Introduction
The gastrointestinal hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), cause glucose-dependent insulin secretion from pancreatic beta cells within minutes of nutrient ingestion [
1,
2]. One characteristic of type 2 diabetes mellitus is impaired incretin effect [
3,
4], although the secretion of GIP and GLP-1 is not always decreased [
4‐
6]. This indicates that the reduced incretin effect is due to defects in incretin receptor signalling pathways, rather than to the concentration of incretin hormones. The insulinotropic activity of GIP is largely impaired in patients with type 2 diabetes [
4,
7]. In contrast, the insulinotropic effects of GLP-1 are partially preserved, which is important for its therapeutic potential, but insulin responses are substantially reduced, especially when studies are done at comparable glucose levels [
7,
8]. Moreover, a growing body of evidence has shown that the glucose-lowering effects of GLP-1 are mediated by various mechanisms, including stimulation of glucose-dependent insulin secretion in pancreatic beta cells [
1,
2], promotion of pancreatic beta cell proliferation and inhibition of beta cell apoptosis [
9‐
11], inhibition of pancreatic alpha cell glucagon release [
12,
13] and regulation of appetite and the central nervous system [
2,
14]. These attributes of GLP-1 provide a strong basis for novel pharmacotherapies in type 2 diabetes. Currently, synthetic versions of GLP-1 mimetics (e.g. exenatide and liraglutide) and dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g. sitagliptin and vildagliptin), which reduce GLP-1 and GIP degradation by DPP-4, have been approved for the treatment of type 2 diabetes [
2,
15].
Type 2 diabetes develops as a result of impaired beta cell function and is closely associated with increased plasma NEFA, which are thought to be an important link between obesity and type 2 diabetes [
16‐
18]. NEFA can result in a state of insulin resistance [
19], induce pancreatic beta cell dysfunction and cause beta cell death [
18,
20]. Although acute exposure to elevated plasma NEFA enhances glucose- and non-glucose-stimulated insulin secretion in vitro and in vivo [
18,
21], long-term exposure to NEFA impairs glucose-stimulated insulin secretion [
22]. Recently, it was reported that while incretin secretion is similar between obese and non-obese type 2 diabetic patients [
23], obesity impairs the incretin effect independently of glucose tolerance [
24]. It has also been reported that loss of the incretin effects was more extensive in obese than in lean type 2 diabetic patients [
25]. More recently, Bando et al reported that obesity may attenuate the HbA
1c-lowering effect of sitagliptin in Japanese type 2 diabetic patients [
26]. This suggests that lipids may be involved in the regulation of incretin responsiveness in pancreatic beta cells. However, little is known about the influence of NEFA on incretin receptor signalling. Our previous study showed that hyperglycaemia downregulates GLP-1 receptor (GLP1R), which potentially contributes to the impaired incretin response in beta cells [
27]. Furthermore, the normalisation of blood glucose concentrations improves the insulin response to GLP-1 and GIP in patients with type 2 diabetes [
28]. In the present study, therefore, we used in vitro and in vivo approaches to investigate the role of NEFA in the impairment of incretin responses.
Discussion
Impaired incretin effects are found in type 2 diabetes [
3,
4]. Our study was designed to investigate the role of hyperlipidaemia in the impairment of the incretin response in vitro and in vivo. In the in vitro models, we found that exposure to palmitate was sufficient for impairment of GLP1R, but not GIPR signalling to occur. This was evidenced by a reduced ability of GLP-1 to stimulate cAMP production, p-CREB and insulin secretion. The specificity of these defects was demonstrated by the partial restoration of signalling after Ad-GLP1R-mediated expression of exogenous
Glp1r. In the in vivo models, we found that hyperlipidaemia was necessary for the downregulation of incretin receptor expression in islets of a mouse model of diabetes. Furthermore, in the
db/
db mouse model of diabetes, normalisation of the lipid profile by bezafibrate dramatically improved the efficacy of incretin-based therapies, including the DPP-4 inhibitor, des-fluoro-sitagliptin, and the GLP1R agonist, exendin-4. These findings, together with the work of others [
34], indicate crucial roles of fatty acids and GLP1R in maintaining incretin signalling, beta cell function and glucose homeostasis.
We and others have reported that GLP1R and GIPR levels were decreased in islets from mouse and rat models of diabetes, and from type 2 diabetic patients [
27,
30,
35]. In the present study, we found that in vitro palmitate treatment resulted in reduced GLP1R levels. In islets isolated from
db/
db mice, we also observed a significant reduction of
Glp1r expression. Furthermore, treatment of
db/
db mice with bezafibrate for 2 weeks, which significantly improved the serum lipid profile, partially restored
Glp1r expression in islets, even though the hyperglycaemic status remained. These findings imply that, apart from hyperglycaemia, hyperlipidaemia is required for downregulation of
Glp1r expression in diabetes. GIPR was less sensitive to regulation by palmitate. This difference in the regulation of incretin receptors by fatty acids is reminiscent of the effects of hyperglycaemia; thus conscious rats receiving glucose infusions and isolated rat islets exposed to high glucose exhibited decreases in
Glp1r but not
Gipr expression [
27]. Although
Gipr expression was unaltered in palmitate-treated INS-1E cells, GIP-stimulated cAMP production and insulin secretion were significantly decreased. This discrepancy in findings for GIP is possibly due to NEFA-induced global beta cell dysfunction via other pathways involving endoplasmic reticulum stress, oxidative stress and Ca
2+ homeostasis [
29,
31]. For example, SREBP1c, which was reported to mediate palmitate-induced impairment of insulin secretion in islets [
36], was increased in INS-1E, but not in MIN6 cells after palmitate treatment. The results probably reflect the complexity of the effects of hyperlipidaemia on beta cell function, with impaired incretin receptor signalling contributing to beta cell glucolipotoxicity in concert with other pathways involving endoplasmic reticulum and oxidative stress [
29,
31].
Although the glucose-lowering efficacy of incretin agonists and DPP-4 inhibitors has been shown in animal models [
37‐
39], it is worth noting that chronic treatment of
db/
db mice with incretin agonists or DPP-4 inhibitors alone only delays the onset of diabetes at the early stages of disease progression [
40]. Exendin-4 treatment does not prevent the ongoing deterioration of glucose intolerance in severely diabetic
db/
db mice [
41]. Likewise, clinical evidence shows that the efficacy of incretin-based drugs for the treatment of type 2 diabetes is variable, and may be affected by various factors such as age [
42], stage and severity of diabetes, differences in responsiveness to GLP-1 in diverse ethnic groups, genetic variance of GIPR and GLP1R [
43,
44], as well as hyperglycaemia [
27]. The current study demonstrates for the first time that hyperlipidaemia should be included as a contributing factor to the reduced efficacy of incretin-based drugs in mouse models of diabetes.
Hyperlipidaemia is closely associated with type 2 diabetes, and glucose-lowering drugs such as thiazolidinediones and metformin improve glucose and lipid metabolism [
45]. Our in vitro data show that elevated NEFA is sufficient to cause impaired GLP1R signalling, prompting us to test the relationship between hyperlipidaemia and the efficacy of incretin-based therapy in animal models of diabetes. The lipid-lowering agent bezafibrate significantly improved the serum lipid profile, without affecting blood glucose levels in
db/
db mice. Strikingly, after lipid lowering, the DPP-4 inhibitor des-fluoro-sitagliptin and the GLP1R agonist exendin-4 both had a more robust effect on glycaemic control than co-treatment with vehicle or treatment with each agent alone. The effects were not due to increases in insulin sensitivity. Rather the improved glucose tolerance was associated with restoration of normal islet morphology and increased beta cell mass. This effect was only apparent with prolonged incretin activation, since the lowering of fatty acids did not enhance glucose disappearance after acute treatment of
db/
db mice with exendin-4. These results suggest that the improved glucose homeostasis induced by chronic administration of exendin-4 and bezafibrate to
db/
db mice is effected via long-term improvements in beta cell mass and function, which may be due to restored expression of
Glp1r and thus GLP1R signalling after lipid lowering. However, although
Gipr expression was also partially restored by bezafibrate treatment, the GIPR agonist
d-GIP did not improve glucose metabolism. Recent reports have demonstrated that GLP1R signalling exerts more robust control of beta cell survival and regeneration than does GIPR signalling in mice [
46]. It has also been reported that GLP-1-stimulated p-CREB plays important roles in the regulation of beta cell survival through GLP1R activation [
33]. In this study, we only observed reduced GLP-1-stimulated p-CREB during in vitro palmitate treatment. Moreover, only combined treatment with exendin-4 plus bezafibrate improved islet morphology and increased beta cell mass, associated with increased beta cell proliferation in
db/
db mice. On the other hand, it has been reported that GIP was associated with impaired insulin sensitivity [
38,
47], and this may partially explain the differences in efficacy of the incretin receptor agonists.
The peroxisome proliferator-activated receptor (PPAR)-α agonist WY14643 has been reported to increase
Gipr expression [
48]; at the same time, PPAR-α activation is associated with improved beta cell survival and function through reduction of lipid accumulation by increased fatty acid oxidation in beta cells and human islets [
49,
50]. It has also been reported that metformin increases
Glp1r expression in INS-1 cells via a PPAR-α-dependent pathway [
51]. Our results demonstrate that palmitate downregulates GLP1R in insulinoma cell lines and bezafibrate can improve the efficacy of des-fluoro-sitagliptin and exendin-4 in mouse models of diabetes. Intriguingly, the related nuclear receptor PPAR-γ has been reported to regulate
Gipr expression by binding to the PPAR response elements within the rat
Gipr promoter [
52]. The question of whether PPAR-α activation regulates incretin receptors in beta cells through direct binding or indirect improvement in lipotoxicity requires further investigation.
In summary, our results show that hyperlipidaemia contributes to impaired beta cell responsiveness to GLP-1, partially through downregulation of GLP1R. Combined treatment with incretin-based drugs (des-fluoro-sitagliptin or exendin-4) and lipid-lowering drugs (bezafibrate) results in synergistic improvements of glucose metabolism and islet morphology and function. These findings reinforce the importance of lipid management in type 2 diabetes and provide important information for the design of new incretin-based therapies for the treatment of type 2 diabetes mellitus.
Funding
This work was supported by the Hong Kong Government Research Grant Committee (478110), the National Natural Science Foundation of China (81170722), the National Health and Medical Research Council of Australia (1030715) and a grant from Merck Sharp & Dohme (C2709; Whitehouse Station, NJ, USA). Z.F. Kang was supported by a studentship from Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, People’s Republic of China.