FOXA1 and IRF-1 intermediary transcriptional regulators of PPARgamma-induced urothelial cytodifferentiation.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that has been implicated in the induction of differentiation of various cell types, including human uroepithelial cells. PPARgamma-mediated differentiation of normal human urothelial (NHU) cells in vitro requires coinhibition of epidermal growth factor receptor (EGFR) signalling and is characterised by de novo expression of late/terminal differentiation-associated genes, including uroplakins (UPK), over a 6-day period. We used gene microarrays to identify intermediary transcription factors induced in direct response to PPARgamma activation of EGFR-inhibited NHU cells. FOXA1 and IRF-1 contained consensus cognate binding sites in UPK1a, UPK2, and UPK3a promoters and transcripts were induced within 12 h of PPARgamma activation; transcription complex formation was confirmed by electromobility shift assays. In urothelium in situ, both FOXA1 and IRF-1 were nuclear and expressed in a differentiation-associated pattern. Knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARgamma-induced uroplakin expression in vitro. This is the first evidence that ligand activation of PPARgamma induces expression of intermediary transcription factors that mediate an epithelial differentiation programme and represents a new paradigm for understanding differentiation, regenerative repair and inflammation in epithelial tissues.

Differential regulation of adipocytokine mRNAs by rosiglitazone in db/db mice.

The precise mechanism by which PPARgamma activation by thiazolidinediones (TZDs) improves insulin sensitivity is still unclear. Recent studies have focused on the role of adipocytokines in metabolic control and their regulation by TZDs. In this study, we compared the chronic effects of antihyperglycemic doses of the TZD rosiglitazone, the beta3-adrenoceptor agonist BRL-35135, and the PPARalpha agonist Wy-14,643 on the mRNA expression of adipocytokines in WAT of db/db mice. Rosiglitazone treatment decreased adiponectin and resistin mRNA levels by 57 and 72%, respectively (P < 0.001), with no effect on the level of TNFalpha or RELMalpha transcripts. In comparison, Wy-14,643 reduced adiponectin transcript levels by 31% (P = 0.015) while BRL-35135 increased RELMalpha mRNA expression by 245% (P < 0.001) without effect on the other transcripts. Our results indicate that although a reduction in adiponectin and resistin mRNA levels in WAT by rosiglitazone treatment of diabetic mice may contribute to the antidiabetic effects, an alteration in TNFalpha, adiponectin, resistin, or RELMalpha mRNA expression is not absolutely required for the regulation of blood glucose concentration in the db/db mouse.

PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer.

The recent development of a novel class of insulin-sensitizing drugs, the thiazolidinediones (TZDs), represents a significant advance in antidiabetic therapy. One key mechanism by which these drugs exert their effects is by activation of the peroxisome proliferator activated receptor gamma (PPAR-gamma), a member of the nuclear receptor family. Evidence supporting this mechanism of action of the TZDs will be reviewed in this article. Recent data suggests that PPAR-gamma agonists might also have therapeutic potential in the treatment of inflammatory diseases and certain cancers.

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS: SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS: SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Non-thiazolidinedione antihyperglycaemic agents. Part 3: The effects of stereochemistry on the potency of alpha-methoxy-beta-phenylpropanoic acids.

Rhizopus delemar lipase catalysed ester hydrolysis of the alpha-methoxy-beta-phenylpropanoate 1 affords the (R)-(+) and (S)-(-) isomers in > 84% enantiomeric excess. Absolute stereochemistry was determined by a single crystal X-ray analysis of a related synthetic analogue. The activity of these two enantiomers on glucose transport in vitro and as anti-diabetic agents in vivo is reported and their unexpected equivalence attributed to an enzyme-mediated stereospecific isomerisation of the (R)-(+) isomer. Binding studies using recombinant human PPARgamma (peroxisomal proliferator activated receptor gamma), now established as a molecular target for this compound class, indicate a 20-fold higher binding affinity for the (S) antipode relative to the (R) antipode.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue.

Insulin stimulated protein kinase B alpha (PKB alpha) more than 10-fold and decreased glycogen synthase kinase-3 (GSK3) activity by 50 +/- 10% in skeletal muscle and adipocytes. Rapamycin did not prevent the activation of PKB, inhibition of GSK3 or stimulation of glycogen synthase up to 5 min. Thus rapamycin-insensitive pathways mediate the acute effect of insulin on glycogen synthase in the major insulin-responsive tissues. The small and very transient effects of EGF on phosphatidylinositol (3,4,5)P3 PKB alpha and GSK3 in adipocytes, compared to the strong and sustained effects of insulin, explains why EGF does not stimulate glucose uptake or glycogen synthesis in adipocytes.

Activators of peroxisome proliferator-activated receptor gamma have depot-specific effects on human preadipocyte differentiation.

Activation of peroxisome proliferator-activated receptor (PPAR) gamma, a nuclear receptor highly expressed in adipocytes, induces the differentiation of murine preadipocyte cell lines. Recently, thiazolidinediones (TZDs), a novel class of insulin-sensitizing compounds effective in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) have been shown to bind to PPARgamma with high affinity. We have examined the effects of these compounds on the differentiation of human preadipocytes derived from subcutaneous (SC) and omental (Om) fat. Assessed by lipid accumulation, glycerol 3-phosphate dehydrogenase activity, and mRNA levels, subcultured preadipocytes isolated from either SC or Om depots did not differentiate in defined serum-free medium. Addition of TZDs (BRL49653 or troglitazone) or 15-deoxyDelta12,14prostaglandin J2 (a natural PPARgamma ligand) enhanced markedly the differentiation of preadipocytes from SC sites, assessed by all three criteria. The rank order of potency of these agents in inducing differentiation matched their ability to activate transcription via human PPARgamma. In contrast, preadipocytes from Om sites in the same individuals were refractory to TZDs, although PPARgamma was expressed at similar levels in both depots. The mechanism of this depot-specific TZD response is unknown. However, given the association between Om adiposity and NIDDM, the site-specific responsiveness of human preadipocytes to TZDs may be involved in the beneficial effects of these compounds on in vivo insulin sensitivity.

Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling.

(+/-)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 mumol/kg diet, which is equivalent to approximately 0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 mumol/kg diet) for 14 days was increased twofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos++ +-4-yloxy) -2-[2-3H]-propylamine was used to measure adipocyte cell-surface-associated glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of human prostate cancer from benign hypertrophy by in vitro 1H NMR.

In vitro 1H NMR spectra were acquired for perchloric acid extracts of tissue samples of human prostate. Seven patients were diagnosed with prostate cancer, 13 with benign prostatic hypertrophy, and 3 with both conditions. Statistically significant differences between the cancer and benign groups were seen for the metabolite peak area ratios of citrate, creatine, and phosphorylcholine to alanine, and citrate to glutamate. There was no correlation of Gleason grade with any of the ratios measured for the cancer samples. Spectra from different sections of large tumors often yielded substantially different area ratios, confirming the heterogeneous nature of these prostate tumors.

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.

The preoperative prevalence and postoperative incidence of thromboembolism in patients with hip fractures treated with dextran prophylaxis.

A prospective preoperative and postoperative venographic study of hip fracture patients has documented a significant preoperative prevalence and postoperative incidence of thromboembolic disease. A statistically higher incidence of deep-venous thrombosis is observed in patients with femoral neck fractures compared to patients with intertrochanteric fractures. A 9% (15 of 176) preoperative prevalence and an 11% (12 of 108) incidence of new postoperative thromboembolic disease were detected. There was a predilection for deep-venous thrombosis in the injured extremity compared to the noninjured extremity both preoperatively and postoperatively.

Fat pad signs in the diagnosis of subtle fractures.

Scrutinizing the soft tissue as well as the bone is essential when reading bone films. Fat pad signs can be invaluable in the diagnosis of subtle fractures and other injuries. At some sites, such as the elbow and the wrist, these signs can be almost pathognomonic of a fracture, even if radiographic findings are otherwise negative.

Synovial osteochondromatosis.

Synovial osteochondromatosis is a benign disorder in which cartilaginous loose bodies develop about the large joints, usually the knee. It is caused by synovial metaplasia of unknown etiology. Symptoms are due either to mechanical problems caused by the loose bodies or to the degenerative arthritis that usually follows in several years. Surgical or arthroscopic removal of the loose bodies appears to be the only effective treatment. Loose bodies may recur, necessitating synovectomy.

Loin pain hematuria syndrome.

Loin pain hematuria syndrome occurs primarily in young women and is manifested by recurrent loin pain, hematuria, and abnormal renal vasculature. This syndrome was first described in 1967 by Little in the British literature. Since that time, approximately 60 cases have been reported. In this paper, we describe a case and provide a pertinent review of the literature concerning diagnosis and treatment.