Thesis (M.Sc.(Med.)--University of the Witwatersrand, Faculty of Health Sciences, 2000.

Thesis advisor: Catherine Y. Read / Background and Significance: The metabolic syndrome is a heterogeneous disorder leading to increased morbidity and mortality. Components of the metabolic syndrome are known to be inherited, however efforts to identify genomic markers in humans have been unsuccessful and a candidate-gene/intermediate phenotype approach may be useful. Evidence supports a relationship between altered metabolic function and three candidate genes, caveolin-1 (CAV1), peroxisome proliferator receptor-activated gamma, and angiotensinogen (AGT). These genes may serve as markers for the co-aggregation of insulin resistance and hypertension. Research Question: To examine whether single nucleotide polymorphisms (SNPs) in the CAV1, PPARg and AGT genes are associated with the co-aggregation of insulin resistance and hypertension. Methods: Three gene association studies were conducted in a Caucasian hypertensive cohort (HyperPATH). The homeostasis assessment model (HOMA-IR), hyperinsulinemic euglycemic clamp, and salt sensitive blood pressure were determined in each subject. Statistical analyses were conducted using a general linear model accounting for relatedness and adjusting for the following covariates: age, gender, body mass index, study site. Replication was assessed in a hypertensive Mexican-American cohort (HTN-IR) for the CAV1 gene and a hypertensive African American cohort (HyperPATH) for the PPARg gene. Results: SNPs of the CAV1 gene were significantly associated with insulin resistance in Caucasians from HyperPATH. These results were replicated in the HTN-IR cohort. A SNP of the PPARg gene was associated with salt sensitive blood pressure and increased plasma renin levels in Caucasians and African Americans from HyperPATH. SNPs of the AGT gene were associated with insulin sensitivity in Caucasians from HyperPATH. Conclusion: CAV1 and AGT are genomic markers for the co-aggregation of insulin resistance and hypertension. The PPARg gene is a potential genomic marker for vascular dysfunction in hypertension. Clinical Perspective: Genomic markers for insulin resistance exist in human populations with hypertension. These markers explain the inter-individual variability of insulin resistance and hypertension and help identify potential underlying mechanisms. Use of these bio-markers in clinical practice may improve individualized prevention and treatment strategies, decreasing the incidence of and improving outcomes for this chronic disease. Promoting health through individualized care makes the incorporation of genomic markers into nursing practice essential. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

Thesis (M.D.)—Boston University

No description available.

A five stage selection protocol originally applied to mouse embryonic stem cells (mESCs) was examined for the derivation of insulin producing cells from human embryonic stem cells (hESCs). Insulin gene expression was observed and insulin protein was measured by radioimmunoassay. However, the radioimmunoassay results were shown to be susceptible to false positive findings due to the presence of exogenous insulin within differentiation media and it was concluded that this particular strategy was not ideal for the derivation of insulin producing cells from hESCs. An investigation was then undertaken regarding the in vivo differentiation of cells derived from hESCs seeded within 3D scaffolds to determine if this would result in the derivation of insulin producing cells. Within scaffolds there were abundant cells which stained positively for ectoderm lineage markers including nestin. Cells which stained positively for markers of endothelial progenitors representing the mesoderm lineage were also observed and rare cells stained for endoderm markers including insulin. These investigations also demonstrated that transplanting scaffolds seeded with cells derived from hESCs between the liver lobules of immunodeficient mice could lead to the formation of teratomas. Factors that may have influence the formation of teratomas were further investigated and it was demonstrated that teratoma formation was inhibited by altering in vitro treatment of cells. An in vitro investigation was then performed to determine the extracellular matrix (ECM) producing capacity of hESCs and differentiated cells derived from hESCs because ECM proteins are required for the formation of 3D structures similar to pancreatic islets. The results from this investigation indicated that differentiated cells produced multiple ECM proteins at substantially higher levels than hESCs. The ECM producing differentiated cells could be useful in the development of surrogate islet like tissue by supplying a suitable ECM structure within a 3D scaffold environment to aid the function of ??-cell surrogates. Furthermore, these differentiated cells derived from hESCs were shown to produce an adhesive basement membrane in vitro, which is derived from human sources, and could be utilized in the derivation, propagation and differentiation of hESCs.

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The Na+-K+-ATPase (pump) is a transmembrane, multi-subunit (α and β) protein that is expressed in all cells, and particularly in skeletal muscle cells. In one cycle, it pumps 3 Na+ ions out of the cell and 2 K+ ions into the cell at the expense of 1 ATP molecule. This enzyme is responsible for maintaining muscle cell excitability. This is of particular importance during contractile activity, when the flux of Na+ and K+ across the cell membrane is high. The activity of the Na+-K+-ATPase is highly regulated and very responsive to hormonal stimuli. Previous research has shown that 20-30 min insulin exposure in vivo induces the translocation of pumps from intracellular stores to the plasma membrane. However, no study has examined the catalytic properties of this enzyme in response to short insulin exposures. The objective of this study was to investigate the response of the Na+-K+-ATPase to short insulin incubation in vitro in muscles of different fibre type. It was hypothesized that the short insulin treatment would result in an increase in pump activity, not only through translocation but also increased intrinsic activity. Using an in vitro model, rat soleus (Sol), red gastrocnemius (RG), and white gastrocnemius (WG) muscle homogenates were incubated at 37°C for 5 min with and without 75μM insulin (Ins). Next, in order to separate mechanisms of translocation and intrinsic activation, the plasma (SLP) and endosomal (EN) membranes were separated through a fractionation procedure. This allowed the investigation of insulin-induced increases in intrinsic activity in SLP and EN fractions of Na+-K+-ATPase; SLP and EN (non-treated) membranes were incubated at 37°C for 5 min with and without 75μM insulin. Lastly, muscle homogenates were insulin-treated for 5 min at 37°C with 625μM insulin prior to fractionation. These SLP and EN fractions (insulin-treated) were then incubated at 37°C for 5 min with and without 75μM insulin. Na+-K+-ATPase maximal activity (Vmax, mmol•mg prot-1•h-1) and km (substrate affinity), α2 content, and tyrosine phosphorylation (Tyr-P) were probed. It was found that insulin increased Vmax (P<0.05) in Sol and RG, but not WG, homogenates (Con vs Ins, Sol=221±17 vs 256±21; RG=190±14 vs 256±18; WG=104±4.6 vs 99±1.8). In non-treated fractions, insulin increased Vmax (P<0.05) in Sol and RG SLP fractions (Con vs Ins, Sol=1710±186 vs 1970±231; RG=1476±128 vs 1655±139). A main effect, Con<Ins (P<0.05) was observed in non-treated WG SLP. Insulin also increased Vmax in non-treated RG EN (Con vs Ins, 246±38 vs 304±43). In insulin-treated fractions, insulin increased Vmax¬ in RG SLP only (Con vs Ins, 1145±119 vs 1426±150). Increased Vmax was not observed in insulin-treated fractions when compared to non-treated fractions. No evidence of translocation or increased Tyr-P was detected with insulin treatment via α2 Western blotting. Short insulin exposure induced increases in Na+-K+-ATPase activity, and these increases were due to stimulation of intrinsic activity and not due to translocation.

The Na+-K+-ATPase (pump) is a transmembrane, multi-subunit (α and β) protein that is expressed in all cells, and particularly in skeletal muscle cells. In one cycle, it pumps 3 Na+ ions out of the cell and 2 K+ ions into the cell at the expense of 1 ATP molecule. This enzyme is responsible for maintaining muscle cell excitability. This is of particular importance during contractile activity, when the flux of Na+ and K+ across the cell membrane is high. The activity of the Na+-K+-ATPase is highly regulated and very responsive to hormonal stimuli. Previous research has shown that 20-30 min insulin exposure in vivo induces the translocation of pumps from intracellular stores to the plasma membrane. However, no study has examined the catalytic properties of this enzyme in response to short insulin exposures. The objective of this study was to investigate the response of the Na+-K+-ATPase to short insulin incubation in vitro in muscles of different fibre type. It was hypothesized that the short insulin treatment would result in an increase in pump activity, not only through translocation but also increased intrinsic activity. Using an in vitro model, rat soleus (Sol), red gastrocnemius (RG), and white gastrocnemius (WG) muscle homogenates were incubated at 37°C for 5 min with and without 75μM insulin (Ins). Next, in order to separate mechanisms of translocation and intrinsic activation, the plasma (SLP) and endosomal (EN) membranes were separated through a fractionation procedure. This allowed the investigation of insulin-induced increases in intrinsic activity in SLP and EN fractions of Na+-K+-ATPase; SLP and EN (non-treated) membranes were incubated at 37°C for 5 min with and without 75μM insulin. Lastly, muscle homogenates were insulin-treated for 5 min at 37°C with 625μM insulin prior to fractionation. These SLP and EN fractions (insulin-treated) were then incubated at 37°C for 5 min with and without 75μM insulin. Na+-K+-ATPase maximal activity (Vmax, mmol•mg prot-1•h-1) and km (substrate affinity), α2 content, and tyrosine phosphorylation (Tyr-P) were probed. It was found that insulin increased Vmax (P<0.05) in Sol and RG, but not WG, homogenates (Con vs Ins, Sol=221±17 vs 256±21; RG=190±14 vs 256±18; WG=104±4.6 vs 99±1.8). In non-treated fractions, insulin increased Vmax (P<0.05) in Sol and RG SLP fractions (Con vs Ins, Sol=1710±186 vs 1970±231; RG=1476±128 vs 1655±139). A main effect, Con<Ins (P<0.05) was observed in non-treated WG SLP. Insulin also increased Vmax in non-treated RG EN (Con vs Ins, 246±38 vs 304±43). In insulin-treated fractions, insulin increased Vmax¬ in RG SLP only (Con vs Ins, 1145±119 vs 1426±150). Increased Vmax was not observed in insulin-treated fractions when compared to non-treated fractions. No evidence of translocation or increased Tyr-P was detected with insulin treatment via α2 Western blotting. Short insulin exposure induced increases in Na+-K+-ATPase activity, and these increases were due to stimulation of intrinsic activity and not due to translocation.

Akt/Protein Kinase B plays an important role in many biological responses including cell survival, proliferation, and nutrient metabolism. Activation of Akt/PKB by growth factors involves PIP3 binding, membrane translocation and the phosphorylation at Thr308 and Ser473 residues by PDK1 and PDK2, respectively. Dephosphorylation and AKT binding proteins were two inhibitory mechanisms for AKT. Carboxyl-terminal modulator protein (CTMP) has been demonstrated to bind to carboxyl-terminus of Akt and as a regulator on plasma membrane. However, the interaction region of CTMP to Akt is still unknown. On the other hand, contradict results of positive and negative effects to AKT activity were reported. In this study, we firstly demonstrated the CTMP protein level shows positive correlation with Akt phosphorylation and cell proliferation in human breast cancer cell. The interaction of CTMP and phosphorylation Akt by co-immunoprecipitation and immunofluorescence experiments shows that CTMP may be involved in regulating Akt phosphrylation. The results of GST pull down assay demonstrated that the direct binding and revealed more then one interaction region of CTMP to Akt. Interestingly, in HeLa cells, transiently or stably expressing CTMP can directly promote Akt phosphorylation even in basal state and significantly increase the phosphorylation at both Thr308 and Ser473 of Akt upon the insulin stimulation in the pre-starvation condition. When compared the cell growth of HeLa cells in 10% serum condition using cell proliferation and soft-agar colony forming assay, rapid cell proliferation and colony number increase were observed in cells stably expressing CTMP suggesting that CTMP may be involved in the regulation of Akt-mediated cell growth. The analogous results were also obtained from colony formation assay with different condition from 10% to 1% serum. The result of cell cycle distribution shows that the increasment of cell proliferation rate may due to the increasment of cell cycle progression in CTMP stable clones. Furthermore, CTMP stable clone promote tumor growth and metastasis in xenograft animal model. Together, these results indicated that the binding of CTMP positively modulate Akt and thus increase insulin sensitivity in HeLa cells.

Insulin resistacne and hyperinsulinemia are important risk factors for development of type 2 diabetes mellitus and hypertension. Recently, accumulating evidence has shown that endothelial dysfunction, increases in peripheral vessel resistnce and overactivation of the sympathetic neruvous system contribute to the development of insulin resistance-associated hypertension. The signigicance of cardiovascular regulatory center in the brain stem in pathophysiology of the insulin resistance-induced hypertension, however, has not been explored. Previously studies have proved that increases in superoxide anion (O2£»−) production in peripheral tissue and suppression of nitric oxide (NO) expression in the endothial cell are involved in insulin resistance and hypertension. The nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM) are involved in neural regulation of blood pressure by serving respectively as the primary baroreceptor afferent terminal sites and the location of sympathetic premotor neurons for cardiovascular regulation in the brain stem. Clinically, the peroxisome proliferator-activated receptor (PPAR) agonist is commonly prescribed for the treatment of type 2 diabetes mellitus by activate PPAR£^ to enhance peripheral tissue insulin sensitizing ability, to maintain blood glucose homeostasis. Intriguingly, both animal and human studies revealed that PPAR£^ agonist also possesses blood pressure lowering effect, although the underlying mechanism is not clear. We therefore investigated in the present study the role of NO and O2£»− in the NTS and RVLM in the pathophysiology of the high fructose diet-induced insulin resistacne and hypertension, and to evaluate the potential central antihypertensive effect of PPAR£^ agonist in rats subjected to high fructose diet. The normotensive male Wistar Kyoto rats (WKY) were divided into 4 groups, including 3 experimental group that received 60% high fructose diet for 8 weeks and one control group that received regular chow diet for the same period of time. Within the 3 experimental groups, two of them received oral administration of rosiglitazone or pioglitazone (10 mg/kg/day) at the last two weeks (from week 6 to week 8) and the third group received saline ingestion. Systemic blood pressure was measured by tail vein sphygmomanometer very week and venous blood was drawn every other week to measure blood sugar and insulin level. At the end of the experiment, oral glucose tolerance test (OGTT) was tested and O2£»− and NO production in the NTS and RVLM were quantified. In adult male WKY rats I found that high fructose diet induced insulin resistance, hypertriglycemia and hypertension. Oral administration of rosiglitazone or pioglitazone significantly blunted the hypertension, hypertriglyceridemia, and ameliorated insulin resistance induced by high fructose diet. The high fructose diet also increased tissue level of O2£»− in the NTS and RVLM. PPAR£^ agonist treatment for two weeks did not affect the induced oxidative stress in these two nuclei. NO production was also increased in the NTS and RVLM after high fructose diet for 6 weeks. Oral treatment of rosiglitazone or pioglitazone significantly attenuated NO production after high fructose diet. At the molecular level, protein expressions of the NADPH oxdase subunits (p40phox, p47phox and gp91phox) and superoxide dismutase (cupper/zinc SOD, mitochondrial SOD, extracellular SOD) were not altered in the NTS or RVLM after high fructose diet alone or in addition with rosiglitazone or pioglitazone treatment. In the RVLM, there was a significant increase in neuronal NO synthase (nNOS) expression with concomitant decrease in inducible NOS (iNOS) expression. Oral treatment of PPAR£^ agonist for two weeks significantly suppressed the induced nNOS upregulation and attenuated the induced downregulation of iNOS expression in the RVLM. Together these results suggest that overproduction of O2£»− and NO in the NTS and RVLM may related to the development of insulin resistance-associated hypertension. Oral treatment of PPAR£^ agonist, including rosiglitazone and pioglitazone, may provide antihypertensive protection by superssing the induced-nNOS expression and increasing the induced-iNOS expression in the RVLM.