A robust experimental model, which permits controlled nutritional
intervention during pregnancy and early life, allows ongoing monitoring of weight gain and physiological parameters, mimics human organ
development and physiology and has precedence of experimental studies in hepatic and adipose tissues is required to optimally study the potential nutritional programming of the obesity-induced cell stress response and inflammation.
1.7.1.1 The sheep as an experimental model for programming
Randomised controlled studies which can control for confounding factors such as genetic predispositions and social status, designed to control for other risk factors such as parity, number of fetuses and gender are best placed to validate findings of epidemiological studies. However, since it is not possible to subject humans to highly invasive physiological or experimental procedures, it would not also be possible to elucidate the molecular biological mechanisms behind the outcome of the interventions. With use of animal models, it is possible to perform interventions in a targeted and controlled fashion and study the effects of these interventions. The outcome of these experimental studies has the potential to be translated to human
applications through a step-wise process.
In order to evaluate the mechanisms behind developmental programming, rodent, pig and sheep models have been extensively used. The sheep has many advantages as an experimental model. At birth, the size of the offspring and the relative weight of the fetus to the mother are comparable in sheep. The ovine pregnancy is of longer gestation as compared to rodent models and the organogenesis and maturation stages of the fetus have largely been characterised [314]. The relatively longer gestation of sheep
pregnancy allows researchers to target specific stages of organ growth and development for nutrient manipulation. Furthermore, the gestational stages in sheep are comparable to humans and the late gestational period (110 days to 148 days gestation) is similar to human third trimester of pregnancy and coincident with maximal fetal growth. The specific organ growth rate and fetal metabolic rate are very similar in sheep and humans and very different to rodents [297]. Importantly, the hypothalamic pituitary adrenal axis maturity is similar in sheep and humans through the late gestation and at the time of birth [298] which is not the case with rodent models.
Experimental studies of maternal undernutrition during pregnancy performed in the sheep model have demonstrated effects on the fetus comparable to human IUGR with identifiable programming effects on insulin sensitivity [315] and cardiovascular system [316] in later life.
The ovine model also has some disadvantages. Unlike the rodent model, which has shorter gestation (21 days) and large litter size enabling a high turnover and generational studies, performance of such generational studies in sheep models would require extensive logistical and financial resources. Sheep have a cotyledonary placenta as compared to the discoid placenta in humans. Being a ruminant animal, the sheep uses volatile fatty acids (VFA) as main energy source as compared to glucose in humans. The VFAs (acetic acid, propionic acid and butyric acid) are metabolised differently in the liver. Propionic acid is the major source of hepatic gluconeogenesis, whilst acetic acid and butyric acid are utilised throughout the body as energy sources. Despite these differences, credible models of ovine obesity have been developed using programmes of manipulation in maternal diet, postnatal feeding and post-weaning activity [68, 267, 279]. The development of
obesity observed in these models shows increased visceral adiposity which is believed to contribute to the development of a low grade chronic
1.7.1.2 Omental adipose tissue characteristics
An excess of visceral adipose tissue deposition is known to be associated with central obesity, insulin resistance and type 2 diabetes [29, 30]. Ovine omental adipose tissue is a major proportion of the visceral adipose tissue depot and is known to rapidly expands soon after birth, a characteristic, which could increase its succeptibility to nutritional programming influences. Macrophage cells have been identified in ovine fetal omentum as early as 72 days of gestation whereas T cells have been identified at 140 days of
gestation spots’ [69]. Adipocyte hypertrophy in omental, but not
subcutaneous depot, in obese women was independently associated with dyslipidemia [317]. The numbers of CLS in omentum correlate with the severity of hepatic fibroinflammatory lesions [318] and liver fat content [319] as well as insulin sensitivity [320]). Several chemotactic cytokines are expressed at higher levels in omentum [321]. Omental adipose tissue
possess distinct characteristics that make it an important subject for investigations into developmental programming of adult disease.
1.7.1.3 Development of sheep omentum and liver
Like humans, the sheep omentum is derived from cells of mesodermal origin (dorsal mesogastrium) in the early fetal period. During this period, it
consists of a gelatinous membrane with evolving blood vessels and no differentiated adipose tissue [322]. However, the cells of immune origin can be identified as early as 72 days of gestation and these rapidly evolve into aggregates known as ‘milky spots’ [69]. These cells have been identified as macrophages. Adipocytes and T cells start appearing at around 125 days of gestation. At birth, the omentum measures approximately 230 x 90mm [69] and, following birth, the weight of the omental adipose tissue depot rapidly expands to 3 g/kg body weight by the age of 7 days and 10 g/kg body weight by the age of 28 days [323].
The earliest identifiable embryonic hepatic structure originates as a ventral outgrowth from the gut endoderm around the seventeenth day of ovine gestation and is called the hepatic diverticulum [324]. Hepatocytes, the principal cell type in the liver, and biliary epithelial cells are derived from the embryonic endoderm, whilst stromal cells, stellate cells, Kupffer cells and blood vessels, are of mesodermal origin and are incorporated as the diverticulum expands into the surrounding mesoderm. With rapid
proliferation and differentiation, hepatic lobes are evident by the twenty first day and hepatic lobules with identifiable structures and endothelial cells are present by the thirtieth day. During fetal development, the liver assumes haematopoietic function until the last 2 months of gestation. The increase in liver size during the pregnancy follows a linear pattern all through the
gestation until birth [325].
1.7.1.4 Experimental approach
The sheep model of obesity and nutritional intervention in early life permits measurement of maternal and offspring physical and constitutional
characteristics, measurement of physical activity, assay of plasma
metabolites and hormones, post-mortem adipose tissue depot size through Dual-energy X-ray absorptiometry (DXA) scan and direct weighing
respectively, and genetic and molecular analysis of hepatic and adipose tissue. Such a comprehensive approach permits scrutiny of the physio- pathological response to experimental intervention throughout the experimental study. The role of each study in the establishment of the experimental model is summarised in Table 1.8.
Process under investigation Physiological parameter
Establishment of model of maternal undernutrition during late gestation, offspring nutrition during suckling period and post weaning obesity
Maternal weight gain
Mid intervention maternal paired plasma glucose, insulin, triglycerides and NEFA Offspring weight
Relative weight gain during early infancy Physical activity measurement
Offspring postweaning weight gain Absolute and relative organ weight
Offspring metabolic outcome of experimental intervention
Glucose tolerance test with timed glucose and insulin assay
Plasma triglycerides, NEFA, cortisol and leptin assay
State of adipose tissue metabolism and distribution
Plasma leptin, triglyceride and NEFA measurement
Dual-energy X-ray absorptiometry of adipose depots
Adipose tissue depot weight
Adipose tissue inflammatory response
Plasma leptin assay
Histological analysis for adipocyte hypertrophy, crown-like structure abundance, macrophage infiltration
Immunohistological analysis for GRP78 (ER stress), IBA1 (macrophage markers) and phosphorylated JNK staining
Gene expression studies
Hepatic structural and metabolic outcome of experimental intervention
Absolute and relative organ weight Hepatic triglyceride assay
Hepatic thiobarbituric acid reactive substances assay for oxidative stress
Histological analysis for features of steatosis and NAFLD
Hepatic inflammatory response
Histological analysis for features of NASH Gene expression analysis
Table 1.8 Overview of experimental approach for study of hepatic and adipose tissue cell stress and inflammatory response to obesity and nutritional programming. NEFA, non esterified fatty acids; NAFLD, non alcoholic fatty liver disease; NASH, non alcoholic steatohepatitis; GRP78, 78kDA glucose regulated protein; JNK, c-Jun N- terminal kinase.
1.7.1.4.1 Gene expression
In selecting the genes of interest for exploration of the cell stress response and metabolic inflammation, transcriptionally regulated genes relevant to the pathway were identified and experiments were performed only when the primers and the experimental products confirmed to the strict quality control criterion described in Chapter 2. Given above constraints, the following gene expression experiments were performed to investigate the effect of obesity and nutritionally mediated developmental programming in sheep liver and omental adipose tissues (Table 1.9):
Process under
investigation Gene of interest
Modulators and effectors of metabolic inflammation CD95 TLR4
Leptin receptor (liver) Leptin (adipose) Adiponectin (adipose)
Cell stress response
Autophagy: components
and regulators BECN1 ATG12 AMPK mTOR ER stress and UPR GRP78
EDEM ATF4 ATF6
Table 1.9 Genes involved in regulation of development of insulin resistance by activation of cell stress response and metabolic inflammation
CD95, cluster of differentiation 95; TLR4, toll-like receptor 4; BECN1, gene encoding Beclin1; ATG12, autophagy related gene 12; AMPK, 5' adenosine monophosphate activated protein kinase; mTOR, mammalian target of rapamycin; ATF4, activating transcription factor-4; ATF6, activating transcription factor-6; EDEM1- ER stress degradation enhancer molecule-1; GRP78, glucose-regulated protein 78.