The ZDF rat is an inbred rat model obtained through selective breeding (fa mutation) that results in shortened leptin receptor which does not effectively interact with leptin and is phenotypically expressed as obesity with high levels of leptin in the blood. When fed with a diet of Purina 5008, homozygote recessive males (fa/fa) develop obesity, hyperlipidemia, fasting hyperglycaemia and non-insulin dependent diabetes (NIDDM).
We observed a trend towards high glucose plasma concentration in all groups since the first time point. At week 10, hyperglycaemia was modestly higher in the vehicle group in which diabetes seems to develop earlier whereas low-calorie diet could delay the onset of a diabetic state similar to obese non-diabetic Zücker rats. Nine weeks later, the plasma glucose level in the vehicle group was up to three times higher than in the non-diabetic one, and almost two times than in the diet restricted group. This rapid increase in glucose level in the vehicle group due to an overt diabetic state was partially controlled by both interventions with telmisartan or low-calorie diet.
On the contrary, greater weight increase rate was found in the non-diabetic animals and minor in the diabetic vehicle group, whereas rats subjected to caloric restriction or telmisartan showed moderate increase during experiment. In work by Corsetti et al. as hyperglycaemia developed with a higher dietary fat-content weight increased slower . Ohneda et al. also showed that when ZDF rats are fed with an unrestricted diet they became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic despite the fact that at 18 weeks of age their mean body weight equalled that of obese rats on an unrestricted diet . As we could see, rats on a moderate caloric restriction diet with a well-adjusted nutrient content showed a normal growth whereas animals on an unrestricted diet had only a markedly increase in body fat content but weight increase was hindered by the severe catabolic state associated with earlier and more severe diabetes as it was suggested by Corsetti and colleagues.
Telmisartan can modulate PPARγ by improving insulin sensitivity without fluid retention . Upton et al. demonstrated that ZDF rats treated with experimental thiazolidinedione MCC-555 improved metabolic status and insulin sensitivity, and had a small but significant increase in body weight gain during experimental period whereas vehicle-treated animals did not . Despite the marked hyperphagia in diabetic ZDF rats, they do not continue to gain weight after 15–20 weeks of age due to heavy losses of energy as glycosuria and higher catabolism. The body weight gain in diabetic ZDF rats following telmisartan treatment is probably the result of increase glucose entry into adipose tissue and the conversion into triglycerides.
Urinary loss of albumin in the vehicle treated diabetic group was significantly higher than groups on caloric restricted diet or telmisartan treatment at week 19, but also since week 14 (data not shown), as well as the increase in albuminuria since the starting point at the experimental period. Albuminuria correlates with endothelial dysfunction and predicts cardiovascular outcomes by its predisposition to systemic atherosclerosis .
With increasing age, fa/fa-rats spontaneously develop proteinuria and focal segmental glomerulosclerosis (FSGS), eventually leading to renal failure . The development of proteinuria and/or FSGS in fa/fa-rats can be ameliorated by treatment with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor . When studying diabetic ZDF, Coimbra et al. showed that the animals exhibited pronounced hyperinsulinemia and hyperlipidaemia at 6 weeks and became diabetic after 14 weeks of age. Significant FSGS nephropathy was first noted in 18-week-old fa/fa-rats and tubulointerstitial damage and proteinuria in 40-week-old fa/fa-rats but progressive glomerular hypertrophy was already detected in diabetic fa/fa rats after 10. They suggested that early progressive podocyte damage and macrophage infiltration was associated with type II diabetes mellitus but also with hyperlipidaemia . Noda et al.  examined the role of angiotensin II in obese Zücker rats and suggested that angiotensin II is the main cause of diabetic nephropathy as is the case in diabetic patients. Increase in proteinuria and albuminuria are inhibited by ACE-I or ARB in animals . Therefore, as in the work by Ohmura et al. , it is possible that the renoprotective effect of telmisartan may be induced at least in part by inhibition of the renal hypertensive effect of angiotensin II based on the antagonistic activity to the AT1 receptor, but also by its non-hemodynamic effects in relation to cell proliferation, renal sclerosis and fibrosis stimulated by angiotensin II . They showed that even at low doses, telmisartan inhibited the increase in albuminuria and was associated with a significant decrease in the progression of glomerulosclerosis. Our findings are consistent with their conclusions regarding those renal changes.
Our results suggest that a low-calorie diet can prevent urinary loss of albumin and therefore early renal damage probably by its lipid lowering effect and slowing the rapid decline in insulin sensitivity. Works by Sparks- Corsetti et al. showed a significant increase over time in serum cholesterol concentration in the higher dietary fat diet relative to the lowest fat-content diet and triglycerides concentration in male ZDF rats. Previously that group had reported an extensive characterization of the diabetic dyslipidaemia in obese ZDF rats that was initially due to triglyceride and free fatty acids but followed by an additionally rise in serum total cholesterol (LDL and HDL cholesterol) in the insulinopenic state, by 20 weeks of age [20, 31]. In this study, telmisartan did not show the same effect as with restricted diet, probably due to low dose of treatment and short experimental period. According to Ohmura et al. telmisartan did only inhibit the increase in blood cholesterol and triglycerides with higher doses of telmisartan and longer treatment periods .
We also found moderate correlation between serum triglyceride concentration and albumin urinary excretion. As mentioned above, treatment of hyperlipidaemia can reduce glomerular injury in obese Zücker rats. Work by Joles et al.  showed that analbuminemic rats with hypertriglyceridemia developed podocyte injury and glomerulosclerosis, and that hypertriglyceridemia, proteinuria, and the increase in desmin staining (podocyte injury) were largely prevented by ovariectomy.
Regarding APOA1 either in serum or in hepatic tissue, to our knowledge, this is the first report on plasma APOA1 concentration and hepatic mRNA expression in ZDF rats subjected to low-calorie diet or telmisartan. Current data shows a trend towards higher plasma APOA1 concentration as well as mRNA hepatic expression when animals are not treated with telmisartan and are fed a normal chow. A study by Sparks et al.  in hyperinsulinemic and insulinopenic Zücker diabetic fatty rats showed increases both APOA1 and APOA4 levels more than doubled between 10 and 20 weeks in ZDF rats as hyperglycaemia and hypercholesterolemia developed. In our experiment conditions, the effect of PPAR modulation with low dose telmisartan and caloric restriction seems to be independent of serum cholesterol levels since there were no differences between vehicle and telmisartan-treated group. This could be explained by the non-linear relationship between HDL and APOA1 levels depending on the type of HDL-particle analysed . A good correlation between serum APOA1 levels and the increase in glycaemia was observed in the present study. Murao et al.  showed that transcriptional activity of the rat APOA1 promoter in HepG2 cells paralleled the endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin: glucose decreases and insulin increases APOA1 promoter activity, respectively. We found that endogenous mRNA expression paralleled serum apolipoprotein concentration. However, animals with marked hyperglycaemia and severe diabetes showed higher serum APOA1 concentration suggesting a positive regulation in vivo.
Oxidation of LDL is thought to contribute to the development of atherosclerosis. Using the dichlorofluorescein analysis (DCF), the high fluorescent signal generated by LDL-oxidized-phospholipids in the vehicle group suggests a greater pro-inflammatory and pro-atherosclerosis state in the obese and diabetic animal group than the others. On the contrary, LDL from calorie-restricted diet resulted in low fluorescent signal. Navab et al. demonstrated that normal HDL inhibited the fluorescent signal generated by oxidized phospholipids in the cell-free assay using dichlorofluorescein, whereas patient (with definite coronary atherosclerosis) HDL did not. In our study when incubating this oxidized LDL from vehicle group with its own HDL, we saw a decrease in the fluorescent signal, suggesting intact beneficial properties of HDL in this early setting when preventing the formation or inactivating oxidized phospholipids. As mentioned above good correlations were also found between the DCF signal difference between LDL and LDL + HDL incubation and the increase in the urinary albumin excretion, suggesting that either LDL oxidisability or the properties of HDL in this animal model of type II diabetes could be used as marker of albuminuria and endothelial dysfunction. The low calorie diet lowered total serum cholesterol, although no dietary manipulations were made in fatty acid content that could increase the LDL oxidative resistance. The relative content in monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) were the same in both the normal chow and the 30%-less calorie diet. Nevertheless, we have clearly shown an inhibition of oxidative LDL signal in the restrictive dietary group compared with the others. This would be in agreement with the results of Hargrove et al.  who showed differences in LDL oxidative susceptibility between different diets (although the objective of the study was to evaluate the effects of diets high in MUFA from different food sources on LDL oxidative susceptibility). They compared these effects with those of a Step II blood cholesterol-lowering diet (25% total fat, 7% saturated) as well as a higher fat average American diet (35% total fat, 15% saturated). As regard to the two last diets, they showed that the average American diet tended to cause the shortest to LDL-oxidation lag time compared with the Step II diet.
Telmisartan may mediate cholesterol efflux by activating PPAR. In this regard, Matsumura et al.  demonstrated that telmisartan increased the expression of CD36, ABCA1 and ABCG1, all PPAR-responsive genes, and the activation of LXR, a transcription factor that also regulates ABCA1 and ABCG1 gene transcription in macrophages. This finding corroborated previous reports that telmisartan activated PPARγ target genes including CD36 in monocytes  and that telmisartan increased cholesterol efflux via PPARγ-dependent ABCA1 and ABCG1 expression in macrophages .