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<p>Prediabetes can lead to Type II Diabetes Mellitus, yet Prediabetes is a disease in its own right with its own physiological complications. Despite the pervasiveness of Prediabetes in our society and the negative impact on current and future health the extent of myopathy, short of muscle insulin resistance, and the mechanisms behind development of muscle insulin resistance remains unclear. Animal models of diet-induced obesity (DIO) have been employed to assess development of muscle insulin resistance and changes to muscle health. However there is a lack of clarity as to the molecular mechanisms leading to muscle insulin resistance. The goal of the studies presented here was to elucidate changes to muscle health and potential mechanisms contributing to muscle insulin resistance in response to DIO. Since the ability to perform exercise is to date one of the best therapies for Prediabetes and exercise contributes to a healthy muscle mass, the ability of muscle to undergo proper regeneration was also assessed following DIO. The results presented in this work demonstrate that skeletal muscle tissue adapts to increased dietary lipid by an early increase in functional lipid oxidation, mitigating IMCL deposition, despite glucose intolerance. Unfortunately this adaptive response is reversed with prolonged dietary fat intake and the development of muscle insulin resistance. Of note was the stronger link between IMCLs and muscle insulin resistance, compared to inflammation. DIO also led to decrements in satellite cell functionality which, along with physiological changes to HGF content and signaling, likely resulted in the observed impairment in regenerative ability. The results reported here improve our understanding of changes to muscle health and the mechanisms behind development of muscle insulin resistance with DIO. These findings have implications for therapies and treatments for Prediabetes.</p> / Doctor of Philosophy (Medical Science)

Résumé : Le diabète gestationnel (DG), qui peut entraîner des conséquences importantes pour la mère et l’enfant, résulte d’un défaut de compensation de la sécrétion d’insuline par rapport à la résistance à l’insuline. Comme la grossesse représente en elle-même un modèle d’augmentation physiologique de la résistance à l’insuline, il est intéressant d’étudier et de caractériser les facteurs qui sont impliqués dans la résistance à l’insuline et, ultimement, dans le DG, chez la femme enceinte. Le Tumor necrosis factor alpha (TNFα) est soupçonné d’être un de ces facteurs, suite aux études effectuées chez les animaux et les populations humaines non enceintes, mais les résultats obtenus en grossesse sont encore controversés. Nous avons émis l’hypothèse que les niveaux circulants de TNFα sont associés au DG et à la résistance à l’insuline dans une large cohorte de femmes enceintes. Nous avons aussi investigué les variations des niveaux de TNFα en réponse à l’hyperglycémie provoquée par voie orale (HGPO) chez des femmes enceintes. Nous avons montré que de hauts niveaux de TNFα étaient liés à une résistance à l’insuline augmentée au 2e trimestre de la grossesse et ce, indépendamment de l’âge, de l’adiposité, de l’âge gestationnel, des triglycérides et des niveaux circulants d’adiponectine dans notre cohorte. De plus, les niveaux de TNFα varient différemment au cours de l’HGPO selon le statut de résistance à l’insuline. En effet, les niveaux de TNFα augmentent à 1h puis diminuent à 2h chez les femmes les plus sensibles à l’insuline, alors qu’ils diminuent tout au long du test chez les femmes les plus résistantes à l’insuline, mais restent en tout temps supérieurs aux niveaux mesurés chez les femmes les plus sensibles à l’insuline. Toutefois, les niveaux de TNFα n’étaient pas différents entre les femmes avec DG et celles normoglycémiques. De façon intéressante, la variation du TNFα pendant l’HGPO chez les femmes DG est similaire à celle chez les femmes avec haute résistance à l’insuline. Ces résultats suggèrent donc que le TNFα est indépendamment associé à la résistance à l’insuline en grossesse et que les voies inflammatoires peuvent contribuer aux dysfonctions glycémiques retrouvées en DG. // Abstract : Gestational diabetes mellitus (GDM), which can exert important impacts on mothers and offspring, results from an imbalance between insulin secretion capacity and insulin resistance. Pregnancy is a state of physiologically increased insulin resistance, providing a unique model to study and characterize biological factors linked to insulin resistance in humans and, ultimately, GDM, in pregnant women. Based on animal studies and analyses in non-pregnant populations, tumor necrosis factor alpha (TNFα) is suspected of being involved in insulin resistance, but results obtained from pregnant populations are still controversial. Our hypothesis was that circulating TNFα would be associated with GDM and insulin resistance in a large cohort of pregnant women. We also investigated dynamic variations of TNFα levels over the course of an oral glucose tolerance test (OGTT) in pregnant women. We showed that higher TNFα levels were associated with higher insulin resistance at 2nd trimester of pregnancy, independent of age, adiposity, gestational age, triglycerides and adiponectin levels in our cohort. Furthermore, TNFα levels varied differently over the course of the OGTT according to insulin resistance status: they rose at 1h and then decreased at 2h in insulin sensitive women, whereas they consistently decreased in insulin resistant women over the course of the test (even though they remained statistically higher than insulin sensitive women’s levels at each time point throughout the OGTT). However, TNFα levels were not different between GDM and non-GDM women. Interestingly, variation of TNFα levels over the course of the OGTT in GDM women followed the same pattern as the variation observed in OGTT in women classified with high insulin resistance. Those results suggest that circulating TNFα is independently associated with insulin resistance in pregnancy and that inflammatory pathways might contribute to glycemic dysregulation observed in GDM.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity. / AFRIKAANSE OPSOMMING: INLEIDING: Die meganisme verantwoordelik vir vetsug-verwante kardiomiopatieë is huidiglik nie bekend nie maar kardiale insulienweerstandigheid word geïmpliseer as een van die hoof bemiddelaars van vetsug-verwante hartsiektes. Verskeie studies het versteurings in die insulien-gemediëerde PI3K/PKB/Akt pad geassosieer met die bevordering van hierdie insulienweerstandigheid. Daarbenewens is dit getoon dat hierdie pad betrokke is in die regulering van miokardiale apoptose, wat op sy beurt geïmpliseer is in 'n aantal kardiovaskulêre siektes. Daar is tans min studies beskikbaar wat die vroeë en laat gevolge van obesiteit op die hart vergelyk. DOELWITTE: Om die vroeë en gevorderde stadiums van vetsug te vergelyk in terme van miokardiale (i) PI3K/PKB/Akt seintransduksie, (ii) apoptotiese seintransduksie en (iii) mitokondriale integriteit. Verder, het die studie ten doel om die kardiale mitokondriale (i) PI3K/PKB/Akt en (ii) apoptotiese seintransduksie en (iii) integriteit in die gevorderde stadiums van vetsug te bepaal. METODES: Manlike Wistar rotte is ewekansig toegewys aan óf 'n kontrole of dieet-geïnduseerde vetsug (DIO) groep. Kontroles is met 'n normale rotkos dieet en die DIO groep met 'n hoë kalorie dieet (normale rotkos aangevul met sukrose en kondensmelk) gevoed. Die dieet is vir 8 of 20 weke volgehou en daarna was die liggaamsgewig, intra-peritoneale vet massa, en vastende bloed glukose en insulien vlakke (insluitende intra-peritoneale glukose toleransie toets (IPGTT`s)) bepaal. Gevriesklampte harte van beide groepe is onderwerp aan sitosoliese WB-analise vir die PI3K p85 subeenheid, PKB / Akt, GSK-3α/β, Bad, Bax en Bcl-2. `n Fraksie van hierdie harte is ook onderwerp aan westerse klad analise (WK-analise) van die mitokondriale elektron vervoer ketting (EVK) komplekse (I-V). Daarna is bogenoemde proteïene ondersoek in mitokondrieë geïsoleer uit die 20 weke groep ná die toediening van insulien en die blootstelling van die harte aan iskemie. Die oksigraaf mitokondriale oksidatiewe fosforilering (OXPHOS) kapasiteit analise is dan op mitokondrieë van 20 weke DIO en kontrole groepe uitgevoer en daarna is 'n sitraatsintase (SS) aktiwiteitstoets gedoen. RESULTATE: Na die 8 en 20 weke dieet, het die intra-peritoneale vet massa, vastende plasma glukose en insulien vlakke in die DIOs aansienlik toegeneem, in vergelyking met hul kontroles. Sitosoliese WK-analise: Die tp85, pp85 en pPKB/Akt vlakke was beduidend hoër in die DIOs in vergelyking met die kontroles, na 8 weke van die dieet. Verder is die pBad en Bax vlakke beduidend verhoog in hierdie diere. Na 20 weke van die dieet, het die pp85, tPKB/Akt en pPKB/Akt vlakke beduidend afgeneem in die DIOs, in vergelyking met die kontroles. Die tBad was beduidend verhoog, terwyl die Bad verhouding van gefosforileerde oor die totale proteïen uitdrukking (P/T)-verhouding) beduidend verminder het in hierdie diere. SS aktiwiteitstoets: SS aktiwiteit is beduidend verminder in die DIOs, teenoor die kontroles, op 20 weke. Mitokondriale EVK WK-analise: Die subeenheid uitdrukking in komplekse I-III en V was nie beduidend verskillend na 8 weke nie. Na 20 weke egter, was die uitdrukking aansienlik laer in komplekse I en II. Interessant genoeg, is die uitdrukking aansienlik verhoog in komplekse III en V. Mitokondriale OXPHOS analise: Die ADP/O verhouding met (1) glutamaat of (2) palmitiel-L-karnitien as substraat, het beduidend afgeneem in die DIOs teen 20 weke. Mitokondriale WK-analise: Die pp85 subeenheid was beduidend verhoog in die kontrole en DIO groepe, blootgestel aan insulien en iskemie, in vergelyking met die onbehandelde kontroles. Die Bcl-2 vlakke was beduidend verminder in die insulien en isgemie DIOs, in vergelyking met onbehandelde DIOs. Die tBad uitdrukking het nie beduidend verskil tussen die insulien en onbehandelde kontroles nie, terwyl die tBad beduidend verhoog was in die isgemie kontroles versus onbehandelde kontroles. Alle beduidende verskille is geneem as p<0.05. GEVOLGTREKKING: Die resultate dui daarop dat die eerste fase van dieet-geïnduseerde obesiteit geassosieer is met kardiale beskerming want `n toename in PI3K/PKB/Akt seintransduksie en 'n afname in apoptotiese merkers is waargeneem. In teenstelling, in die gevorderde stadium van vetsug is daar 'n afname in aktiwiteit in die PI3K/PKB/Akt pad wat verband hou met verhoogde miokardiale apoptose en verminderde mitokondriale funksie en integriteit.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical (HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter protein function and contribute to the onset of myocardial insulin resistance and impaired contractile function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation, and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4 translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose. Although our results were not conclusive, the data suggest a potential novel link between dexamethasone exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and related metabolic derangements and diseases), these results provide new targets for potential therapeutic agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier (HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie. Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak, die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê. Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4) translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4 translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie. Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.

Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCb) is induced in a glucose-dependent manner. ACCb produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake. Previous studies show that increased flux through the hexosamine biosynthetic pathway (HBP) under hyperglycaemic conditions may contribute to the development of insulin resistance. In light of this, we hypothesised that increased HBP flux induces cardiac ACCb gene expression thereby contributing to the onset of insulin resistance. We tested our hypothesis by transiently transfecting cardiac-derived rat H9c2 myoblasts with a 1,317 bp human ACCb promoter-luciferase construct (pPIIb-1317) and an expression construct encoding the rate-limiting step of the HBP i.e. glutamine: fructose 6-phosphate amidotransferase (GFAT). Overexpression of GFAT increased ACCb gene promoter activity by 75 ± 23% versus controls (n=6, p<0.001). When cotransfection experiments were repeated in the presence of varying concentrations of L-glutamine (0 mM, 4 mM, 8 mM), a substrate for the HBP, ACCb promoter activity was dose-dependently increased. To further corroborate these findings, we employed two inhibitors of GFAT, i.e. 40 μM azaserine and 40 μM 6-diazo-5-oxo-Lnorleucine were administered to transfected cells for a period of 24 hours. Here both azaserine and 6-diazo-5-oxonorleucine attenuated ACCb gene promoter activity. In agreement, co-transfections with two dominant negative GFAT constructs also diminished ACCb gene promoter activity. We next inhibited two enzymes of the HBP acting downstream of GFAT, i.e. O-GlcNAc transferase and O-GlcNAcase using alloxan (0.1 mM, 1 mM and 2 mM) and streptozotocin (5 mM and 10 mM), respectively, for a period of 24 hours. Addition of alloxan attenuated ACCb gene promoter activity by 35.6 ± 1.9% (n=16, p<0.001) and streptozotocin increased activity by 32 ± 12% (n=12, p<0.001). We also investigated USF1 and USF2 as transcriptional regulatory candidates for HBP-induced ACCβ promoter regulation. Our data implicates USF2 as an important transcriptional regulator of HBP-induced ACCβ promoter regulation. In summary, this study demonstrates that increased flux through the hexosamine biosynthetic pathway induces ACCb gene promoter activity. We further propose that such an induction would reduce cardiac fatty acid oxidation, thereby leading to intracellular lipid accumulation due to a mismatch between sarcolemmal FA uptake and mitochondrial FA oxidation in the insulin resistant setting (i.e. hyperlipidaemia). / AFRIKAANSE OPSOMMING: Geen uitdrukking van die kardiale isoform asetiel-KoA karboksilase (ACCb) word in ‘n glukose afhanklike wyse geïnduseer. ACCb produseer maloniel-KoA, ‘n kragtige inhibeerder van mitochondriale vetsuuropname. Vorige studies toon aan dat verhoogde fluks deur die heksosamien biosintestiese weg (HBW) onder hiperglukemiese toestande bydra tot die ontwikkeling van insulienweerstand. In die lig hiervan, word daar gehipotetiseer dat verhoogde HBP fluks kardiale ACCb geenuitdrukking induseer en so bydra tot die ontstaan van insulienweerstand. Ons hipotese is getoets deur die kardiale afkomstige rot H9c2 mioblaste met ‘n 1.317 bp mens ACCb-lusiferase promotor konstruk (pPII-1317) te transfekteer en ‘n uitdrukking te konstrueer wat die tempo bepalende stap van HBP i.e. glutamien: fruktose-6-fosfaat amidotransferase (GFAT) kodeer. Ooruitdrukking van GFAT verhoog ACCb geenpromotor aktiviteit deur 75 ± 23% teenoor kontrole (n=6, p<0.001). Die herhaling van ko-transfeksie eksperimente is herhaal in die teenwoordigheid van variëerbare L-glutamienkonsentrasies (0 mM, 4 mM, 8 mM), ’n substraat vir die HBP, ACCb promotor aktiwiteit is dosisafhanglik verhoog. Om die bevindinge verder te staaf, is twee inhibeerders van GFAT, i.e. 40 μM azaserien en 40 μM 6-diazo-5-oxo-L-norleusien aan transfeksie selle toegedien vir ’n tydperk van 24 uur. Beide azaserien en 6-diazo-5-oxo-L-norleusien verlaag ACCb geenpromotor aktiwiteit. In ooreenstemming met die bogenoemde het ko-transfeksies met twee dominante negatiewe GFAT konstrukte ook ACCb geenpromoter aktiwiteit verminder. Die volgende stap is om twee ensieme van die HBP wat stroomaf van GFAT aktief is, vir ‘n periode van 24 uur te inhibeer i.e. O-GlcNAc transferase en O-GlcNAcase deur alloxan (0.1 mM, 1 mM en 2 mM) and streptozotosien (5 mM en 10 mM) onderskeidelik vir ‘n 24 uur periode te gebruik. Toevoeging van alloxan het die ACCb geenpromotor aktiwiteit by 35.6 ± 1.9% (n=16, p<0.001) verlaag en streptozotosien aktiwiteit verhoog by 32 ± 12% (n=12, p<0.001). Ons het ook die USF1 en USF2 as transkripsie regulerings kandidate vir HBP-geïnduseerde ACCβ promotor regulering ondersoek. Ons data impliseer dat USF2 as ‘n belangrike transkripsie reguleerder van HBP-geïndiseerde ACCβ promotor regulering is. Samevattend het hierdie studie demonstreer dat verhoogde fluks deur die hexosamien biosintetiese weg ACCb geenpromotor aktiwiteit induseer. Ons stel verder voor dat hierdie induksie die kardiale vetsuuroksidasie verlaag wat daartoe lei dat intrasellulêre lipied akkumulasie as gevolg van onparing tussen sarkolemma vetsuuropname en mitochondriale vetsuuroksidasie in ’n insulien weerstandige situasie (i.e. hiperlipidaemia).

<p>Obesity is a major health problem in the Western world. Mean birth weight has increased during the last 25 years. One explanation is that the proportion of large for gestational age (LGA) infants has increased. Such infants risk developing obesity, cardiovascular disease and diabetes later in life. Despite the risk of neonatal hypoglycemia, their postnatal metabolic adaptation has not been investigated. Our data, obtained with stable isotope labeled compounds, demonstrate that newborn LGA infants have increased lipolysis and decreased insulin sensitivity. After administration of glucagon, the plasma levels of glucose and the rate of glucose production increased. The simultaneous increase in insulin correlated with the decrease in lipolysis, indicating an antilipolytic effect of insulin in these infants.</p><p>We also demonstrated an intergenerational effect of being born LGA, since women born LGA, were at higher risk of giving birth to LGA infants than women not born LGA. Further, the LGA infants formed three subgroups: born long only, born heavy only, and born both long and heavy. Infants born LGA of women with high birth weight or adult obesity were at higher risk of being LGA concerning weight alone, predisposing to overweight and obesity at childbearing age. In addition we found that pregnant women with gestational diabetes were at increased risk of giving birth to infants that were heavy alone. This could explain the risk of both perinatal complications and later metabolic disease in infants of this group of women.</p><p>To identify determinants of fetal growth, 20 pregnant women with a wide range of fetal weights were investigated at 36 weeks of gestation. Maternal fat mass was strongly associated with insulin resistance. Insulin resistance was related to glucose production, which correlated positively with fetal size. The variation in resting energy expenditure, which was closely related to fetal weight, was largely explained by BMI, insulin resistance, and glucose production. Lipolysis was not rate limiting for fetal growth in this group of women. Consequently, high maternal glucose production due to a high fat mass may result in excessive fetal growth.</p>

Prevalensen av fetma ökar drastiskt i stora delar av världen och utgör en stor riskfaktor för att utveckla insulinresistens och typ 2 diabetes. Fettväven kan bli mycket stor om för mycket energi tas upp av kroppen. Vid extrem övervikt är fettväven i kroppen i ett stresstillstånd, vilket gör att risken för att utveckla metabola sjukdomar som t.ex. typ 2 diabetes ökar. Fett lagras i olika fettdepåer i kroppen. Inlagringen i djupare kroppsdelar, runt och i inre organ s.k. visceralt fett, skiljer sig från fettväven som lagras direkt under huden s.k. subkutant fett. Nyare rön visar att mer visceral fettväv ökar risken för att utveckla insulinresistens och typ 2 diabetes. Fettcellen är tillsammans med muskel- och leverceller de viktigaste för glukosmetabolismen. Fettcellen är en stor cell, som man lätt kan se med blotta ögat. Storleken på ellerna varierar dock kraftigt i en och samma fettvävnad. Upptag av glukos från maten vi äter regleras av hormonet insulin. Insulinresistens är ett tillstånd då cellerna svarar dåligt på insulin, vilket gör att glukoshalten i blodet ökar. Detta förekommer vid typ 2 diabetes, men även vid andra tillstånd där cellerna blir stressade, t.ex. kirurgiska ingrepp. Insulinsignaleringen i fettcellen är komplex och signalöverföringen inne i cellen sker främst via en kaskad av fosforyleringar, där olika proteiner i en signalkedja fosforyleras eller defosforyleras. Slutligen leder denna fosforyleringskaskad till insulinets sluteffekter som t.ex. upptag av glukos, proteinsyntes och celltillväxt. Efter att insulin bundit till och fosforylerat/aktiverat insulinreceptorn delas signalen upp inne i cellen i två huvudvägar; den metabola signalvägen och den mitogena signalvägen. Insulinreceptorsubstrat 1, IRS1, är ett stort protein som insulinreceptorn verkar direkt på. Fosforylering av aminosyran tyrosin på IRS1 är mycket viktigt för fortsatt insulinsignalering i fettcellen. IRS1 fosforyleras även på aminosyran serin som svar på bl.a. insulin. Serinfosforyleringen av IRS1 hämmar eller stimulerar insulinsignaleringen, ofta genom återkoppling av insulinsignalen. Syftet med den här avhandlingen är att beskriva möjliga cellulära mekanismer i insulinsignaleringen vid insulinresistens som resultat av kirurgisk stress eller vid typ 2 diabetes i fettceller från människa. Häri har upptaget av glukos analyserats och jämförts i fettceller från olika fettdepåer. Viscerala fettceller har högre basalt och insulinstimulerat glukosupptag och mer glucostransportörprotein än subkutana fettceller. Däremot är det ingen skillnad i insulinkänslighet angående glukosupptaget i de olika typerna av fettceller. Vidare fann vi att den kirurgiskt orsakade insulinresistensen hos subkutana fettceller från människa återgår till det normala efter övernattinkubering av cellerna i odlingsmedium. Insulinresistensen vid typ 2 diabetes är däremot permanent och har en annan mekanism än den reversibla, stress-relaterade insulinresistensen. Insulinresistansen vid typ 2 diabetes beror på att signalöverföringen mellan olika proteiner i cellen är defekt. Insulinreceptorns förmåga att fosforylera IRS1 på aminosyran tyrosin är nedsatt hos patienter med typ 2 diabetes. Fosforyleringen av IRS1 på serin 307 (i den humana sekvensen) ökar snabbt hos icke-diabetiska fettceller som svar på insulin. Denna serinfosforylering verkar behövas för att IRS1 effektivt ska tyrosinfosforyleras och därmed leda insulinsignalen vidare inne i cellen. Fosforyleringen av IRS1 på serin 307 är kraftigt nedsatt hos subkutana fettceller från patienter med typ 2 diabetes. Fosforyleringen av IRS1 på serin 312 är däremot liknande i fettceller från icke-diabetiker och diabetiker (Öst et.al. (2007) Faseb.J. doi: 10.1096/fj.07-8173com). Fosforyleringen av IRS1 på serin 312 är mest involverad i insulinsignaleringens negativa återkoppling. Fosforyleringen av serin 307 sker snabbt och vid låga insulinkoncentrationer, medan fosforyleringen på serin 312 sker först efter lång inkubering och vid höga insulinkoncentrationer. Detta är en ny mekanism på cellulär nivå som möjligen kan beskriva insulinresistansen i fettceller från människa. Tillsammans styrs återkopplingen via den stimulerande fosforyleringen (serin 307) eller den hämmande fosforyleringen (serin 312) och kontrollerar insulinsignaleringen i cellen. Fosforyleringarna sker möjligen via samma proteinkinas och/eller proteinfosfatas och kan bli mål för terapeutiska läkemedel mot typ 2 diabetes i framtiden. / The prevalence of obesity is increasing in most parts of the world and is a strong risk factor for the development of insulin resistance and type 2 diabetes. Adipose tissue is important in whole body energy balance and grows in size with excess energy intake. Adipose tissue in different regions of the body has different characteristics and adipocytes coming from intraabdominal fat depots, are more associated with insulin resistance than adipocytes from subcutaneous fat depots. Insulin signalling is complex and consists of two major signalling pathways in the cell; the metabolic signalling pathway and the mitogenic signalling pathway. After insulin binding to the insulin receptor a cascade of protein phosphorylations and dephosphorylations is started, eventually leading to the target effects of the hormone. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), a protein directly downstream of the insulin receptor, is essential for further insulin signalling. Serine phosphorylation of IRS1 also affects insulin signalling through inhibitory or stimulatory effects. Adipocytes are together with muscle cells and liver cells central in the development of type 2 diabetes. The focus of this thesis is to describe mechanisms in insulin signalling in primary human adipocytes in insulin resistant states, surgical stress or type 2 diabetes. Visceral adipocytes from humans were analysed and compared to subcutaneous adipocytes. Visceral adipocytes were slightly bigger than subcutaneous adipocytes. Furthermore, visceral adipocytes had an increased level of the glucose transporterprotein GLUT4 and a higher basal and insulin-stimulated glucose uptake, but the sensitivity to insulin was the same. Here it was found that surgical insulin resistance is reversible after overnight incubation of the adipocytes and the impaired insulin sensitivity is at the level between IRS1 and PKB/Akt in insulin signalling. In contrast, the insulin resistance in type 2 diabetes is irreversible and the impaired insulin sensitivity is at the level of insulin receptor-mediated tyrosine phosphorylation of IRS1. Adipocytes from patients with type 2 diabetes were investigated and it was found that diabetic adipocytes have an attenuated insulin-stimulated phosphorylation of IRS1 at serine 307 (corresponding to serine 302 in the mouse sequence). In adipocytes from non-diabetic individuals, the phosphorylation of IRS1 at serine 307 occurred rapidly at low concentrations of insulin. This phosphorylation was associated with the tyrosine phosphorylation of IRS1. The phosphorylation of IRS1 at serine 312 (corresponding to serine 307 in the mouse sequence) in response to insulin was similar in adipocytes from non-diabetic individuals and from patients with type 2 diabetes (Öst et.al. (2007) Faseb.J. doi: 10.1096/fj.07-8173com) and occurred only at high concentrations after prolonged incubation with insulin. This thesis reports the investigation of mechanisms in insulin signalling at a cellular and molecular level in primary human adipocytes. The insulin resistance resulted from surgical stress is different from that in type 2 diabetes and adipocytes from patients with type 2 diabetes have impaired insulin sensitivity at the level of IRS1. Together, the phosphorylation of IRS1 at serine 307 and serine 312 may control insulin signalling through feedback mechanisms in primary human adipocytes.

L’expansion des maladies métaboliques dans les sociétés modernes exige plus d’activités de recherche afin d’augmenter notre compréhension des mécanismes et l’identification de nouvelles cibles d’interventions cliniques. L’obésité, la résistance à l’insuline (RI) et la dyslipidémie, en particulier sont tous des facteurs de risque associés à la pathogenèse du diabète de type 2 (DT2) et des maladies cardiovasculaires. Ainsi, la dyslipidémie postprandiale, notamment la surproduction des lipoprotéines hépatiques et intestinales, contribue d’une façon significative à l’hypertriglycéridémie. Quoique plusieurs études cliniques et fondamentales chez l’homme et les modèles animaux aient mis en évidence les rôles importants joués par le foie et l’intestin dans la dyslipidémie, les mécanismes moléculaires en cause ne sont pas bien élucidés. L’une des voies principales régulant le métabolisme lipidique est la voie de la protéine kinase AMPK. L’épuisement de l’ATP intracellulaire entraîne une activation de l’AMPK qui va œuvrer pour rétablir l’équilibre énergétique en stimulant des voies génératrices d’ATP et en inhibant des voies anaboliques consommatrices d’ATP. Les effets positifs de l’activation de l’AMPK comprennent l’augmentation de la sensibilité à l’insuline dans les tissus périphériques, la réduction de l’hyperglycémie et la réduction de la lipogenèse, d’où son importance dans les interventions cliniques pour la correction des dérangements métaboliques. Il est à souligner que le rôle de l’AMPK dans le foie et l’intestin semble plus complexe et mal compris. Ainsi, la voie de signalisation de l’AMPK n’est pas bien élucidée dans les situations pathologiques telles que le DT2, la RI et l’obésité. Dans le présent projet, notre objectif consiste à caractériser le rôle de cette voie de signalisation dans la lipogenèse hépatique et dans le métabolisme des lipides dans l’intestin chez le Psammomys obesus, un modèle animal d’obésité, de RI et de DT2. À cette fin, 3 groupes d’animaux sont étudiés (i.e. contrôle, RI et DT2). En caractérisant la voie de signalisation de l’AMPK/ACC dans le foie, nous avons constaté une augmentation de l’expression génique des enzymes clés de la lipogenèse (ACC, FAS, SCD-1 et mGPAT) et des facteurs de transcription (ChREBP, SREBP-1) qui modulent leur niveau d’expression. Nos analyses détaillées ont révélé, par la suite, une nette augmentation de l’expression de l’isoforme cytosolique de l’ACC, ACC1 (impliqué dans la lipogenèse de novo) concomitante avec une invariabilité de l’expression de l’isoforme mitochondrial ACC2 (impliqué dans la régulation négative de la β-oxydation). En dépit d’un état adaptatif caractérisé par une expression protéique et une phosphorylation (activation) élevées de l’AMPKα, l’activité de la kinase qui phosphoryle et inhibe l’ACC reste très élevée chez les animaux RI et DT2. Au niveau de l’intestin grêle des animaux RI et DT2, nous avons démontré que l’augmentation de la lipogenèse intestinale est principalement associée avec une diminution de la voie de signalisation de l’AMPK (i.e. expression protéique et phosphorylation/activation réduites des deux isoformes AMPKα1 et AMPKα2). La principale conséquence de la diminution de l’activité AMPK est la réduction de la phosphorylation de l’ACC. Étant donné que le niveau d’expression totale d’ACC reste inchangé, nos résultats suggèrent donc une augmentation de l’activité des deux isoformes ACC1 et ACC2. En parallèle, nous avons observé une réduction de l’expression protéique et génique de la CPT1 [enzyme clé de la β-oxydation des acides gras (AG)]. L’ensemble de ces résultats suggère une inhibition de l’oxydation des AG concomitante avec une stimulation de la lipogenèse de novo. Enfin, nous avons démontré que l’intestin grêle est un organe sensible à l’action de l’insuline et que le développement de la résistance à l’insuline pourrait altérer les deux voies de signalisation (i.e. Akt/GSK3 et p38MAPK) essentielles dans plusieurs processus métaboliques. En conclusion, nos résultats indiquent que l’augmentation de la lipogenèse qui contribue pour une grande partie à la dyslipidémie dans la résistance à l’insuline et le diabète serait due, en partie, à des défauts de signalisation par l’AMPK. Nos observations illustrent donc le rôle crucial du système AMPK au niveau hépatique et intestinal, ce qui valide l’approche thérapeutique consistant à activer l’AMPK pour traiter les maladies métaboliques. / Understanding the cellular mechanisms involved in the development of insulin resistance, and later on the occurrence of type 2 diabetes and its metabolic complications, is a perquisite step toward the identification of new therapeutic targets to fight against the development of these metabolic diseases. In the present studies, we used the gerbil Psammomys obesus, a well-established animal model of obesity, insulin resistance (IR) and type 2 diabetes (T2D), to characterize the hepatic and intestinal signaling abnormalities associated with lipid metabolism disorders during the pathogenesis of IR and T2D. Thus, we are able to demonstrate that the development of these metabolic diseases in Psammomys obesus animals, is accompanied by increased hepatic and intestinal lipogenesis with very high efficiency to form triglycerides rich-lipoproteins. In the liver, we observed an increase in mRNA levels of key lipogenic enzymes (ACC, FAS, SCD-1 and mGPAT) and transcription factors (SREBP-1, ChREBP), which modulate the expression level of lipogenic enzymes. Thereafter, our detailed analysis of the AMPK/ACC signaling pathway revealed a rise in the gene expression of the cytosolic ACC1 isoform of ACC(involved in de novo lipogenesis) concomitant with a constant expression of the mitochondrial ACC2 (negative regulator of β-oxidation). In spite of an adaptive state characterized by higher protein expression and phosphorylation (activation) of AMPKα, the kinase that phosphorylates and inhibits ACC, the activity of the later remains very high in IR and T2D animals. In the small intestine of IR and T2D animals, we demonstrated that the increase in intestinal lipogenesis is mainly associated with a decrease of AMPK signaling pathway (i.e. reduced expression and protein phosphorylation/activation of the two AMPKα1 and AMPKα2 isoforms). The main consequence of the decline in AMPK activity is the reduction of ACC phosphorylation. Given that, the expression levels of ACC remain unchanged; our results thus suggest an increased activity of both ACC isoforms, ACC1 and ACC2. Next, we observed a reduction in protein and gene expression of CPT1 [key enzyme in fatty acid (FA) β-oxidation]. Taken together, these results suggest an inhibition of FA β-oxidation concomitant with a stimulation of de novo lipogenesis. Finally, we demonstrated that the small intestine is an insulin sensitive organ and that the development of IR affects two signaling pathways (i.e. Akt/GSK3 and p38MAPK) essentials for several metabolic processes. In conclusion, our results indicate that increased lipogenesis, in IR and T2D, which exacerbate the dyslipidemia associated with these diseases, might be, at least partially, a result of AMPK signaling defects. In addition, our observations illustrate the crucial role of AMPK/ACC in the liver and intestine and validate AMPK as a potential target to treat the metabolic diseases.

Background: Insulin resistance may play a pathogenic role in cardiovascular disease (CVD). Resistance to insulin has been associated with obesity, hypertension, and abnormal glucose and lipid metabolism. The constellation of these features among insulin resistant subjects has been called the metabolic syndrome. Prevalence of the metabolic syndrome increases with age and is most common in the elderly. Different criteria have been proposed to define the metabolic syndrome (ATP, WHO, AACE, EGIR). Current management of metabolic syndrome focuses on the specific risk factors that the patient may have without targeting the underlying insulin resistance. Angiotensin Converting Enzyme Inhibitors (ACEI) and Angiotensin Receptor Blockers (ARB) are widely used antihypertensive medications that may improve insulin sensitivity. We hypothesize that they can be used to reduce the long term cardiovascular complications in elderly hypertensive subjects with evidence of insulin resistance. In this study, we determined the effect of ACEI/ARB on the long term development of CVD in hypertensive non-diabetic elderly patients with the metabolic syndrome, as well as in patients with insulin resistance. Methods: Our research project utilizes the Cardiovascular Health Study (CHS) dataset. This dataset is a community based observational study where elderly participants were randomly selected and followed up for 11 years and the time to any cardiovascular event was recorded. In our project, we included hypertensive, non-diabetic individuals, with evidence of metabolic syndrome or insulin resistance, but had not experienced cardiovascular events at baseline. Cox regression model was used to evaluate the effect of ACEI/ARB on the time to the first cardiovascular event compared to the other antihypertensive medications adjusting for possible confounders such as age, race, gender, smoking status, triglycerides, LDL levels, systolic blood pressure, development of diabetes, congestive heart failure (CHF) and the number of anti-hypertensives. Results: In elderly hypertensive non-diabetic subjects with the metabolic syndrome according to the ATP and the WHO criteria, the hazard ratio for CVD associated with the use of ACEI/ARB was 0.65 or 0.68 (with 95 % C.I. of [0.45, 0.98], and [0.48, 0.96]) respectively when compared to the group exposed to the other anti-hypertensives. When the metabolic syndrome was defined according to the AACE and EGIR, the use of ACE/ARB was associated with hazard ratios for CVD equal to 0.74 and 0.899, respectively (with 95 % C.I. of [0.54, 1.09] and [0.61, 1.34]) compared to the use of the other anti-hypertensives. Hypertensive non-diabetic elderly subjects who were insulin resistant as evidenced by a HOMA-IR in the upper quartile, had a hazard ratio for CVD of 0.78 (95 % C.I. [0.56, 1.09]) associated with the use of ACEI/ARB compared to the use of other anti-hypertensives. Conclusions: The effect of ACEI/ARB on the development of cardiovascular events differs according to the definition of the metabolic syndrome. Elderly hypertensive patients with the metabolic syndrome, defined by ATP and WHO, seem to have lower risk of CVD with ACEI/ARB compared to the other antihypertensive medications. However, this association is not significant in elderly hypertensive patients in the upper quartile of HOMA and in patients with the metabolic syndrome as defined by AACE and EGIR criteria.