Introduction
It is widely accepted that beta cell function progressively deteriorates in individuals with type 2 diabetes mellitus as described, for example, by the UK Prospective Diabetes Study [
1]. To prevent diabetic complications, maintenance of strict glycaemic control is necessary, but this requires preservation of beta cell function. On the other hand, human diabetic patients exhibit an early defect in glucose-stimulated insulin secretion (GSIS) [
2] and autopsy studies have demonstrated reduced beta cell mass in diabetic patients [
3]. Thus, development of treatment strategies has focused on ways to improve beta cell function and to prevent beta cell death. Hence, there is increased interest in determining whether newer drugs such as insulin-sensitising agents produce sustained improvements in beta cell function [
4,
5]. Likewise, agents associated with glucagon-like peptide-1 (GLP-1) have also attracted considerable attention because they may alter the natural history of type 2 diabetes by preserving pancreatic beta cell mass [
6‐
8] and function [
9‐
11].
GLP-1 has several beneficial effects that counteract the pathophysiology of diabetes mellitus. First, GLP-1 is a potent glucose-lowering polypeptide that induces glucose-dependent insulin secretion [
12,
13] while suppressing glucagon secretion [
14,
15]. Second, GLP-1 has extra-pancreatic effects, such as delayed gastric emptying [
16,
17], appetite suppression [
18] and improved insulin sensitivity [
19]. Third and last, GLP-1 stimulates beta cell replication [
20,
21], neogenesis [
22] and differentiation [
22], as well as inhibiting beta cell apoptosis via reduction of cellular stress [
23‐
26]. Consequently, GLP-1-related agents are currently regarded as a powerful treatment option for type 2 diabetes [
27,
28]. Liraglutide, a novel long-acting GLP-1 derivative, is resistant to dipeptidyl peptidase-IV. Its prolonged effects result from the substitution of Lys for Arg34 and the addition of a glutamic acid and a 16C NEFA to the Lys26 residue of native GLP-1 [
29].
To investigate the molecular mechanism by which liraglutide preserves pancreatic beta cell mass, we treated obese diabetic db/db mice with liraglutide for 2 days or 2 weeks. We also treated normoglycaemic m/m mice with liraglutide for 2 weeks.
Discussion
In the present study, we investigated the effects of liraglutide on obese diabetic mice. We found that the beta cell mass of
db/db mice was increased by long-term liraglutide treatment. These results are essentially consistent with previous reports describing a stimulatory effect on beta cell mass of exendin-4, a potent GLP-1 receptor agonist, in a partial pancreatectomy rat model of type 2 diabetes [
20] and of liraglutide, the long-acting GLP-1 derivative, in diabetic
db/db mice [
7].
The present results demonstrate that short- and long-term treatment with liraglutide affected mRNA expression of
Hlxb-9,
Hes1 and
Neurod in the core of islets in diabetic
db/db mice. The roles of several genes with well defined functions in pancreatic development, such as those mentioned above (
Hlxb9,
Hes-1 and
Neurod) have not been previously studied in the adult pancreas. On the other hand, previous studies have suggested that the Notch signal through hairy and enhancer of split-1 (HES1) is activated even in the adult pancreas in conditions associated with cell regeneration, such as inflammation and neoplasia in the pancreas [
33,
34]. In addition, a recent in vitro study also demonstrated that HES1 is involved in determining the beta cell fate of adult human beta cells [
35]. These findings suggest the possibility that genes associated with an early stage of endocrine pancreas development are expressed in adult
db/db mice and that liraglutide affects the expression of those genes. However, the present results cannot be regarded as conclusive, and further studies will be required to elucidate this issue.
In this study, cell proliferation related to
CycD and
Erk-1 gene expression was readily upregulated in diabetic and normoglycaemic mice treated with liraglutide for 2 days. Immunohistochemical analysis of the pancreatic islets suggested that enhancement of cellular proliferation might be an underlying mechanism that accounts for the restorative effects of GLP-1 on pancreatic beta cell mass. These results strongly support the hypothesis that liraglutide affects pancreatic beta cell mass by directly stimulating cellular proliferation. Interestingly, a previous study demonstrated that pancreatic beta cell mass in non-diabetic rats was significantly increased after 1 week of liraglutide treatment, but no different from that in control animals after 6 weeks of treatment, indicative of a temporary effect on beta cell mass [
36]. This result suggests that the effect of liraglutide on beta cell kinetics is acute and temporary in non-diabetic conditions.
The binding of GLP-1 to its receptors activates adenylate cyclase and the cyclic AMP/protein kinase A signalling pathway. Additionally, GLP-1 activates phosphoinositide 3-kinase (PI3K), p42 mitogen-activated protein kinase (MAPK) and the epidermal growth factor receptor [
37‐
39]. Furthermore, activation of the transcription factor for
Pdx1 [
38], p38 MAPK and protein kinase C-zeta [
40] reportedly plays a role in GLP-1-induced DNA synthesis and replication. Thus there is emergent evidence for extensive cross-talk between the G-protein-coupled receptor and tyrosine kinase-coupled receptor signalling pathways in beta cells. The results of the present study show that modulation of the
CycD gene is involved in the liraglutide-induced increase in beta cell mass, presumably through the MAPK pathway. We also found that cellular differentiation is affected by liraglutide via upregulation of
Pdx1, which is located downstream of the PI3K–protein kinase B–forkhead box O1 pathway.
Liraglutide modified the expression of genes related to cell apoptosis such as
Bcl2,
Casp8,
Casp3 and
Cad in
db/db mice during short- and long-term treatment, whereas mRNA levels of these genes were not altered in normoglycaemic
m/m mice, suggesting that liraglutide directly suppressed beta cell apoptosis in mice under hyperglycaemic conditions. In a previous report, extracellular signal-regulated kinase (ERK)1/2 regulated
Bcl2 gene expression and protein production, as well as the activity of caspase 3 through the regulation of cyclic AMP-responsive element-binding protein [
41]. Furthermore, the extracellular matrix directly suppressed caspase 8 activity through regulation of the ERK but not the PI3K pathway in pancreatic beta cells [
42]. These results indicate that liraglutide may have a direct anti-apoptotic effect, which is at least partly mediated by ERK.
With regard to the effect of GLP-1 on oxidative stress, Tews and co-workers [
26] reported that exendin-4 directly reduces oxidative stress through counterregulation of the reduced abundance of electron transport chain proteins in INS-1 beta cells. In the present study, immunohistological analysis with anti-4-HNE staining and the TUNEL assay showed that 2 weeks of liraglutide treatment inhibited cellular oxidative stress and apoptosis of the pancreatic islets in diabetic
db/db mice. We also showed that 2 weeks, but not 2 days of liraglutide treatment suppressed expression of
Cat and
Gpx. The expression of
Srebp-1c and
Fas mRNAs, as well as islet triacylglycerol content were also decreased only by the 2 week liraglutide treatment regimen. These results suggest that, in contrast to the action of exendin-4 in cultured cell lines, the improvements in oxidative stress observed with liraglutide treatment in
db/db mice are secondary to improvements in glucolipotoxicity.
The inhibitory effect of long-term liraglutide treatment on expression of
Xbp1 in the core area of pancreatic islets, which should reflect a decrease in the unfolded protein accumulated in the ER of pancreatic beta cells, is essentially consistent with the findings of Yusta et al., who reported that chronic administration of exendin-4 decreased expression of
Chop (also known as
Ddit3) and spliced
Xbp1 in the pancreatic islets of
db/db mice [
43]. They also found that exendin-4 potentiated the induction of activating transcription factor 4, a transcription factor mediated by phosphorylated eukaryotic translation initiation factor 2 α (eIF2α), in the INS-1 beta cell in a manner that is dependent on protein kinase A, accelerating recovery from ER stress-mediated translational repression by growth arrest and DNA-damage-inducible 34, which promotes eIF2α dephosphorylation [
43]. Contrary to the in vitro findings reported by Yusta et al. [
43], the present in vivo study demonstrated that even short-term liraglutide treatment did not affect expression of
Xbp1 in hyperglycaemic mice. The different study conditions, in vivo and in vitro experiments, may have led to these different results.
Fehmann and Habener demonstrated a stimulatory effect of GLP-1 on proinsulin biosynthesis in insulinoma beta TC-1 cells [
9]. In the present study, we found that liraglutide enhanced the responsiveness of beta cells to a glucose challenge in diabetic and normoglycaemic mice. These results are essentially consistent with previous studies that have reported an improved responsiveness of the pancreatic beta cells to glucose when isolated mouse islets were incubated with liraglutide [
10].
In conclusion, we have demonstrated that liraglutide restores pancreatic beta cell mass due to acute effects on cell kinetics and chronic effects on oxidative and ER stress that are secondary to improvements in glucolipotoxicity. These conclusions were mainly obtained by analysing the changes of gene expression in pancreatic islet cells. To fully understand the effect of liraglutide on pancreatic beta cell mass, further investigations, e.g. assessing the effect of liraglutide on peptide expression and function, as well as its role in neogenesis from the ducts, should be conducted.