Pharmacological reduction of NEFA restores the efficacy of incretin-based therapies through GLP-1 receptor signalling in the beta cell in mouse models of diabetes
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Type 2 diabetes mellitus is associated with reduced incretin effects. Although previous studies have shown that hyperglycaemia contributes to impaired incretin responses in beta cells, it is largely unknown how hyperlipidaemia, another feature of type 2 diabetes, contributes to impaired glucagon-like peptide 1 (GLP-1) response. Here, we investigated the effects of NEFA on incretin receptor signalling and examined the glucose-lowering efficacy of incretin-based drugs in combination with the lipid-lowering agent bezafibrate.
We used db/db mice to examine the in vivo efficacy of the treatment. Beta cell lines and mouse islets were used to examine GLP-1 and glucose-dependent insulinotropic peptide receptor signalling.
Palmitate treatment decreased Glp1r expression in rodent insulinoma cell lines and isolated islets. This was associated with impairment of the following: GLP-1-stimulated cAMP production, phosphorylation of cAMP-responsive elements binding protein (CREB) and insulin secretion. In insulinoma cell lines, the expression of exogenous Glp1r restored cAMP production and the phosphorylation of CREB. Treatment with bezafibrate in combination with des-fluoro-sitagliptin or exendin-4 led to more robust glycaemic control, associated with improved islet morphology and beta cell mass in db/db mice.
Elevated NEFA contributes to impaired responsiveness to GLP-1, partially through downregulation of GLP-1 receptor signalling. Improvements in lipid control in mouse models of obesity and diabetes increase the efficacy of incretin-based therapy.
KeywordsDipeptidyl peptidase-4 Exendin-4 Glucagon-like peptide 1 Glucose-dependent insulinotropic polypeptide Islet NEFA Non-esterified fatty acid Receptor
Adenoviral vector with green fluorescent protein (GFP)
cAMP-responsive elements binding protein
Green fluorescent protein
Glucose-dependent insulinotropic polypeptide
Glucagon-like peptide 1
Peroxisome proliferator-activated receptor
Sterol regulatory element binding protein 1c
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, 5, 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, 10, 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, 17, 18]. NEFA can result in a state of insulin resistance , 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 . Recently, it was reported that while incretin secretion is similar between obese and non-obese type 2 diabetic patients , obesity impairs the incretin effect independently of glucose tolerance . It has also been reported that loss of the incretin effects was more extensive in obese than in lean type 2 diabetic patients . More recently, Bando et al reported that obesity may attenuate the HbA1c-lowering effect of sitagliptin in Japanese type 2 diabetic patients . 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 . Furthermore, the normalisation of blood glucose concentrations improves the insulin response to GLP-1 and GIP in patients with type 2 diabetes . 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.
Chemicals and reagents
For details, see electronic supplementary material (ESM), Chemicals and reagents.
Cell culture and treatment
INS-1E cell line (passages 65 to 75) was a kind gift from P. Maechler (Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland). INS-1E and MIN6 cells were grown as described previously [11, 29]. Cells were cultured in 6- or 12-well plates for 24 to 48 h before treatment with palmitate or 1.05% (wt/vol) BSA (in RPMI 1640 or DMEM with 1% FBS). GLP-1, GIP, exendin-4 and [d-Ala2]GIP(1-30) (d-GIP) were dissolved in PBS and stored at −80°C. To achieve expression of exogenous Glp1r, cells were infected with an adenoviral vector (Ad), Ad-GLP1R, for 6 h prior to treatment with palmitate or 1.05% BSA.
Islet isolation and culture, construction of Ad-GLP1R, RT-PCR, western blotting and measurement of cAMP production, and insulin secretion
Pancreatic islets were isolated from 8- to 9-week-old male C57BL/6J mice as previously described (ESM Methods, Isolation and cell culture). The gateway-compatible adenoviral expression system was used to generate the recombinant adenoviruses (ESM Methods, Construction of Ad-GLPR1). Real-time PCR (ESM Methods, RNA extraction and quantitative RT-PCR) and western blotting (ESM Methods, Analysis of phosphorylation of CREB) were performed with standard procedures. Intracellular cAMP content was determined using a kit (Cyclic AMP EIA; Cayman, Ann Arbor, MI, USA) (ESM Methods, Measurement of cAMP production) and protein levels were assayed by BCA for the correction. Insulin secretion was measured in INS1-E cells or mouse islets after exposure to 3 or 16.7 mmol/l glucose (ESM Methods, Measurement of insulin secretion).
Animals and experimental protocols
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals and approved by The Animal Subjects Committee of The Chinese University of Hong Kong. Male db/db and db/+ (control) mice (aged 7 to 8 weeks) were obtained from The Chinese University of Hong Kong and housed in specific pathogen-free conditions with a 12 h light–dark cycle and free access to water and food. Experiments were performed after 1 week of acclimatisation. For drug treatments, des-fluoro-sitagliptin (200 mg/kg) and bezafibrate (100 mg/kg) were dissolved in 0.5% (wt/vol) CMC and given by gavage; exendin-4 (10 nmol/kg) and d-GIP (24 nmol/kg) were dissolved in PBS and given by intraperitoneal injection. Mice were treated daily (16:00 to 18:00 hours) by gavage or intraperitoneal injection for the indicated time. Fed random blood glucose was monitored weekly at 09:00 to 10:00 hours. For measurement of the acute glucose-lowering actions of exendin-4 and d-GIP, db/db mice were treated with vehicle or bezafibrate for 2 weeks and then injected intraperitoneally with saline, exendin-4 or d-GIP. Glucose levels were determined at 0, 30, 60 and 240 min after injection.
OGTT, insulin tolerance test and serum lipid profile measurement
For the OGTT, mice were fasted overnight (~17 h). Glucose levels were determined using a glucometer (Johnson & Johnson, Milpitas, CA, USA) at 0, 30, 60 and 120 min after oral administration of 0.3 g/kg glucose. For the insulin tolerance test (ITT), done after 6 h of fasting, mice were intraperitoneally injected with 2 IU/kg human insulin (Novo Nordisk, Bagsvaerd, Denmark). Glucose levels were measured at 0, 30, 60 and 120 min after the injection. Triacylglycerol, NEFA and total cholesterol concentrations were measured using related kits (Wako Lab Assays, Richmond, VA, USA). HDL-cholesterol was determined by enzymatic assays using an automated analyser (Olympus, Tokyo, Japan).
Pancreases were quickly dissected from mice and fixed in 4% (wt/vol.) paraformaldehyde, after which paraffin-embedded 4-μm sections were immunostained overnight at 4°C with guinea pig anti-insulin (Dako, Glostrup, Denmark) and mouse anti-glucagon (1:200; Accurate Chemical & Scientific, Westbury, NY, USA), or with mouse anti-BrdU (BD Biosciences, Franklin Lakes, NJ, USA) antibodies. Following this, staining with cy2-goat anti-guinea pig or cy3-donkey anti-mouse (1:400; Jackson, West Grove, PA, USA) was done at room temperature for 2 h. The sample slides were washed three times with 0.1% PBS Tween (vol./vol., PBST) and stained with DAPI (Invitrogen, Grand Island, NY, USA) before microscopic analysis. The insulin-positive area vs total pancreas or total islet area was evaluated using Image J (NIH, Bethesda, Maryland, USA) .
Animal data are expressed as means ± SEM. Differences between the groups were examined for statistical significance using one-way or two-way ANOVA, followed by Dunnett’s post tests or t tests (as appropriate). For in vitro experiments, quantitative RT-PCR data are expressed as means ± SEM; other data are presented as means ± SD. Statistical significance was determined by Student’s t test. A value of p < 0.05 was considered to be statistically significant.
Reduced expression of Glp1r and reduction of GLP-1-stimulated insulin secretion in palmitate-treated beta cells and mouse islets
In islets isolated from db/db mice, we also found that Glp1r and Gipr mRNA were significantly reduced compared with expression in control mice (Fig. 1d, e). Interestingly, db/db mice that were treated with the lipid-lowering agent bezafibrate for 2 weeks displayed partial restoration of Glp1r and Gipr mRNA to levels not different from control mice (Fig. 1d, e), with lowered triacylglycerol and NEFA, but no obvious changes in glucose levels .
To further assess the functional consequences of the reductions in Glp1r and Gipr expression, we assayed insulin secretion in INS-1E cells and isolated islets. In INS-1E control cells, both GLP1R agonists (GLP-1, exendin-4) and the GIP receptor (GIPR) agonists (GIP, d-GIP) markedly increased insulin secretion in the presence of 16.7 mmol/l glucose (Fig. 1f). However, after pre-incubation with palmitate, this response was significantly attenuated (Fig. 1f). Similar results were found in isolated mouse islets, with palmitate treatment decreasing the fold induction of GLP-1-stimulated glucose-dependent insulin secretion (Fig. 1g).
Palmitate impairs GLP-1-stimulated cAMP production and phosphorylation of cAMP-responsive elements binding protein in rodent insulinoma cell lines
Expression of exogenous Glp1r restores GLP-1-stimulated cAMP production and p-CREB in palmitate-treated rodent insulinoma cell lines
Lipid lowering enhances the efficacy of the DPP-4 inhibitor, des-fluoro-sitagliptin, in db/db mice
Lipid lowering enhances the efficacy of an agonist to GLP1R (exendin-4) but not to GIPR (d-GIP) in db/db mice
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 , 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 . 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 Ca2+ homeostasis [29, 31]. For example, SREBP1c, which was reported to mediate palmitate-induced impairment of insulin secretion in islets , 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, 38, 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 . Exendin-4 treatment does not prevent the ongoing deterioration of glucose intolerance in severely diabetic db/db mice . 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 , 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 . 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 . 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 . It has also been reported that GLP-1-stimulated p-CREB plays important roles in the regulation of beta cell survival through GLP1R activation . 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 ; 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 . 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 . 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.
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.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
ZK, RF, YZ, JL and DRL performed experiments, analysed the data and contributed to the revision of the article. YD and JC contributed to the acquisition of data and revision of the article. ZK, YD and DRL analysed the data and wrote the manuscript. GX conceived and designed the experiments, performed experiments and wrote the manuscript. All authors approved the final version of the article.
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