GW501516 acts as an efficient PPARα activator in the mouse liver.

The peroxisome proliferator-activated receptor (PPAR) subtype specificity of GW501516, a well-known PPARδ-specific agonist, was studied by examining its effects on the expression of endogenous genes in primary hepatocytes and the liver of wild-type and PPARα-null mice. GW501516, like the PPARα-specific agonist Wy14,643, induced the expression of several PPAR target genes in a dose-dependent manner but this action was mostly absent in the cells and liver of PPARα-null mice. Results indicated that GW501516 acts as an efficient PPARα activator in the mouse liver.

Expression of resistin in the adipose tissue is modulated by various factors including peroxisome proliferator-activated receptor alpha.

AIM: Resistin has been suggested to link obesity to diabetes by antagonizing insulin action. However, this model is based on limited observations and how resistin links the two complex processes is not known. In this study, we investigated the effects of various factors on the expression of resistin and examined the generality of the proposal. METHODS: Total RNA was isolated from the adipose tissues of lean, obese and peroxisome proliferator-activated receptor (PPAR)alpha-null mice fed a control diet or that contained a PPAR ligand, and analysed by Northern blotting using cDNAs for resistin, leptin, aP2 and other mRNAs as probes. For quantitative analysis, an image analyser was used. RESULTS: Basal expression of resistin mRNA was suppressed by obesity, but the extent of suppression differed significantly among the mouse strains and types of adipose tissue examined. Anti-diabetic thiazolidinediones induced resistin expression in the lean mice and showed smaller effects in obese mice. Furthermore, PPARalpha was shown to play an important role in constitutive expression of resistin in the adipose tissue. CONCLUSION: Our results indicated that diverse factors modulate the expression of resistin in the adipose tissues of mice, and suggested that resistin is not a master hormone linking obesity to diabetes.

Inhibition of RXR and PPARgamma ameliorates diet-induced obesity and type 2 diabetes.

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance.

Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARgamma and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARgamma by PPARgamma agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARgamma activity by heterozygous PPARgamma deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARgamma deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARgamma deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor alpha, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARgamma deficiency and PPARgamma agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy.

[Suppression of stress proteins, GRP78, GRP94, calreticulin and calnexin in liver endoplasmic reticulum of rat treated with a highly toxic coplanar PCB].

The present study was addressed on the effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) to the expression of molecular chaperon proteins, glucose regulated protein (GRP) 78, GRP94, calreticulin and calnexin in liver endoplasmic reticulum of rat by treatment with acute exposure. Male Wistar rats received PenCB in corn oil at once a dose of 10 mg/kg i.p., then at 5 days after treatment the microsomes were prepared. Free-fed and pair-fed control groups were given the vehicle. The microsomal proteins were separated on SDS-PAGE, transferred to membrane and blotted using antibody towards respective chaperone proteins. The protein levels of GRP78, GRP94, calreticulin and calnexin were significantly decreased with the acute exposure. In addition, albumin level in the microsomes was also significantly reduced by the PenCB treatment. The transferrin level was significantly higher than pair-fed but not from free-fed group. These chaperone proteins have important physiological functions against synthesized and/or denatured proteins, which include assembling, folding of proteins. PenCB-treatment may alter the extent of biosynthesis of secretory protein such as albumin through the decreasing levels of chaperone proteins in endoplasmic reticulum. Interestingly, reduced GRP78 protein level by PenCB was not due to decreased mRNA level. Our results suggested that a part of the toxicity of PenCB is associated to significant decrease of the chaperone proteins in the endoplasmic reticulum.

Technique for calculation of the propagation constant in an optical planar waveguide with a gaussian profile.

We performed analysis of a planar waveguide with arbitrary index variations. We obtained numerical results for the propagation coefficient by using first-order Langer and Liouville transformations. The accuracy of the numerical results is confirmed by a comparison with those obtained by other methods.

Differential effects of PPARalpha activators on induction of ectopic expression of tissue-specific fatty acid binding protein genes in the mouse liver.

The effect of a potent peroxisome proliferator-activated receptor (PPAR) alpha activator Wy14,643 on tissue-specific expression of fatty acid binding (FABP) genes was studied. Wy14,643 immediately induced liver-, intestine- and FABP but not PPARgamma-regulated adipose-FABP (or aP2) mRNAs in respective mouse tissues. Moreover, it gradually induced ectopic expression of heart- and adipose-FABP mRNAs to significant levels in the liver. However, ectopic expression was not induced in the liver of PPARalpha-null mouse, indicating an obligatory role of the receptor in the modulated expression. Among the four PPARalpha activators examined, only Wy14,643 induced ectopical expression of heart-FABP in the liver. Thus, tissue-specificity of the FABP gene expression is not absolute and, with a potent activator, can be distorted by PPARalpha.

Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells.

The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 microM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 microM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the beta isoform of phosphatidylinositol 3-kinase regulatory subunit.

A half-type ABC transporter TAPL is highly conserved between rodent and man, and the human gene is not responsive to interferon-gamma in contrast to TAP1 and TAP2.

TAPL is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that is transcribed in various rat tissues [Yamaguchi, Y., Kasano, M., Terada, T., Sato, R., and Maeda, M. (1999) FEBS Lett. 457, 231-236]. Primary structures of the human and mouse orthologous counterparts were deduced from cDNAs cloned by means of polymerase chain reaction, and they were compared with that of the rat. The mammalian TAPLs (rat, mouse, and human) are highly conserved, since about 95% of the amino acid residues are identical between rodents and man. Phylogenetic analysis demonstrated that the evolutional rate of TAPL is much slower than those of TAP1 and TAP2, although TAPL could have diverged from an ancestor of TAP1 or that of TAP1 and TAP2. The TAPL-GFP fusion protein transiently expressed in Cos-1 cells was co-localized with PDI, suggesting that TAPL is inserted into endoplasmic reticulum membrane. The conservation of the peptide-binding motifs of TAP proteins in TAPL raises the possibility that the TAPL might be a peptide transporter. The gene for human TAPL is assigned to chromosome 12q24.31-q24.32, while those for TAP1 and TAP2 are located at the MHC locus of chromosome 6p21.!3. Furthermore, the transcription of TAPL gene is not responsive to interferon-gamma, in contrast to TAP1 and TAP2. These results indicate that the gene regulation of TAPL is different from those of TAP1 and TAP2.

A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice.

Thiazolidinediones (TZDs) reduce insulin resistance in type 2 diabetes by increasing peripheral uptake of glucose, and they bind to and activate the transcriptional factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Studies have suggested that TZD-induced activation of PPAR-gamma correlates with antidiabetic action, but the mechanism by which the activated PPAR-gamma is involved in reducing insulin resistance is not known. To examine whether activation of PPAR-gamma directly correlates with antidiabetic activities, we compared the effects of 4 TZDs (troglitazone, pioglitazone, BRL-49653, and a new derivative, NC-2100) on the activation of PPAR-gamma in a reporter assay, transcription of the target genes, adipogenesis, plasma glucose and triglyceride levels, and body weight using obese KKAy mice. There were 10- to 30-fold higher concentrations of NC-2100 required for maximal activation of PPAR-gamma in a reporter assay system, and only high concentrations of NC-2100 weakly induced transcription of the PPAR-gamma but not PPAR-alpha target genes in a whole mouse and adipogenesis of cultured 3T3L1 cells, which indicates that NC-2100 is a weak PPAR-gamma activator. However, low concentrations of NC-2100 efficiently lowered plasma glucose levels in KKAy obese mice. These results strongly suggest that TZD-induced activation of PPAR-gamma does not directly correlate with antidiabetic (glucose-lowering) action. Furthermore, NC-2100 caused the smallest body weight increase of the 4 TZDs, which may be partly explained by the finding that NC-2100 efficiently induces uncoupling protein (UCP)-2 mRNA and significantly induces UCP1 mRNA in white adipose tissue (WAT). NC-2100 induced UCP1 efficiently in mesenteric WAT and less efficiently in subcutaneous WAT, although pioglitazone and troglitazone also slightly induced UCP1 only in mesenteric WAT. These characteristics of NC-2100 should be beneficial for humans with limited amounts of brown adipose tissue.

Insulin-degrading enzyme exists inside of rat liver peroxisomes and degrades oxidized proteins.

Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.

PPAR-mediated diverse responses in various tissues of mouse.

Peroxisome proliferator-activated receptors (PPARs) play the essential role in transcriptional modulations of the genes involved in lipid metabolism. In vitro studies have shown that all the mechanisms of the modulations are similarly mediated by PPAR and the response elements through heterodimerization with retinoid X receptors (RXRs). However, PPARs mediate the diverse effects induced in various tissues of mouse by their activators. First, PPAR alpha also plays an obligatory role in the activator-induced transcriptional modulations of various genes, some of which are not related to lipid metabolism. Second, responsiveness of various genes to several activators varies considerably. Third, some of the activator-induced transcriptional activation of several genes is strictly tissue-specific. Fourth, the time courses of the activator-induced transcriptional modulations are diverse. Following brief review of these diverse responses, emphasis is laid on the need for studies of these diversely regulated genes to understand the species-general and coordinated interplay between PPARs and other factors to maintain lipid homeostasis at the body level.

Relationship between signal transduction and PPAR alpha-regulated genes of lipid metabolism in rat hepatic-derived Fao cells.

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of 32P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, alpha isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.

Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner.

Regulation of gene expression of three putative long-chain fatty acid transport proteins, fatty acid translocase (FAT), mitochondrial aspartate aminotransferase (mAspAT), and fatty acid transport protein (FATP), by drugs that activate peroxisome proliferator-activated receptor (PPAR) alpha and gamma were studied using normal and obese mice and rat hepatoma cells. FAT mRNA was induced in liver and intestine of normal mice and in hepatoma cells to various extents only by PPARalpha-activating drugs. FATP mRNA was similarly induced in liver, but to a lesser extent in intestine. The induction time course in the liver was slower for FAT and FATP mRNA than that of an mRNA encoding a peroxisomal enzyme. An obligatory role of PPARalpha in hepatic FAT and FATP induction was demonstrated, since an increase in these mRNAs was not observed in PPARalpha-null mice. Levels of mAspAT mRNA were higher in liver and intestine of mice treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy obese mice, thiazolidinedione PPARgamma activators (pioglitazone and troglitazone) induced FAT and FATP more efficiently than the PPARalpha activator, clofibrate. This effect was absent in brown adipose tissue. Under the same conditions, levels of mAspAT mRNA did not change significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPARalpha and -gamma. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus, peroxisome proliferators not only influence the metabolism of intracellular fatty acids but also cellular uptake, which is likely to be an important regulatory step in lipid homeostasis.

Peroxisome proliferator-activated receptor (PPAR)-dependent and -independent transcriptional modulation of several non-peroxisomal genes by peroxisome proliferators.

To better characterize peroxisome proliferator-induced non-peroxisomal responses, we searched the mRNAs of which the levels were modulated by proliferators. We used the PCR-based methods including differential display. The mRNAs were divided into at least four groups by their time-courses of induction and repression: group 1 very rapidly increased then decreased; group 2 increased after a time lag (well-characterized peroxisomal mRNAs belonged to this group); group 3 decreased reciprocally compared with group 2 mRNAs; group 4 increased after group 2 mRNAs, with a much longer lag period. All of these modulations cannot be explained by peroxisome proliferator action through PPAR and RXR dimerization on the target genes to activate transcription. Another unidentified transcription factor may be involved in some of these modulations. It will also be important to consider PPAR-independent pathways when studying the diverse effects of peroxisome proliferators.

PPAR alpha mediates peroxisome proliferator-induced transcriptional repression of nonperoxisomal gene expression in mouse.

The strain difference, peroxisome proliferator specificity and role of PPAR alpha in peroxisome proliferator-induced transcriptional repression of nonperoxisomal transthyretin and alpha2u-globulin genes were examined. The genes were repressed by four peroxisome proliferators in all seven mouse strains tested. The extent of repression was strongly dependent on both the mouse strains and type of proliferator, although the mRNA levels of PPAR alpha and its partner in heterodimerization, RXR alpha were not different. The role of PPAR alpha in repression was confirmed by the finding that PPAR alpha-null mice were not responsive to transcriptional repression. These results indicate that PPAR alpha plays an obligatory role in transcription of various genes, some of which are not related to lipid metabolism.

Insulinoma: correlation of short-TI inversion-recovery (STIR) imaging and histopathologic findings.

We studied the value of short-TI inversion-recovery (STIR) imaging for the localization of pancreatic insulinoma. Four patients (three women and one man aged 35-65 years) with surgically proven insulinoma were included in this study. All patients were examined by MR imaging with spin echo (SE) and STIR sequences. The STIR images were compared with the histopathologic findings in each case. In two patients, the tumors were markedly hyperintense on STIR images, and a 5-mm insulinoma was depicted only by this imaging method in one of the two. In the other two patients, 10-mm insulinomas were only slightly hyperintense on STIR images. The latter tumors had a higher content of collagen fibers than the former, indicating that the amount of collagen influences the signal intensity of insulinoma. Despite some limitations, STIR imaging is a useful noninvasive method for the localization of pancreatic insulinoma.