The emerging evidence on the interconnectedness between the gut microbiome and host metabolism has led to a paradigm shift in the study of metabolic diseases such as obesity and type 2 diabetes with implications on both underlying pathophysiology and potential treatment. Mounting preclinical and clinical evidence of gut microbiota shifts, increased intestinal permeability in metabolic disease, and the critical positioning of the intestinal barrier at the interface between environment and internal milieu have led to the rekindling of the “leaky gut” concept. Although increased circulation of surrogate markers and directly measurable intestinal permeability have been linked to increased systemic inflammation in metabolic disease, mechanistic models behind this phenomenon are underdeveloped. Given repeated observations of microorganisms in several tissues with congruent phylogenetic findings, we review current evidence on these unanticipated niches, focusing specifically on the interaction between gut permeability and intestinal as well as extra-intestinal bacteria and their joint contributions to systemic inflammation and metabolism. We further address limitations of current studies and suggest strategies drawing on standard techniques for permeability measurement, recent advancements in microbial culture independent techniques and computational methodologies to robustly develop these concepts, which may be of considerable value for the development of prevention and treatment strategies.

Thesis (M. Sc. (Biochemistry)) --University of Limpopo, 2017 / Diabetes mellitus is a chronic metabolic disorder characterised by perpetual hyperglycaemia. Various oral pharmacological theraputic management strategies currently exist but are too expensive and having a host of undesirable side effects. Therefore people resort to the use of traditional medicinal plants as they offer a cost effective and readily available health care avenue. Despite the wide-spread use of traditional medicinal plants, several worrisome concerns about their effectiveness, clinical modes of action and safety have been raised. Leaves of five selected plants (Toona celliata, Seriphium plumosum, Schkuhria pinnata, Olea africana, Opuntia ficus-indica) were collected from Mankweng area, Capricon Local Municipality, Limpopo province, South Africa. Ground plant materials were exhaustively extracted by maceration in methanol, acetone or hexane. The presence of different plant secondary metabolites in the crude extracts was determined using various standard chemical tests and thin layer chromatography (TLC). A myriad of compounds which represented various secondary plant metabolites groups were observed on the TLC plates and were best resolved in the non-polar (BEA) and intermediate (CEF) mobile phases. The total phenolic content and total flavonoids of the different extracts were determined spectrophotometrically using the Folin-Ciocalteu`s phenol reagent method and Aluminium chloride colorimetric assay respectively. The plants contained comparatively higher amounts of total phenolic compounds as compared to the flavonoids. The antiglycation activity of the plant extracts were determined using the bovine serum albumin assay. The acetone extract of Seriphium plumosum (SPlA) exhibited the most glycation inhibitory activity among all the examined extracts, as it resulted in 2,22% glycation. The antioxidant potential of each of the different extracts was quantitatively determined spectrophotometrically using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and the ferric ion reducing power assay. The methanol extract of Seriphium plumosum showed the best antioxidant activity among all the extracts in this study. It exhibited the lowest EC50 values of 0.72 mg/ml and 2.31 mg/ml for the DPPH scavenging activity and the ferric reducing power assay respectively. The cytotoxicity profiles of the different plant extracts on C2C12 cell line were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium xiii bromide (MTT) assay. It was concluded that since the all the extracts investigated had CC50 values greater than 50 μg/ml they were generally non-toxic. The amount of glucose taken up by differentiated C2C12 cells was quantified using the glucose uptake assay. Treatment of the C2C12 cells with the hexane extract of Seriphium plumosum resulted in the best glucose utilisation effect of 35,77% which was higher than that of insulin which was 26,06% after 6 hours. The translocation assay was used to determine the effect of the plant extract on GLUT4 translocation while the expression of various mitogen activated protein kinases in the cells was determined using the human MAPK profiler assay. It was established that treatment with Seriphium plumosum hexane extract resulted in increased GLUT4 translocation from the intracellular vesicular stores to the cell surface membrane. The increase in GLUT4 translocation may have resulted from the upregulation of expression of phosphorylated Akt-1, Akt-2, GSK3β, ERK1, ERK2 p70S kinase and MKK3 under the influence of Seriphium plumosum hexane extract. The study documents a probable insulin-mimetic activity of the hexane extract of Seriphium plumosum. This activity may be responsible for its hypoglycaemic capability and may occur via the augmentation of proximal mitogen activated protein kinases involved in the GLUT4 translocation pathway. Further investigations need to be conducted to ascertain this novel finding which may help provide a cost-effective and readily available antidiabetic therapeutic agent. / National Research Foundation (NRF)

La croissance de la migration dans le monde, intensifiée par des crises politiques et économiques, ainsi que par des catastrophes naturelles dans plusieurs pays, amène des défis dans la prestation des soins de santé dans les pays à revenu élevé, qui doivent s’assurer d’offrir des soins de santé adéquats aux populations migrantes. Il est reconnu que des personnes migrantes présentent un taux élevé de diabète de type 2 (DT2) par rapport aux personnes natives des pays d’accueil, notamment à cause des facteurs génétiques et des facteurs liés à la migration. De plus, il a été prouvé que les personnes migrantes conservent des liens transnationaux avec leurs proches et leurs pays d’origine. Ces liens peuvent constituer des leviers ou s’ériger en obstacle pour la gestion et la prise en charge du DT2 dans le pays d’accueil. Nous avons mené une métasynthèse qualitative dans le but de déterminer comment le transnationalisme se manifeste et influence l’expérience de santé des personnes migrantes vivant avec le DT2 dans les pays à revenu élevé. Un total de 21 études qualitatives pertinentes menées dans des pays à revenu élevé, dont la Norvège (n=1), la Suède (n=1), l’Australie (n=6), le Canada (n=2), le Pays-Bas (n=1), les États-Unis (n=7), le Royaume-Uni (n=2) et la Belgique (n=1), a été repéré dans cinq différentes bases de données. Le transnationalisme a servi de cadre de référence ayant guidé l’analyse des données de l’étude. Plusieurs liens transnationaux (sociaux, culturels, linguistiques, religieux, affectifs, économiques, recherche des soins de santé, etc.) ont été identifiés et l’analyse thématique a permis de formuler des thèmes et des sous-thèmes. Il en est ressorti que les personnes migrantes maintiennent des liens avec leurs cultures, ainsi qu’avec leurs proches (familles, amis) et membres de leur communauté culturelle. Ces liens peuvent exercer une influence importante sur leur vie et sur leur expérience de vivre avec le DT2. En effet, le diagnostic de la maladie (DT2), les changements à apporter aux habitudes de vie (alimentation saine, pratique d’activité physique), la prise des médicaments, ainsi que la gestion quotidienne du DT2 sont influencés par les liens transnationaux. En conclusion, les liens transnationaux ont un impact positif et négatif sur la gestion et la prise en charge du DT2 dans les pays à revenu élevé. Des recommandations pour la pratique, la formation et la recherche ont été émises. / The growth of migration around the world, intensified by political and economic crises and natural disasters in many countries, is creating challenges in the delivery of health care in high-income countries, which must ensure that adequate health care is provided to migrant populations. It is recognized that migrant individuals have a higher rate of type 2 diabetes (T2DM) than native-born individuals in host countries, due in part to genetic and migration-related factors. In addition, it has been shown that migrants maintain transnational ties to their relatives and countries of origin. These links can be levers or barriers for the management and care of T2DM in the host country. We conducted a qualitative metasynthesis to determine how transnationalism manifests itself and influences the health experience of migrants living with T2D in high-income countries. A total of 21 relevant qualitative studies conducted in high-income countries, including Norway (n=1), Sweden (n=1), Australia (n=6), Canada (n=2), the Netherlands (n=1), the United States (n=7), the United Kingdom (n=2), and Belgium (n=1), were identified in five different databases. Transnationalism was used as a framework to guide the analysis of the study data. Several transnational ties (social, cultural, linguistic, religious, emotional, economic, health care seeking, etc.) were identified and thematic analysis was used to formulate themes and sub-themes. It was found that migrants maintain ties with their cultures, as well as with their relatives (family, friends) and members of their cultural community. These ties can have an important influence on their lives and on their experience of living with T2DM. Indeed, the diagnosis of the disease (T2DM), the changes to be made to lifestyle habits (healthy eating, physical activity), the taking of medication, as well as the daily management of T2DM are influenced by transnational ties. In conclusion, transnational ties have a positive and negative impact on the management and care of T2DM in high-income countries. Recommendations for practice, education, and research were made.

Les îlots de Langerhans, principalement composés de cellules bêta sécrétant l’insuline, jouent un rôle majeur dans l’homéostasie glucidique grâce à leur sécrétion hormonale finement régulée. Dans un contexte d’insulino-résistance associée à l’obésité, la masse fonctionnelle des cellules bêta pancréatiques augmente, en partie grâce à une prolifération accrue. Le diabète de type 2 survient lorsque les mécanismes de compensation échouent et que la sécrétion d’insuline devient insuffisante. Par conséquent, augmenter la prolifération des cellules bêta a été proposée comme approche thérapeutique afin de retarder l’apparition du diabète de type 2. Parmi les différents facteurs pouvant moduler la prolifération des cellules bêta, les nutriments, en particulier le glucose et les acides gras, jouent un rôle important et plusieurs études chez le rongeur montrent que les nutriments augmentent la prolifération et la masse des cellules bêta avant l’apparition de l’insulino-résistance. De plus, des travaux de notre laboratoire ont montré que l’infusion d’un mélange d’acide gras, le ClinOleic (65% oléate, 20% linoléate et 15% palmitate) et de glucose provoquait une augmentation marquée de la prolifération des cellules bêta chez le rat. L’objectif de cette thèse est donc d’évaluer les mécanismes par lesquels les acides gras stimulent la prolifération des cellules bêta. Dans un premier article, seul l’oléate, parmi plusieurs acides gras testé, a démonté un effet significatif sur l’augmentation de la prolifération des cellules bêta en présence de glucose ex vivo. La prolifération induite par l’oléate nécessite la formation de sphingolipides à très longue chaîne monoinsaturée, tandis que la perturbation de leur synthèse provoque une diminution de la réponse proliférative. Dans une seconde étude, l’analyse par séquençage d’ARN sur cellules uniques a mis en évidence le rôle important des espèces réactives de l’oxygène, des peroxyrédoxines et du proto-oncogène MYC dans le processus prolifératif des cellules bêta induit par l’oléate. Dans l’ensemble, les travaux présentés dans cette thèse apportent un éclairage nouveau sur le potentiel prolifératif encore énigmatique des cellules bêta pancréatiques et soulignent le rôle des sphingolipides et des espèces réactives de l’oxygène dans ce processus. / The islets of Langerhans, mainly composed of insulin-secreting beta cells, plays a major role in glucose homeostasis due to their finely regulated hormone secretion. In a context of insulin resistance associated with obesity, the functional mass of pancreatic beta cells increases, in part due to increased proliferation. Type 2 diabetes occurs when these compensatory mechanisms fail and insulin secretion becomes insufficient. Therefore, increasing beta cell proliferation has been proposed as a therapeutic approach to delay the onset of type 2 diabetes. Among the various factors that can modulate the proliferation of beta cells, nutrients, in particular glucose and fatty acids, play an important role, and several studies in rodents show that nutrients increase beta-cell proliferation before insulin resistance can be detected. Previous work from our laboratory has shown that infusion of the fatty-acid mixture ClinOleic (65% oleate, 20% linoleate and 15% palmitate) in the presence of glucose markedly increases beta-cell proliferation in rats. The objective of this thesis is to evaluate the underlying mechanisms by which fatty acids stimulate beta-cell proliferation. In a first study, among several fatty acids tested, only oleate increased beta cell proliferation in presence of glucose ex vivo. Oleate-induced beta-cell proliferation requires the formation of monounsaturated very long chain sphingolipids, while blockade of their biosynthesis dampens the proliferative response. In a second study, single-cell RNA sequencing analysis highlighted the role of reactive oxygen species, peroxiredoxins, and the proto-oncogene MYC in oleate-induced beta cell proliferation. Overall, the work presented in this thesis sheds new light on the enigmatic proliferative potential of pancreatic beta cells and identifies a role for sphingolipids and reactive oxygen species in this process.

<p dir="ltr">Obesity is a worldwide epidemic that is partially linked to changing lifestyles within the modern world, including increased access to calorically dense foods and decreased energy output due to more sedentary jobs. Obesity can lead to many different health complications, such as cardiovascular diseases or Type 2 Diabetes (T2D). Obesity-induced T2D is caused by dysfunction of the insulin-producing beta cells of the pancreas. However, mechanisms that promote obesity and the mechanisms by which obesity leads to beta cell dysfunction are not fully known.</p><p dir="ltr">Spinophilin is a filamentous (F)-actin binding, protein scaffolding, and protein phosphatase 1 (PP1)-targeting protein that can regulate protein. Spinophilin has multiple actions. Spinophilin can bundle filamentous actin to modulate the cellular cytoskeleton. Spinophilin also mediates substrate phosphorylation by targeting and modulating PP1 activity. In addition, spinophilin interacts with multiple proteins, including certain G-protein coupled receptors and can scaffold them with F-actin and/or PP1. Previous studies established that spinophilin KO mice have decreased fat mass, increased lean mass, and improved glucose tolerance. Yet, how spinophilin modulates the above metabolic parameters is unclear. We found that spinophilin is expressed in hypothalamic tissue and appears to also be expressed in the feeding center of the hypothalamus, as well as in other glucose-sensing cells known as tanycytes that neighbor the arcuate nucleus and the third ventricle. We found that loss of spinophilin limited weight gain observed in both a leptin receptor db/db mouse line (Leprdb/db<i>)</i> and mice fed a high-fat diet. Moreover, we found that the decreased fat mass seen in global spinophilin KO mice, at least in the Leprdb/db mice, was not due to major differences in feeding behaviors, consistent with what was observed by other groups using high-fat diet-fed mice. </p><p dir="ltr">As spinophilin was not associated with alterations in feeding, we posited that its ability to modulate glucose homeostasis may be linked to non-neuronal actions of the protein. Previous studies have found that spinophilin may regulate adipose tissue function and <i>in vitro</i> pancreatic beta cell function; however, its role in the pancreas and beta cells <i>in vivo</i> is not well characterized. We found that spinophilin is expressed in mouse pancreas. Using proteomics-based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas, including: PP1, the spinophilin homolog neurabin, and myosin-9. KEGG pathway analysis of proteomic proteins identified multiple pathways regulating ER stress, such as the unfolded protein response, and cytoskeletal arrangement. We observed decreased associations of spinophilin with PP1 and neurabin and increased association with myosin-9 in obese, Leprdb/db mice as early as 6 weeks, as well as significant decreases in body weight when spinophilin was knocked out in Leprdb/db mice. Moreover, we confirmed a robust and specific increased interaction of spinophilin with myosin-9, and other cytoskeletal proteins. Additionally, we found specific spinophilin interactions with ribosomal proteins, and exocrine and digestion proteins in high-fat diet-fed mice. Using our recently generated pancreatic beta cell-specific spinophilin KO mice, we found that loss of spinophilin in mice on a high-fat diet significantly reduces weight gain and improves whole- body glucose tolerance, and loss of spinophilin specifically within the beta cells also improves whole-body glucose tolerance, with no effect on body weight, further suggesting cell type-specific and independent roles for spinophilin on body weight and glucose homeostasis.</p>

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