Orientador: Amanda Martins Baviera / Resumo: O diabetes mellitus (DM) é uma síndrome metabólica crônica caracterizada por deficiência na produção pancreática de insulina e/ou prejuízos em suas ações teciduais, culminando principalmente em hiperglicemia. A manutenção da hiperglicemia por longos períodos é um dos principais fatores envolvidos no estabelecimento e manutenção do estresse glico-oxidativo, o qual está implicado no desenvolvimento das complicações micro e macrovasculares do DM, resultando em morbidade e mortalidade significativas. A metformina tem sido um dos principais agentes antidiabéticos orais utilizados para o tratamento do DM; entretanto, é crescente o interesse no uso de produtos naturais como terapia complementar, seja por seus benefícios aditivos aos efeitos da terapia clássica, seja pelo potencial antioxidante. O licopeno é um carotenoide encontrado em diversos vegetais, em especial tomates frescos e seus derivados. Diversos estudos vêm demonstrando vários benefícios atribuídos ao licopeno: redução da glicemia, dislipidemia, inflamação e em biomarcadores relacionados ao estresse glico-oxidativo. Diante do exposto, o licopeno surge como uma opção terapêutica complementar interessante aos fármacos já utilizados no tratamento do DM. O objetivo deste estudo é investigar o potencial antidiabético do iogurte enriquecido com metformina ou licopeno, isolados ou co-administrados, em ratos diabéticos, avaliando parâmetros fisiológicos, bioquímicos, marcadores do estresse glico-oxidativo e potencial antioxidan... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Obesity leads to many severe metabolic disorders, e.g. dyslipidemia, insulin resistance, ectopic fat accumulation in the liver and skeletal muscles, non-alcoholic fatty liver disease and finally diabetes mellitus type 2. Metformin (1,1-dimethylbiguanide) is the most favored medicament for the treatment and prevention of these disorders. It stimulates cellular glucose uptake and normalizes blood levels of lipid metabolites without triggering insulin secretion. Research on insulin resistance and diabetes is often realized through developing diet- induced obesity in laboratory animals. The aim of this project is to compare metabolic effects of two different high-fat diets named HFD and HSD. The HFD diet consists chiefly of n-6 polyunsaturated fatty acids (corn oil) and starch (100% glucose). The HSD diet contains mainly saturated fatty acids (lard) and sucrose (50% glucose and 50% fructose). I also studied metabolic effects of metformin by adding it continuously to the drinking water given to obese mice fed with the HFD or the HSD diet. Methods: Intraperitoneal glucose tolerance test (IPGTT), blood and tissue levels of lipid metabolites assessment, radio-immunological assessment of blood levels of insulin, assessment of AMPK activity in liver by western blotting. Results: Increased consumption of the...

Despite progress being made in our understanding of triple negative breast cancer (TNBC), the overall survival and disease-free survival for TNBC patients continues to be considerably poorer than their ER/PR/HER2+ counterparts. Metastasis and chemoresistance are the pivotal issues holding back the long-term success of TNBC treatments. In addition to the bulk tumor cells, cancer stem cells (CSCs) have emerged as important targets for alleviating TNBC progression and relapse. Cisplatin, a platinum based chemotherapeutic agent, has shown promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with tumor hypoxia that in turn promotes CSC enrichment and drug resistance. My work is to develop a combinational treatment to improve the long-term therapeutic potential of cisplatin that not only targeted the bulk TNBC population but also ALDHhigh and CD44+/CD24- CSC populations. Through clinical dataset analysis, I found that patient TNBC tumors expressed high levels of epidermal growth factor receptor (EGFR) and hypoxia genes. A similar expression pattern was demonstrated in cisplatin-resistant ovarian cancer. I therefore developed a combinational therapeutic to co-inhibit EGFR and hypoxia using metformin (an AMPK activator) and gefitinib (an EGFR inhibitor), which sensitized bulk TNBC cells to cisplatin and also led to the effective inhibition of both CD44+/CD24- and ALDHhigh CSCs. I obtained similar results by using clinically relevant TNBC patient samples ex vivo. Since these drugs are already frequently used in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to improve the long-term therapeutic outcome of cisplatin for TNBC treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19057号 / 人博第710号 / 新制||人||171(附属図書館) / 26||人博||710(吉田南総合図書館) / 32008 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

<p>Osteoarthritis (OA) is one of the most prevalent diseases in the United States and also in the world. Cartilage plays a vital role in articular joints and its structural integrity and mechanical properties are diminished by OA. Post traumatic osteoarthritis (PTOA) is a prevalent type of OA and occurs after a significant joint injury. Currently, no treatments are available to prevent or delay the progression of any form of OA.</p><p>Collagen crosslinking improves the material properties of cartilage and has been proposed as a potential treatment for OA. The wear resistance of cartilage that had been crosslinked with CASPc, a light activated crosslinking agent, was tested. Results suggested that photo-initiated crosslinking improves the wear characteristics of cartilage.</p><p>Another treatment for PTOA is through biological intervention. Preliminary data from our lab showed that metformin rescues the chondrocyte response to injurious impact overloading in the initial 24 hours following impact. However, whether this treatment maintained cartilage integrity for an extended duration had not been investigated. Material properties of cartilage were analyzed with an indentation test at different time points post-impact to determine the functional effect of cartilage injury and metformin treatment. Changes in the composition of the cartilage were investigated through biochemical techniques.</p><p>Having an in vivo model for PTOA is key for testing any new therapeutic intervention. In this study a model was developed to deliver a consistent impact load to the posterior aspect of medial condyle of a rabbit knee. A drop tower was designed for impacting the rabbit knee, and load and acceleration were measured during the impact. A k-wire was passed through the condyles in the medial-lateral direction under the impact site to secure the condyle during the impact. Whether the impact parameters were affected by the location of the k-wire was evaluated. The location of the k-wire was varied in the anterior/posterior and proximal/distal directions in a knee joint of cadaveric rabbits and impact parameters were recorded. Multiple linear regression showed a correlation between the location of the k-wire and peak stress, loading rate, impact duration and work. Moreover a correlation was found between the damage induced to the cartilage and loading rate, impact duration and peak stress. This study indicated that k-wire location is critical to prevent fracture of the subchondral bone.</p><p>A pilot study was designed to investigate the in-vivo effect of the metformin treatment on PTOA. Impacted knee joints in rabbits were treated with intraarticular metformin or were untreated controls. At 12 weeks post-injury, the progression of OA in the rabbit knees was quantified by histology, and OA severity was assessed using OA Research Society International (OARSI) scoring. Although the number of animals in the study were limited, intraarticular metformin appeared to prevent the development of PTOA in the impacted rabbit knees.</p>

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Introduction: Overweight and obesity are among the most serious public health problems in the world. Today, about half of adult men, one third of adult women and one in five children are estimated to be overweight or obese. Being overweight is not considered as a disease, but it can lead to obesity, which puts the individual at high risk of suffering from several diseases such as diabetes type 2, high blood pressure. To define whether an individual is overweight, normal weight, obese or underweight, BMI value is used. Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects women in fertitlity age. The syndrome is associated with several of risk factors such as insulin resistance, obesity, cardiovascular problems, and infertility. Imbalances in the hormones produced by ovaries can be a cause of PCOS.Currently, there are three prescription drugs in Sweden to treat obesity such as liraglutide (Saxenda®), bupropion/naltrexone (Mysimba®) and orlistat (Xenical®).Objectives: The purpose of this study was to investigate the effectiveness of using liraglutide as monotherapy or in combination with metformin to treat women who were overweight or obese and affected by polycystic ovarian syndrome.Method: This study is based on four randomized controlled clinical trials studies, which are taken from the PubMed database. Using these keywords “liraglutide, obesity, weight loss, BMI, treatment, metformin, PCOS”.Results: The greatest weight loss was observed in study 4 in obese women with PCOS, whichwere treated with the combination of both liraglutide 1.2 mg/day and metformin 1000 BID/day. A weight reduction of -9.23 ±1.23 after 12 weeks of treatment was observed. While less weight loss of -3.0 ± 0.6 kg was observed in study two, where obese women with PCOS were treated with liraglutide 1.2 mg/day and metformin 1000 mg BID/day as a monotherapy. In study 1, obese women with PCOS treated with the combination of both liraglutide 1.2-± mg/day and metformin 1000 BID/day had a weight loss of -6.5 ± 2.8 kg. The obese women with PCOS in study 3 were treated with liraglutide 3 mg/day and had a weight loss of -6.3 ± 3.7 kg butexperienced more severe GI adverse events.Conclusion: The four studies showed that short-term treatment with liraglutide, which is a GLP-1 receptor agonist, 1.2 mg/day together with metformin 1000 mg BID/day as a combination therapy is associated with significantly greater weight loss, BMI changes and with changes in waist circumference compared to when treated with liraglutide or metformin as a monotherapy

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Liver injury instigates a proinflammatory response in tissue-resident macrophages, called Kupffer cells (KCs), resulting in the recruitment of monocytes and neutrophils. The high energy demand required for a rapid proinflammatory response in macrophages like KCs is achieved through metabolic reprogramming. This is supported by increased glycolysis. On the other hand, injury resolution requires hepatic macrophages to undergo an anti-inflammatory polarization, which relies on oxidative phosphorylation (OXPHOS). In addition to shifts in mechanisms of adenosine triphosphate (ATP) production, lipid metabolic reprogramming supplies metabolic intermediates and lipids for membrane remodeling and the production of inflammatory mediators. AMP-activated protein kinase (AMPK) is a master metabolic regulator that influences the metabolic reprogramming of macrophages. While AMPK activation promotes an anti-inflammatory polarization, disruption of activity exacerbates proinflammatory signaling. For this thesis work, we addressed whether macrophage AMPK is protective against liver injury by altering immunometabolism. Specifically, we investigated this question in the context of chronic (nonalcoholic steatohepatitis (NASH)) and acute (acetaminophen (APAP) overdose) liver injury. While APAP overdose is a robust and directly translational model of acute injury, models of NASH-induced hepatic fibrosis rely on nutrient-deficient diets like the choline-deficient high-fat diet (CDAHFD) or genetic manipulation. Despite the utility of these models, they seldom mirror the pathogenesis of human NASH, with diets like CDAHFD being completely dissociated from metabolic syndrome. Moreover, models are required to address the divergence between male and female mice. Recently, there has been a shift towards addressing other variables that drive inflammation and metabolism. At room temperature (RT) (22 °C), mice experience cold stress that alters various biological functions. Cold stress drives brown adipose tissue (BAT) activation and upregulates corticosterone production and immunosuppression, all processes that blunt NASH progression. Giles et al. (2016) demonstrated that housing mice at thermoneutrality (TN) (30 °C) exacerbated metabolic-dysfunction associated fatty liver disease (MAFLD) progression toward NASH in both male and female mice. Since then, we and others have implemented TN housing with different dietary interventions and mice strains. We determined that 16-week Western diet (WD) feeding of male and female mice at 29 °C was insufficient to drive hepatic fibrosis, however alterations in glucose tolerance and elevated liver injury enzymes as well as profibrotic gene expression in male mice may indicate that a longer timeline is necessary (24 weeks). Given that our TN NASH model did not produce hepatic fibrosis, we implemented the CDAHFD to investigate macrophage AMPK in chronic liver injury. Male and female AMPK Flox (Prkaa1 fl/fl/Prkaa2 fl/fl) and MacKO (Flox-LysM-Cre+) mice were fed CDAHFD for 8 weeks. In this time frame, CDAHFD produces a lean euglycemic phenotype with hepatic steatosis, inflammation, and fibrosis, to which AMPK MacKO had no influence. Moreover, intervention with a low dose of metformin had no effect, contrary to the reduction in hepatic steatosis observed in HFD-fed mice. Although macrophage AMPK is dispensable in the CDAHFD model of chronic liver injury, acute liver injury needed to be addressed. We found that priming with systemic activation of a direct AMPK activator MK-8722 did not influence hepatic injury and necrosis in our model of APAP-induced liver injury (AILI). Moreover, deletion of hepatocellular AMPK (Flox-Alb-Cre+) or AMPK MacKO did not influence injury at 24 hours post overdose. Despite the lack of effect of systemic AMPK activation, we were interested in a nanoparticle-based targeting of direct AMPK activator MK-8722 (NP-MK8722) delivery. We determined that PLGA-PEG nanoparticles (NPs) accumulated in hepatic macrophages as early as 2 hours post-injection, but NP-MK8722 did not alter hepatic necrosis, injury, or immune infiltration. Overall, my thesis work has advanced our knowledge of the effects of housing temperatures on NASH pathogenesis. Moreover, we are the first to address the effects of macrophage AMPK signaling in NASH and AILI. This is especially true for assessing how AMPK deficiency and targeted activation influences KC immunometabolism during injury.

<p><strong><em>Purpose</em></strong></p> <p>To examine the potential of the anti-diabetic agent Metformin (MET) to enhance responses of NSCLC to ionizing radiation (IR).</p> <p><strong><em>Experimental Design</em></strong></p> <p>Human NSCLC A549, H1299 and SK-MES cells were treated with IR, MET or the mTOR inhibitor rapamycin and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays. A549 and H1299 cells were grafted into flanks of immunosuppressed mice and treated with MET and/or IR. Tumours were analyzed by immunoblotting and immunohistochemistry.</p> <p><strong><em>Results</em></strong></p> <p>MET(2.5uM-5mM) caused dose-dependent inhibition of proliferation (10-70%)in all lines, inibited clonogenic survival and sensitized cells to IR. In A549 cellsMET caused inhibition of proliferation comparable to rapamycin, stimulated expression and activation of the ATM and AMPK-p53-p21^cip1and inhibited the Akt-mTOR-4-EBP1 pathway.MET caused G1 arrest of cell cycle, enhanced apoptosis and induced sustained DNA repair foci of gH2AX. MET and IR alone inhibited xenograft growth and combined treatment enhanced that further. IR and MET induced sustained enhancement of expression and activity of ATM-AMPK-p53-p21^cip1and inhibitionof Akt-mTOR-4-EBP1 pathways in tumours also. MET reduced expression of angiogenesis and enhanced expression of apoptosis markers in both control and radiated tumours.</p> <p><strong><em>Conclusions</em></strong></p> <p>Clinically achievable(uM) doses ofMET inhibit human NSCLC cell and tumour growth and sensitize them to IR.This is accompanied by desirable modulation of molecular signals, inhibition of angiogenesis and induction of apoptosis. Our results suggest that MET could be a clinically useful adjunct to radiotherapy in NSCLC and support clinical investigation of MET in combination with radiotherapy.</p> / Master of Science (MSc)