The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

Independent, marked associations of alleles of the insulin receptor and dipeptidyl carboxypeptidase-I genes with essential hypertension

1. There is evidence to suggest that essential hypertension is a polygenic disorder and that it arises from yet-to-be-identified predisposing variants of certain genes that influence blood pressure. The cloning of various hormone, enzyme, adrenoceptor and hormone receptor genes whose products are involved in blood pressure control and the identification of polymorphisms of these has permitted us to test their genetic association with hypertension. 2. Cross-sectional analyses of a number of candidate gene markers were performed in hypertensive and normotensive subjects who were selected on the basis of both parents being either hypertensive or normotensive, respectively, and the difference in total alleles on all chromosomes for each polymorphism between the hypertensive and normotensive groups was test by χ analysis with one degree of freedom. 3. A marked association was observed between hypertension and insertion alleles of polymorphisms of the insulin receptor gene (INSR) (P<0.0040) and the dipeptidyl carboxypeptidase-1 (angiotensin I-converting enzyme; kininase II) gene (DCP1) (P<0.0018). No association with hypertension was evident, however, for polymorphisms of the growth hormone, low-density lipoprotein receptor, renal kallikrein, α2- and β1-adrenoreceptor, atrial natriuretic factor and insulin genes. 4. All but one of the hypertensive subjects had at least one of the hypertension-associated alleles, and although subjects homozygous for both were three times more frequent in the hypertensive group, examination of the nine possible genotypes suggested that the INSR and DCP1 alleles are independent markers for hypertension. 5. The present results suggest that genetic variant(s) in close linkage disequilibrium with polymorphisms at INSR and DCP1 may be involved in part in the aetiology of essential hypertension.

Collaboration and Engagement Throughout The Supply Chain : The Role of Government

Collaboration is acknowledged as a key to continued growth in the Australian construction industry. Government, as a major industry client, has an important role to play with respect to fostering collaboration and ensuring the global competitiveness of the industry. The paper draws upon data collected for the Construction 2020 study and aims to demonstrate that government can a) help to break down the adversarial situation that currently exists between clients, project managers and subcontractors; and b) allow the supply chain to collaborate more effectively in terms of satisfying the relational and financial needs of all parties. Government can also provide a clear set of guidelines (backed up by a functional dispute resolution system) that will promote confidence with respect to forging relationships. Thus, the paper will discuss the way in which public policy can be more closely aligned with actual industry needs in order to promote greater collaboration.

Crystallographic characterization of the first reported crystalline form of the potent hallucinogen (R)-2-amino-1-(8-bromobenzo[1,2-b;5,4-b']difuran-4-yl)propane or `bromodragonfly': the 1:1 anhydrous proton-transfer compound with 3,5-dinitrosalicylic acid

The 1:1 proton-transfer compound of the potent substituted amphetamine hallucinogen (R)-1-(8-bromobenzo[1,2-b; 4,5-b']difuran-4-yl)-2-aminopropane (common trivial name 'bromodragonfly') with 3,5-dinitrosalicylic acid, 1-(8-bromobenzo[1,2-b;4,5-b']difuran-4-yl)-2-mmoniopropane 2-carboxy-4,6-dinitrophenolate, C13H13BrNO2+ C7H3N2O7- forms hydrogen-bonded cation-anion chain substructures comprising undulating head-to-tail anion chains formed through C(8) carboxyl O-H...O(nitro) associations and incorporating the aminium groups of the cations. The intra-chain cation-anion hydrogen-bonding associations feature proximal cyclic R33(8) interactions involving both a N+-H...O(phenolate) and the carboxyl O--H...O(nitro)associations. Also present are aromatic pi-pi ring interactions [minimum ring centroid separation, 3.566(2)A; inter-plane dihedral angle, 5.13(1)deg]. A lateral hydrogen-bonding interaction between the third aminium proton and a carboxyl O acceptor link the chain substructures giving a two-dimensional sheet structure. This determination represents the first of any form of this compound and confirms that it has the (R) absolute configuration. The atypical crystal stability is attributed both to the hydrogen-bonded chain substructures provided by the anions, which accommodate the aminium proton-donor groups of the cations and give cross-linking, and to the presence of cation--anion aromatic ring pi-pi interactions.

Human preadipocyte proliferation and differentiation

Obesity represents a major health, social and economic burden to many developing and Westernized communities, with the prevalence increasing at a rate exceeding almost all other medical conditions. Despite major recent advances in our understanding of adipose tissue metabolism and dynamics, we still have limited insight into the regulation of adipose tissue mass in humans. Any significant increase in adipose tissue mass requires proliferation and differentiation of precursor cells (preadipocytes) present in the stromo-vascular compartment of adipose tissue. These processes are very complex and an increasing number of growth factors and hormones have been shown to modulate the expression of genes involved in preadipocyte proliferation and differentiation. A number of transcription factors, including the C/EBP family and PP ARy, have been identified as integral to adipose tissue development and preadipocyte differentiation. Together PP ARy and C/EBPa regulate important events in the activation and maintenance of the terminally differentiated phenotype. The ability of PP ARy to increase transcription through its DNA recognition site is dependent on the binding of ligands. This suggests that an endogenous PP ARy ligand may be an important regulator of adipogenesis. Adipose tissue functions as both the major site of energy storage in the body and as an endocrine organ synthesizing and secreting a number of important molecules involved in regulation of energy balance. For optimum functioning therefore, adipose tissue requires extensive vascularization and previous studies have shown that growth of adipose tissue is preceded by development of a microvascular network. This suggests that paracrine interactions between constituent cells in adipose tissue may be involved in both new capillary formation and fat cell growth. To address this hypothesis the work in this project was aimed at (a) further development of a method for inducing preadipocyte differentiation in subcultured human cells; (b) establishing a method for simultaneous isolation and separate culture of both preadipocytes and microvascular endothelial cells from the same adipose tissue biopsies; (c) to determine, using conditioned medium and co-culture techniques, if endothelial cell-derived factors influence the proliferation and/or differentiation of human preadipocytes; and (d) commence characterization of factors that may be responsible for any observed paracrine effects on aspects of human adipogenesis. Major findings of these studies were as follows: (A) Inclusion of either linoleic acid (a long-chain fatty acid reported to be a naturally occurring ligand for PP ARy) or Rosiglitazone (a member of the thiazolidinedione class of insulin-sensitizing drugs and a synthetic PPARy ligand) in differentiation medium had markedly different effects on preadipocyte differentiation. These studies showed that human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation, and that thiazolidinediones and fatty acids may exert their adipogenic and lipogenic effects via different biochemical pathways. It was concluded that Rosiglitazone is a more potent inducer of human preadipocyte differentiation than linoleic acid. (B) A method for isolation and culture of both endothelial cells and preadipocytes from the same adipose tissue biopsy was developed. Adipose-derived microvascular endothelial cells were found to produce factor/s, which enhance both proliferation and differentiation of human preadipocytes. (C) The adipogenic effects of microvascular endothelial cells can be mimicked by exposure of preadipocytes to members of the Fibroblast Growth Factor family, specifically ~-ECGF and FGF-1. (D) Co-culture of human preadipocytes with endothelial cells or exposure of preadipocytes to either ~-ECGF or FGF-1 were found to 'prime' human preadipocytes, during their proliferative phase of growth, for thiazolidinedione-induced differentiation. (E) FGF -1 was not found to be acting as a ligand for PP ARy in this system. Findings from this project represent a significant step forward in our understanding of factors involved in growth of human adipose tissue and may lead to the development of therapeutic strategies aimed at modifying the process. Such strategies would have potential clinical utility in the treatment of obesity and obesity related disorders such as Type II Diabetes.

Studies of granulosa cell maturation in dominant and subordinate bovine follicles : novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1

During growth of antral ovarian follicles granulosa cells first become associated with a novel type of extracellular matrix, focimatrix, and at larger sizes follicles become either subordinate or dominant. To examine this, bovine subordinate (9.0±s.e.m. 0.4 mm; n=16), partially dominant (12.0±0.6 mm; n=18) and fully dominant (15.0±0.4 mm; n=14) follicles were examined by real time RT-PCR analyses of granulosa cells and by immunohistochemistry of focimatrix. Changes in the expression of FSH receptor, LH receptor, cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase, aromatase (CYP19A1) and inhibin-α and β-B were observed as expected for follicle sizes examined. After adjusting for size differences, only CYP11A1 was significantly different between the groups, and elevated in dominant follicles. Also after adjusting for differences in size there were no significant differences in expression of focimatrix components collagen type IV α-1 (COL4A1), laminin β-2, nidogen 1 (NID1), and perlecan (HSPG2) or the volume density of NID1 and -2 and HSPG2. The volume density of focimatrix components in laminin 111 was significantly elevated in dominant follicles. Adjusting for analysis of more than one follicle per animal and for multiple correlations, CYP11A1 mRNA levels were highly correlated with the focimatrix genes COL4A1, NID1 and -2 and HSPG2. Thus, focimatrix may potentially regulate CYP11A1 expression, and the regulation of both could be important in follicular dominance.

Cyclic imides and an open-chain amide carboxylic acid from the facile reaction of cis-cyclohexane-1,2-dicarboxylic anhydride with the isomeric monofluoroanilines

The structures of the cyclic imides cis-2-(2-fluorophenyl)-3a,4,5,6,7,7a-hexahydroisoindole-1,3-dione, C14H14FNO2, (I), and cis-2-(4-fluorophenyl)-3a,4,5,6,7,7a-hexahydroisoindoline-1,3-dione, C14H14FNO2, (III), and the open-chain amide acid rac-cis-2-[(3-fluorophenyl)carbamoyl]cyclohexane-1-carboxylic acid, C14H16FNO3, (II), are reported. Cyclic imides (I) and (III) are conformationally similar, with comparable ring rotations about the imide N-Car bond [the dihedral angles between the benzene ring and the five-membered isoindole ring are 55.40 (8)° for (I) and 51.83 (7)° for (III)]. There are no formal intermolecular hydrogen bonds involved in the crystal packing of either (I) or (III). With the acid (II), in which the meta-related F-atom substituent is rotationally disordered (0.784:0.216), the amide group lies slightly out of the benzene plane [the interplanar dihedral angle is 39.7 (1)°]. Intermolecular amide-carboxyl N-HO hydrogen-bonding interactions between centrosymmetrically related molecules form stacks extending down b, and these are linked across c by carboxyl-amide O-HO hydrogen bonds, giving two-dimensional layered structures which lie in the (011) plane. The structures reported here represent examples of compounds analogous to the phthalimides or phthalanilic acids and have little precedence in the crystallographic literature.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n = 4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n = 4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and 48 h (n = 4), and in normal control horses (n = 4). The only change in gene expression observed was an upregulation of MMP-9 (p < 0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p < 0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Cyclic Imide and Open-Chain Amide Carboxylic Acid Derivatives from the Facile Reaction of cis-Cyclohexane-1,2-dicarboxylic Anhydride with Three Substituted Anilines

The structures of the compounds from the reaction of cis-cyclohexane-1,2-dicarboxylic anhydride with 4-chloroaniline [rac-N-(4-chlorophenyl)-2-carboxycycloclohexane-1-carboxamide] (1), 4-bromoaniline [2-(4-bromophenyl)-perhydroisoindolyl-1,3-dione] (2) and 3-hydroxy-4-carboxyaniline (5-aminosalicylic acid) [2-(3-hydroxy-4-carboxyphenyl)-perhydroisoindolyl-1,3-dione] (3) have been determined at 200 K. Crystals of the open-chain amide carboxylic acid 1 are orthorhombic, space group Pbcn, with unit cell dimensions a = 20.1753(10), b = 8.6267(4), c = 15.9940(9) Å, and Z = 8. Compounds 2 and 3 are cyclic imides, with 1 monoclinic having space group P21 and cell dimensions a = 11.5321(3), b = 6.7095(2), c = 17.2040(5) Å, β = 102.527(3)o. Compound 3 is orthorhombic with cell dimensions a = 6.4642(3), b = 12.8196(5), c = 16.4197(7) Å. Molecules of 1 form hydrogen-bonded cyclic dimers which are extended into a two-dimensional layered structure through amide-group associations: 3 forms into one-dimensional zigzag chains through carboxylic acid…imide O-atom hydrogen bonds, while compound 2 is essentially unassociated. With both cyclic imides 2 and 3, disorder is found which involves the presence of partial enantiomeric replacement of the cis-cyclohexane-1,2-substituted ring systems.

Insulin and the insulin-like growth factor system : a novel theory for hyperinsulinemic laminitis pathogenesis

Research into hyperinsulinemic laminitis has progressed significantly in recent years with the use of the prolonged-euglycemic, hyperinsulinemic clamp (p-EHC). Previous investigations of laminitis pathophysiology have focused on digital vascular dysfunction, inflammation, altered glucose metabolism within the lamellae, and lamellar basement membrane breakdown by metalloproteinases. The etiopathogenesis of laminitis occurring in association with hyperinsulinemia is yet to be fully characterized, but it may not involve these mechanisms. Insulin stimulates cellular proliferation and can also affect other body systems, such as the insulin-like growth factor (IGF) system. Insulin-like growth factor-1 (IGF-1) is structurally homologous to insulin and, like insulin, binds with strong affinity to a specific tyrosine kinase receptor on the cell surface to produce its effects, which include promoting cell proliferation. Receptors for IGF-1 (IGF-1R) are present in the lamellar epidermis. An alternative theory for the pathogenesis of hyperinsulinemic laminitis is that uncontrolled cell proliferation, mediated through both the insulin receptor (InsR) and IGF-1R, leads to lengthening, weakening, and failure of the lamellae. An analysis of the proliferative activity of lamellar epidermal cells during the developmental and acute phases of hyperinsulinemic laminitis, and lamellar gene expression of the InsR and IGF-1R was undertaken.

The individual and interactive relationship between long chain omega-3 polyunsaturated fatty acids and physical activity as predictors of cognition in cognitively impaired and non-impaired older adults

Alterations in cognitive function are characteristic of the aging process in humans and other animals. However, the nature of these age related changes in cognition is complex and is likely to be influenced by interactions between genetic predispositions and environmental factors resulting in dynamic fluctuations within and between individuals. These inter and intra-individual fluctuations are evident in both so-called normal cognitive aging and at the onset of cognitive pathology. Mild Cognitive Impairment (MCI), thought to be a prodromal phase of dementia, represents perhaps the final opportunity to mitigate cognitive declines that may lead to terminal conditions such as dementia. The prognosis for people with MCI is mixed with the evidence suggesting that many will remain stable within 10-years of diagnosis, many will improve, and many will transition to dementia. If the characteristics of people who do not progress to dementia from MCI can be identified and replicated in others it may be possible to reduce or delay dementia onset, thus reducing a growing personal and public health burden. Furthermore, if MCI onset can be prevented or delayed, the burden of cognitive decline in aging populations worldwide may be reduced. A cognitive domain that is sensitive to the effects of advancing age, and declines in which have been shown to presage the onset of dementia in MCI patients, is executive function. Moreover, environmental factors such as diet and physical activity have been shown to affect performance on tests of executive function. For example, improvements in executive function have been demonstrated as a result of increased aerobic and anaerobic physical activity and, although the evidence is not as strong, findings from dietary interventions suggest certain nutrients may preserve or improve executive functions in old age. These encouraging findings have been demonstrated in older adults with MCI and their non-impaired peers. However, there are some gaps in the literature that need to be addressed. For example, little is known about the effect on cognition of an interaction between diet and physical activity. Both are important contributors to health and wellbeing, and a growing body of evidence attests to their importance in mental and cognitive health in aging individuals. Yet physical activity and diet are rarely considered together in the context of cognitive function. There is also little known about potential underlying biological mechanisms that might explain the physical activity/diet/cognition relationship. The first aim of this program of research was to examine the individual and interactive role of physical activity and diet, specifically long chain polyunsaturated fatty acid consumption(LCn3) as predictors of MCI status. The second aim is to examine executive function in MCI in the context of the individual and interactive effects of physical activity and LCn3.. A third aim was to explore the role of immune and endocrine system biomarkers as possible mediators in the relationship between LCn3, physical activity and cognition. Study 1a was a cross-sectional analysis of MCI status as a function of erythrocyte proportions of an interaction between physical activity and LCn3. The marine based LCn3s eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have both received support in the literature as having cognitive benefits, although comparisons of the relative benefits of EPA or DHA, particularly in relation to the aetiology of MCI, are rare. Furthermore, a limited amount of research has examined the cognitive benefits of physical activity in terms of MCI onset. No studies have examined the potential interactive benefits of physical activity and either EPA or DHA. Eighty-four male and female adults aged 65 to 87 years, 50 with MCI and 34 without, participated in Study 1a. A logistic binary regression was conducted with MCI status as a dependent variable, and the individual and interactive relationships between physical activity and either EPA or DHA as predictors. Physical activity was measured using a questionnaire and specific physical activity categories were weighted according to the metabolic equivalents (METs) of each activity to create a physical activity intensity index (PAI). A significant relationship was identified between MCI outcome and the interaction between the PAI and EPA; participants with a higher PAI and higher erythrocyte proportions of EPA were more likely to be classified as non-MCI than their less active peers with less EPA. Study 1b was a randomised control trial using the participants from Study 1a who were identified with MCI. Given the importance of executive function as a determinant of progression to more severe forms of cognitive impairment and dementia, Study 1b aimed to examine the individual and interactive effect of physical activity and supplementation with either EPA or DHA on executive function in a sample of older adults with MCI. Fifty male and female participants were randomly allocated to supplementation groups to receive 6-months of supplementation with EPA, or DHA, or linoleic acid (LA), a long chain polyunsaturated omega-6 fatty acid not known for its cognitive enhancing properties. Physical activity was measured using the PAI from Study 1a at baseline and follow-up. Executive function was measured using five tests thought to measure different executive function domains. Erythrocyte proportions of EPA and DHA were higher at follow-up; however, PAI was not significantly different. There was also a significant improvement in three of the five executive function tests at follow-up. However, regression analyses revealed that none of the variance in executive function at follow-up was predicted by EPA, DHA, PAI, the EPA by PAI interaction, or the DHA by PAI interaction. The absence of an effect may be due to a small sample resulting in limited power to find an effect, the lack of change in physical activity over time in terms of volume and/or intensity, or a combination of both reduced power and no change in physical activity. Study 2a was a cross-sectional study using cognitively unimpaired older adults to examine the individual and interactive effects of LCn3 and PAI on executive function. Several possible explanations for the absence of an effect were identified. From this consideration of alternative explanations it was hypothesised that post-onset interventions with LCn3 either alone or in interation with self-reported physical activity may not be beneficial in MCI. Thus executive function responses to the individual and interactive effects of physical activity and LCn3 were examined in a sample of older male and female adults without cognitive impairment (n = 50). A further aim of study 2a was to operationalise executive function using principal components analysis (PCA) of several executive function tests. This approach was used firstly as a data reduction technique to overcome the task impurity problem, and secondly to examine the executive function structure of the sample for evidence of de-differentiation. Two executive function components were identified as a result of the PCA (EF 1 and EF 2). However, EPA, DHA, the PAI, or the EPA by PAI or DHA by PAI interactions did not account for any variance in the executive function components in subsequent hierarchical multiple regressions. Study 2b was an exploratory correlational study designed to explore the possibility that immune and endocrine system biomarkers may act as mediators of the relationship between LCn3, PAI, the interaction between LCn3 and PAI, and executive functions. Insulin-like growth factor-1 (IGF-1), an endocrine system growth hormone, and interleukin-6 (IL-6) an immune system cytokine involved in the acute inflammatory response, have both been shown to affect cognition including executive functions. Moreover, IGF-1 and IL-6 have been shown to be antithetical in so far as chronically increased IL-6 has been associated with reduced IGF-1 levels, a relationship that has been linked to age related morbidity. Further, physical activity and LCn3 have been shown to modulate levels of both IGF-1 and IL-6. Thus, it is possible that the cognitive enhancing effects of LCn3, physical activity or their interaction are mediated by changes in the balance between IL-6 and IGF-1. Partial and non-parametric correlations were conducted in a subsample of participants from Study 2a (n = 13) to explore these relationships. Correlations of interest did not reach significance; however, the coefficients were quite large for several relationships suggesting studies with larger samples may be warranted. In summary, the current program of research found some evidence supporting an interaction between EPA, not DHA, and higher energy expenditure via physical activity in differentiating between older adults with and without MCI. However, a RCT examining executive function in older adults with MCI found no support for increasing EPA or DHA while maintaining current levels of energy expenditure. Furthermore, a cross-sectional study examining executive function in older adults without MCI found no support for better executive function performance as a function of increased EPA or DHA consumption, greater energy expenditure via physical activity or an interaction between physical activity and either EPA or DHA. Finally, an examination of endocrine and immune system biomarkers revealed promising relationships in terms of executive function in non-MCI older adults particularly with respect to LCn3 and physical activity. Taken together, these findings demonstrate a potential benefit of increasing physical activity and LCn3 consumption, particularly EPA, in mitigating the risk of developing MCI. In contrast, no support was found for a benefit to executive function as a result of increased physical activity, LCn3 consumption or an interaction between physical activity and LCn3, in participants with and without MCI. These results are discussed with reference to previous findings in the literature including possible limitations and opportunities for future research.

Association of a RFLP for the insulin receptor gene, but not insulin, with essential hypertension

Insulin has cardiovascular actions and patients with essential hypertension display insulin resistance. A cross-sectional study of the R1 RFLP of the insulin receptor gene (INSR) was carried out in 67 hypertensive (HT) and 75 normotensive (NT) subjects whose parents had a similar blood pressure status at age ≥50. The frequency of the minor (+) allele was 0.31 in HTs and 0.44 in NTs, and the difference between observed alleles in all subjects in each group was significant (χ2 = 4.8, P<0.05). Allele frequencies of a BglI RFLP of the insulin gene, however, did not differ between the HT and NT groups. The data thus provide evidence in favour of an association of HT with a polymorphism at the INSR locus (19p 13.3-13.2), so implicating this locus, and possibly a genetic variant of the insulin receptor itself, in HT.

Non-linkage of insulin receptor locus with essential hypertension in an affected pedigree

A recent cross-sectional study has demonstrated a significant association of the R1 RsaI restriction fragment length polymorphism of the insulin receptor gene (INSR) with human essential hypertension. In the present study, an alternative approach, involving linkage analysis, was carried out using 8 hypertensive families with 5 or more affected members. Five of the families were found to be informative and in one of these pedigrees a conclusion of non-linkage of INSR and hypertension could be made on the basis of an obligate recombinant in one generation which yielded a Lod score of - ∞ at a recombination fraction (θ) of zero. In another family, the largest studied, a positive Lod score was obtained at θ = 0, but this was below the level required for a conclusion of linkage. Lod score at θ = 0 for a marker at the insulin locus in this family was negative. The present study has thus demonstrated one pedigree in which hypertension is not linked to the insulin receptor locus.