The pancreatic beta cell mass is dynamic and can increase when exposed to appropriate environmental and physiological changes, such as obesity and pregnancy 
, and growth stimuli such as BTC, glucagon-like peptide-1 
and gastrin 
. As diabetes results from absolute or relative deficiency of the beta cell mass, restoration of the beta cell mass might be one strategy for the treatment of diabetes. For this, various methods have been investigated including differentiation of insulin-producing cells from progenitor/stem cells and regeneration of beta cells.
We previously found that in vivo
BTC expression by delivery of a recombinant adenovirus expressing BTC (rAd-BTC) remitted hyperglycemia in STZ-induced diabetic mice by beta cell regeneration 
, and some studies showed that new beta cells by BTC are generated mainly by replication of pre-existing beta cells 
. BTC is a potent growth factor that has positive effects on beta cell growth, through both increased proliferation and neogenesis. BTC promotes beta cell neogenesis from the pancreatic duct, therefore improving glycemic control after selective beta cell destruction with alloxan or after 90% pancreatectomy 
. In addition to BTC, other EGF receptor ligands have positive effects on beta cells. For example, EGF alone or combination with gastrin increased insulin secretion and beta cell numbers 
, and EGF treatment increased serum insulin levels and lowered blood glucose levels in diabetic mice 
. Transforming growth factor-α increased beta cell growth and differentiation 
, and epiregulin, another EGF receptor ligand, stimulated beta cell proliferation and insulin release in pancreatic beta cell lines 
. The functional redundancies of various EGF ligands suggest that EGF receptor binding with ligands via a conserved motif might be important mediators of beta cell mass regulation.
Because BTC produces physiological and pathological effects through its ErbB receptors, their distribution and activation are crucial for the function of BTC. It was reported that proliferative responses after BTC treatment were different between INS-1 cells and more primitive RINm5F cells, due to differential expression of ErbB family receptors 
. Moreover, a BTC mutant protein, with a single mutation replacing glutamic acid with lysine, changed the binding affinity to ErbB receptors and resulted the differentiation of AR42J cells into insulin-secreting cells 
. These studies indicate that the expression and affinity or activity of ErbBs play important roles for BTC function.
Sundaresan et al. reported that the ability of cells to respond to ligand is dependent on the expression of specific ErbB receptor combinations in the target tissue 
. To investigate which ErbBs are involved in BTC-induced beta cell proliferation, we first checked the expression of ErbBs in MIN-6 insulinoma cells and mouse islets and observed that all known ErbB isoforms (ErbB-1, ErbB-2, ErbB-3, ErbB-4) were expressed both in MIN-6 cells and islets of C57BL/6 mice. Expression of four types of ErbB receptors in the MIN-6 cells suggests that multiple combinations of receptor heterodimers are able to form. However, we found that BTC induced only ErbB-1 and ErbB-2 phosphorylation in MIN-6 cells, suggesting that BTC may bind ErbB-1 and ErbB-2 in beta cells and deliver the signals for biological actions. Because it was reported that homodimers of ErbB-2 or ErbB-3 cannot mediate the growth factor signal and they form heterodimers with ErbB-1 and ErbB-4 
, our results suggest that BTC-induced beta cell proliferation could occur through activation of ErbB-1 homodimers or ErbB-1/ErbB-2 heterodimer. As expected, BTC-induced DNA synthesis and cell cycle progression were suppressed by inhibitors of ErbB-1 or ErbB-2 receptors. These results clearly indicate that ErbB-1 and ErbB-2 activation might be involved in BTC action in MIN-6 cells, although it is not clear whether ErbB-1 homodimer or heterodimer of these receptors binds to BTC.
Signaling pathways mediated by ErbB-1 and ErbB-2 receptors involve the activation of Ras, MAPK, and the PI3K-activated Akt pathway, which activates several nuclear proteins, including cyclin D, a protein required for cell progression from G1 to S phase 
. We confirmed that cyclin D was translocated from cytosol to nucleus by BTC treatment (data not shown). Recently, many studies have shown that various transcription factors are involved in BTC-induced proliferation 
. One such transcription factor, CREB, leads to the expression of genes for beta cell function and survival and plays a role in mediating the effects of exendin-4, and BTC 
. As IRS-2, an important mediator of beta cell proliferation, is one of the CREB-dependent genes 
, we confirmed that the expression of IRS-2 was upregulated by BTC treatment, and downregulation of IRS-2 blocked BTC-induced beta cell proliferation. These results indicate that IRS-2 expression is one mechanism by which BTC enhances beta cell proliferation, and this is mediated through ErbB-1 and ErbB-2 receptors.
We then asked whether ErbB-1 or ErbB-2 plays a role in the regeneration of beta cells and remission of diabetes in rAd-BTC-treated diabetic mice. We found an elevated expression of ErbB-1 and ErbB-2 protein in the pancreatic islets of STZ-induced diabetic mice or STZ-induced diabetic mice treated with rAd-BTC as compared with normal mice. Moreover, islet size and the number of proliferating beta cells in rAd-BTC treated mice was significantly reduced by treatment with ErbB-1 and ErbB-2 inhibitors. These results suggest that ErbB-1 and ErbB-2 expression in the islets of diabetic mice may facilitate BTC-induced regeneration of beta cells to compensate for beta cell loss. Once beta cells are destroyed, ErbB-1 and ErbB-2 expression may increase, and binding of these receptors to BTC may induce regeneration of beta cells.
Not only proliferation of pre-existing beta cells but also differentiation of beta cells from precursor cells residing in the pancreatic duct cells have been proposed as methods to increase beta cell mass 
, and BTC is known to have a role in these processes. A combination of hepatocyte growth factor and BTC-δ4 induced differentiation of pancreatic ductal epithelial cells into insulin-producing cells, and BTC gene transduction promoted beta cell differentiation from ductal cells 
. Kritzik et al. observed substantial expression of ErbB receptors in ductal cells of the regenerating pancreas, especially ErbB-2-positive cells were more prevalent than ErbB-3- or ErbB-4-positive cells 
. Therefore, expression level of ErbBs might be relevant to ductal cell differentiation, which also contributes to beta cell regeneration.
Both ErbB-1 and ErbB-2 inhibitors abrogated the remission effect of BTC in diabetic mice, and the effect was most pronounced with the ErbB-2 inhibitor, which is correlated with the enhancement of ErbB-2 expression in islets of diabetic mice treated with rAd-BTC. It is not clear at the present time how the enhanced islet expression of ErbB-2 relates to beta cell regeneration. It was reported that expression of ErbB-2 was significantly induced in islets adjacent to areas infiltrated by immunocytes during interferon-γ mediated pancreatic regeneration 
. Therefore, infiltrating immune cells might supply signals that mediate ErbB-2 induction in the regenerating islets. In addition, induction of ErbB-1 and ErbB-2 in our study might be part of a protective response elicited by the islet in response to the islet damage, but how this works for regeneration of beta cells requires further investigation.
In summary, our data shows that BTC activated ErbB-1 and ErbB-2 receptors in beta cells, deliver the signal, and consequently induce IRS-2 expression, which contributes to beta cell proliferation. In vivo studies also showed that ErbB-1 and ErbB-2 receptors are involved in BTC-induced beta cell regeneration at least in part by inducing beta cell proliferation.