Browsing by Author "Keswell, Dheshnie"

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    Effects of adipose tissue extracellular matrix components on body fat distribution and insulin sensitivity in black and white South African women
    (2018) Kotzé-Hörstmann, Liske; Goedecke, Julia; Keswell, Dheshnie
    The global burden of non-communicable diseases (NCD’s) is unacceptably high and disproportionately affects developing countries such as South Africa (SA). Black SA women have a higher prevalence of obesity and a greater associated risk for developing metabolic diseases (such as type 2 diabetes mellitus) than their white counterparts. An improved understanding of the ethnic-specific mechanisms underlying the increased risk of T2DM in black SA women is needed to inform future studies aimed at reducing the prevalence of this diseases. One of the major determinants of insulin resistance is android/central body fat partitioning, with visceral adipose tissue (VAT) enlargement, in particular, being closely associated with increased risk. Conversely, lower-body fat accumulation is considered to be protective. However, these relationships between body fat distribution and its metabolic effects are altered by ethnicity. Black SA women have less abdominal and greater gluteal-femoral subcutaneous adipose tissue (SAT) but are more insulin resistant compared to BMI- and waist circumference-matched white SA women. A similar profile has been described in black African-American women. The reduced protective effect of peripheral fat distribution in black women remains to be understood. The primary aim of this thesis was positioned within the context of adipose tissue expandability hypothesis, and aimed to examine the hypothesis that differences in SAT extracellular matrix (ECM)- and hypoxia-related gene expression and their ethnic specific associations with body composition and insulin sensitivity may explain, in part, the higher rates of insulin resistance in black compared to white South African women. Therefore, it was hypothesized that, as a consequence of increased adipose tissue hypertrophy in the gluteal depot of obese black compared to obese white women, gluteal SAT adipose tissue hypoxia and ECM component gene expression is higher in black compared to white women, and associates with their reduced insulin sensitivity (SI) and higher insulin response. In order to address this hypothesis, four research studies were designed. The first study (Chapter 3) in this thesis aimed to compare depot-specific (abdominal vs. gluteal) expression of hypoxia and ECM genes in normal-weight and obese black and white women, and to examine the ethnic-specific associations between these genes and body composition, measures of insulin sensitivity and secretion and inflammatory gene expression in black and white SA women by using a gene expression (Reverse transcription polymerase chain reaction (RT-PCR)) analysis. This thesis showed for the first time that hypoxia inducible factor 1 (HIF-1α), collagen type V α1 (Col5A1) and type VI α1 (COL6A1) gene expression were higher in the gluteal, but not the abdominal SAT depots, of black compared to white women, and associated with reduced insulin sensitivity in black women only. The expression of the hypoxia and ECM genes associated with inflammatory gene expression in both the gluteal and abdominal SAT depots of black women, whereas the expression of these genes associated with the inflammatory gene expression mostly in the abdominal SAT depots of white women. The second study (Chapter 4) tests the hypothesis that higher hypoxia and ECM related gene expression would associate with higher central fat mass accumulation in black women and that the expression of these genes may be associated with changes in the measures of insulin sensitivity in black and white women. Thus, this longitudinal study aimed to determine whether changes in body composition and insulin sensitivity variables over a 5 year follow-up period associated with variations in hypoxia and ECM related gene expression in the gluteal SAT of black and white women. Over the 5-year follow-up period, increased body fat mass in white women associated with increased PPARγ mRNA expression whereas increased body fat mass in black women associated with lower COL5A1 expression. Furthermore, HIF-1α, and COL6A1 expression correlated positively with the change in fasting insulin concentrations in black but not in white women. It is not clear whether high circulating insulin may directly increase HIF-1α expression and contribute to the formation of excess ECM, or whether increased insulin may simply be a concomitant downstream effect of increased insulin resistance, as a consequence of increased fibrosis and the generation of inflammation. By using a cell culture based study, the third study in this thesis (chapter 5), investigated the effects of increasing insulin concentrations on the expression of hypoxia and ECM related genes under normoxic and hypoxic conditions in mature 3T3-L1 adipocytes. It was found that insulin and hypoxia treatment significantly elevated HIF-1α mRNA and protein levels but that the observed effects were not additive. Further, hypoxia, but not insulin treatment, increased the expression of Col5a1 and Col6a1 protein but not mRNA levels in mature 3T3-L1 adipocytes. By using a genotyping analysis, the fourth study (Chapter 6) aimed to determine whether variants within two ECM component gene polymorphisms, collagen type 5α1 (COL5A1) rs12722 (C/T) and type 6α1 (COL6A1) rs35796750 (C/T) associates with body fat distribution and insulin resistance in black and white women. Allele and genotype distributions of the COL5A1 rs12722 and COL6A1 rs35796750 polymorphisms, as well as body fat distribution were significantly different between black and white women, the T- variant of the COL5A1 rs12722 polymorphism was associated with significantly less central fat mass, characterised by a smaller waist circumference and lower VAT, and this effect was independent of ethnicity. In addition, T- variant of the COL5A1 rs12722 polymorphism was associated with lower fasted insulin concentrations and HOMA-IR in white but not in black women. In contrast, no genotype associations between COL6A1 rs35796750 and any of the body fat mass, its distribution and insulin resistance measures in black or white women were reported. This thesis used a hypothesis driven approach to provide preliminary evidence that the gluteal depot of obese black women has higher expression of hypoxia and ECM genes compared to that of obese white women and provides novel insight into the apparent paradox of reduced insulin sensitivity despite lower VAT and greater peripheral SAT accumulation in black compared to white women. An improved understanding of the ethnic-specific mechanisms underlying the increased risk of T2DM in black SA women will enable the development of cost-effective preventative care strategies within the South African demographic
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    Ethnic specific associations between body composition and metabolic risk and the role of sex hormones and aromatase among black and white South African women
    (2015) Tootla, Mehreen; Goedecke, Julia; Keswell, Dheshnie
    Background: Previous evidence has demonstrated ethnic differences in the relationship between body fat distribution and metabolic risk between black and white women. However, the reasons for these differences are not known and may be explained in part by differences in sex hormones. The overall aim of this thesis was therefore to i) examine ethnic-specific associations between body fat and its distribution and cardio-metabolic outcomes (study 1) and ii) examine the associations between sex hormones and subcutaneous adipose tissue (SAT) expression of oestrogen receptors (ERα and ERβ) and aromatase (CYP19A), and body fat distribution and insulin resistance (IR) among black and white women (study 2).Methods: Study 1: In 288 black and 197 white premenopausal women, dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) derived measures of body fat distribution and cardio-metabolic factors including IR (HOMAIR) and lipid levels were measured. Study 2: In a subsample consisting of 13 normal-weight and 15 obese black and 15normal-weight and 12 obese white women, HOMAIR and SI (frequently sampled intravenous glucose tolerance test) and ERα, ERβ and CYP19A gene expression were measured in abdominal and gluteal SAT. Results: Study 1: Compared to white women, black women had less central and greater lower body fat, but had similar IR and lower serum lipid concentrations. Despite these differences, the associations between body fat distribution and measures of IR, as well as TG and HDL-Concentrations were similar in black and white women. Notably, central and peripheral fat deposition was independently associated with IR in both the black and white women, and with TG in the black women. In contrast, the associations between body composition and fasting plasma glucose, TC and LDL-C concentrations differed between black and white women. Fasting glucose concentrations were associated with centralisation of body fat in black but not white women, whereas TC and LDL-C concentrations were associated with centralisation of body fat in white but not black women. In addition to body fat distribution, MVPA was associated with IR in the white women, and contraception use was associated with lipid levels in the black and white women. Study 2: CYP19A expression was positively associated with increased adiposity in black and white women in all three depots. Gluteal ERα was significantly higher and ERβ was significantly lower in the black compared to the white women, irrespective of BMI. Gluteal ERα was negatively associated with trunk fat mass (FM) and HOMAIR in the black women only. Gluteal ERα was significantly lower in obese white compared to normal weight white women. Additionally oestradiol (E2) levels were lower in obese compared to normal-weight white women, but did not differ by ethnicity. Conclusion: Our results indicate that it is important in both black and white populations, to decrease centralisation of body fat. Modifiable risk factors such as MVPA and contraception use should be used as therapeutic targets to prevent and manage CVD. Additionally, oestrogen receptors may be an important determinant of body fat distribution and risk in the black women.
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    The effect of beta-oxidation or TCA cycle inhibition on mitochondrial function and the sensitivity of high resolution respiratory detection
    (2019) Osiki, Prisca Ofure; Keswell, Dheshnie; Mendham, Amy E.
    INTRODUCTION: A dysfunction in fatty acid beta-oxidation (β-oxidation), particularly medium chain acyl-CoA dehydrogenase (MCAD) dysfunction is a major cause of mortality and its diagnosis is usually achieved by measuring specific protein activities or metabolites in blood and/or urine samples. However, these methods do not account for secondary defects that accompany primary deficiency; such as where measures of disruption in fatty acid metabolism do not account for defects in the TCA cycle and oxidative phosphorylation. These metabolic pathways are connected and dysfunction in one pathway (primary) could lead to dysfunction in the other (secondary). We propose the use of methods that combines all aspects of the bioenergetics module (enzyme activity in substrate oxidation within each individual pathway, transfer of electrons through the electron transport system (ETS) and oxidative phosphorylation for ATP generation) may be a more effective assessment technique. High resolution respirometry (HRR) is a recently developed technique that accounts for substrate oxidation, electron transfer via the ETS and oxidative phosphorylation. It measures the rate of oxygen consumption or flux at different respiratory states when appropriate substrates, uncouplers and inhibitors (SUIT protocols) are used. With this method, two substrate combinations are commonly used to assess medium-chain fatty acid β-oxidation; a) Octanoylcarnitine and carnitine, which is partial to the β-oxidation cycle alone, and b) Octanoylcarnitine and malate, which assesses the influence of the TCA cycle. Additionally, a combination of pyruvate, glutamate and malate is used to assess oxidation within the TCA cycle. We investigated the sensitivity of commonly used substrate combinations in HRR assessment to detect changes in mitochondrial respiration and dysfunction induced by the inhibition of either β-oxidation or the TCA cycle in C2C12 myotubes. Furthermore, we assessed MCAD, citrate synthase and aconitase enzyme activities when β-oxidation or TCA cycle was inhibited in C2C12 myotubes. METHODS: C2C12 myotubes were differentiated for 6 days and treated for 12 hours with a high or a low concentration of one of two inhibitors as follows; a) 2 mM or 8 mM 2-mercaptoacetate to inhibit medium chain acyl-CoA dehydrogenase (MCAD); b) 6 mM or 9 mM fluorocitrate to inhibit aconitase. Each treatment was compared to control myotubes grown for the same length of time without the addition of inhibitors. The activities of MCAD, aconitase and citrate synthase were determined. In addition, mitochondrial respiration measured as O2 flux at Routine, Leak, OXPHOS and ETS respiratory states were assessed in an Oxygraph-2K after inhibition or in control treatments using; i) Octanoylcarnitine and carnitine ii) Octanoylcarnitine and malate iii) pyruvate, malate and glutamate substrate combinations. For each assessment we corrected O2 flux recorded at each state to; a) approximate number of cells (pmol O2/s/million cells) b) protein concentration (pmol O2/s/mg protein) c) Flux control ratio (FCR) of each state to the maximum ETS capacity; ETSFAO+CI+CII (convergent electron flow from Fatty acid oxidation (FAO), Complex I (CI) and CII) d) FCR to either FAO-linked ETS capacity; (ETSFAO) or CI-linked ETS capacity (ETSCI). RESULTS: Treatment of cells with either a low or high concentration of 2-mercaptoacetate to inhibit MCAD resulted in no significant difference in MCAD activity. Fluorocitrate treatment decreased aconitase activity with low treatment (p = 0.011) compared to control, and conversely it increased MCAD activity in high treatment compared to control (p = 0.024). Both 2-mercaptoacetate (p = 0.03) and fluorocitrate (p < 0.01) treatment at high concentrations resulted in increased citrate synthase activity, compared to low concentration and control. Mitochondrial respiration with octanoylcarnitine and carnitine substrate combination was not altered with MCAD or aconitase inhibition. Octanoylcarnitine and malate substrate combination showed a decrease in mitochondrial respiration at the following respiratory states with both MCAD and aconitase inhibition; Routine (p = 0.01), LeakFAO (p = 0.029), OXPHOSFAO (p = 0.006), ETSFAO (p = 0.008), ETSFAO+CI (p = 0.017). FCR of each state to the maximum capacity (ETSFAO+CI+CII) revealed a decrease with both MCAD and aconitase inhibition at the following states; routine (p = 0.001), OXPHOSFAO (p = 0.003), ETSFAO (p = 0.018), ETSFAO+CAR (p = 0.008) and ETSFAO+CI (p = 0.027). Pyruvate, malate and glutamate substrate combination showed decreased mitochondrial respiration with MCAD inhibition at the following respiratory states; Routine (p = 0.004), LeakCI (p = 0.007), OXPHOSCI (p = 0.003), ETSCI (p = 0.003), ETSCI+FAO (p = 0.01) and ETSCI+FAO+CII (p = 0.003). FCR of each state to the maximum capacity (ETSCI+FAO+CII) decreased with both MCAD and aconitase inhibition at Routine (p = 0.024), OXPHOSCI (p = 0.024) and ETSCI (p = 0.035) states. DISCUSSION: The main finding of this study was related to two of the SUIT protocols 1) octanoylcarnitine and malate, and 2) pyruvate, malate and glutamate. These protocols were sensitive in showing decreased respiratory capacity and coupling control ratios and may be appropriate for assessing changes in oxidative metabolism when there is a defect in β-oxidation and/ or the TCA cycle. On the other hand, octanoylcarnitine and carnitine substrate combination is not sensitive to detect dysfunction induced by inhibition of either β-oxidation or TCA cycle. Irrespective of the enzyme inhibited, HRR detected dysfunction in complex I (CI), although, when aconitase was inhibited, reduced CI-linked respiration was more pronounced compared to MCAD inhibition. Furthermore, primary inhibition of MCAD to inhibit β-oxidation may have caused secondary inhibition of TCA cycle via aconitase, shown in decreased TCA cycle CI-linked respiration where MCAD was inhibited. In contrast, primary inhibition of aconitase seemed to be compensated for by increased MCAD activity and mitochondrial respiration related to β-oxidation. Lastly, enzyme assaysshould not be used as standalone techniques for assessing metabolic dysfunction at the level of TCA, β-oxidation and the mitochondria since they are not sensitive to low level defects, nor do they account for secondary interactions that influence either TCA or betaoxidation. HRR is useful to assess mitochondrial respiration and dysfunction, when using an appropriate substrate combination and should be used in combination with the more traditional enzyme activity assays.