Modulation of Insulin Resistance, Dyslipidemia and Serum Metabolome in iNOS Knockout Mice following Treatment with Nitrite, Metformin, Pioglitazone, and a Combination of Ampicillin and Neomycin

Oxidative and nitrosative stress plays a pivotal role in the incidence of metabolic disorders. Studies from this lab and others in iNOS-/- mice have demonstrated occurrence of insulin resistance (IR), hyperglycemia and dyslipidemia highlighting the importance of optimal redox balance. The present study evaluates role of nitrite, L-arginine, antidiabetics (metformin, pioglitazone) and antibiotics (ampicillin-neomycin combination, metronidazole) on metabolic perturbations observed in iNOS-/- mice. The animals were monitored for glucose tolerance (IPGTT), IR (insulin, HOMA-IR, QUICKI), circulating lipids and serum metabolomics (LC-MS). Hyperglycemia, hyperinsulinemia and IR were rescued by nitrite, antidiabetics, and antibiotics treatments in iNOS-/- mice. Glucose intolerance was improved with nitrite, metformin and pioglitazone treatment, while ampicillin-neomycin combination normalised the glucose utilization in iNOS-/- mice. Increased serum phosphatidylethanolamine lipids in iNOS-/- mice were reversed by metformin, pioglitazone and ampicillin-neomycin; dyslipidemia was however marginally improved by nitrite treatment. The metabolic improvements were associated with changes in selected serum metabolites-purines, ceramide, 10-hydroxydecanoate, glucosaminate, diosmetin, sebacic acid, 3-nitrotyrosine and cysteamine. Bacterial metabolites-hippurate, indole-3-ethanol; IR marker-aminoadipate and oxidative stress marker-ophthalmate were reduced by pioglitazone and ampicillin-neomycin, but not by nitrite and metformin treatment. Results obtained in the present study suggest a crucial role of gut microbiota in the metabolic perturbations observed in iNOS-/- mice.

The Insulin-Sensitizer Pioglitazone Remodels Adipose Tissue Phospholipids in Humans

The insulin-sensitizer pioglitazone exerts its cardiometabolic benefits in type 2 diabetes (T2D) through a redistribution of body fat, from ectopic and visceral areas to subcutaneous adipose depots. Whereas excessive weight gain and lipid storage in obesity promotes insulin resistance and chronic inflammation, the expansion of subcutaneous adipose by pioglitazone is associated with a reversal of these immunometabolic deficits. The precise events driving this beneficial remodeling of adipose tissue with pioglitazone remain unclear, and whether insulin-sensitizers alter the lipidomic composition of human adipose has not previously been investigated. Using shotgun lipidomics, we explored the molecular lipid responses in subcutaneous adipose tissue following 6months of pioglitazone treatment (45mg/day) in obese humans with T2D. Despite an expected increase in body weight following pioglitazone treatment, no robust effects were observed on the composition of storage lipids (i.e., triglycerides) or the content of lipotoxic lipid species (e.g., ceramides and diacylglycerides) in adipose tissue. Instead, pioglitazone caused a selective remodeling of the glycerophospholipid pool, characterized by a decrease in lipids enriched for arachidonic acid, such as plasmanylethanolamines and phosphatidylinositols. This contributed to a greater overall saturation and shortened chain length of fatty acyl groups within cell membrane lipids, changes that are consistent with the purported induction of adipogenesis by pioglitazone. The mechanism through which pioglitazone lowered adipose tissue arachidonic acid, a major modulator of inflammatory pathways, did not involve alterations in phospholipase gene expression but was associated with a reduction in its precursor linoleic acid, an effect that was also observed in skeletal muscle samples from the same subjects. These findings offer important insights into the biological mechanisms through which pioglitazone protects the immunometabolic health of adipocytes in the face of increased lipid storage.

Pioglitazone Ameliorates Acute Endotoxemia-Induced Acute on Chronic Renal Dysfunction in Cirrhotic Ascitic Rats

Endotoxemia-activated tumor necrosis factor (TNFα)/nuclear factor kappa B (NFκB) signals result in acute on chronic inflammation-driven renal dysfunction in advanced cirrhosis. Systemic activation of peroxisome proliferator-activated receptor gamma (PPARγ) with pioglitazone can suppress inflammation-related splanchnic and pulmonary dysfunction in cirrhosis. This study explored the mechanism and effects of pioglitazone treatment on the abovementioned renal dysfunction in cirrhotic rats. Cirrhotic ascitic rats were induced with renal dysfunction by bile duct ligation (BDL). Then, 2 weeks of pioglitazone treatment (Pio, PPAR gamma agonist, 12 mg/kg/day, using the azert osmotic pump) was administered from the 6th week after BDL. Additionally, acute lipopolysaccharide (LPS, Escherichia coli 0111:B4; Sigma, 0.1 mg/kg b.w, i.p. dissolved in NaCl 0.9%) was used to induce acute renal dysfunction. Subsequently, various circulating, renal arterial and renal tissue pathogenic markers were measured. Cirrhotic BDL rats are characterized by decreased mean arterial pressure, increased cardiac output and portal venous pressure, reduced renal arterial blood flow (RABF), increased renal vascular resistance (RVR), increased relative renal weight/hydroxyproline, downregulated renal PPARγ expression, upregulated renal inflammatory markers (TNFα, NFκB, IL-6, MCP-1), increased adhesion molecules (VCAM-1 and ICAM-1), increased renal macrophages (M1, CD68), and progressive renal dysfunction (increasing serum and urinary levels of renal injury markers (lipocalin-2 and IL-18)). In particular, acute LPS administration induces acute on chronic renal dysfunction (increasing serum BUN/creatinine, increasing RVR and decreasing RABF) by increased TNFα-NFκB-mediated renal inflammatory markers as well as renal M1 macrophage infiltration. In comparison with the BDL+LPS group, chronic pioglitazone pre-treatment prevented LPS-induced renal pathogenic changes in the BDL-Pio+LPS group. Activation of systemic, renal vessel and renal tissue levels of PPARγ by chronic pioglitazone treatment has beneficial effects on the endotoxemia-related TNFα/NFκB-mediated acute and chronic renal inflammation in cirrhosis. This study revealed that normalization of renal and renal arterial levels of PPARγ effectively prevented LPS-induced acute and chronic renal dysfunction in cirrhotic ascitic rats.

Coordinated regulation of gene expression and microRNA changes in adipose tissue and circulating extracellular vesicles in response to pioglitazone treatment in humans with type 2 diabetes

Pioglitazone, a PPARγ agonist, is used to treat type 2 diabetes (T2D). PPARγ is highly expressed in adipose tissue (AT), however the effects of pioglitazone to improve insulin sensitivity are also evident in other tissues. We hypothesized that pioglitazone modifies the cargo of circulating AT-derived extracellular vesicles (EVs) to alter interorgan crosstalk. We tested this in a 3-month trial in which 24 subjects with T2D who were well-controlled with diet/exercise or metformin were randomized to treatment with either pioglitazone 45 mg/day or placebo (NCT00656864). Levels of 42 adipocyte-derived EV-miRNAs were measured in plasma EVs. Levels of 5 miRNAs (i.e., miR-7-5p, miR-20a-5p, miR-92a-3p, miR-195-5p, and miR-374b-5p) were significantly downregulated in EVs in response to pioglitazone treatment relative to placebo. However, the opposite occurred for miR-195-5p in subcutaneous AT from the same participants. Changes in miRNA expression in EVs and AT correlated with changes in suppression of lipolysis and improved insulin sensitivity, among others. DICER was downregulated and exosomal miRNA sorting-related genes YBX1 and hnRNPA2B1 displayed a trend toward downregulation in AT. Furthermore, analysis of EV-miRNA targeted genes identified a network of overtargeted transcripts that changed in a coordinated manner in AT. Collectively, our results suggest that some beneficial pharmacologic effects of PIO are mediated by adipose-specific miRNA regulation and exosomal/EV trafficking.

Chronic PPARγ Stimulation Shifts Amyloidosis to Higher Fibrillarity but Improves Cognition

Background: We undertook longitudinal β-amyloid positron emission tomography (Aβ-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone in Aβ model mice. We thus tested the hypothesis this treatment would rescue from increases of the Aβ-PET signal while promoting spatial learning and preservation of synaptic density. Methods: PS2APP mice (N=23; baseline age: 8 months) and AppNL-G-F mice (N=37; baseline age: 5 months) were investigated longitudinally for five months using Aβ-PET. Groups of mice were treated with pioglitazone or vehicle during the follow-up interval. We tested spatial memory performance and confirmed terminal PET findings by immunohistochemical and biochemistry analyses. Results: Surprisingly, Aβ-PET and immunohistochemistry revealed a shift towards higher fibrillary composition of Aβ-plaques during upon chronic pioglitazone treatment. Nonetheless, synaptic density and spatial learning were improved in transgenic mice with pioglitazone treatment, in association with the increased plaque fibrillarity. Conclusion: These translational data suggest that a shift towards higher plaque fibrillarity protects cognitive function and brain integrity. Increases in the Aβ-PET signal upon immunomodulatory treatments targeting Aβ aggregation can thus be protective.

ASSOCIATION ANALYSIS OF PIOGLITAZONE EFFECTIVENESS IN TREATMENT OF NAFLD PATIENTS WITH OBESITY AND PPARG RS1801282 (PRO12ALA) GENOTYPE

The aim: To study the association between the effectiveness of treatment with pioglitazone non-alcoholic fatty liver disease (NAFLD) in patients with obesity and PPARG rs1801282 (Pro12Ala)-polymorphism in Ukrainians. Materials and methods: 123 patients with NAFLD in combination with obesity 1, 2, 3 classes were included in comprehensive weight loss program (5 visits, 12-weeks). The case group was treated with pioglitazone 15 mg / day, while the control group received only program. Ultrasound (US) steatometry and genetic testing rs1801282 polymorphism in PPARG gene were performed. Results: Pioglitazone, PPARG rs1801282 genotype, CAP before treatment, previous weight loss attempts, and duration of obesity were associated with the change in controlled attenuation parameter (CAP) during treatment. There was a significant association between the target CAP reduction achievement and pioglitazone treatment (adjusted odds ratio 0.23, 95% CI 0.07–0.73; p = 0.01) with the CC genotype of PPARG gene (adjusted odds ratio 92.9, 95% CI 7.4–1159; p < 0.001) compared to patients with the CG genotype. Conclusions: Pioglitazone and PPARG rs1801282 polymorphism could influence on dynamics of CAP reduction during treatment.

Pioglitazone-Mediated Attenuation of Experimental Colitis Relies on Cleaving of Annexin A1 Released by Macrophages

Ulcerative colitis and Crohn’s disease are chronic inflammatory bowel diseases (IBDs) which burden health systems worldwide; available pharmacological therapies are limited and cost-intensive. Use of peroxisome proliferator activated-receptor γ (PPARγ) ligands for IBD treatment, while promising, lacks solid evidences to ensure its efficacy. Annexin A1 (AnxA1), a glucocorticoid-modulated anti-inflammatory protein, plays a key role on IBD control and is a potential biomarker of IBD progression. We here investigated whether effects of pioglitazone, a PPARγ ligand, rely on AnxA1 actions to modulate IBD inflammation. Experimental colitis was evoked by 2% dextran sodium sulfate (DSS) in AnxA1 knockout (AnxA1−/−) or wild type (WT) C57BL/6 mice. Clinical and histological parameters were more severe for AnxA−/− than WT mice, and 10 mg/kg pioglitazone treatment attenuated disease parameters in WT mice only. AnxA1 expression was increased in tissue sections of diseased WT mice, correlating positively with presence of CD68+ macrophages. Metalloproteinase-9 (MMP-9) and inactive 33 kDa AnxA1 levels were increased in the colon of diseased WT mice, which were reduced by pioglitazone treatment. Cytokine secretion, reactive oxygen species generation and MMP-9 expression caused by lipopolysaccharide (LPS) treatment in AnxA1-expressing RAW 264.7 macrophages were reduced by pioglitazone treatment, effects not detected in AnxA1 knockdown macrophages. LPS-mediated increase of AnxA1 cleaving in RAW 264.7 macrophages was also attenuated by pioglitazone treatment. Finally, pioglitazone treatment increased extracellular signal-regulated kinase (ERK) phosphorylation in AnxA1-expressing RAW 264.7 macrophages, but not in AnxA1-knockdown macrophages. Thus, our data highlight AnxA1 as a crucial factor for the therapeutic actions of pioglitazone on IBDs.

P0721PPAR-GAMMA ACTIVATION INHIBITS TGF-BETA INDUCED RENAL COMPLEMENT AND GALECTIN-3 EXPRESSION IN VIVO AND IN VITRO

Background and Aims Reduced peroxisome proliferator-activated receptor-γ (PPARγ) activity has been observed in chronic kidney disease and fibrosis. We have recently shown that the PPARγ agonist pioglitazone dampens TGF-β induced renal pro-fibrotic transcription factors in vivo. Several studies have shown that renal complement expression is associated with fibrosis, yet the role of galectin-3 (Lgals3) remains controversial. However, the effect of PPARγ agonists on renal complement (C3, C4b) and Lgals3 expression has not been studied. We investigated how pioglitazone treatment affects TGF-β–driven renal inflammatory molecule expression in transgenic mice and in primary tubular epithelial cell (PTEC) culture. Method Ten weeks-old male C57Bl6 control (CTL) and TGF-β transgenic mice (with elevated circulating TGF-β1 level) were divided in two sets. The first set of mice received regular chow (CTL and TGFβ, n=4/group). The second set of mice were treated orally with pioglitazone (20mg/kg/day) for 5 weeks (CTL+Pio and TGFβ+Pio, n=5/group), when the kidneys were analyzed for mRNA and protein expression. PTEC were isolated from 4-weels old CTL mouse kidneys using graded sieving and characterized by immunoblot. Isolated cells were maintained in DMEM/F12 medium supplemented with 2% FBS and insulin/transferrin/selenium, and cells of passage 4-6 were used in triplicate for the experiments. Cells were treated for 24h with 10 ng/ml TGF-β and/or 5 µM pioglitazone, then mRNA expression levels were assessed. Statistical significance was verified using Mann-Whitney test. Results TGFβ kidneys depicted 13-fold and 4-fold overexpression of C3 and C4b mRNA (p&lt;0.05), respectively, accompanied by a 1.7-fold Lgals3 expression (p&lt;0.05). Oral treatment with the PPARγ agonist pioglitazone inhibited the TGF-β1 induced overexpression of C3, C4b, Lgals3, as well as IL-6 and CCL2 (MCP-1) mRNA (p&lt;0.01). This was accompanied by reduced alfa-SMA (Actca2) and fibronectin mRNA expression. Renal Lgals3 showed significant correlation with alfa-SMA expression (p&lt;0.01, see Figure). PTEC treated with TGF-β also depicted increased C3aR, alfa-SMA and collagen-1 mRNA expression by 1.7-fold, 2-fold and 2.1-fold, respectively (p&lt;0.01), that were reduced to control levels by pioglitazone treatment (p&lt;0.05). Conclusion Our data indicate that PPARγ activation exert strong anti-inflammatory effects in the renal tubular epithelial cells, implicating its possible therapeutic benefit by attenuating renal EMT and fibrosis in CKD patients.

The Effects of Pioglitazone on Respiratory Function Test in Patients with Asthma and Type 2 Diabetes Mellitus- A before –after Study

Objective: Pioglitazone is one of the oral medications of type 2 diabetes (T2DM). The purpose of this study was to evaluate the effect of pioglitazone on asthma and diabetes treatment outcomes among patients with concurrent asthma and T2DM. Materials and Methods: We conducted a quasi-experimental study on 11 patients with concurrent asthma and T2DM in Yazd Afshar Hospital and Yazd diabetic research center 2014-2017. The inclusion criteria were patients between 20-60 years old, at least one year with concurrent asthma and T2DM (documented with spirometer, bronchodilator test), ejection fraction more than 50%. Patients who were smoker, on oral corticosteroids, phenobarbital, methotrexate, rifampin, phenytoin and gemfibrozil were excluded. Laboratory tests (FBS, HbA1c, 2hpp, leptin), spirometer test, exhaled nitric oxide were done before and after 10 weeks of pioglitazone medication. All patients were visited every two weeks. The before and after pioglitazone treatment differences were checked by paired t-test and Wilcoxon Rank sum test. Results: The mean (± SD) age of participants was 55.81 (±7.66). The median of differences of leptin (p-value: 0.885), FEV1 to FVC ( P -value: 0.185), FEV1 (p-value: 0.386), NO ( P -value: 0.574), FVC percent ( P -value: 0.477), FEV1 percent ( P -value: 0.515) did not differ before and after pioglitazone treatment. Conclusion: Our finding suggested that pioglitazone may not be effective in the treatment and improvement of respiratory function in T2DM with concurrent asthma.