Melatonin synthesis in and uptake by mitochondria: implications for diseased cells with dysfunctional mitochondria

Although there is one exception (red blood cells), the lack of energy (ATP) provided by mitochondrial oxidative phosphorylation (OXPHOS) would not be compatible with the long-term survival of normal cells. During conventional metabolism, pyruvate, the cytosolic glycolysis product, enters mitochondria where it is metabolized to acetyl-coenzyme A (acetyl-CoA) under the influence of the enzyme pyruvate dehydrogenase complex (PDC). Acetyl-CoA makes an important contribution to the tricarboxylic acid (TCA) cycle which feeds NADH and FADH2 to the respiratory chain which benefits ATP´s generation by OXPHOS. In some diseased cells, however, pyruvate metabolism becomes aberrant since its transport into the mitochondria is blunted due to the downregulation of PDC due to its inhibition by pyruvate dehydrogenase kinase (PDK), which is upregulated by hypoxia-inducible factor 1 (HIF-1). Therefore, pyruvate undergoes fermentation to lactate in the cytosol. This alternate pathway of pyruvate metabolism is known as the Warburg effect, named after the individual who discovered it, Otto Warburg [1]. Since pyruvate does not enter the mitochondria, mitochondrial ATP synthesis is depressed. Warburg-type metabolism, however, compensates for this by rapidly, albeit inefficiently, synthesizing ATP in the cytosol. Warburg metabolism (also known as aerobic glycolysis) is almost always associated with pathological cells.Fil: Reiter, Russel. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Sharma, Ramaswamy. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Pires De Campos Zuccari, Debora Aparecida. Faculdade de Medicina de São José Do Rio Preto; BrasilFil: Almeida Chuffa, Luiz Gustavo de. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Manucha, Walter Ariel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Rodriguez, Carmen. Universidad de Oviedo; Españ

Melatonin and pathological cell interactions: mitochondrial glucose processing in cancer cells

Melatonin is synthesized in the pineal gland at night. Since melatonin is produced in the mitochondria of all other cells in a non-circadian manner, the amount synthesized by the pineal gland is less than 5% of the total. Melatonin produced in mitochondria influences glucose metabolism in all cells. Many pathological cells adopt aerobic glycolysis (Warburg effect) in which pyruvate is excluded from the mitochondria and remains in the cytosol where it is metabolized to lactate. The entrance of pyruvate into the mitochondria of healthy cells allows it to be irreversibly decarboxylated by pyruvate dehydrogenase (PDH) to acetyl coenzyme A (acetyl-CoA). The exclusion of pyruvate from the mitochondria in pathological cells prevents the generation of acetyl-CoA from pyruvate. This is relevant to mitochondrial melatonin production, as acetyl-CoA is a required co-substrate/co-factor for melatonin synthesis. When PDH is inhibited during aerobic glycolysis or during intracellular hypoxia, the deficiency of acetyl-CoA likely prevents mitochondrial melatonin synthesis. When cells experiencing aerobic glycolysis or hypoxia with a diminished level of acetyl-CoA are supplemented with melatonin or receive it from another endogenous source (pineal-derived), pathological cells convert to a more normal phenotype and support the transport of pyruvate into the mitochondria, thereby re-establishing a healthier mitochondrial metabolic physiology.Fil: Reiter, Russel. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Sharma, Ramaswamy. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Rosales Corral, Sergio. Instituto Mexicano del Seguro Social; MéxicoFil: Manucha, Walter Ariel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Almeida Chuffa, Luiz Gustavo de. Institute of Biosciences of Botucatu; BrasilFil: Pires de Campos Zuccari, Debora Aparecida. Faculdade de Medicina de Sao Jose Do Rio Preto; Brasi

Exosomes and Melatonin: Where Their Destinies Intersect

Cell-to-cell communication is a broad and complex process associated with regular stimuli to maintain healthy cell interactions. One of the agents capable of cellular communication is known as an exosome, a subset of extracellular vesicles (EVs) released by the cell membrane. The exosome contains a wide range of functional proteins, mRNAs and miRNAs, which have the potential to interact with healthy or diseased cells in the body. On the other hand, melatonin also acts as a cellular communicator, produced and released by the pineal gland in a circadian way and also, non-circadian melatonin is derived from the mitochondria of all normal cells. In addition to exhibiting antioxidant, anti-inflammatory, anti-tumor and anti-aging activities, melatonin has recently been studied by its influence on exosomes. This review summarizes the relationship between exosomes and melatonin in various pathological processes. There is robust evidence that their combination ameliorates inflammation, ischemia-reperfusion injury, hepatic metabolic disturbance, cancer immunosuppression status, degenerative processes like chronic kidney disease, vascular calcification, ageing, ischemic brain injury, neurodegenerative diseases, obesity, colitis, wound healing and even embryonic development. Association of exosomes and melatonin represent a promising therapeutic tool, capable of interfering with basic molecular processes, such as oxidative stress and the inflammatory cascade, which support many pathophysiological aspects of diseases

Melatonin-Loaded Nanocarriers: New Horizons for Therapeutic Applications

The use of nanosized particles has emerged to facilitate selective applications in medicine. Drug-delivery systems represent novel opportunities to provide stricter, focused, and fine-tuned therapy, enhancing the therapeutic efficacy of chemical agents at the molecular level while reducing their toxic effects. Melatonin (N-acetyl-5-methoxytriptamine) is a small indoleamine secreted essentially by the pineal gland during darkness, but also produced by most cells in a non-circadian manner from which it is not released into the blood. Although the therapeutic promise of melatonin is indisputable, aspects regarding optimal dosage, biotransformation and metabolism, route and time of administration, and targeted therapy remain to be examined for proper treatment results. Recently, prolonged release of melatonin has shown greater efficacy and safety when combined with a nanostructured formulation. This review summarizes the role of melatonin incorporated into different nanocarriers (e.g., lipid-based vesicles, polymeric vesicles, non-ionic surfactant-based vesicles, charge carriers in graphene, electro spun nanofibers, silica-based carriers, metallic and nonmetallic nanocomposites) as drug delivery system platforms or multilevel determinations in various in vivo and in vitro experimental conditions. Melatonin incorporated into nanosized materials exhibits superior effectiveness in multiple diseases and pathological processes than does free melatonin; thus, such information has functional significance for clinical intervention.Fil: Chuffa, Luiz Gustavo de Almeida. Universidade de Sao Paulo; BrasilFil: Seiva, Fábio Rodrigues Ferreira. Universidade Estadual do Norte do Paraná; BrasilFil: Novais, Adriana Alonso. Universidade Federal do Mato Grosso do Sul; BrasilFil: Simão, Vinícius Augusto. Universidade de Sao Paulo; BrasilFil: Martín Giménez, Virna Margarita. Universidad Católica de Cuyo. Facultad de Ciencias Químicas y Tecnológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Manucha, Walter Ariel Fernando. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Zuccari, Debora Aparecida Pires de Campos. Faculdade de Medicina de São José do Rio Preto; BrasilFil: Reiter, Russel. University of Texas at San Antonio; Estados Unido

P-MAPA and interleukin-12 reduce cell migration/invasion and attenuate the toll-like receptor-mediated inflammatory response in ovarian cancer SKOV-3 cells : a preliminary study

Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium–ammonium phospholinoleate–palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment2515CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP401040/2016-00708/20182019/00906-6; 2016/03993-9We would like to give a special thanks to Farmabrasilis-Brazil, FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, grant numbers: 2019/00906-6 and 2016/03993-9), CAPES (grant number: 0708/2018), and CNPq (grant number: 401040/2016-0) by providing financial suppor

Structure and biometry of UchA and UchB adult rats'ovary (ethanol voluntary intake)

Orientador: Francisco Eduardo MartinezDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O alcoolismo crônico está inserido no grupo das principais doenças classificadas como oriundas de distúrbios mentais, sendo as mulheres mais atingidas em relação aos homens. Embora, o álcool conduza inicialmente a estímulos emocionais benéficos para o organismo, proporcionando aumento da euforia, do prazer, entre outros, seu uso excessivo e prolongado pode promover efeitos colaterais indesejáveis, inclusive sobre a reprodução. Na literatura existem poucos estudos envolvendo as conseqüências do alcoolismo crônico em fêmeas. O presente trabalho tem como objetivo elucidar as alterações manifestadas no ovário de ratas adultas UChA e UChB (consumidoras voluntárias de etanol a 10%). Após o período de tratamento, 42 ratas subdivididas em três grupos experimentais (UChA, UChB e Wistar) foram eutanasiadas por decapitação, e os ovários coletados e processados para análise em microscopia de luz e eletrônica de transmissão. O material foi corado com Hematoxilina e Eosina, Tricrômico de Masson, Azul de Toluidina, ácido periódico de Schiff, Giemsa, Feulgen e, empregou-se o método enzimológico para atividade da fosfatase ácida e alcalina. Os parâmetros: peso corpóreo e dos genitais, índice de ganho de peso, duração dos ciclos estrais e dosagens hormonais (FSH, LH, _-estradiol e progesterona) foram avaliados. A análise estatística foi realizada com 5% de significância. Não houve diferença significativa entre o peso dos animais no início e final do experimento, embora, ao final do experimento, os animais do grupo UChB apresentaram maior índice de ganho de massa. O peso relativo dos ovários dos animais UChB mostrou-se significativamente menor comparado ao grupo Wistar. As dosagens hormonais não apresentaram diferenças estatísticas entre os grupos. Os animais UChA e UChB revelaram as maiores médias de duração dos ciclos estrais e permanência na fase de estro. Nota-se estágios diferenciados de proliferação celular e atresia folicular avançada nos ovários, variando entre os grupos UChA e UChB. Nas variedades bebedoras, a túnica albugínea apresentou-se fibrosa e o estroma medular predominantemente celular. Estroma fibrocelular e túnica albugínea fibrosa estão presente no grupo controle. O grupo UChA apresentou reações metacromáticas entre as células da granulosa dos folículos em crescimento, enquanto regiões delimitadas na parede dos folículos primários, em desenvolvimento e antrais apresentaram intensa metacromasia nos animais UChB. Os grupos UChA e UChB apresentaram reações PAS-positivo no tecido glandular intersticial, enquanto nos animais controles essas reações ficaram restritas na zona pelúcida de ovócitos e entre as células da granulosa de folículos secundários. Os folículos antrais do grupo UChB apresentaram forte reação à fosfatase ácida (FA), comparados aos grupos UChA e controle. Nos animais UChA, os corpos lúteos hemorrágicos e em regressão, destacaram reação à FA com presença de grumos associados. A fosfatase alcalina (FAL) demarcou ampla vascularização nos corpos lúteos dos animais UCh, enquanto que a teca interna dos folículos secundários do grupo UChB reagiram intensamente com a FAL. A análise ultra-estrutural revelou corpos lúteos com vacúolos autofágicos no citoplasma e início de picnose nuclear nas linhagens UCh. As células da granulosa apresentaram núcleos com marginalização da cromatina e mitocôndrias edemaciadas. Conclui-se que há irregularidades do ciclo estral e, consequentemente, alterações estruturais nos ovários das linhagens UChA e UChBAbstract: Chronic alcoholism belongs to the group of diseases classified as originated from mental disturbs, being the women more affected than men. Although ethanol intake causes benefic stimulus to the organism, like increase in euphoria and pleasure, excessive and prolonged use can cause side effects, even to the reproduction. On the literature, there were few researches involving chronic alcoholism and female. In regard to the current incidence of early and late alcoholism in women, and its consequences to the reproduction, the aim of this study was to evaluate the UChA and UChB (10% ethanol voluntary intake) adult rat ovary structure. After the experimental period, 42 rats divided into three groups (UChA, UChB and Wistar control) were killed, by decapitation method, and their ovaries collected to the light and electronic microscopy analysis. Ovary slides were stained with HE, Masson¿s tricromic, toluidine blue, Periodic Acid Schiff and tissue was cryofrozen to further acid/alkaline phosphatase histochemical techniques. Final body weight, reproductive organs weight, body weight gain index, estrous cycle duration and hormone dosages (FSH, LH, _-oestradiol and progesterone) were analyzed. The statistical analysis was made using 5% of significance. There was no significative difference between the groups as to initial and final body weights, although the UChB rats showed an increased body mass gain at the final treatment period. The UChB ovaries relative weight was significantly lower comparing to the control. The hormonal levels did not differ among the groups. The UChA and UChB groups presented prolonged estrous cycles and persistent oestrous phases. Different stages of atresia and proliferation on follicle cells were found varying in UChA and UChB ovaries. The tunica albuginea showed fibrous tissue and cellular stromal components in ethanol drinking animals. Fibrocellular stroma and fibrous tunica albuginea were present in the control group. The UChA group showed metachromatic reaction between the growing follicles granulosa cells, whereas, in the UChB rats, intense metachromasia appeared on small, growing and antral follicles. The UCh groups presented PAS-positive reaction in the interstitial glandular tissue, while in control animals these reactions were restricted to the zona pelucida of oocytes and among granulosa cells of secondary follicles. The antral follicles of the UChB rats showed a high intensity reaction to acid phosphatase (AP), when compared to UChA and control groups. In UChA animals, the hemorrhagic and regression corpora lutea had AP reaction with the presence of associated clumps. Alkaline phosphatase (ALP) marked a hypervascularization in corpora lutea of UCh rats. In UChB strain, internal theca layers of growing follicles, reacted strongerly with ALP in contrast to UChA and control groups. The ultrastructural analysis revelated in UCh strain, corpus luteum with autofagic vacuoles and pyknotic nuclei at the initial stage. The UCh granulosa cells resented irregular nucleus with chromatin marginalization and edematous itochondria. In conclusion, there were estral cyclicity irregularities, caused by chronic ethanol intake in the UCh groups, which were consequently reflected as morphologic alterations in the ovaries structureMestradoBiologia CelularMestre em Biologia Celular e Estrutura

Effects of exogenous melatonin on sex steroid receptors in the ovary, oviduct and uterus and the ovarian oxidative stress in adult UChB rats (10% (v/v) ethanol voluntary intake) during ovulation

Orientador: Francisco Eduardo MartinezTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O alcoolismo crônico está associado a distúrbios no sistema reprodutor feminino como disfunção hormonal, alteração na expressão dos receptores esteróides, produção de espécies reativas de oxigênio (ERO), entre outros. A melatonina, hormônio secretado pela glândula pineal, possui função moduladora no ciclo reprodutivo e têm papel importante no combate as ERO. Os estudos envolvendo o alcoolismo crônico e sua interação com a melatonina, em fêmeas, são ainda inconclusivos. O presente trabalho tem como objetivo investigar os efeitos da administração exógena da melatonina sobre os hormônios sexuais, os receptores esteróides sexuais (AR, ER-?, ER-?, PRA e PRB) no ovário, oviduto e útero, além do perfil nutricional e o estresse oxidativo nos ovários de ratas adultas UChB (consumidoras voluntárias de etanol a 10%). Foram utilizadas 60 ratas UChB, distribuídas nos seguintes grupos: UChB Co: sem acesso ao etanol; UChB EtOH: consumo diário de 4 - 5 g etanol/100g de peso corpóreo (PC), ambos recebendo solução veículo. Concomitantemente, os grupos UChB Co+M e UChB EtOH+M receberam injeções diárias de melatonina (100?g/100g PC) via i.p, a partir dos 90 dias de idade, durante 60 dias consecutivos. Aos 150 dias de idade, os animais foram eutanasiados em estro (4a.m) e os materiais coletados e processados. A melatonina aumentou os níveis de progesterona, 6-sulfatoximelatonina e reduziu 17?-estradiol, enquanto a combinação entre etanol + melatonina causou uma queda significativa nesses hormônios. Apesar do receptor androgênico (AR) ovariano não ter sido influenciado pela melatonina, os grupos UChB EtOH e UChB EtOH+M mostraram uma diminuição no AR do oviduto. Ambos os receptores de estrogênio (ER-? e ER-?) no oviduto foram pouco expressos em animais recebendo etanol ou melatonina enquanto somente o ER-? uterino foi reduzido. Por outro lado, receptores de progesterona (PRA e PRB) foram positivamente regulados no ovário por etanol ou etanol + melatonina, enquanto PRA foi negativamente regulado no útero e oviduto, exceto quando o etanol e melatonina foram combinados. Os níveis do receptor de melatonina (MT1R) foram maiores no ovário e útero de ratas tratadas com melatonina, independentemente do consumo de etanol. O peso corpóreo dos animais foi reduzido após interação do etanol e melatonina após 40 dias de tratamento. Em ambos os grupos tratados com melatonina, observou-se redução no consumo energético e líquido. Houve diminuição da quantidade de etanol consumida durante o tratamento e o ciclo estral foi maior em ratas que receberam etanol e melatonina, evidenciado por diestro prolongado. Os níveis de hidroperóxido de lipídio foram maiores nos ovários de ratas UChB EtOH e diminuiu após o tratamento com melatonina. Atividades antioxidantes da superóxido dismutase, glutationa peroxidase e glutationa redutase foram aumentadas nos grupos tratados com melatonina. Conclui-se que a melatonina tem efeito oposto ao etanol sobre os hormônios sexuais. Melatonina e etanol regulam diferencialmente os receptores de esteróides sexuais nos tecidos reprodutivos, atuando principalmente através de seu receptor MT1R. Além disso, a melatonina é capaz de alterar a eficiência alimentar, o ciclo estral, e, contudo, protege os ovários contra o estresse oxidativo resultante do consumo de etanolAbstract: Chronic ethanol intake is associated with female reproductive disturbances including hormonal dysfunction, changes in the steroid receptors expression, production of reactive oxygen species (ROS), among others. Melatonin, an indolamine secreted by pineal gland, plays key roles in the reproductive cycle, besides having an important function in scavenging ROS. Studies focusing chronic alcoholism and its interaction with melatonin, in females, are still inconclusive. This study aims to investigate the effects of exogenous melatonin administration on sex hormones, sex steroid receptors (AR, ER-?, ER-?, PRA and PRB) in the ovary, oviduct and uterus, as well as the nutritional profile and oxidative stress in the ovaries of adult UChB rats (10% (v/v) ethanol voluntary intake). 60 UChB female rats were divided into the following groups: UChB Co: without access to ethanol (used as control); UChB EtOH: drinking daily ethanol at 4 - 5 g ethanol/100g body weight (BW), both receiving vehicle solution. Concomitantly, UChB Co + M and UChB EtOH + M groups received daily injections of melatonin (100?g/100g BW) via i.p, starting from 90 days old and during the next 60 consecutive days. At 150 days of age, all animals were euthanized in estrus (4a.m). Melatonin increased progesterone, 6- sulfatoximelatonin and decreased 17?-estradiol, while the ethanol+melatonin combination caused a significant fall in these hormones. Despite androgen receptor (AR) in ovary has not been influenced by melatonin, ethanol and ethanol+melatonin led to a decrease in oviduct AR. Both estrogen receptors (ER-? and ER-?) were underexpressed by either ethanol or melatonin in oviduct and only uterine ER-? was downregulated. Conversely, progesterone receptors (PRA and PRB) were positively regulated in the ovary by ethanol or ethanol+melatonin, whereas PRA was downregulated in uterus and oviduct, except when ethanol+melatonin were combined. Additionally, melatonin receptor (MT1R) was increased in ovary and uterus of melatonin-treated rats, regardless of ethanol consumption. Body weight gain was reduced with ethanol plus melatonin after 40 days of treatment. In both melatonin-treated groups, it was observed a reduction in food-derived calories and liquid intake toward the end of treatment. The amount of consumed ethanol dropped during the treatment. Estrous cycle was longer in rats that received both ethanol and melatonin, with prolonged diestrus. Following to oxidative status, lipid hydroperoxide levels were higher in the ovaries of ethanol-preferring rats and decreased after melatonin treatment. Additionally, antioxidant activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were increased in melatonin-treated groups . We conclude that melatonin has opposite effect on sex hormones to those of ethanol consumption. Together, melatonin and ethanol differentially regulates the sex steroid receptors in the reproductive tissues, mostly acting "in situ" through its MT1R receptor. Finally, melatonin is able to affect feed efficiency and, conversely, it protects the ovaries against the oxidative stress arising from ethanol consumption.DoutoradoBiologia CelularDoutor em Biologia Celular e Estrutura

Combined effects of age and diet-induced obesity on biochemical parameters and cardiac energy metabolism in rats

Obesity is often associated with decreased fat oxidation and aging is a well-recognized risk factor for cardiovascular disease. This study investigated calorimetric and morphometric parameters, as well as the glucose levels, lipid profile and cardiac energy metabolism in young and old, controls and obese rats. The animals were divided into four groups: Group I (GI): young rats fed normal diet for 75 days; Group II (GII): young rats fed hypercaloric diet (HD) for 75 days; Group III (GIII): old rats fed normal diet for 510 days; and Group IV (G IV): old rats fed HD for 510 days. The following analyses were performed: calorimetric, glucose and lipid concentrations, atherogenic index (AI), total antioxidant substances (TAS), fat depots, cardiac lipid hydroperoxide (LH) and cardiac lactate dehydrogenase (LDH), citrate synthase (CS), phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH) activities. Older animals were heavier than young and the hypercaloric animals were heavier than controls. Animals from GIV had lower fat oxidation than GIII, which in turn, had higher fat oxidation than GI. Total cholesterol, LDL-C and all fat depots were higher in the GII, as compared to GI. The GIV rats had higher VLDL, retroperitoneal fat, serum lipids and cardiac glycogen levels than GII. Furthermore, GIV rats had higher fat depots, triacylglycerol, total cholesterol and VLDL than GIII. Animals from GII and -IV showed higher LH and AI than age-matched controls. Older hypercaloric rats also had higher TAS than older control rats, which also had lower LH and TAS than younger control rats. Aged animals had increased CS and LDH and decreased PFK and PDH activities. Additionally, GIV rats exhibited an increase in PDH activity, compared to GIII. We conclude that the consumption of HD coupled with aging leads to impaired basal and cardiac metabolism.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

P-MAPA immunotherapy potentiates the effect of cisplatin on serous ovarian carcinoma through targeting TLR4 signaling

Abstract Background Toll-like receptors (TLRs) are transmembrane proteins expressed on the surface of ovarian cancer (OC) and immune cells. Identifying the specific roles of the TLR-mediated signaling pathways in OC cells is important to guide new treatments. Because immunotherapies have emerged as the adjuvant treatment for patients with OC, we investigated the effect of a promising immunotherapeutic strategy based on protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) combined with cisplatin (CIS) on the TLR2 and TLR4 signaling pathways via myeloid differentiation factor 88 (MyD88) and TLR-associated activator of interferon (TRIF) in an in vivo model of OC. Methods Tumors were chemically induced by a single injection of 100 μg of 7,12-dimethylbenz(a)anthracene (DMBA) directly under the left ovarian bursa in Fischer 344 rats. After the rats developed serous papillary OC, they were given P-MAPA, CIS or the combination P-MAPA+CIS as therapies. To understand the effects of the treatments, we assessed the tumor size, histopathology, and the TLR2- and TLR4-mediated inflammatory responses. Results Although CIS therapy was more effective than P-MAPA in reducing the tumor size, P-MAPA immunotherapy significantly increased the expressions of TLR2 and TLR4. More importantly, the combination of P-MAPA with CIS showed a greater survival rate compared to CIS alone, and exhibited a significant reduction in tumor volume compared to P-MAPA alone. The combination therapy also promoted the increase in the levels of the following OC-related proteins: TLR4, MyD88, TRIF, inhibitor of phosphorylated NF-kB alpha (p-IkBα), and nuclear factor kappa B (NF-kB p65) in both cytoplasmic and nuclear sites. While P-MAPA had no apparent effect on tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, it seems to increase interferon-γ (IFN-γ), which may induce the Thelper (Th1)-mediated immune response. Conclusion Collectively, our results suggest that P-MAPA immunotherapy combined with cisplatin could be considered an important therapeutic strategy against OC cells based on signaling pathways activated by TLR4