Introduction
Glucagon is secreted from pancreatic alpha cells and contributes to promoting hepatic glucose production [
1]. Diabetic patients show a paradoxical secretion of glucagon in response to meal test [
2] and such diabetic hyperglucagonaemia is thought to be due to the relative deficiency of insulin action [
3]. Thus, blockade of glucagon action is considered to be a novel target for glucose-lowering drug development [
3].
Several animal models deficient in glucagon action have been reported, including prohormone convertase 2 knockout mice [
4,
5], glucagon receptor knockout (
Gcgr
−/−) mice [
6], mice treated with glucagon receptor antisense oligonucleotide [
7] and mice having pancreas-specific
Arx ablation [
8]. All of these animal models show lower blood glucose levels, suggesting that glucagon plays a major role in hepatic glucose production and the maintenance of blood glucose levels. Moreover, several studies demonstrated that such animal models do not develop hyperglycaemia after beta cell destruction by streptozotocin (STZ) treatment [
8‐
11], suggesting that glucagon plays an indispensable role in hyperglycaemia caused by beta cell destruction.
Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are incretins released from intestinal K- and L cells, respectively, and potentiate insulin secretion from beta cells in a glucose-dependent manner [
12,
13]. GLP-1 is produced from proglucagon, which also serves as a precursor of glucagon. Several animal models deficient in glucagon action show markedly elevated plasma GLP-1 levels [
5‐
7,
14], suggesting that GLP-1 might contribute to normoglycaemia under STZ-induced beta cell destruction via extra-pancreatic effects. We previously generated mice lacking proglucagon-derived peptides (PGDPs), including glucagon and GLP-1 (
GcgKO mice) [
15].
GcgKO mice display increased insulin sensitivity due to glucagon deficiency and enhanced early-phase insulin secretion in a GIP-dependent manner [
16]. In the present study, we investigated glucose metabolism in
GcgKO mice administered with STZ. We found that
GcgKO mice developed marked hyperglycaemia under the severe insulin deficiency caused by STZ-induced beta cell destruction despite the absence of glucagon. However,
GcgKO mice displayed normoglycaemia under moderate insulin deficiency caused by moderate beta cell damage. We also investigated involvement of GIP in resistance to beta cell damage in
GcgKO mice.
Discussion
Glucagon increases hepatic glucose production; insulin inhibits glucose production [
27]. The relative contribution of dysregulated glucagon secretion and impairment of insulin secretion to hyperglycaemia in diabetic patients has been a matter of debate [
28]. It has been shown recently that administration of STZ to
Gcgr
−/− mice disrupted 90% of the beta cells and abolished glucose-induced insulin secretion yet failed to cause hyperglycaemia [
10]. Based on this observation, it has been proposed that hyperglucagonaemia itself plays an essential role in increasing blood glucose levels.
However, in the present study we observed that
GcgKO mice lacking both glucagon and GLP-1 developed hyperglycaemia upon hSTZ-induced beta cell ablation. This difference between
Gcgr
−/− and
GcgKO is most likely due to the presence or absence of extra-pancreatic GLP-1 action. Several studies have shown that GLP-1 exhibits extra-pancreatic action in increasing insulin sensitivity and modulating glucose metabolism [
9,
20,
29]. This is supported by studies employing
Gcgr
−/−
Glp1r
−/− double knockout mice and mice with diphtheria toxin mediated-ablation of alpha and L cells (Gluc-DTR). Both models are deficient in GLP-1 action and exhibit hyperglycaemia on STZ treatment, underscoring the critical importance of GLP-1 on glycaemic control under STZ-induced beta cell ablation [
30‐
32]. Nevertheless, there are unique characteristics among
Gcgr
−/−
Glp1r
−/−, Gluc-DTR and
GcgKO mice.
GcgKO mice lack all proglucagon-derived peptides throughout life, while there is a decrease in PGDPs in Gluc-DTR mice: GLP-1 synthesis in Gluc-DTR mice returns to normal levels 7 days after injection of diphtheria toxin [
33]. GLP-2 is present in
Gcgr
−/−
Glp1r
−/− mice, but is reduced or absent in Gluc-DTR and
GcgKO mice, respectively. Thus, the presence or absence of GLP-2 and residual glucagon in Gluc-DTR mice does not seem to affect glycaemic control under beta cell ablation.
Nevertheless,
GcgKO mice, which lack GLP-1, maintain normoglycaemia after mSTZ treatment, which causes persistent hyperglycaemia in the control mice. Thus, the requirement for insulin to maintain normal blood glucose levels is lower in both
GcgKO and
Gcgr
−/− mice [
9,
15,
34,
35]. In the present study, plasma insulin levels under ad libitum-fed states were comparable between mSTZ-control and mSTZ-
GcgKO mice (Fig.
2b). Insulin levels in
GcgKO mice before STZ treatment were not significantly different from those after mSTZ treatment. These findings indicate that insulin plays a critical role in the maintenance of glucose levels in mSTZ-
GcgKO mice. Incretins regulate insulin secretion and GIP is the major incretin in
GcgKO mice, which lack GLP-1 [
36].
Several reports suggest that GIP potentiates the early phase of glucose-induced insulin secretion to contribute to improved glycaemic control [
16,
37‐
39]. GIP also has been reported to contribute to beta cell survival in vitro [
40,
41], while GIP overexpression was found to enhance the increment of insulin content induced by high-fat diet feeding by decreasing beta cell apoptosis [
39]. We previously reported that GIP was expressed not only in the gastrointestinal tract but also in pancreatic beta cells in
GcgKO mice and that GIP hypersecretion contributes to the enhanced glucose-induced insulin secretion and improved glucose tolerance under non-diabetic states in
GcgKO mice [
16]. These findings led us to investigate whether GIP contributes to resistance to developing diabetes in mSTZ-
GcgKO mice.
Moderate beta cell damage abolished insulin secretion in DKO but not in
GcgKO mice (Fig.
3). Treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-
GcgKO but not in mSTZ-DKO mice (Fig.
4, ESM Fig.
4). These results indicate that GIP played an important role in protecting mice deficient in PDGPs from diabetes. However, treatment with DPP4i did not significantly reduce the number of apoptotic cells in islets (Fig.
5), and blocking GIP actions did not modify pancreatic insulin content in mSTZ-treated mice (ESM Fig.
6). These results indicate that GIP does not contribute to beta cell protection in mSTZ-
GcgKO mice but that it does contribute to increase insulin secretion from each of the beta cells.
It was reported recently that GIP and GLP-1 are secreted not only from enteroendocrine K- and L cells but also from pancreatic islets [
16,
42‐
45]. In the present study, pancreatic GIP content in control mice decreased neither by hSTZ treatment nor by mSTZ treatment, most likely because GIP is expressed in pancreatic alpha cells [
16,
26,
45]. On the other hand, GIP content in
GcgKO pancreas was decreased by hSTZ treatment, confirming our previous results showing that GIP is expressed in beta cells in
GcgKO. However, pancreatic GIP content was not changed by mSTZ treatment (ESM Fig.
5). The mechanism underlying the lack of change in pancreatic GIP content under the mSTZ-induced beta cell damage, including that of regeneration of GIP-positive cells, remains to be elucidated.
Islet-derived GLP-1 has been shown to enhance glucose-induced insulin secretion in vitro by using DPP4i in non-diabetic human and mouse islets [
46]. In the present study, IPGTT and OGTT analyses suggested that both islet-derived GIP and gut-derived GIP contributes to improving glucose metabolism in mSTZ-
GcgKO mice. In, addition, our results indicate that islet-derived GIP can exert an insulinotropic effect even when islet insulin contents are decreased under glucagon-deficient states. We therefore propose that combination therapy with glucagon antagonist and DPP4i might be considered as a therapeutic option to treat diabetes.