interventions range from caloric restriction to induction of obesity and in many cases, result in similar sequelae to those observed in humans (e.g. Buettner et al., 2007; Panchal & Brown, 2011). In general, their small, manageable size enables studies, such as these, where reproducibility is desired, and replication is necessary to obtain statistical significance. With a relatively short lifespan, they allow for relatively rapid collection of data along multiple parameters of feeding and body weight metabolism. Advantages are that these can be examined at whole-body, integrated levels in vivo, and then at tissue and cellular levels ex vivo. In vivo measures can include energy intake in response to a variety of diet compositions and feeding schedules, resulting body weight, composition, adiposity and fat distribution, and behavioural indices, such as timing and volume of meals consumed. Concurrent intermittent body fluid sampling allows for monitoring of progression of dietary effects, through measurement of key endocrine and metabolic factors (e.g. West & York, 1998; Dobrian et al., 2000; Keenan et al., 2005; Buettner et al., 2007).
Rodents also allow for the convenient study of mammalian body-brain relationships and interactions, the complexity of which cannot be replicated in less sentient species or non-animal alternatives. This is particularly true when behavioural expression of these interactions is of interest, for example in the form of food selection and meal patterns (Farley et al., 2003). Given that one of the main objectives of the work presented in this thesis was to examine associations between brain and body in the context of feeding, specifically, changes in hypothalamic cell proliferation in response to dietary fatty acids (Chapter 1, Section 1.4), animals which could model the sophisticated physiology of other mammals which have shown these relationships (Buettner et al., 2007; Panchal & Brown, 2011), were essential to the work. Sub-mammalian species differ too fundamentally from humans in their systems physiology and brain structure to have more than limited relevance in this context. The alternative of human testing would have been unethical, as post- mortem tissue was required to examine changes in the brain at the level of resolution of individual cells.
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These considerations also clearly show that in vitro work is not suitable in this research area because it cannot answer questions regarding phenotypic identification, number and regional distribution of cells in intact brain regions. It is self-evident that neither does it allow for development of a whole-animal dietary phenotype.
2.1.2.1 Selection of Species
Rodents are the vertebrate group of lowest neurophysiological sensitivity which have also provided the best-characterised models of peripheral and central regulation of appetite (e.g. Schwartz et al., 2000). Some mouse strains, such as the C57BL/6 can be more sensitive to dietary intervention, and weight gain, in particular, than commonly used outbred rats, such as the Wistar and Sprague-Dawley (Rossmeisl et al., 2003). However, as models for examination of, and intervention in, already well- characterised feeding behaviours, they are less apt than rats (Anson et al., 2003). Studies described here involved investigation of feeding behaviour expressed as meal patterns (see Section 2.4, below, and Chapters 3 & 5). Whereas there is no published evidence that mice have been used for this purpose previously, rats are commonly studied in such paradigms; for example, the behavioural satiety sequence (BSS; Halford & Blundell, 1996; Halford et al., 1997; Cooper et al., 2010) and studies of circadian rhythmicity of feeding (Kohsaka et al., 2007; Hariri & Thibault, 2011). This provides an appropriate context into which results generated in this project can be placed.
2.1.2.2 Selection of Strain
In investigating effects of dietary enrichment with PUFAs, it was important to incorporate the correct control diets into experimental design, as discussed in more detail below (Section 2.3.3). These included diets equally enriched in SFAs, and based on extensive literature, were predicted to induce obesity. Outbred rat strains, including Wistar and Sprague-Dawley, when fed such diets, become progressively more obese than their standard chow-fed littermates and develop insulin resistance and hyperleptinaemia (e.g. Storlein et al., 1986; Chang et al., 1990; Kraegan et al., 1991; Buettner et al., 2006; Chen et al., 2010; Hariri et al., 2010), similar to obese humans (Kopelman, 2000; Woods et al., 2003; Hariri & Thibault, 2010). They do not develop overt diabetes, and therefore, do not display β-cell failure or hyperglycaemia
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(Levin et al., 1997; Rossmeisl et al., 2003; Collins et al., 2004; Chatzigeorgiou et al., 2009; Peyot et al., 2010). This is typical of the prolonged period of impaired glucose tolerance observed in insulin-resistant obese humans, which can prevail for many years, preceding eventual development of frank diabetes (Ferrannini et al., 2004). In this way, outbred rat strains with diet-induced obesity (DIO) are more appropriate models of human obesity and its associated metabolic dysfunction than monogenic strains, such as the Zucker or Zucker Fatty Diabetic (ZDF) rat. The current consensus is that most human forms of obesity are diet-induced and occur against a polygenic background; that is, they are multi-factorial, resulting from the interaction between several susceptibility genes and environmental influences, such as sedentary lifestyle (Speakman, 2004). Outbred rats in a given colony or sample population will show variable responses to diets of a given composition. No two randomly selected populations will gain weight to exactly the same extent or within exactly the same time period (Sclafani et al., 1976; Levin et al., 1983; Levin & Sullivan, 1987; Chang et al., 1990; Levin et al., 1997; Reuter, 2007; Schariff & Ziv, 2009), as discussed in Chapter 1, Section 1.1.18.4. In contrast, the monogenic forms of obesity are due to single-gene defects, such as those resulting in the failure to produce a functional form of leptin or its receptor (Montague et al., 1997).
In the studies detailed here, the Wistar rat was the outbred strain of choice because it had been shown in-house to reliably develop DIO when exposed to a highly-palatable diet (Pickavance et al., 1999; 2000; Harrold et al., 2000a; Fatani et al., 2007; Al-Qahtani et al., 2008; Moore et al., 2008). This would enable direct comparison of project data to previous in-house data for the purposes of monitoring body weight and metabolic change. Males were selected over females to avoid the potential influence of hormonal cycling on the parameters of interest and to enable comparison with the majority of the literature in the field (use of males being the convention). Rats were supplied by Charles River U.K. Ltd. (Margate, U.K.) at sexual maturity (about 6 weeks of age; 225-250 g), to restrict parameters to those of an adult model.
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