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Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly due to inadequate pancreatic islet alpha-cell glucagon secretion. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by selective somatostatin receptor type 2 (SSTR2) antagonism of alpha cells to relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes prone (BBDP) rats (D) vs non-diabetic BBDP (N) rats, underwent infusion of vehicle or SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycaemia. D rats, treated with SSTR2a, needed little or no glucose to maintain hypoglycemia. To monitor real-time glucagon secretory response directly, we developed the technique of thin slices of the pancreas from D and N rats as well as normal human pancreas, subjected to perifusion with vehicle vs SSTR2a. SSTR2a treatment enhanced glucagon secretion in N and D rats and human pancreas. We conclude that SSTR2 antagonism can enhance hypoglycemia-stimulated glucagon release sufficient to achieve normoglycemic control.

Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly due to inadequate pancreatic islet alpha-cell glucagon secretion. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by selective somatostatin receptor type 2 (SSTR2) antagonism of alpha cells to relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes prone (BBDP) rats (D) vs non-diabetic BBDP (N) rats, underwent infusion of vehicle or SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycaemia. D rats, treated with SSTR2a, needed little or no glucose to maintain hypoglycemia. To monitor real-time glucagon secretory response directly, we developed the technique of thin slices of the pancreas from D and N rats as well as normal human pancreas, subjected to perifusion with vehicle vs SSTR2a. SSTR2a treatment enhanced glucagon secretion in N and D rats and human pancreas. We conclude that SSTR2 antagonism can enhance hypoglycemia-stimulated glucagon release sufficient to achieve normoglycemic control.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine degradation pathway. The defective fumarylacetoacetate hydrolase enzyme causes the accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate (MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA). In vitro and in vivo studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were developed. Most notably was the fah deficient mouse, whose neonatally lethal phenotype is rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Although, this model most closely resembles the human phenotype with elevated tyrosine levels and the development of hepatocellular carcinoma (HCC), the model is not human genome based. Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1. However, it is not yet clear which DNA repair mechanisms are affected and if only protein functionality is affected, or if expression of DNA repair proteins are also affected. Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism. The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation to wild-type. The general consensus is that the reversion is the result of a true back mutation. However, the mechanism underlying the back mutation is still unresolved. It was suggested that cancer develops either through a chromosomal instability mutator phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator phenotype. In HT1 only chromosomal instability was reported. The aims of this study were to contribute to the understanding of the molecular basis of the genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand nucleotide DNA repair mechanisms are affected and to what extent, and to determine if microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed: i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material. To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression assays, microsatellite instability assays, high resolution melting and dideoxy sequencing techniques were employed. Results from the comet assay showed that the HT1 accumulating metabolites, SA and pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1 or ERCC1. The expression of these genes were, however, low in HT1 patient samples. Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome, and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de novo mutations on other parts of the genome can not be ruled out. To conclude, results presented in this thesis, for the first time show that in HT1 the initiating proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA repair proteins are low, and microsatellite instability is found in HT1. By contributing to the elucidation of the mechanism underlying the development of HT1-associated HCC, and providing evidence for the development of a mutator phenotype, the results presented in this thesis contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in HT1. In addition to these contributions, a hypothesis is posited, which suggests that a point mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions seen in HT1. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine degradation pathway. The defective fumarylacetoacetate hydrolase enzyme causes the accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate (MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA). In vitro and in vivo studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were developed. Most notably was the fah deficient mouse, whose neonatally lethal phenotype is rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Although, this model most closely resembles the human phenotype with elevated tyrosine levels and the development of hepatocellular carcinoma (HCC), the model is not human genome based. Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1. However, it is not yet clear which DNA repair mechanisms are affected and if only protein functionality is affected, or if expression of DNA repair proteins are also affected. Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism. The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation to wild-type. The general consensus is that the reversion is the result of a true back mutation. However, the mechanism underlying the back mutation is still unresolved. It was suggested that cancer develops either through a chromosomal instability mutator phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator phenotype. In HT1 only chromosomal instability was reported. The aims of this study were to contribute to the understanding of the molecular basis of the genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand nucleotide DNA repair mechanisms are affected and to what extent, and to determine if microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed: i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material. To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression assays, microsatellite instability assays, high resolution melting and dideoxy sequencing techniques were employed. Results from the comet assay showed that the HT1 accumulating metabolites, SA and pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1 or ERCC1. The expression of these genes were, however, low in HT1 patient samples. Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome, and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de novo mutations on other parts of the genome can not be ruled out. To conclude, results presented in this thesis, for the first time show that in HT1 the initiating proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA repair proteins are low, and microsatellite instability is found in HT1. By contributing to the elucidation of the mechanism underlying the development of HT1-associated HCC, and providing evidence for the development of a mutator phenotype, the results presented in this thesis contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in HT1. In addition to these contributions, a hypothesis is posited, which suggests that a point mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions seen in HT1. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

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The beneficial effects of angiotensin II type 1 (AT1) receptor blockers (ARB) in cerebrovascular disease have been shown in clinical trials. However, the effects of ARBs vary based on their unique pharmacologic properties. In this study, we focused on telmisartan, a fat-soluble ARB with selective peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity, and investigated its effects on ischemic injury in cerebral vasculature using murine models of both transient and permanent focal ischemia. Analysis by triphenyltetrazolium-staining revealed that pre-treatment of mice with telmisartan reduced stroke volume 72 h after the transient ischemic insult in a dose-dependent manner, though such treatment did not reduce stroke volume due to permanent ischemia. Transient ischemia induced pro-inflammatory adhesion molecules, such as ICAM-1 and P-selectin in the ischemic region, and treatment with telmisartan diminished the expression of these adhesion molecules with diminished infiltration of inflammatory cells. The beneficial effect of telmisartan was attenuated, in part, by administration of a PPARγ antagonist. Treatment with valsartan (an ARB without PPARγ agonist activity) also decreased ischemic injury after transient ischemia, though to a lesser extent than telmisartan. Our findings indicate that telmisartan has a beneficial effect in a murine model of ischemia/reperfusion injury through blockade of AT1 receptors, and, in addition, due to a positive effect via its specific anti-inflammatory PPARγ agonist activity.

L’obésité abdominale est associée à de nombreuses complications métaboliques, telles que la résistance à l’insuline, le diabète de type 2 et les maladies cardiovasculaires. Récemment, il a été suggéré que ces anomalies métaboliques sont étroitement associées à la taille des adipocytes, cellules constitutives du tissu adipeux. En effet, plusieurs travaux suggèrent que plus les adipocytes sont gros, plus ils produisent des adipokines pro-inflammatoires, responsables de l’inflammation chronique associée à l’obésité. Dans ce contexte, le but de ce travail de thèse était, dans un premier temps, d’établir les répartitions en fréquences de tailles des adipocytes dans différentes situations physiologiques et physiopathologiques et dans différents dépôts adipeux à l’aide du Multisizer IV Coulter counter et dans un second temps de corréler la taille des adipocytes à leurs fonctions physiologiques. D’une part, nous avons montré que quels que soient les dépôts adipeux, les répartitions en fréquences de tailles des adipocytes ont toutes le même profil, elles sont bimodales avec une population de petits adipocytes (diamètre < nadir) et une population de gros adipocytes (diamètre > nadir). La privation en insuline (diabète de type I) entraînant une diminution de 60% de la masse du tissu adipeux, altére profondément la répartition bimodale en fréquences de tailles des adipocytes. La supplémentation en insuline de ces animaux diabétiques restaure la cellularité du tissu adipeux et la répartition en fréquences de tailles des adipocytes, suggérant un rôle régulateur majeur de l’insuline sur ces paramètres. La restriction calorique partielle, nous a permis de mettre en évidence un phénomène d’hystérésis dans certains tissus adipeux. De plus, à partir des données de restriction calorique prolongée il a été possible d’établir un modèle prédisant la cinétique des changements de tailles des adipocytes et de la répartition en fréquences de tailles. D’autre part, après avoir séparé les adipocytes en deux populations : les gros (diamètre > 50 µm) et les petits adipocytes (diamètre < 50 µm) nous avons montré que les petits adipocytes sont plus sensibles à l’insuline que les gros. Nous avons aussi constaté que les gros adipocytes sont plus lipolytiques que les petits adipocytes. Ces données sont en accord avec le modèle établi montrant que les échanges avec le micro-environnement tissulaire sont surtout dépendants de la surface des membranes cellulaires. Ainsi, nos résultats mettent en évidence que si la quantité de tissu adipeux est importante, sa qualité, dont la répartition en fréquences de tailles des adipocytes, est également importante et pourrait servir de paramètre prédictif du développement des désordres métaboliques. / Abdominal obesity is associated with several metabolic complications such as insulin resistance, type 2 diabetes and cardiovascular diseases. Recently, it has been suggested that these metabolic abnormalities are closely related to the size of adipocytes, cells constituting adipose tissue. Indeed, numerous studies suggested that the more adipocytes are larger, the more they produce pro-inflammatory adipokines which are responsible for obesity-associated chronic inflammation. In this context, the aim of this work was, in the first time, to establish the size frequency distributions of adipocytes in different physiological and pathological situations and in different fat depots using the Coulter counter Multisizer IV, and in the second time to correlate the size of the adipocytes to their physiological functions. On the one hand, we have shown that, all adipocyte size frequency distributions have the same profile regardless of fat depots, they are bimodal with both the population of small adipocytes (diameter < nadir) and the population of large adipocytes (diameter > nadir). Deprivation of insulin (type I diabetes) resulting in a decrease of 60% of adipose tissue mass, profoundly alters the bimodal distribution of adipocyte size frequency. Insulin supplementation of these diabetic animals restores cellularity of adipose tissue and the size-frequency distribution of adipocytes, suggesting a major regulatory role of insulin on these parameters. Partial caloric restriction has allowed us to a hysteresis phenomenon in some adipose tissues. In addition, following data from extended caloric restriction it has been possible to develop a model predicting the kinetic of changes concerning both the size of adipocytes and the frequency distribution. On the other hand, after separating the adipocytes in two populations: large (diameter > 50 microns) and small adipocytes (diameter < 50 microns), we have shown that small adipocytes are more insulin-sensitive than large ones. We also observed that larger adipocytes are more lipolytic than smaller ones. These data are in agreement with the established model showing that the interactions with the tissue microenvironment are mainly dependent on the surface of cell membranes. Thus, our results show that if the amount of adipose tissue is important, the quality which noticed by the adipocyte size frequency distributions is also important and could be used as a marker to predict the development of metabolic disorders.

Les phosphates de calcium sont des biomatériaux couramment utilisés dans de nombreuses spécialités médicales. L'amélioration de ces biomatériaux vise à augmenter leur ostéointégration et leur bioactivité. Le strontium possédant d'intéressantes capacités de modification de la physiologie osseuse, l'incorporation de ce dernier au sein de phosphates de calcium par substitution ionique pourrait permettre un déplacement de la balance osseuse vers la formation osseuse.Notre travail a permis de démontrer la capacité des particules de phosphates de calcium substitués en strontium à augmenter la prolifération des ostéoblastes en culture et à modifier l'expression et la synthèse des principales protéines impliquées dans la physiologie osseuse (Collagène de type I, Serpine H1, métalloprotéinases matricielles 1 et 2, inhibiteurs tissulaires des MMPs). Par ailleurs, la poudre de phosphates de calcium ne contenant pas de strontium a entrainé une sécrétion accrue de chimiokines pro-inflammatoires (MCP-1 et GRO-?) qui n'a pas été observée pour la poudre substituée. Enfin, des études in-vivo réalisées dans un modèle de défaut osseux murin a permis de démontrer une plus grande résorbabilité de la poudre contenant du strontium et sa plus grande capacité à stimuler la réparation osseuse.

Les phosphates de calcium sont des biomatériaux couramment utilisés dans de nombreuses spécialités médicales. L'amélioration de ces biomatériaux vise à augmenter leur ostéointégration et leur bioactivité. Le strontium possédant d'intéressantes capacités de modification de la physiologie osseuse, l'incorporation de ce dernier au sein de phosphates de calcium par substitution ionique pourrait permettre un déplacement de la balance osseuse vers la formation osseuse.Notre travail a permis de démontrer la capacité des particules de phosphates de calcium substitués en strontium à augmenter la prolifération des ostéoblastes en culture et à modifier l'expression et la synthèse des principales protéines impliquées dans la physiologie osseuse (Collagène de type I, Serpine H1, métalloprotéinases matricielles 1 et 2, inhibiteurs tissulaires des MMPs). Par ailleurs, la poudre de phosphates de calcium ne contenant pas de strontium a entrainé une sécrétion accrue de chimiokines pro-inflammatoires (MCP-1 et GRO-?) qui n'a pas été observée pour la poudre substituée. Enfin, des études in-vivo réalisées dans un modèle de défaut osseux murin a permis de démontrer une plus grande résorbabilité de la poudre contenant du strontium et sa plus grande capacité à stimuler la réparation osseuse. / Calcium phosphate are widely used in medicine. Their upgrade tend to enhance their biocompatibility and their bioactivity. Strontium has interesting capability to modify the bone physiologie. Its incorporation in calcium phosphates could lead to modify the bone balance toward osteogenesis.The present work reveal the capacities of such biomaterials to enhance the replication of osteoblasts ant to modify the expression and the synthesis of proteins implicated in the bone balance (type I collagen, serpinH1, Matrix metalloproteinases 1 and 2, tissular inhibitors of MMPs). Moreover, non substituted calcium phosphate powders enhance the expression and synthesis of inflammatory cytokines (MCP-1 and Gro-a). This fact was not observed with the non substituted powder. In-vivo studies on a mouse model permit us to demonstrate the higher resorbability and the higher bone healing capability of the substituted powder.