Estudio de cortocircuitos

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Figure 5.12 Hypothalamic CRF and NPY expression, (a) CRF mRNA expression in the PVN of 4% (chow) and 30% fat fed male rats, (b) NPY mRNA expression in the ARC o f 4% (chow) and 30% fat fed male rats (n=5-7, */§p<0.05, ***p<0.001).

Chapter 5_________________________________Manipulation o f the SLOB Rat Phenotype

5.4 Effect of Food Restriction in SLOB Males

The development o f most forms o f obesity is associated with hyperphagia (Seidell,

2 0 0 0), therefore the most commonly promulgated treatment for both prevention or amelioration o f the obese condition, regardless of its origin, has been the prevention of hyperphagia by caloric restriction (Danforth, 1985; Wadden, 1993). Such studies investigating the effects of caloric restriction have been carried out for the past 30-40 years. Furthermore, the close association o f the severe hyperplastic-hypertrophic form o f early onset obesity (Hirsch and Knittle., 1970; Bjomtorp et al, 1971; Brook

et al., 1972) has led to the suggestion that infants and children may become hypercellular and obese due primarily to early overfeeding, and that prevention o f this early hyperphagia may prevent the later development o f obesity. However, it is becoming increasingly clear that caloric restriction, while decreasing bodyweight, does not always decrease body fat to the desired extent (Zucker, 1967; Cox and Powley, 1977). For example, it is well known that long-term food restriction has little effect on body composition in mature ob/ob mice (Alonzo et al., 1955; Dubuc et a l,

1976b). Neither caloric restriction from 17d of age nor continuing the pre-weaning milk diet until four weeks of age substantially affects the development o f obesity or delays the onset o f diabetes in these mice (Dubuc et a l, 1981). In the Zucker rat prevention of hyperphagia throughout life by continuous food restriction also has no effect on production of the complete obese phenotype (Cleary et al, 1980).

One of the major differences between these animals and SLOB animals are that severely obese SLOB rats are not hyperphagic. The consequence o f food restriction in SLOB rats is therefore not easy to predict and may be different to that seen in ob/ob

mice and Zucker rats. Food restriction studies can be achieved by adopting various methods. For food restriction from birth, one can limit food intake during the suckling period by increasing litter size in a weaning cage. When an obese animal displays hyperphagia, food restriction can be achieved by pairfeeding to the intake of normal lean animals or reducing the intake to a certain percentage o f daily consumption by normal lean animals. I showed in chapter 3 that old (365 days) SLOB rats are not hyperphagic, even when expressed per lOOg bodyweight. I

Chapter 5_________________________________Manipulation o f the SLOB Rat Phenotype

decided to study old male rats (11 months old) that already show a severe obesity phenotype and assess the effects of any change in obesity, rather than the prevention o f it. Because SLOB and normal animal food intake at this age are the same, food restriction could be chosen as 50% of the mean daily food intake per animal for all the animals. This was chosen as previous researchers had also used this in adult rats and produced a significant decrease in bodyweight with minimum stress to the animal (Moustafa et al, 1980). 50% of normal food intake could also produce a sustained reduction over 2 weeks, rather than a drastic effect over a few days which results from a more pronounced food withdrawal

Four groups of age-matched males were selected, each group containing 6 animals;

(a)SLOB-W libitum^ (b)SLOB-food restricted, (c)normal-W libitum, (d)normal-food restricted. Animals were placed into individual metabolic cages with standard chow diet and tap water to drink. After acclimatisation food intake per animal was recorded daily for four days and mean daily food intake calculated. In this case I found the rats to consume an average of 2 0g of food per day per animal, therefore food restricted animals were allowed lOg/day. Food was rationed out daily at 19.00h, just before the dark period in the hope to maintain the normal rhythm of eating in rats, at the same time food hoppers for ad libitum groups were topped up and bodyweights for all animals recorded. After 2 weeks o f food restriction, animals were culled, measurements taken and blood and organs collected for analysis.

Figure 5.13(a) shows bodyweights for normal and SLOB rats during 2-weeks of food restriction or ad libitum. All groups lost weight, with as expected the greatest loss in bodyweight occurring in food restricted SLOB and normal animals. However, their bodyweight curves were almost identical therefore no difference in the rate o f loss of weight between transgenic and normal animals was seen. The short initial loss of weight in ad libitum SLOB and normal groups was probably due to the fact that at this age these animals are quite large and perhaps take time to adjust to housing and feeding in metabolic cages. However, I ensured they could still access the food hoppers. As SLOB ad libitum animals lost more weight than normal ad libitum

animals, I had to consider the possibility that their greater abdominal adiposity reduced their mobility or adaptation to this housing. However this weight loss had stabilised after 4 days and by the end of the study there was no significant difference

Chapter 5_________________________________Manipulation o f the SLOB Rat Phenotype

in weight loss between ad libitum groups. This was shown in both the final weight at the end o f the study (day 17 Figure 5.13(a)) and the weight loss chart (F ig u re 5.13(b)). Fat pad weights, supra-renal, mesenteric and testicular depots were measured at the end o f the experiment (Figure 5.14(a),(b),(c)). In normal animals, significant weight loss was observed in all three depots but in SLOB animals a significant loss was only observed in the supra-renal fat depot. As the effect o f food restriction in SLOB rats appears to be adipose-depot specific, a measurement of plasma leptin levels would indicate a change in contribution from this depot in SLOB rats. Leptin levels are thought to reflect peripheral rather than central adipose stores (Lonnqvist et a l, 1997a; Friedman and Halaas, 1998), I would therefore expect relatively less change in plasma leptin levels in food restricted compared to ad libitum

SLOB rats. However, plasma leptins were significantly reduced in food restricted SLOB rats compared to ad libitum SLOB rats (Figure 5.14(d)), suggesting that the increased central adipose depot in these rats is responsible for their elevated plasma leptin levels.

I also measured the effect of food restriction on various organ weights (Figure 5.15). For both the heart and soleus muscle, no change was found between ad libitum and food restricted SLOB or normal animals. This indicated both SLOB and normal rats utilised relatively more energy from their adipose depots, although the two-week restriction may not have been enough to mobilise energy away from muscle. Liver weights were significantly decreased in both food restricted SLOB and normal animals, whereas spleen weight was only reduced in food restricted normal animals.

As described in 5.2, SLOB organ weights are reduced in weight compared to normal rats, probably due to GH deficiency. Food intake affects plasma IGF-I levels and this subsequently affects organ growth (Frystyk et al, 1995; Cattaneo et a l, 1996; Clark

et a l, 1996; Nam and Marcus, 2000). I therefore decided to measure plasma IGF-I levels in these animals. Figure 5.16 shows IGF-I levels were significantly reduced in food restricted normal rats, but food restriction had no effect on IGF-I levels in SLOB rats.

(a) B o d y w eig h t change

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Figure 5.13 Weight change in SLOB and normal male rats fed ad libitum or on 50% food restriction, (a) Body weight change over 2 weeks on ad lib. or restricted diet, (b) Overall bodyweight loss after 2 weeks (n=6, **p<0.01, ***p<0.001, n.s=not significant).

□ SLOB ad libitum, O SLOB restricted, 0 normal ad libitum, © norm al restricted

(a) Supra-renal fat pad w eig h t (b) T esticu lar fat p ad w eig h t

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