title: Fatty acid metabolism and adipocyte function in healthy and gestational diabetes mellitus pregnancy
Creators: name: Alrehaili, Amaal Faraj
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: Adipose tissue, gestational diabetes, adipocytes, maternal obesity.

description: Gestational diabetes mellitus (GDM) is defined as glucose intolerance of varying severity with first recognition during pregnancy. The prevalence of GDM is increasing worldwide, largely attributable to the dramatic rise in maternal obesity, resulting in several maternal and fetal complications. The maternal metabolic adaptation during pregnancy is challenged by maternal obesity, resulting in β cell dysfunction and exaggerated insulin resistance in women with GDM. However, the exact cellular mechanisms involved in the development of GDM are not yet completely understood. Increasing evidence from clinical and experimental studies has suggested that adipose tissue dysfunction could be one of the underlying mechanisms for the metabolic abnormalities observed in women with GDM. However, most of the published literature on this topic has been focused on plasma measures of adipocyte-derived adipokines and how they are linked to insulin resistance. Functional measures of adipocytes, such as adipocyte size, lipolysis, insulin sensitivity and direct measurement of adipocyte secretory function, have not been widely studied in GDM. Failure to efficiently increase the adipocyte cell number (hyperplasia) rather than increase size (hypertrophy) in order to store excess free fatty acids (FFA), and the subsequent failure to suppress adipocyte lipolysis when FFA demands are low, is believed to be a key mechanism in the development of type 2 diabetes in the non-pregnant. Similarly, this could be a major candidate pathway for pregnancy complicated by GDM, leading to the observed higher plasma FFA and higher plasma pro-inflammatory cytokine concentrations, which may result from exaggerated adipocyte insulin resistance and inflammation. Therefore, the hypothesis tested in this thesis was that GDM results from defective expansion of SAT adipocytes, resulting in adipocyte hypertrophy. Subsequently, there is increased adipocyte lipolysis and inflammatory adipokine secretion. The aim of this thesis was, firstly, to explore the epidemiological evidence for the role of maternal obesity in the development of GDM and other maternal and fetal complications, specifically in the highly diverse local Greater Glasgow and Clyde population. Data from the Scottish Morbidity Record 2 (SMR02) and the Scottish Care Information – Diabetes Mellitus (SCI-diabetes) databases for pregnant women between 2010 and 2015 was combined, and the prevalence of maternal obesity and GDM established among 38,178 births. The associated risks for several adverse pregnancy outcomes among women with maternal obesity and GDM in the Greater Glasgow and Clyde population were calculated. It was found that in the local population maternal obesity and GDM were at higher prevalence (22.3% and 2.2%, respectively) compared to recent historical studies, and are associated with an increase in the incidence of a range of adverse pregnancy outcomes. Furthermore, the resulting adverse maternal and fetal complications of both first trimester maternal obesity and GDM were likely to be a considerable burden on clinical resources. Assessment of the long-term implications of maternal and fetal complications secondary to GDM and maternal obesity was not included, and a prospective follow-up analysis of this cohort is recommended. The second aim of this thesis was to determine the evidence for the role of failure of adipocyte expansion in the development of underlying metabolic abnormalities in women with GDM. Several functional measures of adipocyte expansion were assessed, including adipocyte size, lipolytic function, adipokine secretion, and the expression of genes involved in adipocyte physiology and biochemistry. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) biopsies from non-labouring women with GDM (n=22) and healthy (n=22) BMI-matched controls, undergoing elective caesarean section, were collected. Maternal blood was collected prior to delivery and maternal phenotyping was carried out by the assessment of plasma glucose, insulin, plasma lipids (triglyceride, cholesterol, glycerol and non-esterified fatty acids), pregnancy hormones (estradiol and progesterone), liver enzymes (gamma-glutamyl transferase, alanine aminotransferase and aspartate aminotransferase), plasma inflammatory cytokines and oxidised low-density lipoprotein (oxLDL), a biomarker of lipotoxicity. Maternal BMI at booking was recorded from the patient notes, along with other demographic information. Fetal weight and sex were recorded after the baby was delivered. Adipocyte isolation and sizing were carried out. The ex vivo lipolytic activity (basal and β adrenergic- stimulated lipolysis, and insulin suppression of lipolysis) and adipokine production (basal and lipopolysaccharide stimulated conditions) were assessed in isolated adipocytes. The adipocyte expression of genes involved in adipocyte differentiation, lipid storage, lipid and glucose metabolism and angiogenesis were also performed by RT-qPCR. In GDM, VAT adipocytes had higher mean adipocyte diameter ((control) 62.9[3.8] vs (GDM) 75.5[11.6] µm, p=0.004) and volume ((control) 0.00007[0.00000002] vs (GDM) 0.00012[0.0000005] mm3, p=0.003) compared to controls. VAT adipocytes from women with GDM had higher basal lipolysis compared to controls (0.02[0.02] vs 0.07[0.07] glycerol mmol/hr/ug of DNA, p=0.001). The fat cell insulin sensitivity index (FCISI), a measure of adipocyte insulin suppression of lipolysis, was six times lower in the VAT adipocytes of women with GDM, but failed to reach statistical significance ((control) 62[204] vs (GDM) 35[52] FCISI, p=0.61). In contrast, there were no differences observed in SAT adipocytes with regard to adipocyte size, lipolysis and FCISI between the two groups. Hypertrophic expansion of VAT adipocytes may be one of the contributors to the increased basal lipolysis rate seen in this depot. This will favour portal release of fatty acids, and could be an important factor in triggering metabolic abnormalities associated with liver fat accumulation in obese pregnant women. Thus, dysfunctional VAT adipocytes may represent an important event contributing to the emergence of metabolic dysfunction in women with GDM. The study of isolated VAT adipocyte adipokine secretion in basal or activated (LPS-stimulated) conditions in a subset of healthy and GDM women did not show any differences in VAT adipocyte adipokine release in GDM compared to controls. However, these results should be interpreted with caution, because of the small sample size. Therefore, further tissue collection is recommended in order to increase the power of the study. Further investigation of macrophage and other immune cell contribution to adipose tissue inflammation in women with GDM is warranted. Isolated adipocyte gene expression analysis in both SAT and VAT showed that women with GDM had significantly higher insulin receptor (INSR) expression in both SAT (9.0(3.1) vs 11.7(3.7) INSR percentage expression relative to PPIA, p= 0.031) and VAT (10.8(3.6) vs 15.8(5.6) INSR percentage expression relative to PPIA, p= 0.022) compared to controls. There were no differences in the expression of other genes involved in adipocyte differentiation, lipid storage, lipid and glucose metabolism and angiogenesis in isolated SAT and VAT adipocytes for women with GDM compared to controls. The final aim was to assess the role, in early pregnancy, of very low-density lipoprotein (VLDL) as a potential maternal plasma carrier of the extremely important long chain polyunsaturated fatty acid (LC-PUFA) docosahexaenoic acid (DHA), required by the fetus for neuronal development. During pregnancy, there is a three-fold increase in VLDL synthesis by the liver. The liver is the primary site for de novo LC-PUFA synthesis, which is shown to be increased in early pregnancy; therefore, VLDL could be the carrier of DHA at early pregnancy. Infants born to mothers with GDM had lower DHA levels, and were shown to have lower cognitive performance, partly attributed to lower placental transfer of DHA in GDM. However, the underlying mechanism for defective DHA metabolism and transport in GDM pregnancy is not fully understood. Using an archival plasma collection of women undergoing frozen embryo transfer (FET), fasting blood samples were collected at approximately day 10 following the last menstrual period (LMP) (pre-luteinizing hormone (LH) surge), and on days 18, 29 and 45 post-LH surge from women who were successful in getting pregnant (n= 27). VLDL FA composition was assessed by gas chromatography in women who were successful in getting pregnant and women who were not. There was no evidence for VLDL being the main carrier of DHA at the critical time of neuronal tube closure, as DHA concentration in VLDL and VLDL DHA enrichment was unchanged over time. A reduction in VLDL particle number (measured by apo-B concentration) by 18 days post-LH surge was observed (p&lt;0.001), which was then recovered to pre-LH surge level by 45 days post-LH surge. It is likely that DHA is carried by other lipoproteins such as HDL, as is observed in the non-pregnant population. Further investigation of the main lipoprotein carrier for DHA during pregnancy, and the study of its metabolism and transport in GDM pregnancy, is needed. In conclusion, GDM and maternal obesity are associated with increased risk of adverse pregnancy outcomes. Effective intervention strategies are required for weight control prior to pregnancy. The data presented in this thesis provides evidence that in GDM, VAT adipocytes expand in a hypertrophic manner, and have increased basal lipolysis. Hypertrophic expansion of VAT adipocytes may be linked to systemic insulin resistance. Thus, defective VAT adipocyte expansion might have a role in the underlying pathophysiology of GDM. Further studies on the role of adipocyte differentiation and ectopic fat storage in normal and complicated human pregnancy are warranted. DHA is not transported by VLDL in early pregnancy. Further investigation of the main carrier of DHA during pregnancy, and the study of DHA metabolism in pregnancies complicated by GDM, is needed.