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.     

Therapeutic roles of peroxisome proliferator-activated receptor agonists

Peroxisome proliferator-activated receptors (PPARs) play key roles in the regulation of energy homeostasis and inflammation, and agonists of PPARalpha and -gamma are currently used therapeutically. Fibrates, first used in the 1970s for their lipid-modifying properties, were later shown to activate PPARalpha. These agents lower plasma triglycerides and VLDL particles and increase HDL cholesterol, effects that are associated with cardiovascular benefit. Thiazolidinediones, acting via PPARgamma, influence free fatty acid flux and thus reduce insulin resistance and blood glucose levels. PPARgamma agonists are therefore used to treat type 2 diabetes. PPARalpha and -gamma agonists also affect inflammation, vascular function, and vascular remodeling. As knowledge of the pleiotropic effects of these agents advances, further potential indications are being revealed, including roles in the management of cardiovascular disease (CVD) and the metabolic syndrome. Dual PPARalpha/gamma agonists (currently in development) look set to combine the properties of thiazolidinediones and fibrates, and they hold considerable promise for improving the management of type 2 diabetes and providing an effective therapeutic option for treating the multifactorial components of CVD and the metabolic syndrome. The functions of a third PPAR isoform, PPARdelta, and its potential as a therapeutic target are currently under investigation.     

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.     

Genetic polymorphisms in peroxisome proliferator-activated receptor delta associated with obesity

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors regulating the expression of genes involved in lipid and glucose metabolism. Three different PPARs, PPAR-alpha, -gamma, and -delta, have been characterized, and they are distinguished from each other by tissue distribution and cell activation. All PPARs are, to different extents, activated by fatty acids and derivatives. Recently, it has been shown that PPAR-delta serves as a widespread regulator of fat burning, suggesting that it might be a potential target in the treatment of obesity and type 2 diabetes. In an effort to identify polymorphic markers in potential candidate genes for type 2 diabetes, we have sequenced PPAR-delta, including -1,500 bp of the 5' flanking region. Nine polymorphisms were identified in PPAR-delta: four in the intron, one in the 5' untranslated region (UTR), and four in the 3' UTR. Among identified polymorphisms, five common sites, including c.-13454G>T, c.-87T>C, c.2022+12G>A, c.2629T>C, and c.2806C>G, were genotyped in subjects with type 2 diabetes and normal control subjects (n = 702). The genetic associations with the risk of type 2 diabetes and metabolic phenotype were analyzed. No significant associations with the risk of type 2 diabetes were detected. However, several positive associations of PPAR-delta polymorphisms with fasting plasma glucose and BMI were detected in nondiabetic control subjects. The genetic information about PPAR-delta from this study would be useful for further genetic study of obesity, diabetes, and other metabolic diseases.     

Apolipoprotein E4 exaggerates diabetic dyslipidemia and atherosclerosis in mice lacking the LDL receptor

OBJECTIVE

We investigated the differential roles of apolipoprotein E (apoE) isoforms in modulating diabetic dyslipidemia—a potential cause of the increased cardiovascular disease risk of patients with diabetes.

RESEARCH DESIGN AND METHODS

Diabetes was induced using streptozotocin (STZ) in human apoE3 (E3) or human apoE4 (E4) mice deficient in the LDL receptor (LDLR^−/−).

RESULTS

Diabetic E3LDLR^−/− and E4LDLR^−/− mice have indistinguishable levels of plasma glucose and insulin. Despite this, diabetes increased VLDL triglycerides and LDL cholesterol in E4LDLR^−/− mice twice as much as in E3LDLR^−/− mice. Diabetic E4LDLR^−/− mice had similar lipoprotein fractional catabolic rates compared with diabetic E3LDLR^−/− mice but had larger hepatic fat stores and increased VLDL secretion. Diabetic E4LDLR^−/− mice demonstrated a decreased reliance on lipid as an energy source based on indirect calorimetry. Lower phosphorylated acetyl-CoA carboxylase content and higher gene expression of fatty acid synthase in the liver indicated reduced fatty acid oxidation and increased fatty acid synthesis. E4LDLR^−/− primary hepatocytes cultured in high glucose accumulated more intracellular lipid than E3LDLR^−/− hepatocytes concomitant with a 60% reduction in fatty acid oxidation. Finally, the exaggerated dyslipidemia in diabetic E4LDLR^−/− mice was accompanied by a dramatic increase in atherosclerosis.

CONCLUSIONS

ApoE4 causes severe dyslipidemia and atherosclerosis independent of its interaction with LDLR in a model of STZ-induced diabetes. ApoE4-expressing livers have reduced fatty acid oxidation, which contributes to the accumulation of tissue and plasma lipids.Cardiovascular disease (CVD) caused by a worsening of atherosclerosis is an important complication of diabetes and is the leading cause of mortality among patients with diabetes (1). Patients with poorly managed type 1 diabetes or type 2 diabetes commonly have elevated VLDL triglycerides (TGs), a reduction of HDL cholesterol, and smaller, dense LDL. This common cluster of harmful changes in lipid metabolism is referred to as diabetic dyslipidemia (2).Apolipoprotein E (apoE) is a small circulating protein associated predominantly with VLDL and HDL. It is the primary ligand for several lipoprotein receptors, making it a crucial component in the clearance of lipid from the circulation and a major determinant of plasma cholesterol and CVD risk (3). In humans, the APOE gene is polymorphic, resulting in production of three common isoforms: apoE2, apoE3, and apoE4. The apoE4 isoform is carried by more than a quarter (28%) of the U.S. population and is associated with higher LDL cholesterol and an increased risk of CVD (3). In addition to its well-established role in CVD, recent findings have implicated a role for apoE in glucose metabolism. Epidemiological studies have suggested that in certain populations, APOE genotype may influence plasma glucose and insulin levels (4,5), postprandial glucose response (6), the development of metabolic syndrome (7,8), and a myriad of diabetes complications (9). In addition, apoE4 carriers with diabetes have been shown to have increased carotid atherosclerosis (10), and elderly apoE4 carriers with diabetes have an increased risk of CVD-associated death (11).Increases in VLDL TGs; decreases in HDL; the accumulation of smaller, more dense LDL; slower clearance of postprandial chylomicrons; and a decrease in LDL receptor (LDLR) expression are all noted phenotypes associated with both type 1 and type 2 diabetes (2). All of these components of diabetic dyslipidemia are areas of normal lipid metabolism in which apoE has previously been shown to play a direct role. The major receptor through which apoE mediates lipoprotein clearance is the LDLR, and the apoE isoforms exhibit differential LDLR binding affinities (12). Therefore, we sought whether apoE isoforms retain differential roles in diabetic dyslipidemia and atherosclerosis in the absence of the LDLR by using a mouse model of diabetes induced by streptozotocin (STZ).In this study, we show that dyslipidemia and atherosclerosis are greatly exaggerated in diabetic LDLR^−/− mice expressing human apoE4 (E4LDLR^−/−) compared with those with human apoE3 (E3LDLR^−/−), despite a similar degree of hyperglycemia. This E4-specific aggravation of diabetic dyslipidemia is central to the liver and is associated with a reduction in hepatic lipid oxidation, an accumulation of liver TGs, and increased rates of VLDL secretion.     

Accelerated diabetes in rat insulin promoter-tumor necrosis factor-alpha transgenic nonobese diabetic mice lacking major histocompatibility class II molecules

The major predisposing genetic component in type 1 diabetes maps to the major histocompatibility complex locus in both mice and humans. To verify the HLA class II association with disease pathogenesis, we adopted the transgenic approach. Expression of HLA-DQ8, the molecule showing the strongest association with human type 1 diabetes, in the diabetes-predisposing milieu of NOD mice in the absence of the endogenous class II molecule I-A(g7) did not render susceptibility to type 1 diabetes. To study if providing a local proinflammatory environment would lead to diabetes in these mice, Abeta(o).NOD.DQ8 were bred with C57BL/6 mice expressing tumor necrosis factor (TNF)-alpha in the beta-cells of the islets of Langerhans. Surprisingly, although diabetes was evident in the F1 intercross expressing rat insulin promoter (RIP)-TNF, offspring lacking either endogenous or transgenic class II molecules developed accelerated diabetes with high frequency in both sexes. Moreover, expression of any functional class II molecule seemed to confer significant protection from diabetes in this model. Thus, neonatal expression of TNF-alpha in an islet-specific manner bypassed the requirement of CD4(+) T-cells and resulted in diabetes that could be mediated by CD8(+) T-cells. We also show for the first time that diabetes in NOD.RIP-TNF mice can occur independent of inheritance of NOD-derived idd1.     

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.     

The human peroxisome proliferator-activated receptor gamma2 (PPARgamma2) Pro12Ala polymorphism is associated with decreased risk of diabetic nephropathy in patients with type 2 diabetes

The peroxisome proliferator-activated receptor gamma2 (PPARgamma2) Pro12Ala polymorphism has been associated with a decreased risk of type 2 diabetes and a lower albumin excretion rate (AER) in patients with established diabetes. We performed a case-control study aiming to evaluate the association between the Pro12Ala polymorphism and diabetic nephropathy. Genomic DNA was obtained from 104 type 2 diabetic patients (case subjects) with chronic renal insufficiency (78 on dialysis and 26 with proteinuria [AER >or=200 microg/min] and serum creatinine >or=2.0 mg/dl) and 212 normoalbuminuric patients (AER <20 microg/min) with known diabetes duration >or=10 years (control subjects). The genotypic distribution of the PPARgamma2 Pro12Ala polymorphism in these diabetic patients was in Hardy-Weinberg equilibrium, and the Ala allele frequency was 9%. The frequency of Ala carriers (Ala/Ala or Ala/Pro) was 20.3% in control subjects and 10.6% in case subjects (P = 0.031). The odds ratio of having diabetic nephropathy for Ala carriers was 0.465 (95% CI 0.229-0.945; P = 0.034). Carriers of the Ala allele were not different from noncarriers (Pro/Pro) regarding sex (38.9 vs. 44.1% males) or ethnicity (77.4 vs. 71.7% white) distribution, age (61 +/- 10 vs. 61 +/- 10 years), known diabetes duration (17 +/- 7 vs. 16 +/- 7 years), BMI (27 +/- 4 vs. 28 +/- 5 kg/m(2)), fasting plasma glucose (184 +/- 81 vs. 176 +/- 72 mg/dl), HbA(1c) (6.7 +/- 2.3 vs. 6.9 +/- 2.4%; high-performance liquid chromatography reference range: 2.7-4.3%), and systolic (145 +/- 27 vs. 0.144 +/- 24 mmHg) or diastolic (87 +/- 14 vs. 85 +/- 14 mmHg) blood pressure, respectively. In conclusion, the presence of the Ala allele may confer protection from diabetic nephropathy in patients with type 2 diabetes.     

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.     

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。     

Amelioration of diabetic nephropathy in SPARC-null mice

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular protein that inhibits mesangial cell proliferation and also affects production of extracellular matrix (ECM) by regulating transforming growth factor-beta1 (TGF-beta1) and type I collagen in mesangial cells. This study is an investigation of the role of SPARC in streptozotocin (STZ)-induced diabetic nephropathy (DN) of 6-mo duration in wild type (WT) and SPARC-null mice. SPARC expression was evaluated by immunohistochemistry (IHC) and by in situ hybridization (ISH). Deposition of type I and IV collagen and laminin was evaluated by IHC, and TGF-beta 1 mRNA was assessed by ISH. Renal function studies revealed no significant difference in BUN between diabetic SPARC-null mice and diabetic WT mice, whereas a significant increase in albumin excretion was detected in diabetic WT relative to diabetic SPARC-null mice. Diabetic WT animals exhibited increased levels of SPARC mRNA and protein in glomerular epithelial cells and in interstitial cells, in comparison with nondiabetic WT mice. Neither SPARC mRNA nor protein was detected in SPARC-null mice. Morphometry revealed a significant increase in the percentage of the glomerular tufts occupied by ECM in diabetic WT compared with nondiabetic WT mice, although there was no difference in the mean glomerular tuft area among groups. In contrast, diabetic SPARC-null mice did not show a significant difference in the percentage of the glomerular tufts occupied by ECM relative to nondiabetic null mice. Tubulointerstitial fibrosis was ameliorated in diabetic SPARC-null mice compared with diabetic WT animals. Further characterization of diabetic SPARC-null mice revealed diminished glomerular deposition of type IV collagen and laminin, and diminished interstitial deposition of type I and type IV collagen correlated with decreases in TGF-beta 1 mRNA compared with WT diabetic mice. These observations suggest that SPARC contributes to glomerulosclerosis and tubulointerstitial damage in response to hyperglycemia through increasing TGF-beta 1 expression in this model of chronic DN.