Anti-Inflammatory Effects of GLP-1-Based Therapies beyond Glucose Control

Glucagon-like peptide-1 (GLP-1) is an incretin hormone mainly secreted from intestinal L cells in response to nutrient ingestion. GLP-1 has beneficial effects for glucose homeostasis by stimulating insulin secretion from pancreatic beta-cells, delaying gastric emptying, decreasing plasma glucagon, reducing food intake, and stimulating glucose disposal. Therefore, GLP-1-based therapies such as GLP-1 receptor agonists and inhibitors of dipeptidyl peptidase-4, which is a GLP-1 inactivating enzyme, have been developed for treatment of type 2 diabetes. In addition to glucose-lowering effects, emerging data suggests that GLP-1-based therapies also show anti-inflammatory effects in chronic inflammatory diseases including type 1 and 2 diabetes, atherosclerosis, neurodegenerative disorders, nonalcoholic steatohepatitis, diabetic nephropathy, asthma, and psoriasis. This review outlines the anti-inflammatory actions of GLP-1-based therapies on diseases associated with chronic inflammation in vivo and in vitro, and their molecular mechanisms of anti-inflammatory action.

C-X-C motif chemokine 10 (CXCL10/IP10), which is induced by IFN-, has an important role in recruiting activated T cells into the islets in type 1 diabetes. Exendin-4 (100 nM) decreased IFN--induced signal transducer and activator of transcription-1 (STAT1), which is important for CXCL10 expression in the pancreatic beta-cell line, MIN6 cells, and human islets. Therefore, suppression of CXCL10 production by exendin-4 could reduce islet inflammation by decreasing cytotoxic T lymphocyte recruitment into the islets in autoimmune type 1 diabetes [48].
Serine proteinase inhibitor-9 plays an important role in the survival of cells against attack by natural killer cells and cytotoxic T lymphocytes, which play a direct role in the destruction of pancreatic beta-cells in type 1 diabetes. The GLP-1 receptor agonist, exenatide (a synthetic form of exendin-4) (10 nM), induces the expression of serine protease inhibitor-9 in human islets [49]. These results suggest that GLP-1-based therapies not only directly regulate the expression of inflammatory mediators, but also regulate the recruitment of immunocytes and protect from immunocyte attack, contributing to the preservation of pancreatic islets.
The abundance of proinflammatory cytokines and chemokines in adipose tissue is a key contributor to insulin resistance in type 2 diabetes, and blocking of inflammatory signaling pathways or immune cell infiltration in adipose tissue improves insulin sensitivity [50][51][52]. Administration of a recombinant adenovirus producing GLP-1 (4 × 10 9 PFU/mouse) to ob/ob mice reduced the macrophage population and production of TNF-, MCP-1, and IL-6 in adipose tissue via inhibition of nuclear factor-kappa B (NF-B) activation and phosphorylation of ERK1/2 and c-Jun N-terminal kinases [18]. Sitagliptin (4 g/kg) also showed similar effects and reduced the expression of mRNA for inflammatory cytokine genes and macrophage infiltration in adipose tissue of high fat diet-(HFD-) induced obese mice [19]. In patients with type 2 diabetes, sitagliptin (100 mg/day) therapy significantly reduced the plasma levels of C-reactive protein (CRP), IL-6, IL-18, secreted phospholipase-A2, soluble intracellular adhesion molecule-(ICAM-) 1, and E-selectin compared with placebo. The inflammatory score and the homeostatic model assessment index for insulin resistance were significantly reduced in sitagliptin-treated type 2 diabetes patients [7]. Therefore, suppression of inflammatory mediators in adipose tissue by GLP-1-based therapies might contribute to the improvement of insulin sensitivity.
GLP-1-based therapies for diabetes contribute to reduce inflammation and have additional beneficial effects such as islet preservation and improvement of insulin sensitivity in addition to glucose-lowering effects. However, some rare cases of acute pancreatitis and neoplasms have been reported [53][54][55]; thus the establishment of safety of GLP-1-based therapy should be validated by sufficient further studies.

Vascular Disease
Inflammation is known to be a risk factor for vascular diseases such as atherosclerosis. Atherosclerotic cardiovascular disease is caused by proinflammatory stimuli in the vascular endothelial cells and is associated with increased plasma levels of TNF-, IL-6, CRP, and circulating endotoxin (i.e., lipopolysaccharide (LPS)) [56,57]. Atherosclerosis is a chronic inflammatory condition resulting from the invasion and accumulation of white blood cells (foam cells) in the walls of arteries and therefore is a syndrome affecting arterial blood vessels [58].
GLP-1 (5.0 M) perfusion attenuates LPS-induced microvascular permeability via the cAMP protein kinase A (PKA) pathway [59]. Liraglutide (100 M) reduced the mRNA expression of adhesion molecules such as vascular cell adhesion molecule-(VCAM-) 1, ICAM-1, and E-selectin in TNF--or LPS-stimulated human aortic endothelial cells and human umbilical vein endothelial cells [60][61][62]. Liraglutide (100 nM) induced phosphorylation of calcium/calmodulin-dependent protein kinase I and 5 adenosine monophosphate-activated protein kinase (AMPK), and inhibition of calcium/calmodulin-dependent protein kinase kinase (CAMKK ) abolished the inhibitory effect of liraglutide on the expression of VCAM-1 and E-selectin. In addition, knockdown of AMPK with short hairpin AMPK RNA abolished the liraglutide activation of AMPK and antiinflammatory effects. These results demonstrate that the antiinflammatory effects of liraglutide in human aortic endothelial cells is dependent on activation of CAMKK and AMPK, which are cAMP/Ca 2+ signaling pathways [60]. In addition, it was reported that liraglutide (100 nM) inhibited TNF--or hyperglyceamia-mediated induction of plasminogen activator inhibitor type-1 in human vascular endothelial cells [23]. Exendin-4 (50 g/kg/day) treatment resulted in a reduction of atherosclerosis development and the number of monocytes adhering to the endothelium wall in the aortic root in western-type diet-fed APOE * 3-Leiden.CETP(E3L.CETP) mice [63].
The loss of sirtuin 6 (SIRT6), which regulates proinflammatory mediators, in human umbilical vein endothelial cells is associated with upregulation of the expression of proinflammatory genes [71]. Liraglutide (100 nM) treatment increased SIRT6 expression and reduced NF-B expression compared with only high glucose-treated endothelial cells.
In diabetic patients treated with GLP-1-based therapy, the protein level of SIRT6 in asymptomatic plaques was significantly increased and TNF-and MMP-9 levels in lesions were significantly reduced compared with diabetic patients without treatment [8]. This result suggests that GLP-1-based therapy has anti-inflammatory effects by induction of SIRT6 expression in endothelial cells.
Cardiovascular disease is increased in type 2 diabetes, and hyperglyceamia is a critical promoter during the development of cardiovascular diseases. Inflammation is an important pathophysiologic factor in diabetic cardiomyopathy. Exendin-4 protects against cardiac contractile dysfunction in an experimental myocardial infarction model. Exendin-4 (5 g/kg or 1 and 10 nM) inhibited high mobility group box I protein expression, a proinflammatory mediator, in myocardial ischemia and reperfusion in rats [72] and in high glucose-induced myocardial cell injury [73]. Sitagliptin (30 and 50 mg/kg/day) reduced the expression of TNF-and IL-6 in the diabetic heart and had a myocardial protective effect in STZ/HFD-induced diabetic rats [74]. Therefore, GLP-1-based therapy have anti-inflammatory effects on vascular disease and may explain the vasoprotective properties.

Neurodegenerative Brain Disorder
Neurodegenerative central nervous system disorders are associated with chronic neuroinflammation [75][76][77]. Epidemiological and clinical studies have suggested a link between type 2 diabetes and Alzheimer's disease [78]. In patients with Alzheimer's disease, insulin receptors and insulin signaling in the brain are desensitized and impaired as found in type 2 diabetes patients. Therefore, drugs used for treatment of diabetes are expected to have a preventive effect against Alzheimer's disease. GLP-1 is known to be produced in the brain [79] and has many functions including neuroprotection [80][81][82]. In addition, GLP-1 and GLP-1 analogues enter the brain through blood brain barrier [83][84][85][86].
The glia may play a critical role in the central nervous system inflammatory responses including Alzheimer's disease, and GLP-1 receptor was observed in astrocytes and microglia [17,87]. In astrocytes, GLP-1 (1 M) prevented the LPS-induced IL-1 expression by increase of cAMP [17].
Models of Alzheimer's disease include intracerebroventricular injection of STZ [88], intracerebral injection of LPS [88], and the APPSWE/PS1ΔE9 mouse [84]. Exenatide (20 g/kg/day) treatment inhibited brain TNF-levels, which were induced by intracerebroventricular injection of STZ [89]. GLP-1 (7-36) amide (50 nM) protected the synaptic impairments induced by intracerebral injection of LPS in the rat hippocampus [90]. Liraglutide (25 nmol/kg/day) treatment significantly reduced the inflammatory response in the cortex as measured by the number of activated microglia and prevented degenerative processes in a 7-month-old APP SWE /PS1 ΔE9 mouse model of Alzheimer's disease [86]. In addition, in the 14-month-old APP SWE /PS1 ΔE9 mouse, inflammation was also markedly reduced and restorative effects were improved by liraglutide treatment [91]. The GLP-1 receptor agonist, lixisenatide, exerted neuroprotective Mediators of Inflammation 5 effects via reduction of oxidative stress and the chronic inflammation response in the brain of APP SWE /PS1 ΔE9 mouse [92]. In addition, sitagliptin (10 and 20 mg/kg) also showed similar anti-inflammatory effects in APP SWE /PS1 ΔE9 mouse [93]. This suggests that GLP-1-based therapies could have a preventive and restorative effect on the pathophysiology of Alzheimer's disease progression.
Irradiation of the brain causes a chronic inflammatory response. X-ray irradiation of the brain significantly increased IL-6, IL-1 , and IL-12p70 cytokine protein expression. Liraglutide (25 nmol/kg/day) treatment reduced the mRNA expression of proinflammatory cytokine genes, which was induced by X-ray irradiation [24].
Parkinson's disease is a chronic and neurodegenerative brain disorder, and inflammatory activity is one of important features of Parkinson's disease. Microglial activation plays a critical role in the pathogenesis of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyidine-(MPTP-) induced Parkinson's disease model and human Parkinson's disease [27]. Exendin-4 (10 g/kg) treatment significantly decreased MPTP-induced microglial activation and suppressed MPTP-induced expression of TNF-and IL-1 [94]. The inhibitory effect of exendin-4 on microglial activation may have therapeutic potential for the treatment of Parkinson's disease. These anti-inflammatory effects of GLP-1-based therapies on the brain may protect against neurodegenerative central nervous system disorders.

Nonalcoholic Steatohepatitis
Nonalcoholic steatohepatitis is associated with an inflammation of the liver by an aberrant accumulation of fat in the liver. GLP-1 receptor agonists reduced alanine aminotransferase and aspartate aminotransferase levels in patients with nonalcoholic fatty liver disease (or type 2 diabetes) and improved lipid metabolism and reduced fat mass [21]. Liraglutide (50, 100, and 200 g/kg/12 h) treatment protected against nonalcoholic fatty liver disease by inhibition of ER stress-associated apoptosis in HFD-fed rats [28]. Liraglutide or exendin-4 (1 nmol/kg/day) treatment dose-dependently reduced steatosis and lobular inflammation in HFD-fed rats or mice compared with the saline-injected group [28,95], probably due to an increase of SIRT1 [96]. As a matter of fact, exendin-4 (50 g/kg/day) treatment increased the expression of SIRT1 and its downstream factor, AMPK, in exendin-4 treated mouse livers and hepatocytes. Exendin-4 treatment reduced hepatic expression of the inflammatory markers TNF-, IL-1 , and IL-6 and macrophage markers, cluster of differentiation 68 (CD68), and F4/80 in the liver of mice fed a western-type diet [63].
In nonalcoholic steatohepatitis patients with glucose intolerance, liraglutide (0.9 mg/person/day) therapy for 96 weeks resulted in improvement of histological indicators of inflammation in seven subjects out of ten subjects [97]. CRP is produced by the liver and is a marker of inflammation. In a retrospective analysis of 110 obese patients with type 2 diabetes treated with liraglutide, the mean concentration of CRP declined after treatment with liraglutide for a mean duration of 7.5 months [9]. In addition, exenatide plus metformin resulted in a significant reduction in CRP and TNF-compared with baseline [98]. These reports suggest that GLP-1-based therapies improve fatty liver disease by ameliorating inflammation in rodents and humans.

Nephropathy
Diabetic nephropathy is associated with a state of lowgrade inflammation in the microvasculature of the kidney's glomeruli [99,100]. The GLP-1 receptor is expressed in glomerular capillaries and vascular walls of the mouse kidney [14,101] and in the glomerulus and proximal convoluted tubules of the rat and pig [29,102]. GLP-1 receptor deficiency in the diabetic nephropathy-resistant C57BL/6-Akita mouse contributes to the development of diabetic nephropathy, and liraglutide treatment suppressed the progression of nephropathy of the KK/Ta-Akita mouse, which shows high susceptibility to diabetic nephropathy [14], suggesting that GLP-1 action might play an important role in prevention of diabetic nephropathy.

Other Diseases
Asthma is a chronic pulmonary inflammatory disease. Liraglutide (2 mg/kg) reduced immune cell infiltration and protein expression of E-selectin, TNF-, IL-4, IL-5, and IL-13 in the lung tissue or bronchoalveolar lavage fluid in an ovalbumin-induced chronic asthma model. Liraglutide treatment decreased NF-B activation, which was reversed by PKA inhibitor, H-89, suggesting that the cAMP-PKA pathway is involved in inhibition of NF-B activation, and subsequently the inhibition of inflammation [110]. In addition, in mice with bleomycin-induced pulmonary fibrosis, liraglutide treatment inhibited infiltration of immune cells and decreased the content of TGF-1. Liraglutide treatment markedly attenuated bleomycin-induced VCAM-1 and NF-B activation [30]. These results suggest that GLP-1-based therapies might have beneficial effects on asthma but need to be validated by clinical studies.
Obesity can reduce the quality and count of men's sperm [111,112]. The expression of TNF-, MCP-1, and F4/80 mRNA levels is increased in the testis and significantly decreased the sperm motility and activity in diet-induced obesity mice, and exenatide (24 nmol/kg/day) treatment suppressed the expression of TNF-, MCP-1, and F4/80 mRNA levels in testis and improved sperm quality in diet-induced obesity mice [111].
In type 2 diabetes patients, GLP-1 and liraglutide also improve clinical symptoms of psoriasis, a skin inflammatory disease, by downregulation of invariant natural killer T cells [31,113,114].
These reports suggest that GLP-1-based therapies have anti-inflammatory effects in the lung, testis, skin, and eye.

Conclusion
Inflammation is a protective process including immune system, vascular system, and molecular mediators. However out-of-control inflammation and chronic inflammation can cause pathological disease. Inflammation is a risk factor for diabetes, atherosclerosis, cardiovascular disease, neurodegenerative central nervous system disorders, nonalcoholic steatohepatitis, and nephropathy.
GLP-1-based therapies have many attractive and beneficial effects including their antidiabetic actions on pancreatic beta-cells. However, beyond their metabolic effects, GLP-1based therapies have been shown to have anti-inflammatory effects via several molecular pathways ( Figure 1) in several organs, tissues, and cells ( Figure 2). GLP-1-based therapies downregulate proinflammatory responses in inflammatory related diseases. This review concludes that GLP-1-based therapy has beneficial effects on inflammatory disease. Thus GLP-1, GLP-1R agonists, and DPP-4 inhibitors might have important roles as mediators of inflammation.

Competing Interests
The authors declare that there are no competing interests regarding the publication of this paper.