In the present work, we demonstrated that PTX significantly reduces glycemia and triglyceride levels in alloxan-induced diabetic rats, after its daily administration for 1 week. Prolonged treatments bring blood glucose concentrations to normality, while triglycerides decreased by 50%. Our study showed a potentiation of the hypoglycemic effect of PTX by glibenclamide (GLI), after the two drugs association at lower doses. GLI works by inhibiting the ATP-sensitive potassium channels (KATP), in pancreatic beta-cells. This inhibition causes cell membrane depolarization and opening of the voltage-dependent calcium channel, which results in increased intracellular calcium concentration in the beta-cell and subsequent stimulation of insulin release (Luzi & Pozza 1997). PTX, at least in part, shares with GLI this mechanism of action.
Diazoxide (DZD) is used to inhibit inappropriate insulin secretion, causing hypoglycemia (Marks & Samols 1968). Its major mode of action is the opening of KATP channels in the beta-cell membrane, with repolarization, closure of voltage-dependent Ca2+ channels, and lowering of [Ca2+] ic (Henquin & Meissner 1982; Trube et al. 1986; Gilon & Henquin 1992). However, DZD has been proposed to decrease the efficacy of Ca2+ on exocytosis (Flatt et al. 1994; Renström & Rorsman 1996). In order to investigate whether PTX would block DZD hyperglycemia, as GLI does, the effects of both drugs were studied on diabetic and non-diabetic rats administered with DZD.
Our results show that, in diabetic animals, PTX and GLI presented a similar profile and did not prevent DZD-induced increases in glucose levels. However, in non-diabetic animals, while GLI blocked the increase in glycemia levels after DZD, this effect was not observed after PTX, when the animals treated with PTX + DZD behaved similarly to those treated with DZD only. Such results suggest that other factors, besides the KATP-dependent channels blockade, interfere with the PTX action.
The administration of PTX, for 1 and 2 months, to diabetic rats significantly decreased fructosamine and HbA1c levels, as related to untreated diabetic controls. Both fructosamine and glycated hemoglobin are associated with microvascular conditions in diabetic patients, and increased risk for mortality and morbidity in hemodialysis patients (Selvin et al. 2011; Shafi et al. 2013).
Our histological studies showed that changes in diabetic pancreas were partly reversed after PTX treatment. Interestingly, the diabetic group treated with the association of lower doses of PTX and GLI showed a pancreas with diminished damage, as related to that of diabetic rats treated with PTX alone. Similar results were also observed in diabetic liver and kidney. Previously (MartonJ et al. 1998), a beneficial effect of PTX treatment was demonstrated in rats submitted to acute pancreatitis and showed that PTX very effectively decreased TNF-alpha and IL-6 production under this condition.
Evidences (Coelho et al. 2012) indicate that the administration of PTX, after the onset of acute pancreatitis, decreased levels of pro-inflammatory cytokines.
Other studies have shown that PTX exerts a protective effect in animal models of diabetes (Stosic-Grujicic et al. 2001a; Stosic-Grujicic et al. 2001b). These authors showed that the administration of PTX to diabetic animals reduced the production of inflammatory mediators and prevented the development of hyperglycemia. They concluded that these effects of PTX involve down-regulation of the pro-inflammatory cytokine-mediated nitric oxide (NO) synthase pathway. Furthermore, inducible nitric oxide synthase (iNOS) was shown to play a role in fasting hyperglycemia, contributing to hepatic insulin resistance, in a model of obese diabetic mice (Fujimoto et al. 2005). These findings are very similar to ours, except that we used another model and lower PTX doses. Despite these differences, we demonstrated that PTX decreased not only hyperglycemia but also TG levels, in alloxan-induced diabetic rats.
In the present work, we demonstrated that the number of iNOS immune-stained cells was lower in pancreas of diabetic rats, after the PTX treatment. It is largely accepted that the induction of iNOS in pancreatic islets leads to increased NO production associated with dysfunctional beta-cells. Furthermore, a recent study (Muhammed et al. 2012) with diabetic islets culture, exposed to phosphodiesterase (PDE) inhibitors, showed a marked suppression of iNOS mRNA, reduced nitrite production and increased insulin secretion. Considering inflammatory cytokines and NO as potential mediators of pancreatic beta-cell destruction in diabetes, and since PTX is also a PDE inhibitor, these data point out to the same direction as ours and emphasize the potential benefit of PTX in this pathologic condition.
We also demonstrated that PTX treatment decreases the number of cyclooxygenase (COX-2) immunopositive cells in diabetic pancreas. COX-2 has been previously reported (Persaud et al. 2004) to be the dominant isoform and insulin-secretor in beta-cells under basal conditions. The observation that hyperglycemia increases the production of IL-1beta in human beta-cells and induces COX-2 expression, led these authors to suggest this to be a route by which hyperglycemia contributes to beta-cell dysfunction. Another work (Ling et al. 2005) demonstrated that the exposure of pancreatic beta-cells to IL-1beta induces the expression of iNOS and COX-2, and the subsequent formation of NO and prostaglandin E2 (PGE2) may impair beta-cell function. Therefore, the authors concluded that NO-affected COX-2 activity is directly linked to COX-2 gene transcription and protein expression in pancreatic beta-cells, providing a new therapeutic strategy for the management of diabetes mellitus.
In addition, PTX also decreases TNF-alpha immunoreactivity in diabetic pancreas, liver and kidney. Increased TNF-alpha production has been observed in adipose tissue derived from obese rodents or humans, and has been implicated as a causative factor in obesity-associated insulin resistance and diabetes mellitus pathogenesis. Furthermore, current evidence suggests that the administration of exogenous TNF-alpha to animals can induce insulin resistance, whereas its neutralization can improve insulin sensitivity (Moller 2000). Since plasma TNF-alpha is associated to insulin resistance, one can assume that this cytokine plays a significant role in the pathogenesis of chronic insulin resistance in humans (Plomgaard et al. 2007). In addition, PTX (50 mg/kg) administered to rats for 8 weeks was shown to inhibit insulin resistance and prevent TNF-alpha elevation, leukocyte infiltration and endothelial pyknosis (El-Bassossy et al. 2011).
A recent work (Francés et al. 2013) studied the contribution of TNF-alpha intracellular pathway and oxidative stress for the development of apoptosis, in the liver of diabetic rats. Interestingly, iNOS inhibition significantly reduced TNF-alpha levels. These data indicate that the regulation of TNF-alpha and oxidative stress in the diabetic state could be of therapeutic relevance for the improvement of complications linked to chronic hyperglycemia. Furthermore, results from most animal studies and randomized controlled trials on diabetic kidney disease consistently demonstrated that short-term use of PTX produces a significant reduction of proteinuria and microalbuminuria (Badri et al. 2011).
In conclusion, we showed that pentoxifylline decreases blood glucose and TG levels in diabetic rats. The pentoxifylline hypoglycemic effects are similar to those of glibenclamide and, at least partly, related to the inhibition of ATP-sensitive K+ channels. It also improves histological changes and decreases the immunoreactivity to iNOS, COX-2 and TNF-alpha, in diabetic pancreas, liver and kidney. Thus, pentoxifylline by its anti-inflammatory and antioxidant properties is a potential and alternative drug for the treatment of diabetes mellitus and its related complications.