Effects of pioglitazone and fenofibrate co-administration on bone biomechanics and histomorphometry in ovariectomized rats.

Pioglitazone, the peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist is an effective therapy for type 2 diabetes, but has been associated with increased risk for bone fracture. Preclinical studies suggest that PPAR-α agonists (e.g., fenofibrate) increase bone mineral density/content, although clinical data on bone effects of fibrates are lacking. We investigated the effects of pioglitazone (10 mg/kg/day) and fenofibrate (25 mg/kg/day) on bone strength and bone histomorphometric parameters in osteopenic ovariectomized (OVX) rats. An additional group of rats received a combination of pioglitazone + fenofibrate to mimic the effects of a dual PPAR-α/γ agonist. The study consisted of a 13-week treatment phase followed by a 6-week treatment-free recovery period. Pioglitazone significantly reduced biomechanical strength at the lumbar spine and femoral neck compared with rats administered fenofibrate. Co-treatment with pioglitazone + fenofibrate had no significant effect on bone strength in comparison with OVX vehicle controls. Histomorphometric analysis of the proximal tibia revealed that pioglitazone suppressed bone formation and increased bone resorption at both cancellous and cortical bone sites relative to OVX vehicle controls. In contrast, fenofibrate did not affect bone resorption and only slightly suppressed bone formation. Discontinuation of pioglitazone treatment, both in the monotherapy and in the combination therapy arms, resulted in restoration of bone formation and resorption rates, demonstrating reversibility of effects. The above data support the concept that dual activation of PPAR-γ and PPAR-α attenuates the negative effects of PPAR-γ agonism on bone strength.

Rhesus macaques develop metabolic syndrome with reversible vascular dysfunction responsive to pioglitazone.

BACKGROUND: The metabolic syndrome (MetS) is a constellation of clinical features that include central obesity, hypertension, atherogenic dyslipidemia, and insulin resistance. However, the concept remains controversial; it has been debated whether MetS represents nothing more than simultaneous co-occurrence of individual risk factors or whether there are common shared pathophysiological mechanisms that link the individual components. METHODS AND RESULTS: To investigate the emergence of metabolic and cardiovascular components during the development of MetS, we identified MetS-predisposed animals (n=35) in a large population of rhesus macaques (Macaca mulatta, 12.7±2.9 years old, n=408), acclimated them to standardized conditions, and monitored the progression of individual component features over 18 months. In 18 MetS animals with recently developed fasting hyperinsulinemia, central obesity, hypertension, and atherogenic dyslipidemia, we found that individual metabolic and cardiovascular components track together during the transition from pre-MetS to onset of MetS; MetS was associated with a 60% impairment of flow-mediated dilation, establishing the mechanistic link with vascular dysfunction. Pioglitazone treatment (3 mg/kg body weight/d for 6 weeks), a peroxisome proliferator-activated receptor γ agonist, reversibly improved atherogenic dyslipidemia and insulin resistance and fully restored flow-mediated dilation with persistent benefits. CONCLUSIONS: Coemergence of metabolic and cardiovascular components during MetS progression and complete normalization of vascular dysfunction with peroxisome proliferator-activated receptor γ agonists suggest shared underlying mechanisms rather than separate processes, arguing for the benefit of early intervention of MetS components. Predictive nonhuman primate (NHP) models of MetS should be highly valuable in mechanistic and translational studies on the pathogenesis of MetS in relation to cardiovascular disease and diabetes mellitus.

Effects of aleglitazar, a balanced dual peroxisome proliferator-activated receptor α/γ agonist on glycemic and lipid parameters in a primate model of the metabolic syndrome.

BACKGROUND: Glycemic control and management of dyslipidemia to reduce cardiovascular risk are major therapeutic goals in individuals with type 2 diabetes mellitus (T2DM). This study was performed to evaluate the effects of aleglitazar, a balanced dual peroxisome proliferator-activated receptor α/γ (PPARα/γ) agonist, on both lipid and glycemic parameters in obese, hypertriglyceridemic, insulin-resistant rhesus monkeys. METHODS: A 135-day efficacy study was performed in six rhesus monkeys. After a 28-day baseline assessment (vehicle only), monkeys received oral aleglitazar 0.03 mg/kg per day for 42 days, followed by a 63-day washout period. Plasma levels of markers of glycemic and lipid regulation were measured at baseline, at the end of the dosing period, and at the end of the washout period. RESULTS: Compared with baseline values, aleglitazar 0.03 mg/kg per day reduced triglyceride levels by an average of 89% (328 to 36 mg/dL; P = 0.0035 when normalized for baseline levels) and increased high-density lipoprotein cholesterol levels by 125% (46 to 102 mg/dL; P = 0.0007). Furthermore, aleglitazar reduced low-density lipoprotein cholesterol levels (41%) and increased levels of apolipoprotein A-I (17%) and A-II (17%). Aleglitazar also improved insulin sensitivity by 60% (P = 0.001). Mean body weight was reduced by 5.9% from baseline values with aleglitazar at this dose (P = 0.043). CONCLUSIONS: Aleglitazar, a dual PPARα/γ agonist, has beneficial effects on both lipid and glucose parameters and may have a therapeutic role in modifying cardiovascular risk factors and improving glycemic control in patients with T2DM.

Discovery of carmegliptin: a potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Design, synthesis, and SAR are described for a class of DPP-IV inhibitors based on aminobenzo[a]quinolizines with non-aromatic substituents in the S1 specificity pocket. One representative thereof, carmegliptin (8p), was chosen for clinical development. Its X-ray structure in complex with the enzyme and early efficacy data in animal models of type 2 diabetes are also presented.

Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors.

Synthesis and SAR are described for a structurally distinct class of DPP-IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)aromatic substituents in the S1 specificity pocket. The m-(fluoromethyl)-phenyl derivative (S,S,S)-2g possesses the best fit in the S1 pocket. However, (S,S,S)-2i, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.

Medium-chain fatty acids ameliorate insulin resistance caused by high-fat diets in rats.

BACKGROUND: High dietary intake of saturated fat impairs insulin sensitivity and lipid metabolism. The influence of fatty acid chain length, however, is not yet fully understood, but evidence exists for different effects of saturated long-chain (LC) versus saturated medium-chain (MC) fatty acids (FA). METHODS: To investigate the effects of the FA chain length, male Wistar rats were fed high-fat diets containing triacylglycerols composed of either MC- or LCFA for 4 weeks; rats fed maintenance diet served as a control. The animals underwent euglycemic hyperinsulinemic clamping or oral metabolic tolerance testing respectively; enzyme activities of mitochondrial (EC2.3.1.21 carnitine palmitoyl transferase) and peroxisomal (EC1.3.3.6 acyl-CoA oxidase) FA oxidation were measured in liver and muscle. RESULTS: LCFA consumption resulted in higher fasted serum insulin and glucose concentrations compared to controls, while MCFA-fed animals did not differ from controls. Insulin sensitivity was reduced by 30% in the LCFA group while the MCFA group did not differ from controls. Feeding MCFA resulted in the controls' lowered fasted and post-prandial triacylglycerol concentration compared to LCFA, while triacylglycerol concentrations in muscle were higher in both high-fat groups compared to controls. No diet-induced changes were found in acyl-CoA oxidase (ACO) activity (liver and muscle), while LCFA feeding significantly raised carnitine palmitoyltransferase activity. CONCLUSIONS: The chain length of saturated fatty acids in isocaloric diets affects insulin sensitivity, lipid metabolism and mitochondrial fatty acid oxidation without influencing body weight. While dietary LCFA clearly impair insulin sensitivity and lipid metabolism, MCFA seem to protect from lipotoxicity and subsequent insulin resistance without caloric restriction.

Possible effects of environmental nitrates and toxic organochlorines on human thyroid in highly polluted areas in Slovakia.

Heavy environmental pollution resulting from uncontrolled industrial and agricultural activities has occurred in several areas of Slovakia. So far, field surveys focused mainly on the thyroid have been conducted in one area polluted by nitrates and in a large area polluted mainly by organochlorinated toxicants. In children from the high nitrate area (HNA, n = 324) significantly higher thyroid volume (ThV) by ultrasound was found compared with age-matched children from surrounding areas with low nitrate (LNA, n = 764). In blood samples of 324 children from the HNA and of 100 children from the LNA no difference between areas was found in the level of total thyroxine (T4) and free triiodothyronine (T3). However, positive thyroid peroxidase antibodies (TPOAb) were found in 7/324 (2.2%) and thyrotropin (TSH) levels > 4.0 mIU/L in 13/324 (4.0%) of children from the HNA area, while no positive values were obtained in the LNA. In the area heavily polluted by an organochlorine (OC) cocktail consisting of polychlorinated biphenyls (PCBs), 2,2'-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), and dioxins and furans (polluted area) and in the background pollution area (background area) a total of 2046 adults were examined. In polluted area very high blood levels of OCs were found as well as increased ThV and prevalence of thyroid hypoechogenicity by ultrasound. For the evaluation of data the level of PCBs was used as a marker of all OCs. Increasing PCB levels were significantly associated with the increase of free T4 (p < 0.001) and total T3 (p < 0.05) in blood, while slight but not significant negative association of PCBs was observed with the level of TSH. In both women and men the prevalence of TPOAb was significantly higher in polluted area. Although the absolute TPOAb prevalence in both areas was higher in women than that in men, the increase in polluted vs. background area was more striking in men. From these data it appears that the effects of environmental pollution on the thyroid cannot be neglected.

A novel partial agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride accumulation, and potentiates insulin signaling in vitro.

Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.

Fish from industrially polluted freshwater as the main source of organochlorinated pollutants and increased frequency of thyroid disorders and dysglycemia.

In a certain area of Michalovce district in East Slovakia, heavy industrial pollution by polychlorinated biphenyls (PCBs) developed in 1955-1984 and very high PCB levels in environmental and human samples are still persisting. Recently, a total of 2045 adults from this and the surrounding background pollution area have been examined using questionnaire data, thyroid volume by ultrasound (ThV), urinary iodine and serum levels of 15 PCB congeners, hexachlorobenzene (HCB), 2,2'-2-bis(4-chlorobiphenyl)-1,1-dichloroethylene (DDE), 2,2'-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT), alpha-, beta- and gamma-hexachlorocyclohexane (HCH), thyrotropin (TSH), free thyroxine (FT4), anti-thyroperoxidase antibodies (TPOab) and fasting glucose. As based on our previous findings of strikingly high level of PCBs in fish from high pollution area (e.g. mean level of 375430 ng/g lipid) and considerably lower, but still relatively high level in background pollution area (e.g. mean PCB level of 5150 ng/g), the information on the frequency of fish meals and approximate annual consumption of fish from local waters was obtained by questionnaires. The association of contaminated fish consumption with very high blood levels of PCBs, DDE and HCB and increased ThV as well as with increased frequency of positive TPOab, high values of FT4 and impaired fasting glucose (IFG) was found. These associations were also confirmed in 16 marital pairs from high pollution area with very high PCB level in both members associated with high fish consumption. It was concluded that, due to persistent heavy pollution of waters, soil and food chain namely by PCBs, but also by pesticides (e.g. DDE and HCB) resulting from their previous extensive use in agriculture, the fish from local waters still remains the most important source of these toxic pollutants which results in considerable adverse health effects.

Possible effects of persistent organochlorinated pollutants cocktail on thyroid hormone levels and pituitary-thyroid interrelations.

In polluted district of Michalovce in East Slovakia (POLL) and two districts with background pollution (BCGR) 2046 adults (834 males and 1212 females aged 20-75 years) were examined. Serum levels of thyrotropin (TSH), free thyroxine (FT4), total triiodothyronine (TT3) and antithyroperoxidase antibodies (TPOab) were estimated by electrochemiluminiscent assay and also these of 15 polychlorinated biphenyl congeners (PCBs), p,p'-DDE, p,p'-DDT, hexachlorobenzene (HCB) and hexachlorocyclohexane were measured by high resolution gas chromatography/mass spectrometry. In addition, also dioxins, furans, coplanar- and mono-ortho-PCBs as well as selected hydroxylated and methylsulphonated PCBs and DDE metabolites were measured by appropriate methods based on gas chromatography/mass spectrometry principle. In POLL significantly higher levels of all organochlorines were found than these in BCGR. When pooled values from both areas were stratified in terms of PCBs level and treated as continuous variables, positive association of PCBs with FT4 and TT3 was found, the latter two being also mutually associated. However, within the category of PCBs level <530 ng/glipid (n=232) the association between PCBs and both the FT4 (p<0.09) and TT3 (p<0.03) was negative and any association of these was not found within the category of PCBs level of 531-1000 ng/g (n=691). In contrast, in the category of 531-2000 ng/g (n=1307) positive association appeared between PCBs and FT4 (p<0.001) as well as TT3 (p<0.05). Highly significant association of PCBs with FT4 (p<0.001) was further found in the categories with PCBs level of 1001-101414 ng/g (n=1307) and 2001-101414 (n=1123), while significant association with TT3 was observed only in the category of 531-2000 ng/g. Such findings suggest possible threshold level in positive effect of PCBs on FT4 and TT3 level which seems to be individual and located somewhere around the PCBs level of 1000 ng/g. However, highly significant negative association of both FT4 and TT3 with TSH was found in each of above indicated PCBs categories. Considerable difference in FT4 and TT3 level between large groups of subjects with the same range of PCBs level was also found suggesting different individual susceptibility to the effects of organochlorines. Among a total of 26 cases from POLL with very low TSH level (<0.5 mU l(-1)) 13 cases showed very high level of PCBs, FT4 and TT3, thus supporting a hypothesis on a novel sporadic form of high PCBs related peripheral subclinical hyperthyroidism possibly resulting from the long-term disruption of equilibrium between bound and free thyroxine in plasma by high PCBs level followed by a striking inhibition of TSH release from the pituitary.