Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

Background: The augmented whole body glucose uptake response to insulin during the postprandial state is described as meal-induced sensitization (MIS). MIS occurs when the presence of food in the upper gastrointestinal tract (GIT) activates two feeding signals (activation of hepatic parasympathetic nerves and elevation of hepatic glutathione level), and causes insulin to release hepatic insulin sensitizing substance (HISS), which stimulates glucose uptake in peripheral tissues. The impairment of HISS release results in the absence of meal-induced insulin sensitization (AMIS), causing progression to a cluster of metabolic, vascular, and cardiac dysfunction, which we refer to as components of the AMIS syndrome. Objectives: The objective of my doctoral research was to study the manipulation of the HISS-pathway, in age- and diet-induced AMIS models, with exercise ± antioxidants. Also, in a separate project I studied the signaling pathways involved with the HISS action in skeletal muscle. Methods: The 7-day voluntary running was used as exercise intervention to manipulate the HISS pathway in healthy and prediabetic rats. The interaction of an antioxidant cocktail, SAMEC (S-adenosylmethionine + vitamin E + vitamin C), with the effects of exercise on postprandial insulin response was studied. Moreover, in the signaling studies the insulin and 5'-adenosine monophosphate activated protein kinase (AMPK) pathways were examined to test their possible involvement with the HISS action in skeletal muscle. Results: Voluntary running-wheel exercise for 7 days increases the postprandial glucose uptake response to insulin in health and in prediabetes through enhancement/restoration of HISS action. Supplementation with SAMEC during 7 days of exercise does not either harm or add benefits to the positive effects of exercise on insulin sensitivity. Finally, the signaling studies indicate that HISS increases the rate of glycogen synthesis in muscle through an insulin/AMPK-independent pathway.

Despite inconsistent results in the literature, high protein diets are being promoted for the management of metabolic syndrome parameters primarily due to their proposed favorable effects on weight loss. Therefore, lean and fa/fa Zucker rats were given normal and high protein diets with varying protein sources for 12 weeks. A high protein diet with a mixture of animal and plant protein sources was the most effective for improving metabolic syndrome parameters, specifically insulin resistance and hepatic steatosis. A high protein soy diet was the second most effective diet, while a high protein casein diet demonstrated no benefits compared to the other two high protein diets and minimal benefits compared to a normal protein casein diet. Interestingly, high protein diets did not affect body weight regardless of protein source. These findings suggest that the source of protein within a high protein diet is critical for improving metabolic syndrome parameters and that improvements can be observed independent of weight loss.

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

Background: The augmented whole body glucose uptake response to insulin during the postprandial state is described as meal-induced sensitization (MIS). MIS occurs when the presence of food in the upper gastrointestinal tract (GIT) activates two feeding signals (activation of hepatic parasympathetic nerves and elevation of hepatic glutathione level), and causes insulin to release hepatic insulin sensitizing substance (HISS), which stimulates glucose uptake in peripheral tissues. The impairment of HISS release results in the absence of meal-induced insulin sensitization (AMIS), causing progression to a cluster of metabolic, vascular, and cardiac dysfunction, which we refer to as components of the AMIS syndrome. Objectives: The objective of my doctoral research was to study the manipulation of the HISS-pathway, in age- and diet-induced AMIS models, with exercise ± antioxidants. Also, in a separate project I studied the signaling pathways involved with the HISS action in skeletal muscle. Methods: The 7-day voluntary running was used as exercise intervention to manipulate the HISS pathway in healthy and prediabetic rats. The interaction of an antioxidant cocktail, SAMEC (S-adenosylmethionine + vitamin E + vitamin C), with the effects of exercise on postprandial insulin response was studied. Moreover, in the signaling studies the insulin and 5'-adenosine monophosphate activated protein kinase (AMPK) pathways were examined to test their possible involvement with the HISS action in skeletal muscle. Results: Voluntary running-wheel exercise for 7 days increases the postprandial glucose uptake response to insulin in health and in prediabetes through enhancement/restoration of HISS action. Supplementation with SAMEC during 7 days of exercise does not either harm or add benefits to the positive effects of exercise on insulin sensitivity. Finally, the signaling studies indicate that HISS increases the rate of glycogen synthesis in muscle through an insulin/AMPK-independent pathway.

Norint diagnozuoti kepenų funkcijos pažeidimus yra atliekami biocheminiai kraujo tyrimai. Net mažiausius paktimus kepenyse parodo kepenų fermentų padidėjimas, arba sumažėjimas kraujo serume. Mūsų tikslas buvo įvertinti 3 fermentų kitimo dinamiką (laktatdehydrogenazę (LDH), šarminę fosfatazę (ALPL) , aspartatamino transferazę (AST)) ganykliniu ir tvartiniu laikotarpiais. Rezultatai parodė, kad šarminės fosfatazės rodikliai buvo didžiausi tvartiniu laikotarpiu, o ganykliniame laikotarpyje ženkliai krito. Tačiau aspartatamino transferazės ir laktatdehydrogenazės aktyvumas svyravo nepriklausomai nuo laikotarpio. / In order to diagnose a hepatic disorder a biochemical blood test was carried out. Even slight changes in hepatic function show a decrease or an increase in the concentration of hepatic enzymes. Our aim was to monitor 3 enzymes: alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and aminotransferase (AST). From the results of the blood test we deduced hepatic enzyme funcionality of local Lithuanian cow breed („ Lietuvos šėmųjų“). The experiment was carried out during indoor and pasture periods. The greatest activity of alkaline phosphatase was observed during the pasture period and during the indoor period its activity was greatly reduced. The activity of aminotransferase and lactate dehydrogenase was independent on the season.

The incidences of obesity and type 2 diabetes are reaching epidemic proportions worldwide. A cardinal feature of these conditions is resistance to the effects of the hormone insulin and a resulting hepatic overproduction of glucose. Insulin resistance is also implicated in a range of liver diseases including non-alcoholic fatty liver disease (NAFLD) and hepatitis C infection. Insulin is released after a meal and acts on liver, skeletal muscle and adipose tissue to reduce blood glucose concentration. In the liver, insulin inhibits the production and release of glucose into the circulation and stimulates its storage as glycogen. Glucagon, on the other hand, is present in the fasting state and causes breakdown of hepatic glycogen along with production of new glucose. This glucose is released from hepatocytes into the circulation. For the studies in this thesis, functional assays to measure various aspects of hepatic glucose metabolism in vitro were developed. This included measuring glucose output into culture medium, hepatocyte uptake of radiolabelled glucose and incorporation into glycogen, and total cellular glycogen content. These assays were used to investigate glucose metabolism in primary rat hepatocytes and FaO rat hepatoma cells. Both cell types responded to physiological concentrations of insulin, showing decreased glucose output and increased glycogen synthesis. Glucagon increased glucose output and reduced glycogen synthesis in primary cells but had no effect on FaO cells. Factors that have been identified that may inhibit or potentiate insulin action were investigated. Increased body iron stores have been linked with insulin resistance. De-ironing patients improves insulin sensitivity, suggesting a causal relationship between iron and insulin resistance. Hepatocytes store the majority of the body’s excess iron. This project investigated the effects of increasing hepatocyte iron stores, through addition of ferric ammonium citrate (FAC), or depleting iron stores by chelation with dipyridyl. Small increases or decreases of iron in primary cells had negative effects on cell viability, resulting in significantly reduced glucose output and glycogen synthesis. Dipyridyl treatment had similar effects on FaO cells as on primary cells but FAC treatment increased FaO glucose output, although significant iron loading was not achieved. With concentrations of FAC and dipyridyl low enough to not significantly influence cell viability, insulin sensitivity was not affected. Adiponectin is an insulin sensitiser and appears to exert this effect primarily through the liver. Adiponectin can also reduce hepatic glucose output (HGO) independent of insulin. It is believed adiponectin mediates its effects in liver, skeletal muscle and adipose tissue through activation of AMP-activated protein kinase (AMPK). In muscle, p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a downstream component of adiponectin signalling. In this study, recombinant human adiponectin was produced and collected in culture medium which was then concentrated. Despite the presence of both high molecular weight (HMW) and low molecular weight (LMW) adiponectin multimers, the concentrated medium had no effect on HGO in the presence or absence of insulin. Concentrated adiponectin medium did not affect AMPK or p38 MAPK phosphorylation in hepatocytes or other cell types previously shown to respond to adiponectin. However, commercially-sourced purified recombinant adiponectin also failed to elicit any observable responses. AICAR and metformin are pharmacological activators of AMPK and were used to treat primary rat hepatocytes and FaO cells. These treatments reduced HGO independent of insulin in both cell types. In primary cells, these reductions were partially inhibited with Compound C, an AMPK inhibitor, suggesting that both AICAR and metformin action is at least partly AMPK dependent. In FaO cells, Compound C only inhibited the AICAR-mediated reduction of glucose output, indicating that metformin may act independently of AMPK in these cells. Compound C significantly inhibited AICAR and metformin-mediated increases in AMPK phosphorylation in primary hepatocytes and FaO cells. There was a trend towards inhibition of AICAR-mediated p38 MAPK phosphorylation with Compound C treatment, suggesting that p38 MAPK may lie downstream of AMPK in hepatocytes. Adenoviral expression of constitutively active (CA) and dominant negative (DN) AMPK in primary rat hepatocytes was used to further study the role of AMPK in hepatic glucose metabolism. Despite significant expression of CA AMPK, phosphorylation of downstream acetyl-CoA carboxylase (ACC) was not affected nor was HGO. CA AMPK did, however, increase phosphorylation of p38 MAPK. DN AMPK completely inhibited AICAR-mediated AMPK phosphorylation and partially inhibited phosphorylation of ACC. In addition, AICAR-mediated phosphorylation of p38 MAPK was inhibited by DN AMPK. Taken together, these results suggest that p38 MAPK is downstream of AMPK in hepatocytes. The implication that p38 MAPK is involved in hepatic AMPK signalling is a novel finding. A greater understanding of this pathway in the liver may identify novel therapeutic targets, leading to improved treatment strategies for metabolic disorders linked to obesity and type 2 diabetes.

[Truncated abstract] Hepatic progenitor cells (HPC) are becoming increasingly recognized as facultative stem cells capable of regenerating the liver during chronic liver injury and also as targets of malignant transformation. Similar markers are expressed by hepatocellular carcinoma (HCC) and HPC, and a precursor-product relationship is well established. This thesis focuses on the ways in which the HPC population can be controlled under circumstances of chronic liver injury, and in this manner, reduce the risk of progression to HCC reduced. The major aim of Chapters 3 to 5 was to elucidate the effect of interferon α (IFNα) therapy on HPC. Chronic hepatitis C affects approximately 250 million individuals world wide. Approximately 80% of infections progress to chronicity, which places the individuals at greater risk of developing HCC. The gold standard of treatment of chronic hepatitis C is a combination of pegylated IFNα and ribavirin. ...The results were surprising. While IFNγ exerted a pro-apoptotic and antiproliferative effect on HPC in vitro, administration of IFNγ to CDE-fed mice for 14 days increased fibrosis, enhanced inflammatory infiltration and exacerbated the HPC response, with concurrent hepatocyte cell death. In addition, increased morbidity and mortality were observed in the IFNγ-treated mice compared to control. IFNγ treatment was found to prime the liver for the HPC response by recruiting inflammatory cells and altering the hepatic cytokine profile, both of which may facilitate an increased HPC response. Numbers of activated HSC were also increased in the IFNγ-treated, CDE-fed mice, correlating with the increased fibrosis seen in these animals. This data contradicts the current experimental use of IFNγ for treatment of fibrosis. Based on our results, we suggest that IFNγ promotes HPC proliferation in the CDE model, by encouraging inflammatory infiltration and hepatocyte damage and this initiates pro-fibrotic events. Concurrent proliferation of HPC and activated HSC further supports the view that there is a close relationship between the two cell types, and thus, a link between the HPC response and fibrosis. In conclusion, findings documented in this thesis suggest that administration of IFNα and IFNγ can contribute to shaping the HPC response. IFNα therapy may reduce HCC risk in chronic hepatitis C patients by bringing the HPC population under control. In contrast, IFNγ treatment can exacerbate the HPC response, liver fibrosis and parenchymal damage, illustrating the need to approach this method of fibrosis treatment with caution.

Les objectifs de ce travail étaient d’évaluer qualitativement et quantitativement l’effet d’antipaludiques « classiques » (primaquine, Malarone®, amino-4-quinoléines) et « d’avenir » (artésunate, ferroquine, seuls ou associés) sur les formes hépatiques et les stades sexués du parasite responsable de la transmission du paludisme. Le modèle expérimental comprend des souris swiss femelle infestées par Plasmodium yoelii et Anopheles stephensi comme moustique vecteur. L’action de la Malarone® (proguanil-atovaquuone) sur les stades hépatiques est quasi totale et plus importante que celle, incomplète, de la primaquine, de la ferroquine ou de l’artésunate. Si les molécules précédentes (ferroquine, artésunate), prescrites à doses subcuratives, entraînent souvent une augmentation de la gamétocytogénèse, elles altèrent certains stades de gamétocytes et inhibent statistiquement chez le moustique la formation d’oocystes et leur nombre et, par là même, interviennent négativement dans la transmission du parasite. / The objective of this study is to evaluate qualitatively and quantitatively the effect of "classic" (primaquine, Malarone®, amino-4-quinoline) and "future" (artesunate, ferroquine, alone or associated) antimalarials on the liver forms and sexual stages of the parasite responsible for malaria transmission. The experimental model was : swiss mouse female infected with Plasmodium yoelii and Anophelesstephensi as the vector. The action of Malarone® (proguanil-atovaquuone) on liver stages is almost complete and more than that, incomplete, primaquine, the ferroquine or artesunate. If the previous molecules (ferroquine, artesunate), prescribed at subcurative doses, often lead to an increase in gametocytogenesis, they alter certain stages of gametocytes and statistically inhibit the formation of oocysts in the mosquito; hence, their number involve negatively in the transmission of the parasite.

p. 1-79 / Submitted by Antonio Geraldo Couto Barreto (ppgms@ufba.br) on 2013-10-02T17:02:51Z No. of bitstreams: 1 TESE_Consuêlo Padilha-1.pdf: 9442449 bytes, checksum: 72ab1d2cb912c235e58236ec944e7b16 (MD5) / Approved for entry into archive by Patricia Barroso (pbarroso@ufba.br) on 2013-10-30T19:36:47Z (GMT) No. of bitstreams: 1 TESE_Consuêlo Padilha-1.pdf: 9442449 bytes, checksum: 72ab1d2cb912c235e58236ec944e7b16 (MD5) / Made available in DSpace on 2013-10-30T19:36:47Z (GMT). No. of bitstreams: 1 TESE_Consuêlo Padilha-1.pdf: 9442449 bytes, checksum: 72ab1d2cb912c235e58236ec944e7b16 (MD5) Previous issue date: 2013 / A Doença Hepática Gordurosa Não Alcoólica (DHGNA) representa a causa mais comum de doença do fígado na atualidade. Está associada com síndrome metabólica e, mais recentemente, às doenças cardiovasculares. Objetivo: avaliar a associação entre DHGNA e doença arterial coronariana (DAC) e os fatores preditivos desta associação em indivíduos submetidos à cineangiocoronariografia (CAG). Metodologia: Foi realizado estudo transversal, que avaliou indivíduos submetidos à CAG com suspeita de DAC. Os pacientes realizaram avaliação clínico–laboratorial e ultrassonografia abdominal (USAB). Critérios para DAC: presença de lesão obstrutiva em artérias coronárias epicárdicas ou seus principais ramos. Critérios para DHGNA: presença de esteatose na USAB; ingestão alcoólica ≤20 g/dia; ausência de outras doenças hepáticas. As variáveis contínuas foram avaliadas pelos testes t de Student ou teste de Mann-Whitney e as categóricas pelo teste do qui-quadrado com nível de significância (p) < 0,05. Análise de regressão mediu a força da relação entre os fatores de risco e a presença concomitante de DAC e DHGNA. Resultados: Foram estudados 244 pacientes com média de idade de 61,5±9,3 anos. A DAC foi observada em 63,5%, e a DHGNA, em 42,2% dos pacientes. Aqueles com DAC eram, predominantemente, do gênero masculino (p<0,01); diabéticos (p<0,05) e tabagistas (p=0,045), e 43,9% deles tinham DHGNA. A DHGNA esteve associada à DM2, resistência insulínica, síndrome metabólica, obesidade central, índice de massa corpórea, triglicérides e alanina aminotransferase (p<0,05). A ocorrência concomitante de DAC e DHGNA foi positivamente correlacionada a sobrepeso/obesidade e HOMA-IR ≥3,0. Conclusões: A amostra teve elevada frequência de ambas, DAC e DHGNA; DHGNA foi mais elevada em pacientes com DAC, porém não houve associação estatisticamente significante entre as duas doenças; pacientes com associação DAC e DHGNA apresentavam sobrepeso/obesidade, e resistência insulínica. Os resultados sugerem a relevância clínica de investigar a DHGNA em pacientes com DAC, no sentido de diminuir a morbimortalidade desses pacientes. / Salvador