William T. L. Festuccia

Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)γ agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPARγ agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPARγ activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPARγ activation.

Omega-3 fatty acids protect from diet-induced obesity, glucose intolerance, and adipose tissue inflammation through PPARγ-dependent and PPARγ-independent actions

SCOPE We tested herein the hypothesis that peroxisome proliferator activated receptor γ (PPARγ) is a major mediator of omega-3 (n-3) protective actions against high-fat diet (HFD) induced obesity, glucose intolerance, and adipose tissue inflammation. METHODS AND RESULTS C57BL6 wild-type and fat-1 transgenic (fat-1) mice were fed a low-fat diet (LFD) or HFD, treated or not with PPARγ antagonist, and evaluated for energy balance, adiposity, glucose tolerance, and adipose tissue inflammation. Fat-1 mice were protected from obesity, fasting hyperglycemia, glucose intolerance, and adipose tissue inflammation. PPARγ inhibition completely abolished fat-1 protection against HFD-induced glucose intolerance, but not obesity or adipose tissue inflammation. To investigate the role of myeloid cell as mediator of n-3 beneficial metabolic actions, mice with deletion (LyzM-PPARγ(KO)) or nondeletion (LyzM-PPARγ(WT)) of PPARγ in myeloid cells were fed either LFD or HFD (lard) or an HFD rich in n-3 (fish oil). Our findings indicate that myeloid cell associated PPARγ is not involved in the attenuation of HFD-induced glucose intolerance and adipose tissue inflammation induced by n-3. CONCLUSION High endogenous n-3 fatty acid levels protect from HFD obesity, glucose intolerance, and adipose tissue inflammation. Among these, only protection against glucose intolerance is mediated by non-myeloid cell PPARγ.

High‐fat diet‐induced hypertension and autonomic imbalance are associated with an upregulation of CART in the dorsomedial hypothalamus of mice

We evaluated herein whether diet‐induced obesity alters sympathovagal balance, blood pressure, and neuropeptides levels at the hypothalamus and brainstem of mice. Male C57BL6J mice fed with a high‐fat (HFD) or a high‐fat high‐sucrose (HFHSu), or a regular chow diet (C) for 8 weeks were evaluated for metabolic parameters and blood pressure, the latter being performed in conscious freely moving mice. Spectral analysis from the records of systolic blood pressure (SBP) and cardiac pulse intervals (PI) was performed to analyse the autonomic balance in the cardiovascular system. HFD‐fed mice developed two distinct hemodynamic phenotypes: hypertensive mice (HFD‐H) with high systolic and diastolic BP levels and hypertension‐resistant mice (HFD‐R) whose BP levels were similar to C group. Spectral analysis of SBP and PI variabilities indicate that the low‐frequency (LF)/high‐frequency (HF) ratio, which is an index of sympathovagal balance, is higher in HFD‐H compared to HFD‐R. Along with hypertension and higher LF/HF ratio, HFD‐H mice presented increased hypothalamic mRNA levels of cocaine‐ and amphetamine‐regulated transcript (CART), and increased CART‐positive neurones in the dorsomedial hypothalamus (DMH) by high‐fat diet when compared to C group. Despite developing obesity to similar levels than HFD feeding, intake of a HFHSu was not associated with hypertension in mice neither CART levels increase. Collectively, our main findings indicate that high‐fat diet induced‐hypertension and autonomic imbalance are associated to an upregulation of CART levels in the DMH of mice.

Abstract P1-08-08: Reduction of HER2-associated lipogenic phenotype by docosahexaenoic acid (DHA) induces apoptosis in breast tumor cells harboring HER2 overexpression

The lipogenic phenotype is associated with oncogenic transformation and malignancy in the cancer setting. Our hypothesis is that the oncogenic nature of lipogenesis depends on the expression of HER2 oncogene, and its modulation by docosahexaenoic acid-DHA, might induce apoptosis in breast tumor cells with HER2 overexpression. To evaluate if the lipogenic phenotype might be induced by HER2 overexpression, we used a oncogenic transformation cell model in which, cells were engineered to overexpress HER-2 receptor (HB4aC5.2), but are identical to their parental strain (HB4a) in all other aspects, permitting an specific analysis of enhanced HER-2 effects. Toward this end the lipogenesis profiling was characterized, evaluating several molecular features, including synthesis (FASN), uptake(CD36), transport(FABP4) and storage(DGAT) of FA by RT-PCR and lipogenic regulatory pathways (mTOR, DEPTOR, SREBP1 and PPARγ) in both cells. Lipogenic contribution to lipid rafts formation, which is necessary to HER2 receptor location and activation in the cell membrane, was evaluated by gas chromatography and confocal microscopy. The influence of HER2 overexpression and lipogenic phenotype on proteins activated by HER-2 (AKT, ERK1/2 and FASN) was analyzed by western blot. Next, HB4a and HB4aC5.2 cells were treated, alone or in combination, with DHA, Trastuzumab (anti-HER2), and GW9662 (PPARγ inhibitor) for 72h, and the above experiments were repeated. Cell death was analyzed by flow cytometry and confocal microscopy. Results: In HB4aC5.2 cells, the oncogenic transformation by HER2 overexpression was associated with a lipogenic phenotype, which contributed to increase of lipid rafts formation, activation of survival and proliferation signals, as compared to HB4a normal cells (p Citation Format: Graziela R Ravacci, Maria M Brentani, William Festuccia, Tharcisio Tortelli, Angela F Waitzberg, Dan L Waitzberg. Reduction of HER2-associated lipogenic phenotype by docosahexaenoic acid (DHA) induces apoptosis in breast tumor cells harboring HER2 overexpression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-08.