforming growth factor-beta1, and the number of leukocytes adherent to cultured mesangial cells. Adherence of leukocytes was inhibited by the PPARalpha agonist fenofibrate. Taken together, PPARalpha deficiency appears to aggravate the severity of diabetic nephropathy through an increase in extracellular matrix formation, inflammation, and circulating free fatty acid and triglyceride concentrations. PPARalpha agonists may serve as useful therapeutic agents for type 1 diabetic nephropathy.     

Identification of functions of peroxisome proliferator-activated receptor alpha in proximal tubules

Peroxisome proliferator-activated receptor alpha(PPARalpha) is a member of the steroid/nuclear receptor superfamily that is intensively expressed in the kidney, but its physiologic function is unknown. In this study, PPARalpha-null mice were used to help clarify the function. Starved PPARalpha-null mice were found to secrete significantly more quantities of urine albumin than starved wild-type mice. Furthermore, the appearance of giant lysosomes, marked accumulation of albumin, and an impaired ability concerning albumin digestion were found only in proximal tubules of the starved PPARalpha-null mice. These abnormalities were probably derived from ATP insufficiency as a result of the starvation-induced decline of carbohydrate metabolism and a lack of PPARalpha-dependent fatty acid metabolism. It is interesting that these abnormalities disappeared when glucose was administered. Taken together, these findings demonstrate important functions of PPARalpha in the proximal tubules, the dynamic regulation of the protein-degradation system through maintenance of ATP homeostasis, and emphasize the importance of the fatty acid metabolism in renal physiology.     

Role of ATP production and uncoupling protein-2 in the insulin secretory defect induced by chronic exposure to high glucose or free fatty acids and effects of peroxisome proliferator-activated receptor-gamma inhibition

In rat pancreatic islets chronically exposed to high glucose or high free fatty acid (FFA) levels, glucose-induced insulin release and mitochondrial glucose oxidation are impaired. These abnormalities are associated with high basal ATP levels but a decreased glucose-induced ATP production (Delta of increment over baseline 0.7 +/- 0.5 or 0.5 +/- 0.3 pmol/islet in islets exposed to glucose or FFA vs. 12.0 +/- 0.6 in control islets, n = 3; P < 0.01) and, as a consequence, with an altered ATP/ADP ratio. To investigate further the mechanism of the impaired ATP formation, we measured in rat pancreatic islets glucose-stimulated pyruvate dehydrogenase (PDH) activity, a key enzyme for pyruvate metabolism and for the subsequent glucose oxidation through the Krebs cycle, and also the uncoupling protein-2 (UCP-2) content by Western blot. In islets exposed to high glucose or FFA, glucose-stimulated PDH activity was impaired and UCP-2 was overexpressed. Because UCP-2 expression is modulated by a peroxisome proliferator- activated receptor (PPAR)-dependent pathway, we measured PPAR-gamma contents by Western blot and the effects of a PPAR-gamma antagonist. PPAR-gamma levels were overexpressed in islets cultured with high FFA levels but unaffected in islets exposed to high glucose. In islets exposed to high FFA concentration, a PPAR-gamma antagonist was able to prevent UCP-2 overexpression and to restore insulin secretion and the ATP/ADP ratio. These data indicate that in rat pancreatic islets chronically exposed to high glucose or FFA, glucose-induced impairment of insulin secretion is associated with (and might be due to) altered mitochondrial function, which results in impaired glucose oxidation, overexpression of the UCP-2 protein, and a consequent decrease of ATP production. This alteration in FFA cultured islets is mediated by the PPAR-gamma pathway.     

Impact of oxidative stress and peroxisome proliferator-activated receptor gamma coactivator-1alpha in hepatic insulin resistance

OBJECTIVE—Recent studies identified accumulation of reactive oxygen species (ROS) as a common pathway causing insulin resistance. However, whether and how the reduction of ROS levels improves insulin resistance remains to be elucidated. The present study was designed to define this mechanism.RESEARCH DESIGN AND METHODS—We investigated the effect of overexpression of superoxide dismutase (SOD)1 in liver of obese diabetic model (db/db) mice by adenoviral injection.RESULTS—db/db mice had high ROS levels in liver. Overexpression of SOD1 in liver of db/db mice reduced hepatic ROS and blood glucose level. These changes were accompanied by improvement in insulin resistance and reduction of hepatic gene expression of phosphoenol-pyruvate carboxykinase and peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), which is the main regulator of gluconeogenic genes. The inhibition of hepatic insulin resistance was accompanied by attenuation of phosphorylation of cAMP-responsive element-binding protein (CREB), which is a main regulator of PGC-1α expression, and attenuation of Jun NH₂-terminal kinase (JNK) phosphorylation. Simultaneously, overexpression of SOD1 in db/db mice enhanced the inactivation of forkhead box class O1, another regulator of PGC-1α expression, without the changes of insulin-induced Akt phosphorylation in liver. In hepatocyte cell lines, ROS induced phosphorylation of JNK and CREB, and the latter, together with PGC-1α expression, was inhibited by a JNK inhibitor.CONCLUSIONS—Our results indicate that the reduction of ROS is a potential therapeutic target of liver insulin resistance, at least partly by the reduced expression of PGC-1α.Accumulation of reactive oxygen species (ROS) plays a critical role in the pathogenesis of various diseases. ROS are generated by the electron transport chain in mitochondrial respiration and are thus increased in conditions associated with enhanced oxidation of energy substrate such as glucose and free fatty acids. Furthermore, ROS is produced by NADPH oxidase, which is activated by various cytokines. The state of insulin resistance is accompanied by increases in the levels of blood glucose, free fatty acids, and adipocytokines and is thus regarded as a state of increased exposure to ROS (1,2). Although the exact mechanism of insulin resistance is not fully understood, recent data implicate ROS in the pathogenesis of multiple forms of insulin resistance (3–5). However, there is little or no information on how ROS induce insulin resistance in vivo.The tissue ROS level in each organ depends on the production and elimination of ROS. Superoxide dismutases (SODs) are major antioxidant enzymes that degrade superoxide into hydrogen peroxide. At present, three distinct isoforms of SOD have been identified in mammals (6). SOD1, or CuZn-SOD, is a copper- and zinc-containing homodimer. Although this enzyme had been regarded to be expressed exclusively in the cytoplasm, at least in rodent liver, it is found both in the intermembrane space of mitochondria and in the cytosol (7). SOD2, or Mn-SOD, is a manganese-containing enzyme found almost exclusively in the mitochondria. SOD3, or EC-SOD, is the most recently characterized SOD; it exists as a copper- and zinc-containing tetramer and contains a signal peptide that directs this enzyme exclusively to extracellular spaces.The present study was designed to explore the effect of ROS on hepatic insulin resistance. For this purpose, we injected an adenovirus encoding human SOD1 (AdSOD1) into db/db mice, a genetic model of type 2 diabetes. The results demonstrated that reduction of ROS in liver improved glucose tolerance with reduced expression of gluconeogenic genes. The reduced expression of peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α), independent of insulin signaling at the Akt phosphorylation level, seems to be involved in this mechanism.     

Plasma fatty acids and the control of lipolysis by cathecholamines.

The role of lipolysis in adipose tissue is described in relation to the maintentance of plasma levels of fatty acids. The molecular details of the hormonal control of lipolysis in the human adipocyte are discussed.     

Expression of peroxisome proliferator-activated receptor (PPAR) in human prostate cancer

BACKGROUND: Recent studies have demonstrated that peroxisome proliferator activator-receptors (PPAR)-gamma is expressed in some cancer cells such as breast, lung, and gastric cancer, and its ligand induces growth arrest of these cancer cells through apoptosis. However, the expression and localization of PPARs in prostate have not been examined. In this study, PPARs expression was investigated in human prostate cancer (PC), prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), and normal prostate (NP) tissues. METHODS: Tumor specimens were obtained from 156 patients with PC, 15 with PIN, 20 with BPH, and 12 patients with NP tissues. The expressions were investigated by RT-PCR and immunohistochemical methods. RESULTS: Immunoreactive PPAR-alpha and -beta were significantly apparent in PC tissues. Marked expressions of PPAR-alpha and -beta were also detected in PIN, BPH, and NP groups. However, very weak or no expression of immunoreactive PPAR-gamma was found in BPH and NP cases. In contrast, we found significant expression of immunoreactive PPAR-gamma in cancer cells in PC group and in PIN group. CONCLUSIONS: Our results demonstrated that PPAR-gamma is induced in PC, and suggest that PPAR-gamma ligands may mediate its own potent antiproliferative effect against PC cells through differentiation.     

过氧化物酶体增殖物激活受体γ激动剂对肝星状细胞增殖的影响

目的 观察过氧化物酶体增殖物激活受体γ（PPARγ）激动剂对肝星状细胞增殖的影响，探讨其抗肝纤维化的可能作用机制。方法 利用胶原酶原位灌注梯密度离心方法分离大鼠肝星状细胞（HSC），利用噻唑蓝（MTT）比色法、流式细胞技术检测曲格列酮、15-脱氧-前列腺素J2（15-d-PGJ2）对HSC增殖及细胞周期的作用。结果 MTT检测表明曲格列酮、15-d—PGJ2在5～100μmoL／L浓度范围内可显著抑制HSC的增殖，与对照组比较P〈0．01；流式细胞检测表明25、50μmoL／L的曲格列酮可显著降低HSCS期细胞数量，降低细胞增殖指数，与对照组比较，差异有统计学意义（P〈0．05）。结论 PPARy激动剂通过影响HSC的增殖而发挥其抗肝纤维化作用。     

Plasma and muscle free amino acids in maintenance hemodialysis patients without protein malnutrition

To investigate how uremia modified by maintenance hemodialysis treatment influences the extra- and intracellular amino acid pattern, we collected muscle samples by percutaneous muscle biopsy and plasma samples for determination of free amino acids in 11 functionally anephric patients (creatinine clearance less than 1 ml/min), who had been treated with hemodialysis for greater than 6 months and had no clinical or laboratory signs of protein malnutrition. Five patients had mild acidosis (standard bicarbonate pre-dialysis 18 to 21 mmol/liter). The amino acid results were compared with data from age- and sex-matched healthy controls and with data obtained earlier from non-dialyzed patients with chronic uremia. In the hemodialysis patients threonine, serine and valine were significantly reduced in plasma compared to the controls, whereas the plasma concentrations of aspartate, glycine, citrulline, cysteine and arginine were elevated. In aspartate, glycine, citrulline, cysteine and arginine were elevated. In muscle, valine, serine and the tyrosine to phenylalanine ratio were low. Compared with the untreated uremic patients the hemodialysis patients exhibited fewer significant abnormalities, but the general pattern was similar, demonstrating that hemodialysis is unable to fully correct the amino acid abnormalities of chronic uremia. There was a significant positive correlation between both pre-dialysis and post-dialysis plasma bicarbonate and the muscle valine concentration, suggesting that mild acidosis may be causally related to the inbalance of the branched-chain amino acids in uremia. Extra- and intracellular serine depletion in the presence of high plasma glycine may reflect a defect in the metabolism of glycine to serine in hemodialysis patients, related to a lack of metabolizing renal tissue.     

脂质过氧化物酶体增殖物激活受体α在人肝癌多药耐药中的作用

目的研究脂质过氧化物酶体增殖物激活受体α（peroxisome proliferator-activated receptord，PPARct）在人肝癌多药耐药中的作用机制。方法利用透射电镜观察人肝癌多药耐药（multidrug resistance，MDR）细胞HepG2／ADM及其亲本细胞HepG2的超微结构；荧光定量PCR技术检测PPARαmRNA在两种细胞系中的表达水平；免疫印记法检测PPARα蛋白的在两种细胞中的表达情况。结果人肝癌多药耐药细胞与其亲本细胞相比，耐药细胞的核膜上绒毛样突起增多，胞浆内有大量空泡，粗面内质网增多；HepG2／ADM细胞的PPARαmRNA表达和蛋白表达均有下降。结论PPARα与HepG2／ADM细胞的MDR现象有关，PPARα表达下调可能是肿瘤细胞MDR形成的机制之一。     

Human metabolic syndrome resulting from dominant-negative mutations in the nuclear receptor peroxisome proliferator-activated receptor-gamma

We previously reported a syndrome of severe hyperinsulinemia and early-onset hypertension in three patients with dominant-negative mutations in the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-gamma. We now report the results of further detailed pathophysiological evaluation of these subjects, the identification of affected prepubertal children within one of the original families, and the effects of thiazolidinedione therapy in two subjects. These studies 1) definitively demonstrate the presence of severe peripheral and hepatic insulin resistance in the affected subjects; 2) describe a stereotyped pattern of partial lipodystrophy associated with all the features of the metabolic syndrome and nonalcoholic steatohepatitis; 3) document abnormalities in the in vivo function of remaining adipose tissue, including the inability of subcutaneous abdominal adipose tissue to trap and store free fatty acids postprandially and the presence of very low circulating levels of adiponectin; 4) document the presence of severe hyperinsulinemia in prepubertal carriers of the proline-467-leucine (P467L) PPAR-gamma mutation; 5) provide the first direct evidence of cellular resistance to PPAR-gamma agonists in mononuclear cells derived from the patients; and 6) report on the metabolic response to thiazolidinedione therapy in two affected subjects. Although the condition is rare, the study of humans with dominant-negative mutations in PPAR-gamma can provide important insight into the roles of this nuclear receptor in human metabolism.     

Association between peroxisome proliferator-activated receptor gamma haplotypes and the metabolic syndrome in French men and women

We assessed the association of four polymorphisms (promoter P3 -681C>G, P2 -689C>T, Pro12Ala, and 1431C>T) in peroxisome proliferator-activated receptor gamma (PPARgamma) with the metabolic syndrome risk in a large, French population study (n = 1,155). In this sample, 279 men and women presented with metabolic syndrome according to the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) criteria. When taken individually, none of the polymorphisms was significantly associated with the metabolic syndrome. Haplotype analyses, in contrast, revealed a significant enrichment of the GTGC haplotype frequency (corresponding to the P3 -681C>G, P2 -689C>T, Pro12Ala (C/G), and 1431C>T polymorphisms in this order) among those with metabolic syndrome compared with control subjects. Compared with the most common CCCC haplotype, the adjusted odds ratio (OR) (95% CI) of the metabolic syndrome for bearers of the GTGC haplotype was 2.37 (1.42-3.95; P = 0.002), 1.92 (1.00-3.72; P = 0.05), and 2.47 (1.09-5.62; P = 0.045) in the whole sample of men and women, respectively. Similar results were obtained when using another haplotype (GCCC, GTGT, CCCT, or GCCT) as a reference. Furthermore, when the GTGC haplotype frequency was tested alone (i.e., versus the frequency of the five other haplotypes together), the OR (95% CI) of the metabolic syndrome was 2.30 (1.05-5.00; P = 0.022). These data show that only the frequency of the GTGC haplotype was different between subjects with and without metabolic syndrome. Further analyses stratified on the 1431C>T single nucleotide polymorphism (SNP) indicated that the rare alleles of the P2 -689C>T and Pro12Ala SNPs were associated with an increased risk of the metabolic syndrome when combined to the 1431CC genotype. In conclusion, a specific haplotype of PPARgamma polymorphisms is associated with an increased risk of the metabolic syndrome in a French general population.     

Elevated levels of free fatty acids in lung injury associated with acute pancreatitis in rats

Pulmonary injury is associated with acute pancreatitis (AP) in up to 70% of the cases, and may even progress to the adult respiratory distress syndrome (ARDS). Some authors discuss the socalled “free fatty acids” (FFA) to play a certain role in the pathogenesis of AP and in the subsequent systemic complications. We therefore tried to find out what correlations may exist between FFA, pancreatitis, and lung injury in a rat model. AP was induced by infusing 2 different concentrations of glycodeoxycholic acid (GDOC 17 mmol and 34 mmol) into the cannulated pancreatic duct. At set times (2, 6, 12, 24 and 48 hours), the animals were sacrificed, and blood was collected for determining FFA, amylase and pO₂. Pancreas and lungs were removed for histologic examination, and the lung weight was determined. The 2 treatment groups and the control group, which received no infusion after cannulation, were of equal size. GDOC-34 animals developed severe pancreatitis with hemorrhage and necrosis as well as lung injury with edema, inflammatory infiltrates including “foamy macrophages”, hemorrhage, and thickening of the alveolar membrane. In contrast to this, the GDOC-17 animals showed pancreatic edema only until 24 hours, they obviously suffered less severe histologic changes in the lungs. These results together with the corresponding changes in amylase, FFA, pO₂, and lung weight show that AP can be experimentally induced at different levels of severity associated with different degrees of morphologic changes and dysfunctions in the lungs. The clinical course of the disease, the rising amylase levels and lung weights, and the decreasing pO₂ correlate significantly with the FFA values.