Antidiabetic Thiazolidinediones Research Articles

Quantification of Lobeglitazone Sulfate in Bulk and Tablet Dosage Form by a Validated UV Spectroscopy Method: A New Thiazolidinedione Anti-diabetic Drug

Background: Lobeglitazone sulfate is a novel thiazolidinedione drug used for the therapy of type 2 diabetes. Recent pre-clinical studies show that the drug is also effective in attenuating mucus hypersecretion and airway inflammation caused due to ovalbumin-induced asthma. Objective: This study aimed to develop an accurate, simple, and cost-effective analytical method for the quantification of lobeglitazone sulfate in bulk and pharmaceutical dosage forms by UV spectrophotometric technique. Methods: The drug was identified by FTIR (ATR) instrument and the method development was performed with methanol as the solvent. In compliance with the ICH Q2(R2) guidelines, all the important validation parameters, such as linearity, accuracy, Limit of Detection (LOD), precision, specificity, Limit of Quantification (LOQ), and robustness were deter-mined. Results: The maximum absorption wavelength of lobeglitazone sulfate was found to be 248 nm. The linearity range was established from 2-14 μg/mL with a linearity equation of y = 0.0361x + 0.0024 and r² = 0.9987. The accuracy of the analytical method was found to be in the range of 99.37-100.41%. The precision studies were performed under three subsets of re-peatability, intra-day, and inter-day, and the results recorded were within the acceptance lim-its, i.e., %RSD (<2%). The developed analytical method's Limit of Detection (LOD) and Limit of Quantification (LOQ) values were 0.07 μg/mL and 0.22 μg/mL, respectively. Conclusion: The developed analytical method has been found to be valuable in regular qual-ity control analysis of lobeglitazone sulfate in pharmaceutical industries. The adopted ap-proach of the developed method has been found to be facile, accurate, precise, and economi-cal.

Peroxisome Proliferator-Activated Receptor γ (PPAR γ): A Systemic Insulin Sensitizer Associated with Decreased Risk of Type 2 Diabetes

The peroxisome proliferator-activated receptors (PPARs) are three members of the nuclear receptor prearranged by separate genes as PPARα, PPARβ/δ, and PPARγ that differ in tissue distribution and specific function. PPARγ is a nuclear receptor highly expressed in the colon, and plays a key role in bacterial induced inflammation. It is a mediator of adipocyte differentiation that regulates transcription of several genes involved in fatty acid and energy metabolism. It is known as the glitazone reverse insulin resistance receptor or nuclear receptor subfamily 1, group C, member 3 (NR1C3). It is activated by anti-diabetic Thiazolidinedione (TZD) drugs and is used as insulin sensitizers and also a favorite option for the treatment of T2D patients. It is used as monotherapy or in combination with other oral hypoglycaemic agents, which are generally safe and well-tolerated. The aim of this mini review is to discuss the potential roles of PPARγ for the prevention and treatment of T2D.

Targeting the Alternative Vitamin E Metabolite Binding Site Enables Noncanonical PPARγ Modulation.

The lipid-sensing transcription factor PPARγ is the target of antidiabetic thiazolidinediones (TZD). At two sites within its ligand binding domain, it also binds oxidized vitamin E metabolites and the vitamin E mimetic garcinoic acid. While the canonical interaction within the TZD binding site mediates classical PPARγ activation, the effects of the second binding on PPARγ activity remain elusive. Here, we identified an agonist mimicking dual binding of vitamin E metabolites and developed a selective ligand of the second site, unveiling potential noncanonical regulation of PPARγ activities. We found that this alternative binding event can simultaneously occur with orthosteric ligands and it exerted different effects on PPARγ-cofactor interactions compared to both orthosteric PPARγ agonists and antagonists, indicating the diverse roles of the two binding sites. Alternative site binding lacked the pro-adipogenic effect of TZD and mediated no classical PPAR signaling in differential gene expression analysis but markedly diminished FOXO signaling, suggesting potential therapeutic applications.

Isoform-specific functions of PPARγ in gene regulation and metabolism.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that is a vital regulator of adipogenesis, insulin sensitivity, and lipid metabolism. Activation of PPARγ by antidiabetic thiazolidinediones (TZD) reverses insulin resistance but also leads to weight gain that limits the use of these drugs. There are two main PPARγ isoforms, but the specific functions of each are not established. Here we generated mouse lines in which endogenous PPARγ1 and PPARγ2 were epitope-tagged to interrogate isoform-specific genomic binding, and mice deficient in either PPARγ1 or PPARγ2 to assess isoform-specific gene regulation. Strikingly, although PPARγ1 and PPARγ2 contain identical DNA binding domains, we uncovered isoform-specific genomic binding sites in addition to shared sites. Moreover, PPARγ1 and PPARγ2 regulated a different set of genes in adipose tissue depots, suggesting distinct roles in adipocyte biology. Indeed, mice with selective deficiency of PPARγ1 maintained body temperature better than wild-type or PPARγ2-deficient mice. Most remarkably, although TZD treatment improved glucose tolerance in mice lacking either PPARγ1 or PPARγ2, the PPARγ1-deficient mice were protected from TZD-induced body weight gain compared with PPARγ2-deficient mice. Thus, PPARγ isoforms have specific and separable metabolic functions that may be targeted to improve therapy for insulin resistance and diabetes.

Additive Effects of Omega-3 Fatty Acids and Thiazolidinediones in Mice Fed a High-Fat Diet: Triacylglycerol/Fatty Acid Cycling in Adipose Tissue.

Long-chain n-3 polyunsaturated fatty acids (Omega-3) and anti-diabetic drugs thiazolidinediones (TZDs) exhibit additive effects in counteraction of dietary obesity and associated metabolic dysfunctions in mice. The underlying mechanisms need to be clarified. Here, we aimed to learn whether the futile cycle based on the hydrolysis of triacylglycerol and re-esterification of fatty acids (TAG/FA cycling) in white adipose tissue (WAT) could be involved. We compared Omega-3 (30 mg/g diet) and two different TZDs—pioglitazone (50 mg/g diet) and a second-generation TZD, MSDC-0602K (330 mg/g diet)—regarding their effects in C57BL/6N mice fed an obesogenic high-fat (HF) diet for 8 weeks. The diet was supplemented or not by the tested compound alone or with the two TZDs combined individually with Omega-3. Activity of TAG/FA cycle in WAT was suppressed by the obesogenic HF diet. Additive effects in partial rescue of TAG/FA cycling in WAT were observed with both combined interventions, with a stronger effect of Omega-3 and MSDC-0602K. Our results (i) supported the role of TAG/FA cycling in WAT in the beneficial additive effects of Omega-3 and TZDs on metabolism of diet-induced obese mice, and (ii) showed differential modulation of WAT gene expression and metabolism by the two TZDs, depending also on Omega-3.

WSF-7 Inhibits Obesity-Mediated PPARγ Phosphorylation and Improves Insulin Sensitivity in 3T3-L1 Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ), the molecular target for antidiabetic thiazolidinediones (TZDs), is a master regulator of preadipocyte differentiation and lipid metabolism. The adverse side effects of TZDs, arising from their potent agonistic activity, can be minimized by PPARγ partial agonists or PPARγ non-agonists without loss of insulin sensitization. In this study, we reported that WSF-7, a synthetic chemical derived from natural monoterpene α-pinene, is a partial PPARγ agonist. We found that WSF-7 binds directly to PPARγ. Activation of PPARγ by WSF-7 promotes adipogenesis, adiponectin oligomerization and insulin-induced glucose uptake. WSF-7 also inhibits obesity-mediated PPARγ phosphorylation at serine (Ser)273 and improves insulin sensitivity of 3T3-L1 adipocytes. Our study suggested that WSF-7 activates PPARγ transcription by a mechanism different from that of rosiglitazone or luteolin. Therefore, WSF-7 might be a potential therapeutic drug to treat type 2 diabetes.

PPARγ-Independent Side Effects of Thiazolidinediones on Mitochondrial Redox State in Rat Isolated Hearts.

The effect of anti-diabetic thiazolidinediones (TZDs) on contributing to heart failure and cardiac ischemia/reperfusion (IR) injury is controversial. In this study we investigated the effect of select TZDs on myocardial and mitochondrial function in Brown Norway rat isolated hearts. In a first set of experiments, the TZD rosiglitazone was given acutely before global myocardial IR, and pre- and post-IR function and infarct size were assessed. In a second set of experiments, different concentrations of rosiglitazone and pioglitazone were administered in the presence or absence of the specific PPARγ antagonist GW9662, and their effects on the mitochondrial redox state were measured by online NADH and FAD autofluorescence. The administration of rosiglitazone did not significantly affect myocardial function except for transiently increasing coronary flow, but it increased IR injury compared to the control hearts. Both TZDs resulted in dose-dependent, reversible increases in mitochondrial oxidation which was not attenuated by GW9662. Taken together, these data suggest that TZDs cause excessive mitochondrial uncoupling by a PPARγ-independent mechanism. Acute rosiglitazone administration before IR was associated with enhanced cardiac injury. If translated clinically, susceptible patients on PPARγ agonists may experience enhanced myocardial IR injury by mitochondrial dysfunction.

Natural human genetic variation determines basal and inducible expression of PM20D1, an obesity-associated gene

PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene's transcription start site (TSS) in human but not mouse adipocytes. The -4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.

Design, synthesis, and evaluation of potent novel peroxisome proliferator-activated receptor γ indole partial agonists.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor important for glucose homeostasis and insulin sensitivity. The anti-diabetic drugs thiazolidinediones improve insulin sensitivity by blocking PPARγ phosphorylation at S273; however, their full agonism on PPARγ also causes significant unwanted side effects. The indole derivative UHC1 displays insulin-sensitizing effect by acting as a partial agonist through the inhibition of PPARγ S273 phosphorylation, but without full agonist-associated side effects; however, its potency leaves much to be desired. Herein we report the design and synthesis of potent indole analogs as partial PPARγ agonists via the structure-activity relationship studies. Our studies revealed that vanillylamine and piperonyl benzylamine at Site 1 are favored to bind PPARγ with either biphenyl or 3-trifluoromethyl benzyl group at Site 2. In particular, compound WO91A with vanillylamine at Site 1 displays highly potent PPARγ binding affinity (IC50 = 16.7 nM), over 30-fold more potent than the parental compound UHC1, yet with less side effect-associated transactivation activity.

Establishment of a mouse model of troglitazone-induced liver injury and analysis of its hepatotoxic mechanism.

Drug-induced liver injury is a major problem in drug development and clinical drug therapy. Troglitazone (TGZ), a thiazolidinedione antidiabetic drug for the treatment of type II diabetes mellitus, was found to induce rare idiosyncratic severe liver injury in patients, which led to its withdrawal in 2000. However, in normal experimental animals in vivo TGZ has never induced liver injury. To explore TGZ hepatotoxic mechanism, we established a novel mouse model of TGZ-induced liver injury. Administration of BALB/c female mice with a single intraperitoneal TGZ dose (300mg/kg) significantly elevated alanine aminotransferase and aspartate aminotransferase levels 6hours after the treatment. The ratio of oxidative stress marker glutathione/disulfide glutathione was significantly decreased. The increased hepatic mRNA levels of inflammation- and oxidative stress-related factors were observed in TGZ-treated mice. Subsequently, hepatic transcriptome profiles of TGZ-exposed liver were compared with those of non-hepatotoxic rosiglitazone. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway was activated in TGZ-induced liver injury. The activation of the JAK/STAT pathway promoted phosphorylation of STAT3 in TGZ-treated mice. Consequently, upregulation of STAT3 activation increased mRNA levels of its downstream genes. In conclusion, a single intraperitoneal dose of TGZ exposure could induce liver injury in BALB/c female mice and, by a hepatic transcriptomic analysis, we found that the activation of JAK/STAT pathway might be related to TGZ-induced hepatotoxicity.

The Furan Fatty Acid 9M5 Acts as a Partial Ligand to Peroxisome Proliferator-Activated Receptor gamma and Enhances Adipogenesis in 3T3-L1 Preadipocytes.

A food that has been praised for its beneficial effects on overall health is fish, particularly its polyunsaturated n-3 fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). However, it has recently been suggested that minor fatty acids such as furan fatty acids are needed in combination with DHA and EPA to exert these positive effects of fish and fish oils. Only recently have furan fatty acids become available in quantities that allow the investigation of their biofunctional properties. In this study, the uptake and effect of the furan fatty acid 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5) as a sole component and in combination with DHA and EPA on adipogenesis were analyzed using the 3T3-L1 cell model. 9M5 is taken up and metabolized into 7M5, 5M5, and 3M5 in 3T3-L1 adipocytes during a 24-h period as shown with gas chromatography with mass spectrometry (GC/MS). Furthermore, 9M5 significantly increased lipid accumulation during the differentiation process of 3T3-L1 preadipocytes into adipocytes. In addition, the combinations of DHA + 9M5 and EPA + DHA + 9M5 also exerted a significant increase compared to control adipocytes. 3T3-L1 cells incubated with 9M5 resulted in an increased protein expression of PPARγ, C/EBPα, FABP4, and adiponectin, although not to the extent that DHA as a sole component or DHA + 9M5 did. Earlier studies have shown that DHA is a natural ligand for PPARγ, thus being a potential alternative to the antidiabetic thiazolidinediones. We show that 9M5 activates a PPARγ-responsive reporter gene and could therefore be a natural ligand for PPARγ.

CORR® ORS Richard A. Brand Award: Disruption in Peroxisome Proliferator-Activated Receptor-γ (PPARG) Increases Osteonecrosis Risk Through Genetic Variance and Pharmacologic Modulation.

The pathophysiology of osteonecrosis of the femoral head (ONFH) is poorly understood, and the diagnosis is idiopathic in as many as 40% of patients. Genetic and epigenetic etiologies have been postulated, yet no single nucleotide polymorphisms (SNPs) with intuitive biologic implications have been elucidated. (1) Do individuals with ONFH share common biologically relevant genetic variants associated with disease development? (2) What is the mechanism by which these SNPs may impact the expression or function of the affected gene or protein? This retrospective genome-wide association study (GWAS) evaluated participants from the Mayo Clinic Biobank and Mayo Clinic Genome Consortium between August 2009 and March 2017. We included every patient with atraumatic ONFH in each of these respective registries and every control patient in a previous GWAS with an acceptable platform to perform statistical imputation. The study was performed in two phases, with an initial discovery cohort and a subsequent validation cohort. The initial discovery cohort consisted of 102 patients with ONFH and 4125 controls. A logistic regression analysis was used to evaluate associations between SNPs and the risk of ONFH, adjusted for age and sex. Seven SNPs were identified in a gene of biological interest, peroxisome proliferator-activated receptor gamma (PPARG), which were then evaluated in a subsequent validation cohort of 38 patients with ONFH and 464 controls. Age, sex, race, and previous steroid exposure were similar between patients with ONFH and controls in both the discovery and validation cohorts. Separate from the two-phase genetic investigation, we performed targeted pharmacosurveillance to evaluate the risk association between the use of antidiabetic thiazolidinediones, a class of PPARG agonists, and development of ONFH by referencing 9,638,296 patient records for individuals treated at Mayo Clinic. A combined analysis of the discovery and validation cohorts revealed that seven SNPs were tightly clustered adjacent to the 3' end of PPARG, suggesting an association with the risk of ONFH (p = 1.58 x 10-5.50 x10). PPARG gene-level significance was achieved (p = 3.33 x 10) when all seven SNPs were considered. SNP rs980990 had the strongest association with the risk of ONFH (odds ratio [OR], 1.95; 95% CI, 1.46-2.59; p = 5.50 x 10).The seven identified SNPs were mapped to a region near the PPARG gene and fell in a highly conserved region consisting of several critical transcription factor binding sites. Nucleotide polymorphisms at these sites may compromise three-dimensional chromatin organization and alter PPARG 3' end interactions with its 5' promoter and transcription start site. Pharmacosurveillance identified that patients who were exposed to thiazolidinediones had an increased relative risk of developing ONFH of 5.6 (95% CI, 4.5-7.1). We found that disruption of PPARG regulatory domains is linked to an increased risk of ONFH. Mechanistically, aberrant regulation of PPARG compromises musculoskeletal differentiation because this master regulator creates a proadipogenic and antiosteogenic state. Furthermore, PPARG alters steroid metabolism and vasculogenesis, processes that are inextricably linked with ONFH. Pharmacologically, predisposition to ONFH was further exposed with thiazolidinedione use, which upregulates the expression of PPARG and is known to alter bone metabolism. Collectively, these findings provide a foundation to perform confirmatory studies of our proposed mechanism in preclinical models to develop screening diagnostics and potential therapies in patients with limited options. Level III, prognostic study.

MON-LB017 Natural Genetic Variation in Humans Determines Basal and PPAR-Inducible Expression of PM20D1, a Putative Thermogenic Gene

Thermogenesis by brown and beige adipocytes is a potential avenue to increased energy expenditure and thus management of obesity and metabolic syndrome. In mice, Pm20d1 has been previously identified as a candidate thermogenic gene, with its mRNA levels cold-induced and enriched in brown and beige versus white adipocytes, and with the potential mechanism of generating a novel uncoupling metabolite. Thiazolidinedione (TZD) antidiabetic drugs, which activate the PPARγ nuclear receptor, are potent stimuli for adipocyte browning, yet they fail to induce Pm20d1 expression mouse adipocytes or adipose tissue. In contrast, we show here that PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, though not in samples from all individuals. Two putative PPARγ binding sites were identified near the gene’s transcription start site (TSS) in human but not mouse adipocytes. The ~4kb upstream binding site falls in a segmental duplication of a nearly identical intronic region ~2.5kb downstream of the TSS, and this duplication event occurred in the primate lineage and is absent in other mammals like mice. We demonstrate by chromatin immunoprecipitation and reporter assays that PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site has genetic variation in the human population, with one single nucleotide polymorphism (SNP) allele disrupting a PPAR response element. We show that this allele gives less activation by PPARγ and TZDs in reporter assays and reduced TZD activation of PM20D1 in patient-derived cultured adipocytes. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, genotype at a distinct and unlinked variant >40kb downstream of the TSS strongly correlates with overall levels of PM20D1 expression in human fat as well as multiple other tissues. Seven tightly linked downstream SNP alleles correlate with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These low PM20D1 expression variants may have phenotypic consequences, accounting for human genetic associations in this region with metabolic and neurodegenerative diseases. Overall, human PM20D1 expression is genetically variable at two levels, with genotype at downstream SNPs correlating with overall PM20D1 expression across tissues (an “on/off switch”), while the upstream PPARγ site SNP determines PPARγ and TZD regulation in adipose tissue (a “rheostat”). This human genetic variation in PM20D1 expression may ultimately inform precision medicine approaches. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

The impact of water molecules on binding affinity of the anti-diabetic thiazolidinediones for catalase: Kinetic and mechanistic approaches

Water molecules play a vital role in efficient drug binding to its target. Thiazolidinediones (TZDs), a class of anti-diabetic drugs, are widely used for treatment of type 2 diabetes mellitus. In the present study, the possible contribution of water molecules to the binding of TZDs to catalase, a potential target in the liver, was investigated by different experimental and theoretical methods. These studies indicated that TZDs could significantly improve the catalase catalytic function with a significant contribution from water molecules. As a probe for the differential number of released water molecules during the catalase transition from E to E* states, the activity of TZDs-catalase complexes was demonstrated to be mainly dependent on water activity. However, free catalase decomposed the substrate more independently. In addition, the spectrofluorimetry studies showed that the binding of TZDs to catalase needed the release of water molecules from the enzyme's binding pocket. The thermodynamic studies indicated that the binding enthalpy and entropy of TZDs for catalase were decreased with lower water activity. The favorable process contributes to release of water molecules from the binding pocket through the formation of hydrophobic interactions between catalase and TZDs in an enthalpic manner. Molecular docking simulations confirmed that the depletion of water molecules from the binding cavity is essential for effective interactions between TZDs and catalase.

Effects of lobeglitazone on insulin resistance and hepatic steatosis in high-fat diet-fed mice.

Lobeglitazone (Lobe) is a novel thiazolidinedione antidiabetic drug that reduces insulin resistance by activating peroxisome proliferator-activated receptor-gamma (PPARγ). However, the exact mechanisms of antidiabetic effects of Lobe have not been established in an animal model. The aim of this study was to evaluate the hypoglycemic effects of Lobe and investigate possible factors involved in Lobe-enhanced hepatic steatosis in high-fat diet (HFD)-fed mice. Mice were fed an HFD for 15 weeks. Lobe was administrated orally during the last 9 weeks. Lobe treatment significantly reduced insulin resistance and increased expression of hepatic glucose transporter 4 (GLUT4) and PPARs in HFD-fed mice. However, increased body weight and hepatic steatosis were not reduced by Lobe in these mice. Metabolomics fingerprinting showed that several lipogenesis-related hepatic and serum metabolites in HFD-fed mice had positive or negative correlations with Lobe administration. In particular, increased leptin levels during HFD were further increased by Lobe. HFD-induced signaling transducer and activator of transcription 3 (STAT3) phosphorylation in the hypothalamus was increased by Lobe. In addition, immunohistochemical analysis showed more proopiomelanocortin (POMC)-positive neurons in the hypothalamus of HFD-fed mice (with or without Lobe) compared with normal diet-fed mice. Despite improving leptin signaling in the hypothalamus and enhancing insulin sensitivity in HFD-fed mice, Lobe increased body weight and steatosis. Further research is necessary regarding other factors affecting Lobe-enhanced hepatic steatosis and hyperphagia.

Synthesis and biological evaluation of dihydropyrano-[2,3-c]pyrazoles as a new class of PPARγ partial agonists.

Peroxisome proliferator-activated receptor γ (PPARγ) is a well-known target for thiazolidinedione antidiabetic drugs. In this paper, we present the synthesis and biological evaluation of a series of dihydropyrano[2,3-c]pyrazole derivatives as a novel family of PPARγ partial agonists. Two analogues were found to display high affinity for PPARγ with potencies in the micro molar range. Both of these hits were selective against PPARγ, since no activity was measured when tested against PPARα, PPARδ and RXRα. In addition, a novel modelling approach based on multiple individual flexible alignments was developed for the identification of ligand binding interactions in PPARγ. In combination with cell-based transactivation experiments, the flexible alignment model provides an excellent analytical tool to evaluate and visualize the effect of ligand chemical structure with respect to receptor binding mode and biological activity.

Evaluation of Biochemical Toxicity and Antioxidant Properties of Pioglitazone on Albino Wistar Rats

Pioglitazone is one of the thiazolidinedione anti-diabetic drugs which have been used for the treatment of non-insulin dependent diabetes mellitus. This study aims at investigating the biochemical effects and safety of pioglitazone (PIO) at various concentrations in female Wistar rats. A total of 28 rats were randomly divided into four groups of seven animals each. Groups 1-4 were given 0.5 mL kgG1 b.wt., dayG1 of distilled water as normal control; 15, 30 and 45 mg kgG1 b.wt., dayG1 of PIO, respectively as treatment groups 2, 3 and 4, respectively for 28 days. Using standard biochemical kits and reported chemical procedures, plasma biochemical parameter and organ lipid peroxidation effects were determined in all the groups. There was significant increase (p<0.05) in plasma total protein concentration of group 3 and 4 in comparison with control. There was also significant (p<0.05) reduction in total and LDL cholesterols in PIO-treated groups and concentration of TBARS was reduced in the liver and heart of PIO-treated groups in comparison with normal control. There was no significant alteration in the concentrations and activities of liver and kidney function markers of PIO treated groups in comparison with normal control groups. Pioglitazone at highest concentration of 45 mg kgG1 b.wt., for the duration of 28 days did not elicit any measurable biochemical toxicity on non-diabetic rat model

Increased Circulating Adiponectin in Response to Thiazolidinediones: Investigating the Role of Bone Marrow Adipose Tissue.

Bone marrow adipose tissue (MAT) contributes to increased circulating adiponectin, an insulin-sensitizing hormone, during caloric restriction (CR), but whether this occurs in other contexts remains unknown. The antidiabetic thiazolidinediones (TZDs) also promote MAT expansion and hyperadiponectinemia, even without increasing adiponectin expression in white adipose tissue (WAT). To test the hypothesis that MAT expansion contributes to TZD-associated hyperadiponectinemia, we investigated the effects of rosiglitazone, a prototypical TZD, in wild-type (WT) or Ocn-Wnt10b mice. The latter resist MAT expansion during CR, leading us to postulate that they would also resist this effect of rosiglitazone. Male and female WT or Ocn-Wnt10b mice (C57BL/6J) were treated with or without rosiglitazone for 2, 4, or 8 weeks, up to 30 weeks of age. MAT content was assessed by osmium tetroxide staining and adipocyte marker expression. Circulating adiponectin was determined by ELISA. In WT mice, rosiglitazone caused hyperadiponectinemia and MAT expansion. Compared to WT mice, Ocn-Wnt10b mice had significantly less MAT in distal tibiae and sometimes in proximal tibiae; however, interpretation was complicated by the leakage of osmium tetroxide from ruptures in some tibiae, highlighting an important technical consideration for osmium-based MAT analysis. Despite decreased MAT in Ocn-Wnt10b mice, circulating adiponectin was generally similar between WT and Ocn-Wnt10b mice; however, in females receiving rosiglitazone for 4 weeks, hyperadiponectinemia was significantly blunted in Ocn-Wnt10b compared to WT mice. Notably, this was also the only group in which tibial adiponectin expression was lower than in WT mice, suggesting a close association between MAT adiponectin production and circulating adiponectin. However, rosiglitazone significantly increased adiponectin protein expression in WAT, suggesting that WAT contributes to hyperadiponectinemia in this context. Finally, rosiglitazone upregulated uncoupling protein 1 in brown adipose tissue (BAT), but this protein was undetectable in tibiae, suggesting that MAT is unlikely to share thermogenic properties of BAT. TZD-induced hyperadiponectinemia is closely associated with increased adiponectin production in MAT but is not prevented by the partial loss of MAT that occurs in Ocn-Wnt10b mice. Thus, more robust loss-of-MAT models are required for future studies to better establish MAT's elusive functions, both on an endocrine level and beyond.

AMP-Activated Protein Kinase Mediates the Antiplatelet Effects of the Thiazolidinediones Rosiglitazone and Pioglitazone.

The thiazolidinedione antidiabetic drugs rosiglitazone and pioglitazone exert antiplatelet effects. Such effects are known to be mediated by the peroxisome proliferator-activated receptor γ (PPARγ), an acknowledged target of the thiazolidinediones, although the molecular mechanism is elusive. Recently, AMP-activated protein kinase (AMPK) signaling was reported to inhibit platelet aggregation. Because AMPK is another target of the thiazolidinediones, the impact of rosiglitazone and pioglitazone on platelet AMPK and its involvement in aggregation were investigated to assess the contribution of AMPK to the antiplatelet activity of these agents. Treatment with rosiglitazone stimulated both AMPK and PPARγ in isolated rat platelets. However, the concentration and the treatment time required for activation were distinct from each other. Indeed, stimulation of AMPK and PPARγ were discrete events without any cross-activation in platelets. Activation of AMPK or PPARγ by rosiglitazone rendered platelets less responsive to aggregatory stimuli such as collagen, ADP, and thrombin. However, the resultant efficacy caused by activating AMPK was higher than that attributable to PPARγ stimulation. Similar results were obtained with pioglitazone. Taken together, rosiglitazone and pioglitazone inhibit platelet aggregation by activating AMPK. AMPK functions as a potential target of rosiglitazone and pioglitazone for their antiplatelet activity, although the in vivo or clinical relevance remains to be assessed.

Abstract 84: Expression of the Transcription Factors PPAR Alpha and Gamma, and Their Target Genes in the Heart of Obese Mice

The Peroxisome-Proliferator Activated Receptors (PPARs) have been identified as key regulators of energy metabolism by sensing fatty acid availability and activating the transcription of enzymes involved in fatty acid and glucose metabolism. In the heart, PPARs have been proven to participate in the development of diseases, such as cardiac hypertrophy and diabetic cardiomyopathy, conditions that have been associated to obesity. Furthermore, the use of PPARs activator drugs is frequent for the treatment of obesity-dependent diseases, such as hypoglycemiant (fibrates) and anti-diabetic (thiazolidinediones) drugs. Therefore, the objective of this project was to characterize the expression of PPARs and some of their target genes involved in fatty acid and glucose metabolism in the heart of obese mice. Mice (C57BL/6) were fed with either a standard chow diet (control group) or a high fat diet (obese group) for three months. Hearts were weighed prior to quantifying protein expression of two PPARs (alpha and gamma), and both mRNA and protein expression of three enzymes (phosphofructokinase (PFK), glycerol-3-phosphate dehydrogenase (GPD1) and glycerol-3-phosphate acyltransferase (GPAT)). Protein levels were measured by western blot, while mRNA levels were quantified by real-time PCR. The obese group presented 41% increase in body weight compared with control mice. However, no change was observed in obese hearts weight compared with control group, meaning that hypertrophy had not been developed yet. The expression of PPAR-alpha was increased 42% in obese mice compared with the control group, suggesting a possible activation of the fatty acid oxidation pathway. PPAR-gamma together with GPD1 and GPAT showed no differences at any level; however, PFK presented 90% increase in mRNA level and 54% increase in protein level in obese mice. Interestingly, decrease in PFK has been reported in any altered PPAR-alpha condition, contrary to our results. These results suggest that there is a dysregulation of the heart’s energetic pathways mediated by PPARs in earlier stages of obesity.