No description available.

No description available.

No description available.

The object of the present study was to determine simultaneously aspects of hepatic and peripheral glucose metabolism in the intact baboon. Isotopic techniques were used to study glucose turnover rates, glucose recycling, glucose pool and space, and the forearm technique to study peripheral exchange of glucose. The results obtained in the normal animals acted as reference values for each animal. Thereafter diabetes mellitus was produced with streptozotocin, a drug causing destruction of the beta cells of the pancreatic islets. Experiments were then repeated in the acutely diabetic baboon and the nature and extent of the abnormalities in glucose metabolism documented. Lactate metabolism and peripheral lipid metabolism were included in the study in order to establish any interrelationships with glucose metabolism and to determine the abnormalities resulting from the production of diabetes. In the normal animal the turnover rate of lactate was greater than glucose although the lactate pool was much smaller than the glucose pool. After producing diabetes glucose turnover rates increased threefold and correlated with the severity of hyperglycaemia. A significant increase in lactate turnover rates was noted but the increase was less than in the case of glucose turnover rates. The formation of glucose from lactate increased significantly but the fraction of the lactate turnover rate converted to glucose was unchanged. The glucose pool increased nearly threefold and correlated with the increase in glucose turnover rate. There was a significant but smaller increase in lactate pool which correlated with the increase in lactate turnover rate. Both glucose and lactate space decreased after diabetes but the decrease did not correlate with the severity of hyperglycaemia. In the majority of diabetic animals there was no glucose utilization in the forearm, and in fact glucose release was observed. Increased production of lactate occurred in the forearm of the diabetic baboon, despite decreased glucose utilization.Arterial levels of triglyceride and free fatty acid increased threefold after diabetes while the free glycerol level doubled. In the normal animal the general pattern of exchange in the forearm consisted of triglyceride and free fatty acid uptake and free glycerol release. In the diabetic animal triglyceride and free fatty acid release was observed, while the release of free glycerol was decreased. The pattern of forearm metabolism in the diabetic animals was variable and not as consistent as before the production of diabetes. Several interrelationships between glucose, lactate and lipid metabolism were noted. The baboons used in this study showed extreme sensitivity to the metabolic effects of Streptozotocin Diabetes. Hyperglycaemia increased in severity and ketoacidosis invariably developed in the second week. The animals were not treated with insulin and death from severe uncontrolled diabetes occurred in nearly all animals within two weeks. This study has demonstrated the severe abnormalities in hepatic and peripheral glucose metabolism in diabetes. The simultaneous pathogenesis of these abnormalities and their importance in the development of the acute diabetic syndrome have been defined. Associated abnormalities in lactate metabolism and lipid metabolism have also been documented. / Thesis (M.D.)-University of Natal, 1979.

by Wai-kit Hon. / Bibliography: leaves 129-140 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1985

Familial Glucocorticoid Deficiency (FGD) is an autosomal recessive form of adrenal failure characterized by isolated glucocorticoid deficiency with preserved mineralocorticoid secretion. I studied two cohorts of patients who presented with late onset FGD. Firstly I investigated 2 families of Turkish ethnicity who presented with delayed onset adrenal insufficiency and identified two novel missense mutations in the melanocortin 2 receptor accessory protein (MRAP). I characterised both these mutations in vitro and confirmed that both mutant MRAPs demonstrated reduced rather than absent function consistent with the phenotype of delayed presentation. Secondly I studied 3 families from a genetically isolated Irish population who had an interesting variant of adrenal failure. Patients had typical biochemical features of FGD with isolated glucocorticoid deficiency, raised ACTH and normal renin and aldosterone levels. Unlike other forms of FGD, cortisol deficiency was often not as severe and onset was usually in childhood following a period of normal adrenal function. Affected children develop hypocortisolaemia but also growth failure, increased chromosomal breakage and natural killer (NK) cell deficiency. Targeted exome sequencing identified a variant (c.71-1insG) in mini chromosome maintenance homologue 4 (MCM4) that segregated with the disease in all 3 families. RT-PCR of patient leucocyte RNA revealed this mutation leads to aberrant splicing of 3 exon 2 and a foreshortened ORF encoding a prematurely terminated translation product (p. Pro24ArgfsX4). Western blotting of patient lymphocytes revealed loss of the full length MCM4 protein but two smaller MCM4 isoforms were preserved. Histological examination of the adrenals of an MCM4 depletion mouse model revealed an abnormal adrenal morphology. Small, spindle-shaped cells were present throughout the adrenal cortex. These cells did not express either CYP11A1 or CYP11B1 and significantly reduced the number of steroidogenic cells in the zona fasciculata. Further staining showed these cells expressed GATA4, a transcription factor expressed in foetal but not adult adrenals, and capsular markers, indicating that they may be non-steroidogenic capsular cells infiltrating the cortex. MCM4 is one part of a heterohexameric complex essential for normal DNA replication and genome stability in all eukaryotes and no MCM mutation has ever been described in humans. I have identified a mutation in MCM4 that results in adrenal failure, growth retardation, increased chromosomal fragility and NK cell deficiency. Animal models indicate that loss of MCM4 is lethal, but it is likely that the smaller isoforms I observe may rescue the patients from a lethal phenotype. This research has revealed a novel mechanism of adrenal failure and potentially a novel function of MCM4. The seemingly specific impact on adrenal function may reflect a defect in adrenal stem cell differentiation and the inability of capsular cells to differentiate into steroidogenic cells.

Background - The alternatively spliced insulin receptor (IR) exon 11 (36 nucleotides) and the constitutively spliced Apolipoprotein B (APOB) exon 26 (7572 nucleotides) are examples of the shortest and longest exons in the genome. Aim - The aim of this study was to investigate the regulation of splicing of these 2 exons in cell culture using 2′-O-methyl RNA antisense oligonucleotides (ASOs) and peptide nucleic acid (PNA)-peptide hybrid ASOs. Methods - ASOs were designed to target key sequences involved in the splicing of the IR exon 11 and exonic splicing silencer sequences (ESS) in APOB exon 26. HepG2 cells were reverse-transfected with the ASOs for 48 hours, mRNA harvested and RT-PCR was performed to amplify the IR isoform and APOB cDNAs which were separated by PAGE and quantified. Results Insulin receptor exon 11 - 2′-O-methyl RNA ASOs targeted to two intronic sites, the 3′ half of exon 11 and spanning the entire exon caused significant exon skipping. PNA-peptide hybrids predicted to increase exon 11 splicing, paradoxically caused exon skipping. PNA-peptide hybrids with 3′ tails caused exon 11 skipping more effectively than hybrids with 5′ tails. Apolipoprotein B exon 26 - Only combinations of 2′-O-methyl RNA ASOs targeting multiple ESSs in APOB exon 26 caused a small proportion of aberrant splicing. This consisted of complete exon 26 skipping and the selection of a downstream cryptic 3′ splice site in preference to the native 3′ splice site. Discussion - Exclusion of the IR exon 11 can be induced by targeting a combination of intronic or exonic sequences. PNA-peptide hybrid ASOs were unable to increase exon 11 splicing. The aberrant splicing of large constitutive exons such as APOB exon 26 can be induced by targeting multiple ESS sites along its course.

Type 2 diabetes, as well as other metabolic diseases, is an increasing global health concern and many of the mechanisms of both the disease and its current drug treatments have not been fully described. It has been shown that the anti-diabetic class of drugs, the thiazolidinediones, work via both a known PPARγ-dependent, and a lesser known PPARγ-independent mechanism of action. This PPARγ-independent mechanism likely involves the metabolic regulatory molecule AMPK, which has a newly elucidated inhibitory site of phosphorylation at Ser485/Ser491. In this study we sought to determine if the thiazolidinedione troglitazone affects AMPK in HepG2 liver cells via phosphorylation at both the known Thr172 site as well as the letter understood Ser485 site. We also looked for potential upstream kinases of the Ser485 site by comparing our results to recently proposed mechanisms of phosphorylation here. HepG2 cells were cultured in the lab and treated with troglitazone to determine time- and dose- dependent effects on AMPK. We also treated cultured HepG2 cells with PMA as well as troglitazone and PMA in order to compare mechanisms of action of troglitazone on AMPK. Results were analyzed using common western blot techniques and statistical analysis. Our data found that troglitazone increased AMPK activity by increasing phosphorylation at Thr172 in a time- and dose- dependent manner. The inhibitory site Ser485 was also increasingly phosphorylated with troglitazone treatments, although the net result of troglitazone treatment remained AMPK activation. The recently elucidated results from our laboratory showing the mechanism of p-AMPK Ser485 phosphorylation via PKD after PMA treatment also occurred in HepG2 cells, although this did not appear to be the mechanism by which troglitazone phosphorylated AMPK at Ser485. These data support the current research that there is an AMPK mediated PPARγ-independent mechanism of troglitazone treatment for type 2 diabetes and other metabolic diseases. The results do however bring into question the full effects of the drug on AMPK at a molecular level and leaves room for new research in this area, specifically the exact mechanism by which troglitazone phosphorylates AMPK at Ser485. Our data also brings up new questions as to the simultaneous phosphorylation of AMPK at both Thr172 and Ser485 and what this means for the activity of the molecule as a whole, a current area of critical research. Lastly our data support the newly elucidated mechanism of AMPK phosphorylation at Ser485 via PKD1, an exciting and novel discovery and potential target for therapeutic intervention.

Misregulation of Dlk1, a paternally expressed imprinted gene, is known to cause adipose phenotypes in both mice and humans. It is now known that the type of fat a person has, the location of and type of expansion are all important factors for the epidemic of obesity-related diseases, yet there is little understanding of the factors that influence which depots expand, and how. This project investigates how Dlk1, expressed primarily during embryogenesis, effects adulthood adiposity, which has shed light on the mechanism by which embryonic insults affect adulthood adipose physiology and resultant metabolic disease. Much previous work has been done on the role of Dlk1 in adipogenesis in vitro, however little is known of its role in this process in vivo. To achieve an in vivo investigation of its role in adipogenesis, adipose tissue has been measured in mice with deleted Dlk1 from embryo through early life and into adulthood. Gross measurement has been supported by mechanistic interrogation of adipose expandability using a triple transgenic adipocyte labelling mouse model, results from which are the most comprehensive to date in a wild type context and reveal insight into the Dlk1 knock-out phenotype. Results indicate a complex and dynamic role of Dlk1 that is interlinked with overall growth in mice. Moreover new evidence is presented here for tissue specific c imprinting of Dlk1 in some adipose cell types with consequential growth and adipose alterations in Dlk1 heterozygote mice that do not follow the expected phenotype of imprinted gene knock-out models.

The genetic control of puberty remains an important but mostly unanswered question. Late pubertal timing affects over 2% of adolescents and is associated with adverse health outcomes. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern and is highly heritable; however, its neuroendocrine pathophysiology and genetic regulation remain unclear. The genetic control of puberty remains an important but mostly unanswered question. Late pubertal timing affects over 2% of adolescents and is associated with adverse health outcomes. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern and is highly heritable; however, its neuroendocrine pathophysiology and genetic regulation remain unclear. Our large, accurately phenotyped cohort of patients with familial self-limited DP is a unique resource with a relatively homogeneous genetic composition. I have utilised this cohort to investigate the genetic variants segregating with the DP trait in these pedigrees. Whole exome sequencing in eighteen probands and their relatives, and subsequent targeted sequencing in an extended subgroup of the cohort, has revealed potential novel genetic regulators of pubertal timing. In ten unrelated probands, I identified rare mutations in IGSF10, a gene that is strongly expressed in the nasal mesenchyme during embryonic migration of gonadotropin-releasing hormone (GnRH) neurons. IGSF10 knockdown both in vitro and in a transgenic zebrafish model resulted in perturbed GnRH neuronal migration. Loss-of-function mutations in IGSF10 were also identified in five patients with absent puberty due to hypogonadotropic hypogonadism (HH). Additionally, I have identified and investigated one rare, pathogenic mutation in HS6ST1 - a gene known to cause HH - in one family with DP, and two rare variants in FTO - a gene implicated in the timing of menarche in the general population - in 3 families. Further potentially pathogenic variants have emerged from investigating candidate genes identified from microarray studies (LGR4, SEMA6A and NEGR1) and from related clinical phenotypes (IGSF1). Our large, accurately phenotyped cohort of patients with familial self-limited DP is a unique resource with a relatively homogeneous genetic composition. I have utilised this cohort to investigate the genetic variants segregating with the DP trait in these pedigrees. Whole exome sequencing in eighteen probands and their relatives, and subsequent targeted sequencing in an extended subgroup of the cohort, has revealed potential novel genetic regulators of pubertal timing. In ten unrelated probands, I identified rare mutations in IGSF10, a gene that is strongly expressed in the nasal mesenchyme during embryonic migration of gonadotropin-releasing hormone (GnRH) neurons. IGSF10 knockdown both in vitro and in a transgenic zebrafish model resulted in perturbed GnRH neuronal migration. Loss-of-function mutations in IGSF10 were also identified in five patients with absent puberty due to hypogonadotropic hypogonadism (HH). Additionally, I have identified and investigated one rare, pathogenic mutation in HS6ST1 - a gene known to cause HH - in one family with DP, and two rare variants in FTO - a gene implicated in the timing of menarche in the general population - in 3 families. Further potentially pathogenic variants have emerged from investigating candidate genes identified from microarray studies (LGR4, SEMA6A and NEGR1) and from related clinical phenotypes (IGSF1).