Metformin treatment restores the altered microvascular reactivity in neonatal streptozotocin-induced diabetic rats increasing NOS activity, but not NOS expression
Introduction
Impaired endothelium-dependent and endothelium-independent relaxations have been reported in different types of blood vessels and in different animal models of diabetes (De Vriese et al., 2000). However, experimental research has been mainly focused on large conduit arteries such as the aorta and in adult chemical-induced diabetic rats.
Functional changes in the behavior of microvessels are observed in experimental model of type 1 diabetes mellitus (Garcia-Leme, 1981, Garcia-Leme, 1989). Decreased responses of mesenteric microvessels to histamine and bradykinin, vasodilators with permeability-increasing properties, but not acetylcholine, a vasodilator devoid of such action, are observed in alloxan-diabetic rats (Fortes et al., 1984, Fortes et al., 1989). In addition, there is a widespread dysfunction of microvessels leading to ischaemia in some tissues and hyperperfusion in others (Tomlinson et al., 1994).
The natural history of vascular disease in type 2 diabetes may differ substantially from that in type 1, as commented by De Vriese et al. (2000). Furthermore, studies investigating endothelial dysfunction in animal models of type 2 diabetes are scarce and have yielded conflicting results. Both impaired (Sakamoto et al., 1998) and preserved endothelium-dependent (Bohlen and Lash, 1995) responses have been reported. In clinical studies, impaired endothelial-dependent vasodilatation was found but dyslipidemia and hypertension were often present in patients with type 2 diabetes (De Vriese et al., 2000). In addition, responses to endothelium-independent vasodilator agents, such as sodium nitroprusside (SNP), have been found to be impaired in some (Caballero et al., 1999, Woodman et al., 2002) but not in all (Goodfellow et al., 1996, Avogaro et al., 1997) studies.
The neonatal STZ rat model of non-insulin-dependent diabetes mellitus (NIDDM) (Bonner-Weir, 1994, Movassat et al., 1997), firstly described by Portha et al., 1974, Weir et al., 1981 is an alternate model that presents pancreatic beta-cell destruction followed by beta-cell regeneration and glucose intolerance. Other authors confirmed these findings and showed that neonatally-STZ treated rats in adulthood display the typical characteristics of NIDDM (Iwase et al., 1986, Grill et al., 1987a, Grill et al., 1987b, Welsh and Hellerstroöm, 1990, Hemmings and Spafford, 2000). Also reduced insulin response to an acute increase in glucose concentrations has been found (Leahy and Weir, 1985). We recently demonstrated, in n-STZ diabetic rats, a reduced microvascular response to inflammatory mediators and endothelium-dependent vasodilators histamine, bradykinin and platelet-activating factor (PAF), using intravital microscopy (Rastelli et al., 2005). These agents induce endothelium-dependent vasodilatation releasing endothelium-dependent relaxing factors such as nitric oxide (NO) and endothelium-dependent hyperpolarizing factor (De Vriese et al., 2000).
Metformin, a biguanine, has been used in the management of type 2 diabetes for more than 40 years and is available in the United States for nearly 10 years ago. Metformin ameliorates hyperglycemia without stimulating insulin secretion (Wiernsperger and Bailey, 1999). Decreased hepatic glucose production (Hundal et al., 2000) and increased skeletal myocyte glucose uptake (Galuska et al., 1994) have been implicated as major contributors to metformin glucose-lowering efficacy. Matthaei et al. (1991) suggest that metformin acts at the level of glucose transport. Metformin also decreases hepatic lipids in obese mice (Lin et al., 2000) and has beneficial effects on circulating lipids linked to increased cardiovascular risk (Wu et al., 1990). However, no information is available on the effects of metformin treatment on the metabolic parameters, microvascular responses to endothelium-dependent and independent agents and the possible mechanism involved mainly the NO system in n-STZ diabetic rats.
The aim of this study was to assess the effect of metformin treatment (15 days) on mesenteric microvascular response to endothelium-dependent and independent vasodilators, to evaluate whether a positive relationship between hyperglycemia or impaired glucose tolerance and microvascular dysfunction can be found. The possible mechanisms involved in the effects of metformin in n-STZ diabetic rats will also be investigated.
Section snippets
Induction of diabetes
Diabetes mellitus was induced in male newborn (2-day-old, weighing 8 to 10 g) Wistar rats with bolus injection of STZ (160 mg/kg, i.p.) dissolved in citrate buffer (10 mM, pH 4.5). Control rats were sham injected with the same volume of citrate buffer. The n-STZ diabetic rats and matching controls were housed according to institutional guidelines (room temperature 22 ± 0.5 °C, 12 h light/dark cycle, 60% humidity, standard rat chow and water ad libitum). The experimental protocols were approved
General characteristics of the animals
Forty percent of the animals that received STZ died within 48 h of the injection. Twenty percent of the survivors did not present characteristics of diabetes.
Two weeks after metformin treatment, the body weight and the body weight gain of n-STZ diabetic and metformin-treated n-STZ diabetic rats were significantly less than those of the control animals (Table 1). Blood (Fig. 1) and urine (Table 1) glucose concentrations were found to be similarly elevated in n-STZ diabetic and metformin-treated
Discussion
In the present study, we demonstrated that metformin restores the reduced mesenteric arteriolar and venular responses to endothelium-dependent vasodilators, mediators or not of the inflammatory response and to endothelium-independent vasodilator agent, without correcting the metabolic alterations found. Increased NOS activity without alteration of NOS expression might contribute to the improvement of microvascular reactivity in n-STZ diabetic rats.
The administration of STZ (160 mg/kg, i.p.) to
Acknowledgements
This work had the financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), SP, Brazil, and PRONEX/CNPq (Conselho Nacional de Pesquisa, Brazil. Grants: 01/11616-5 and 579. We also gratefully acknowledge Larissa de Sá Lima, Tiyeko Anna Eliza Vieira de Moraes Urakawa and Rosangela Aparecida dos Santos for their expert technical assistance.
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