Obesity is a condition in which fat accumulation in adipose tissue is in excess to an extent that health may be impaired. Insulin resistance is integral to the pathophysiology of obesity-related metabolic complications. Central adiposity and skeletal muscle mass and function determine insulin sensitivity and metabolic risk. A high visceral fat-to-skeletal muscle mass-ratio contributes to an unfavourable metabolic profile. Epidemiological and experimental studies suggest that high dietary saturated fat intake is deleterious while polyunsaturated fatty acids (PUFAs), in particular n-3 PUFAs of marine origin, may be advantageous to metabolic health. The aim was to determine the effect of subcutaneous (SC) and visceral (IAB) fat, and long-chain saturated, n-3 and n-6 PUFAs, and the interactions between them, on insulin sensitivity and the pathways regulating energy metabolism in skeletal muscle. Thereby an adipose tissue-conditioned media-skeletal muscle myotube co-culture system was developed. Adipose tissue-conditioned medium (CM) was generated from SC and IAB fat biopsy of obese humans. Viability of the tissue explants was confirmed by the measurement of lactate dehydrogenase activity in the CM and nuclear DNA fragmentation of tissue explants. The concentrations of cytokines (leptin, adiponectin, interleukin (IL)-1β, IL 6, IL-8, tumor necrosis factor-α, resistin and plasminogen activator inhibitor-1) and long-chain fatty acids were determined in CM. CM from IAB but not SC fat reduced insulin-stimulated glucose uptake. The effect of IAB fat was predominantly mediated by IL-6 via the activation of a nuclear factor kappa B/mammalian target of rapamycin complex 1 (NFκB/mTORC1)-dependent pathway. Palmitic acid (PA; 16:0) reduced insulin-stimulated glucose uptake, an effect mediated by intramuscular accumulation of ceramide and the activation of NFκB and mTORC1. The effects of fatty acids were similar in the presence of CM from either fat depot, where the effect of PA was partially reversed by docosahexaenoic acid (DHA; 22:6n-3) and completely by linoleic acid (LA; 18:2n-6). The effect of each fatty acid in the presence or absence of CM from each fat depot on mRNA expression of key genes regulating muscle energy metabolism was determined. Protein phosphorylation of adenosine monophosphate-activated protein kinase (AMPK)-α and acetyl-coenzyme A carboxylase (ACC)-β were also determined. PA increased SCD1 mRNA. DHA and LA increased AMPKα2 mRNA and AMPKα and ACCβ protein phosphorylation. Microarray analysis was used to determine the global gene expression changes in PAand DHA-treated L6 myotubes. DHA down-regulated lipogenic genes and upregulated genes which were involved in β-oxidation and mitochondrial function. When compared to PA, DHA down-regulated genes which were involved in lipid synthesis, endoplasmic reticulum metabolism and mitogen-activated protein kinase activity. Taken together, pro-inflammatory cytokines from IAB fat and PA induced insulin resistance in skeletal muscle and both were at least partly mediated by a NFκB/mTORC1-dependent pathway. In contrast, DHA and LA may improve insulin sensitivity by diverting fatty acids towards oxidation and subsequently reducing substrate availability for the formation of lipid metabolites including ceramide. A reduction in PA intake and substitution (rather than addition) of DHA and LA, together with a reduction in overall energy intake and increase in physical activity, is optimal for metabolic health. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1523054 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010

Insulin stimulates glucose uptake in muscle and adipose tissue by translocating glucose transporter 4 (GLUT4) from an intracellular storage sige to the cellsurface. Studies have shown that insulin-activation of PI3K is required for stimulatin of GLUT4 translocation, however, platelet-derived growth factor (PDGF) also activates PI3K in adipocytes without significant effects on GLUT4 localisation. The aim of this study is to examine the hypothesis that insulin stimulates anunique signalling pathway that regulates GLUT4 translocation in adipocytes, and to identify novel signalling candidates in this pathway. A proteomic approach was employed to compare the effect of insulin versus PDGF on protein phosphorylation in 3T3-L1 adipocytes, using high-resolution two-dimensional gel electrophoresis (2-DE) in combination with 32P-metabolic labelling and autoradiography. The effect of wortmannin, an inhibitor of PI3K, on insulin-induced phosphorylation was also examined as a further screen for selection of candidate phosphoproteins for insulin-stimulation of GLUT4 translocation. Three subcellular Insulin stimulates glucose uptake in muscle and adipose tissue by translocating glucose transporter 4 (GLUT4) from an intracellular storage site to the cell surface. Studies have shown that insulin-activation of PI3K is required for stimulation of GLUT4 translocation, however, platelet-derived growth factor (PDGF) also activates PI3K in adipocytes without significant effects on GLUT4 localisation. The aim of this study is to examine the hypothesis that insulin stimulates an unique signalling pathway that regulates GLUT4 translocation in adipocytes, and to identify novel signalling candidates in this pathway. fractions were extensively examined, the plasma membrane (PM) fraction, the cytosol fraction and a high speed pellet (HSP) fraction that contains the insulin-responsive pool of GLUT4. These studies showed that phosphorylation of 18 proteins were consistently increased by insulin treatment, with 8 proteins in the cytosol, 10 proteins in the HSP and none in the PM. The phosphorylation of 6 cytosolic and 2 HSP insulin-stimulated phosphoproteins were also significantly increased by PDGF treatment. Wortmannin inhibited the insulin-stimulated phosphorylation of all but 2 proteins. Ten insulin-stimulated phosphoproteins were wortmannin-sensitive and PDGF-insensitive, and were selected as potential candidates for involvement in insulin-stimulated GLUT4 translocation. The molecular identity of insulin-stimulated phosphoproteins were initially examined by immunoblotting. Two cytosolic 2D gel spots (C65 and C79) were identified as mitogen-activated protein kinase (MAPK) isoforms by immunoblotting. Importantly, treatment-induced changes in the phosphorylation of these spots matched those reported for MAPK in adipocytes, confirming that our phosphoprotein mapping accurately reflects cellular phosphorylation. As the observed molecular mass and isoelectric point (pI) of most insulin-regulated phosphoproteins did not match known insulin-signalling proteins, we attempted to identify these by protein sequencing. In agreement with the low abundance of signalling proteins, most of the insulin-regulated phosphoprotein spots were not Coomassie-stained, even on preparative 2D gels. Thus, corresponding spots were pooled from numerous preparative 2D gels, before tryptic digestion and anlaysis by liquid chromatography-coupled tandem mass spectrometry (LC-MS). One of the more abundant candidate phosphoproteins was identified as ATP-citrate lyase (ACL), an enzyme involved in fatty acid synthesis previously shown to be an insulin-regulated phosphoprotein. Insulin treatment also results in the dephosphorylation of several phosphoproteins, one of which was identified as eukaryotic translation elongation factor 2 (eEF2). ACL and eEF2 phosphorylation were found to be regulated in an insulin-specific and wortmannin-sensitive manner in adipocytes. Recent studies suggest that protein kinase B (PKB) is activated downstream of PI3K, and participates in insulin-stimulated glucose uptake. However, the molecular mass and pI of phosphoproteins mapped in this study did not match any of the known PKB isoforms. To investigate the regulation of PKB isoform in adipocytes, studies were performed with PKB antibodies. PKBb expression was found to be induced upon differentiation of 3T3-L1 adipocytes, where insulin, but not PDGF stimulated its phosphorylation. In contrast, PKBa expression was high in fibroblasts, where PDGF was more efficacious than insulin in inducing its phosphorylation. These results suggest that PKBb, rather than PKBa, is the most pertinent isoform for insulin action in adipocytes. PDGF exerted no detectable effect on the phosphorylation of PKBb in 3T3-L1 adipocytes, as determined by phospho-specific antibodies, 32P-labelling and shifts in electrophoretic mobility and pI. Furthermore, insulin stimulated the translocation of PKBb to membrane fractions of adipocytes, whereas PDGF was without effect. Together, these results strongly suggest that PDGF does not activate PKBb in 3T3-L1 adipocytes. Using 2-DE, we have obtained data which support the hypothesis that insulin stimulates an unique signalling pathway in adipocytes. Three potential models are proposed to explain the insulin specificity: targeting of PI3K to pre-assembled signalling complexes in the HSP, activation of specific PI3K isoforms, and activation of two PI3K-dependent signalling pathways. These models remain to be validated in future studies. Candidate phosphoproteins for insulin regulation of glucose metabolism have been mapped using 2-DE. While only abundant metabolic enzymes have been identified so far, methods for phosphoprotein purification and identification have been established. The candidate phosphoproteins mapped in the HSP fraction are likely to be low abundance signalling proteins, and further preparative isolation of these phosphoproteins is required in order to obtain enough protein for identification of these candidates. In conclusion, the present study has demonstrated the successful application of proteomic techniques in cell signalling research.

Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "December, 2007." Includes bibliographical references. Online version available on the World Wide Web.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005. / Also available in print.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Thesis (M.A.)--University of Hawaii at Manoa, 2001. / Includes bibliographical references (leaves 92-101). Also available on microfiche.

Thesis (M.A.)--University of Adelaide, 1989. / Includes bibliographical references (leaves 191-204).

Thesis (Ph.D.) from the Dept. of Physical and Inorganic Chemistry, University of Adelaide, 1972.