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.

Characterization of liver-specific expression of rat uricase using monoclonal antibodies and cloned cDNAs.

Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.

Cloning of rabbit uricase cDNA reveals a conserved carboxy-terminal tripeptide in three species.

cDNA clones encoding uricase have been isolated from a rabbit liver cDNA library. The nucleotide sequences of the cDNAs have been determined and those of the rat uricase cDNA have been revised. In all three uricases, the carboxy-terminal tripeptides are Ser-Arg/Lys-Leu sequences, which have recently been suggested as an essential element of peroxisomal targetting signals for many but not all peroxisomal proteins.

cDNA cloning and sequence of rat ribonuclease inhibitor, and tissue distribution of the mRNA.

A cDNA clone encoding ribonuclease inhibitor was isolated from a rat lung cDNA library. The deduced amino acid sequence revealed a high degree of conservation of a repeated structure. The mRNA was detected in all seven tissues of rat examined, the amount being highest in the lung and lowest in the heart.

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.

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.

Organization of rat uricase chromosomal gene differs greatly from that of the corresponding plant gene.

The rat chromosomal gene for uricase was cloned. The gene spans more than 20 kilobases and the coding region is divided into 8 exons by 7 introns. The organization of the rat uricase gene is so greatly different from that of the soybean gene that the difference may not have been caused only by the removal of some ancestral introns during the period of widely separated evolution.

Two rabbit uricase mRNAs and their tissue-specific expression.

Presence of two uricase mRNA species in rabbit was revealed by cDNA cloning. They were different only in the 5'-non-coding region. Their hepatic and extra-hepatic expression in a tissue-specific manner was demonstrated by Northern blot and primer extension analyses.

Rat liver BiP/GRP78 is down-regulated by a peroxisome-proliferator, clofibrate.

Administration of clofibrate in rat results in down-regulation of several liver proteins and a vast induction of peroxisomal proteins. One protein was identified as BiP/GRP78 using antibodies and cDNA cloning. The level of mRNA was reduced by the drug.

Brain-specific expression of transthyretin mRNA as revealed by cDNA cloning from brain.

cDNAs for rat transthyretin mRNA were cloned from a brain cDNA library. Sequencing analyses showed the presence of an additional 5' sequence that had not been reported for the liver mRNA corresponding to the flanking promoter region of the gene. This additional sequence was expressed only in the brain, suggesting the presence of a brain-specific promoter.

A protein histidine kinase induced in rat liver by peroxisome proliferators. In vitro activation by Ras protein and guanine nucleotides.

A novel protein kinase is induced in rat liver plasma membrane by the administration of peroxisome proliferators. A 36 kDa protein (P36) on the membrane was rapidly phosphorylated in vitro by the kinase and the phosphorylated amino acid was identified as phosphohistidine. Histidine phosphorylation of P36 was activated in vitro by recombinant Ras protein and GTP; both decreased Michaelis constant (Km) for ATP from 1.25 to 0.25 microM. The novel histidine kinase, products of which have been overlooked due to their acid lability, may participate in cellular signaling and peroxisome proliferators may perturb the pathway.

Transient induction of fatty acid synthase in rat liver after removal of a peroxisome proliferator.

Removal of a peroxisome proliferator from the diet triggered the degradation of peroxisomes and induced the transient expression of a 220 kDa soluble protein in rat liver. The 220 kDa protein was purified by conventional methods and analyzed by amino acid sequencing. A total of 99 amino acid residues in 4 lysylendopeptidase-digested peptides completely matched those in rat fatty acid synthase. The transient induction of fatty acid synthase mRNA during peroxisome degradation was confirmed by Northern blotting.

Control region and gastric specific transcription of the rat H+,K(+)-ATPase alpha subunit gene.

The rat gastric H+,K(+)-ATPase alpha subunit gene was cloned and the nucleotide sequence of its 5'-upstream region was determined. Sequence comparison with the corresponding part of the human gene indicated the presence of highly conserved regions which may be important for specific transcription of the alpha subunit in gastric parietal cells. The amino-terminal sequence (Met-Gly-Lys-Ala-Glu-) of the rat enzyme was similar to those of the pig and human enzymes. The gene organization of the rat enzyme was also similar to that of the human gene: introns 1, 2 and 9 were located in exactly the same positions as those in the human gene, and, as in the latter, exon 6 was not separated by an intron. The sequences of introns 1 and 2 were highly conserved among the rat, human and pig genes, but were entirely different from those of Na+,K(+)-ATPase catalytic subunit genes. Northern blot hybridization indicated that the gene was transcribed only in gastric mucosa.

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.

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.

Bile secretion and histology of liver autotransplanted into the pancreatic parenchyma.

The pancreatic parenchyma was evaluated as a potential recipient site for hepatic fragment autotransplantation. Histologic and functional studies of hepatic autografts in the pancreas were performed in 15 mongrel dogs. Approximately 10 g of liver parenchyma was resected from each left lateral lobe. The remnant liver remained in situ. Bile secretion from the hepatic tissues implanted into the pancreas was estimated by measuring the indocyanine green (ICG)* concentration in pancreatic juice following intravenous ICG injection. One month following implantation, the hepatocytes in the pancreatic parenchyma were histologically colorless and did not have sinusoids. However, by the second month following implantation, hepatic nodules had grown extensively to become normal liver tissue, with sinusoids and a single liver cell-plate structure. At 4 months following intrapancreatic implantation the transplanted hepatic masses consisted of several hepatic lobules. Furthermore, ICG could be detected in the pancreatic juice of dogs surviving more than 2 months after implantation but no ICG could be detected in the pancreatic juice of normal controls. The present study provides direct evidence that hepatic grafts transplanted into the pancreas that has a ductal drainage system for bile secretion can reconstitute histologically normal liver tissue capable of secreting bile. This model can be used to understand the early steps of hepatic regeneration.

DR antigen expression on vascular endothelium and duct epithelium in fresh or cultured human fetal pancreata in the presence of gamma-interferon.

The cells expressing MHC class II antigens play an important role in allograft rejection. We examined the expression of HLA DR antigens in human fetal pancreata ranging in gestational ages from 8 to 24 weeks. DR antigens were detected by immunohistochemical techniques using H4 and 40D MoAbs with immunoperoxidase staining and the ABC procedure. Vascular endothelium was identified by goat antibodies directed to human factor VIII-related antigens. DR expression on endothelium was further confirmed by double staining of tissue sections with H4 and anti-FVIII antibodies. In pancreata younger than 18 weeks of gestation, DR antigens were found on single cells that were randomly distributed throughout the pancreatic parenchyma. These cells resembled dendritic cells in morphology, as reported previously. Some vascular endothelial cells located in the intralobular connective tissues expressed DR antigens, but those in the pancreatic parenchyma were DR-negative. In 18-22 weeks, some endothelia of small vessels in the parenchyma became DR-positive. By 24 weeks, DR antigens were also expressed on medium-sized blood vessels, whereas intraislet capillaries and duct epithelia were DR-negative. The number of DR-positive cells increased rapidly from 11.2 cells/field (x400) in 12-14 week pancreata to 42.8 cells/field in 18-22 week pancreata. Most DR-positive cells in pancreata earlier than 18 weeks were dendritic-like cells, while those in older pancreata were endothelial cells. When pancreatic tissue fragments were cultured for two days in the presence of rIFN-gamma, vascular endothelium and duct epithelium, both of which were otherwise DR-negative, had become DR-positive. Even with this increase, DR-positive cell numbers were fewer in pancreata younger than 18 weeks of gestation as compared with those in older pancreata. We also detected clusters of epithelial-like cells that were clearly stained for DR antigens. These cells were located in the parenchyma where insulin positive cells were generally found. Their DR-antigens had not changed during the 3-day rIFN-gamma culture.

Prevention of insulitis and diabetes in nonobese diabetic mice by administration of FK506.

We investigated the preventive effect of the immunosuppressive agent FK506 on autoimmune insulitis in nonobese diabetic mice. The mice were given FK506 in a dose of 1.0 mg/kg, every other day, from age 2 to 12, 2 to 6, and 4 to 12 weeks, respectively; after which, the incidence of insulitis and overt diabetes was monitored. Effects of FK506 on immune reactions to beta cells were also investigated by using both syngeneic and allogeneic islet transplants. Treatment with FK506 in mice from age 2 weeks prevented completely the onset of overt diabetes, and the incidence of insulitis was reduced to less than 10% at age 30 weeks. Treatment of mice with FK506 from age 4 weeks was less effective in preventing insulitis and the onset of diabetes. In case of islet transplantation, FK506 treatment of NOD mice from age 2 to 6 weeks prevented autoimmune responses both in syngeneic islets and in allogeneic islets, which share the same H-2 antigen with the nonobese diabetic mouse. These results also indicate that the recognition of islet antigens and the generation of autoimmune-reactive T lymphocytes start between 2 and 4 weeks of age, and FK506 prevents an autoimmune reaction.

A comparison of endocrine and exocrine function after pancreatic fragment autotransplantation into splenic pulp, portal vein, and hepatic parenchyma.

Three sites were evaluated for potential pancreatic fragment autotransplantation. Both endocrine and exocrine functions were evaluated following autotransplantation into splenic pulp, portal vein, or hepatic parenchyma in 44 pancreatectomized dogs. Cholecystic bile amylase concentrations in the hepatic parenchyma group surviving more than 2 months were elevated significantly, and choledochal bile amylase concentrations increased markedly following pancreozymin-secretin injection. In contrast, bile amylase concentrations in dogs with intrasplenic or intraportal implants were low and did not respond to PS injection. Histologically pancreatic autografts in hepatic parenchyma revealed marked proliferation of exocrine tissue with abundant zymogen granules and reconstruction of the acinar lobules with a few islets. These acinar cells in the hepatic parenchyma were ultrastructurally normal. Transplant endocrine function, estimated by K values, was significantly better after splenic pulp and portal vein than after a hepatic parenchyma implantation, but no group improved during 1-year follow-up. Glucose-stimulated initial insulin responses were abnormally low in all recipients. Islet B cells lacked mature insulin granules, such as seen in normal resting B cells. This ultrastructural finding implies a persistent demand on the B cells and may explain the spontaneous recurrence of hyperglycemia and the diminished initial insulin response to a glucose load. This study indicates that euglycemic recipients of pancreatic fragment autotransplantation remain unstable and prediabetic.

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.