This paper focuses on the molecular mechanisms by which tungstate administration modulates pancreatic plasticity in diabetic animals. We selected the streptozotocin rat model, which shows a high degree of pancreatic damage at both endocrine and exocrine levels [20
]. In this situation, tungstate administration partially restores glycemia, insulinemia and amylasemia. Moreover, the morphological results clearly demonstrate that treatment increased beta cell mass in the pancreas and increased insulin and Pdx-1 positive cells in the islets. This increased beta cell mass can be ascribed, at least in part, to a combination of a decrease in beta cell apoptosis and an increase in islet proliferation.
In agreement with the metabolic phenotype, the pancreatic microarray analysis found no differences in the gene expression pattern between HU and HT animals, but it showed a very different pattern in DU animals as compared with both healthy samples. These changes are found not only in genes linked to endocrine function but also in those linked to exocrine function, probably due to the toxic effects of STZ. Finally, DT animals presented a specific pattern which was different from both DU animals and healthy animals, due to the effects of sodium tungstate on the diabetes background.
The analysis of the microarray data led us to propose a model of tungstate action (Figure ) which could explain the general improvement in the exocrine and endocrine pancreatic function of the diabetic animals treated with sodium tungstate. To understand these effects, we should bear in mind that tungstate is able to inhibit phosphatase activity due to its chemical properties [21
]. Our group and others have shown that, probably as a result of this property, tungstate treatment increases the phosphorylation of key proteins of different pathways [9
] and is probably responsible for the effects of tungstate in diabetic animals. Thus, the combination of these two actions – modification of gene expression and enhancement of phosphorylation – may cooperate synergistically to increase beta cell mass, which, together with the improvement of hepatic metabolism [5
], would lead to a decrease in hyperglycemia.
Figure 5 Pathways involved in the effects of tungstate on the pancreas. Microarrays from diabetic treated animals show that tungstate is acting through many interconnected pathways, which lead to an overall improvement of pancreas function. Some of the genes found (more ...)
In relation to the recovery of exocrine function, we should mention the possible role of Xbp1
. This gene plays a key role in the unfolded protein response [22
], and Xbp1
knockout animals display abnormalities exclusively in secretory organs such as the exocrine pancreas, which lead to early postnatal lethality [23
]. In our model, we found Xbp1
to be decreased in the DU animals and increased in the DT animals concomitantly to the recovery of the amylase expression, a finding that stresses the important role of this gene in the correct function of the exocrine machinery.
Several genes whose expression was modified by tungstate administration to diabetic animals may be responsible for the recovery of pancreatic function. Those genes are involved in different pathways which control a wide range of physiological functions, covering growth and differentiation to morphogenesis and angiogenesis. Therefore, it is probably the combination of tungstate effects upon all these pathways that leads to the recovery of pancreatic function. Since we performed microarrays using the total pancreas, we cannot identify the exact sites of these changes (exocrine, endocrine or ducts). Nevertheless, it seems that their modulation may contribute to the pancreatic regeneration observed in the diabetic treated animals. Fgf13
is one of the genes modified in the diabetic treated animals. Fgf13 binds to islet-brain 2, recruiting p38δ and increasing the activity of this kinase [24
]. Interestingly, it has already been shown that tungstate increases the phosphorylation of p38 in both MIN6 beta cells [25
] and islets of nSTZ-rats [9
], which may lead to the improvement observed in the pancreatic beta cell population. Another modified gene was Tspn8
, which may exert tumor-promoting activities by increasing cell motility and by inducing angiogenesis [26
]. Therefore, bearing in mind that STZ damage provokes severe microcirculatory disturbances within pancreatic islets [27
], we propose that an increase in the Tspn8
expression may improve pancreas revascularization and enhance islet function. Finally, other interesting genes are Tgfb3, Usag-1
plays a key role in complex processes including epithelial to mesenchymal transition [28
], which is relatively important in pancreas regeneration [29
modulates Wnt and Bone Morphogenic Protein signaling [31
] in different ways. Sel1h
has been implicated as a Notch inhibitor in exocrine [34
] and endocrine replication [35
] and modifies the expression of genes involved in cell-matrix interactions and in the cell cycle [36
]. Although the exact role of these genes in pancreatic regeneration has not been investigated in detail, we hypothesize that the combined action of these multiple pathways leads to the regeneration of the pancreas observed in the DT rats.
Although many pathways are involved in tungstate action, the results clearly show that the MAPK pathway plays a key role. The array analysis showed normalization in Rkip
expression in the DT animals. This protein binds to v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1), leading to the blockage of Mek and extracellular-signal-regulated kinase 1 (Erk) activation. It has been shown that, in the pancreas, Rkip localizes specifically in the islets and is involved in the regulation of beta cell growth. When this gene is up-regulated, as in the case of the DU animals, it acts as a brake on the MAPK signaling and beta cell proliferation [18
]. The normalization of Rkip
in the DT rats would permit the unblocking of the MAPK signaling and thus allowing the proliferative signals through this pathway. In the liver, it has been shown that tungstate enhances glycogen synthesis through MAPK activation [10
]. Here we demonstrate that tungstate activates MAPK both directly and indirectly and is crucial for the tungstate-induced increase observed in beta cell replication. On the one hand, tungstate enhances MAPK phosphorylation; on the other, it normalizes Rkip
expression which also leads to an increase in the MAPK pathway tone. The combination of these two mechanisms is the cause of the increased beta cell proliferation observed in our study.