3.3 CARACTERIZACIÓN BÁSICA DE LA LÍNEA DE TRANSMISIÓN
3.3.1 SELECCIÓN DE LA RUTA DE LA LÍNEA DE TRANSMISIÓN
F i^ r e 3.11 Plasma corticosterone levels in SLOB and normal rats over 24 hours. Individual corticosterone levels are shown for 5 normal and 4 SLOB male rats. Animals were sampled every 30 minutes over 24 hours. Corticosterone was measured by radioimmunoassay in half of the samples (every hour).
Chapter 3______________________________Characterisation o f the SLOB Rat Phenotype
observed in the early morning, the so-called ‘dawn phenomenon’ (Bolli et a l, 1984) and the insulin resistance following hypoglycemia (Fowelin et ah, 1989). GH also has a direct effect on adipose tissue where it promotes lipolysis (Richelsen, 1997). When abdominally obese individuals are treated with GH it is thought that although their fat pad depots may be reduced their diabetes may be worsened. In fact, their insulin resistance is lessened due to the GH effect on removal o f fat over-riding the GH effect on muscle and other tissues (Bemeis and Keller, 1996). I considered the possibility that reduced levels of rGH might have a role in protecting SLOBs from diabetes. In Chapter 5 I will show that GH treatment has no effect on abolishing fat mass in the GH-deficient SLOB rat. Subsequently, it would be interesting to measure plasma insulin levels in GH-treated animals to assess if replacing GH alters the diabetic status of SLOB rats. 20-22 week old SLOB and normal rats were treated for 4 weeks with recombinant growth hormone at a dose o f 200pg per day, delivered using an osmotic minipump. Following treatment, blood was collected and analysis of plasma insulin revealed no change between SLOB sham and SLOB treated rats, that is GH had no effect on plasma insulin levels (SLOB sham 6.02±l.lng/m l vs treated 6.85±Q.9ng/ml).
3.6.1 Insulin tolerance test (ITT) and Glucose tolerance test (GTT)
The resting glucose and insulin measurements shown above are static measures. A more insightful test of diabetic status is to carry out insulin tolerance tests (ITT) and glucose tolerance tests (GTT). An ITT was performed on 6 SLOB and 6 normal rats of just over a year in age when SLOB rats show severe obesity. Each received a dose of insulin at a concentration of 0.4U/kg bodyweight. This dose of insulin was chosen as it lowers blood glucose by 50% in dw arf rats (Clark et a l, 1996). Animals were sampled for 90 minutes following insulin administration, and for each time-point glucose was measured immediately following centrifugation to separate plasma (Figure 3.12 (a)). For the first 30 minutes both normal and SLOB animals appear to respond the same, at 30 minutes plasma glucose concentrations are equal in both groups (55mg/% glucose). After 30 minutes, normal animals start to clear the insulin load and glucose levels start returning to normal. However, in SLOB animals glucose levels remain low for a further 30 minutes after which they too increase. These results indicated SLOB rats were not insulin resistant and if anything were more
Chapter 3______________________________Characterisation o f the SLOB Rat Phenotype
sensitive to the effects of insulin. It must be noted GH deficiency together with IGF-I deficiency may have a role in this blunted insulin response. To investigate this the ITT was repeated using GH treated SLOB rats; these rats were treated using osmotic minipumps as previously described. However, GH treatment had no effect on glucose response and was comparable to that in the untreated SLOB animals (Figure 3.12(b)).
Glucose tolerance tests were also performed in normal and SLOB animals of the same age, each receiving 0.5g/kg of D-glucose. This dose was chosen as it had previously been tested in corpulent {cp) rats (Russell et al., 1999). In response to an intravenous glucose bolus, both SLOB and normal rats responded the same and shortly after administration began to clear the glucose load (Figure 3.13 (a)). Glucose levels continued to fall in both groups, although normal rat levels decreased at a faster rate than SLOB animals and had returned to normal by 60 minutes. However, there was no significant difference between SLOB and normal animals for any of the timepoints measured. Analysis of plasma insulin levels also showed little difference between SLOB and normal animals, a significant difference was only observed 5 minutes after treatment (Figure 3.13 (b)). From both ITT and GTT I concluded SLOB rats were neither diabetic nor insulin resistant. Thus, SLOB rats were able to respond to glucose and insulin loads in a normal manner and GH treatment did not have an effect on this response.
3 .7 L ip id a n a ly ses
Although the relationship between heart disease, obesity and diabetes has long been recognised, the underlying mechanisms for the increased risk are poorly understood. However, perturbations in plasma lipids, which have been associated with the development of heart disease, are observed in some obese individuals (Sirtori and Vega, 1997). These include elevated levels of plasma triglyceride and low-density lipoprotein cholesterol (LDL-C) and decreased levels o f high-density lipoprotein cholesterol (HDL-C). Several rodent models with varying severity’s o f obesity and diabetes have also been examined to determine whether they have high levels o f plasma triglycerides and combined very-low-density lipoprotein and low-density lipoprotein cholesterol (VLDL/LDL-C) and decreased HDL-C levels, similar to those
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i.v. insulin 0.4U/kg bodyweightI
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Figure 3.12 Insulin tolerance tests. Plasma glucose levels after 0.4U/kg bodyweight insulin challenge administered i.v. in (a) SLOB and normal maie rats (n=6), (b) SLOB, SLOB-GH treated and normal maie rats (n=6) (*p<0.05,
**p<0.01) I SLOB, □ Normal, # SLOB GH-treated.