Decreased expression of Glucagon-like peptide-1 receptor and Sodium-glucose co-transporter 2 in patients with proliferative diabetic retinopathy.

Purpose: To investigate the expression of Glucagon-like peptide-1 receptor (GLP-1R), sodium-glucose co-transporter (SGLT) 1, SGLT2, Glucose transporter type 1 (GLUT1) and GLUT2 in patients with diabetic retinopathy (DR). Methods: We obtained peripheral blood mononuclear cells (PBMCs) and vitreous samples from 26 proliferative DR (PDR) patients, 25 non-proliferative DR (NPDR) patients, 25 non-DR (NDR) patients, and 26 nondiabetic patients with idiopathic epiretinal membranes (ERMs, control). The protein level and mRNA expression level of GLP-1R were quantified by immunoblot and qRT-PCR and the levels of SGLT1, SGLT2, GLUT1, and GLUT2 expression were determined by PCR. Their association with clinical parameters and PBMCs/vitreous cytokine was analyzed. Furthermore, immunofluorescence staining of GLP-1R and SGLT2 was carried out on samples of fibrovascular membranes (FVMs) retrieved from 26 patients with PDR and 26 patients with ERMs. Results: The transcriptional levels of GLP-1R and SGLT2 in PBMCs were significantly more decreased in PDR patients than in patients without DR and controls, which was simultaneously associated with an increased level of expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The expression levels of GLUT1 and GLUT2 were tightly correlated with their SGLT partners, respectively. Further, Immunofluorescence staining showed no positive staining of GLP-1R and SGLT2 was detected in the FVMs from PDR. Conclusions: GLP-1R and SGLT2 were significantly decreased in PDR patients which was associated with an increased level of expression of TNF-α and IFN-γ. These findings implicate that defective GLP-1R and SGLT2 signaling may potentially correlate with immune response cytokines in patients with PDR.

Expression Profile of the GLP-1 Receptor in the Gastrointestinal Tract and Pancreas in Adult Female Mice.

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all ß-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Cardiovascular Safety and Benefits of Semaglutide in Patients With Type 2 Diabetes: Findings From SUSTAIN 6 and PIONEER 6.

To exclude an excess risk of cardiovascular (CV) events, CV outcomes trials (CVOTs) have assessed the effects of new glucose-lowering therapies, including glucagon-like peptide-1 receptor agonists (GLP-1RAs), in patients with type 2 diabetes and established CV disease or CV risk factors. The CV safety of semaglutide vs. placebo, when added to standard care, was evaluated in the SUSTAIN 6 trial for the formulation administered once-weekly subcutaneously and in PIONEER 6 for the new once-daily oral formulation. In SUSTAIN 6 and PIONEER 6, both powered to demonstrate noninferiority (upper 95% confidence interval [CI] of the hazard ratio [HR] <1.8), there were fewer first major adverse CV events with semaglutide vs. placebo, with HRs of 0.74 (95% CI 0.58-0.95) and 0.79 (0.57-1.11), respectively. In SUSTAIN 6, the results were significant for noninferiority and superiority, although the latter was not prespecified. Surprisingly, CV and all-cause mortality were significantly reduced by oral semaglutide in PIONEER 6. The ongoing SOUL CVOT will further inform about CV outcomes with oral semaglutide vs. placebo (NCT03914326). Findings from SUSTAIN 6 and PIONEER 6 fall within the spectrum reported with other GLP-1RA CVOTs: noninferiority vs. placebo for major CV events was seen with lixisenatide and exenatide extended-release, while superiority was demonstrated with liraglutide, albiglutide, and dulaglutide. Beneficial outcomes have been recognized in international guidelines, which recommend subcutaneous liraglutide, semaglutide, and dulaglutide to reduce the risk of CV events in high-risk patients. Both indirect mechanisms via risk factor modification and direct effects via GLP-1 receptors in the CV system have been proposed to be responsible for CV event reductions. The exact mechanism(s) remains to be characterized, but appears to be mainly linked to anti-atherosclerotic effects. Further research is needed to elucidate the relevant mechanisms for CV benefits of GLP-1RAs.

Exendin-4 enhances the sensitivity of prostate cancer to enzalutamide by targeting Akt activation.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) and its analogs are first-line choices for the treatment of type 2 diabetes mellitus. Recent studies have shown that they exhibit antitumor properties in some tumors. We previously found that a GLP-1 analog, exendin-4 (Ex-4), inhibited the growth of prostate cancer cells through suppressing the PI3K/Akt/mTOR pathway, which is activated in response to enzalutamide treatment and reported to be closely related to resistance to enzalutamide. So we speculated that exendin-4 may enhance the sensitivity of prostate cancer to enzalutamide through inhibiting Akt activation. METHODS: LNCap and CWR22RV1 cell lines, as well as mice bearing xenografts formed from the two cells, were used. RESULTS: Exendin-4 in combination with enzalutamide dramatically suppressed tumor growth of prostate cancer cells compared to enzalutamide alone; exendin-4 is capable of antagonizing enzalutamide-induced invasion and migration of both prostate cancer cells (P < .05). Furthermore, the combination treatment significantly reduced Akt and mTOR levels that were triggered by enzalutamide administration, caused a further decrease in nuclear AR localization compared with the enzalutamide as a monotherapy (P < .5), though exendin-4 treatment alone showed no effect on nuclear AR. CONCLUSION: Our study demonstrated that exendin-4 alleviated resistance to enzalutamide, and suggested that exendin-4 combined with enzalutamide may be a more efficacious treatment for patients with advanced prostate cancer.

Effect of hCMSCs and liraglutide combination in ALI through cAMP/PKAc/ß-catenin signaling pathway.

BACKGROUND: ALI/ARDS is the major cause of acute respiratory failure in critically ill patients. As human chorionic villi-derived MSCs (hCMSCs) could attenuate ALI in the airway injury model, and liraglutide, glucagon-like peptide 1 (GLP-1) agonist, possesses anti-inflammatory and proliferation promotion functions, we proposed to probe the potential combinatory effect of hCMSCs and liraglutide on ALI. METHODS: We examined the time- and dose-dependent manner of GLP-1R, SPC, Ang-1, and FGF-10 with LPS via western blot and qRT-PCR. Western blot and chromatin immunoprecipitation assay detected the effects of liraglutide on GLP-1R, SPC, Ang-1, and FGF-10 through PKAc/ß-catenin pathway and cAMP pathway. In the ALI animal model, we detected the effects of MSC and liraglutide combination on ALI symptoms by H&E staining, western blot, ELISA assays, calculating wet-to-dry ratio of the lung tissue, and counting neutrophils, leukocytes, and macrophages in mouse bronchoalveolar lavage fluid (BALF). RESULTS: The data demonstrated that LPS reduced hCMSC proliferation and GLP-1R, SPC, Ang-1, and FGF-10 levels in a dose- and time-dependent manner. Liraglutide significantly dampened the reduction of GLP-1R, SPC, Ang-1, and FGF-10 and reversed the effect of LPS on hCMSCs, which could be regulated by GLP-1R and its downstream cAMP/PKAc/ß-catenin-TCF4 signaling. Combination of hCMSCs with liraglutide showed more therapeutic efficacy than liraglutide alone in reducing LPS-induced ALI in the animal model. CONCLUSIONS: These results reveal that the combination of hCMSCs and liraglutide might be an effective strategy for ALI treatment.

D-Ser2-oxyntomodulin ameliorated Aß31-35-induced circadian rhythm disorder in mice.

INTRODUCTION: The occurrence of circadian rhythm disorder in patients with Alzheimer's disease (AD) is closely related to the abnormal deposition of amyloid-ß (Aß), and d-Ser2-oxyntomodulin (Oxy) is a protease-resistant oxyntomodulin analogue that has been shown to exert neuroprotective effects. AIMS: This study aimed to explore whether Oxy, a new GLP-1R/GCGR dual receptor agonist, can improve the Aß-induced disrupted circadian rhythm and the role of GLP-1R. METHODS: A mouse wheel-running experiment was performed to explore the circadian rhythm, and western blotting and real-time PCR were performed to assess the expression of the circadian clock genes Bmal1 and Per2. Furthermore, a lentivirus encoding an shGLP-1R-GFP-PURO was used to interfere with GLP-1R gene expression and so explore the role of GLP-1R. RESULTS: The present study has confirmed that Oxy could restore Aß31-35-induced circadian rhythm disorders and improve the abnormal expression of Bmal1 and Per2. After interfering the GLP-1R gene, we found that Oxy could not improve the Aß31-35-induced circadian rhythm disorder and abnormal expression of clock genes. CONCLUSION: This study demonstrated that Oxy could improve Aß31-35-induced circadian rhythm disorders, and GLP-1R plays a critical role. This study thus describes a novel target that may be potentially used in the treatment of AD.

Endothelial GLP-1 (Glucagon-Like Peptide-1) Receptor Mediates Cardiovascular Protection by Liraglutide In Mice With Experimental Arterial Hypertension.

OBJECTIVE: Cardiovascular outcome trials demonstrated that GLP-1 (glucagon-like peptide-1) analogs including liraglutide reduce the risk of cardiovascular events in type 2 diabetes mellitus. Whether GLP-1 analogs reduce the risk for atherosclerosis independent of glycemic control is challenging to elucidate as the GLP-1R (GLP-1 receptor) is expressed on different cell types, including endothelial and immune cells. Approach and Results: Here, we reveal the cardio- and vasoprotective mechanism of the GLP-1 analog liraglutide at the cellular level in a murine, nondiabetic model of arterial hypertension. Wild-type (C57BL/6J), global (Glp1r-/-), as well as endothelial (Glp1rflox/floxxCdh5cre) and myeloid cell-specific knockout mice (Glp1rflox/floxxLysMcre) of the GLP-1R were studied, and arterial hypertension was induced by angiotensin II. Liraglutide treatment normalized blood pressure, cardiac hypertrophy, vascular fibrosis, endothelial dysfunction, oxidative stress, and vascular inflammation in a GLP-1R-dependent manner. Mechanistically, liraglutide reduced leukocyte rolling on the endothelium and infiltration of myeloid Ly6G-Ly6C+ and Ly6G+Ly6C+ cells into the vascular wall. As a consequence, liraglutide prevented vascular oxidative stress, reduced S-glutathionylation as a marker of eNOS (endothelial NO synthase) uncoupling, and increased NO bioavailability. Importantly, all of these beneficial cardiovascular effects of liraglutide persisted in myeloid cell GLP-1R-deficient (Glp1rflox/floxxLysMcre) mice but were abolished in global (Glp1r-/-) and endothelial cell-specific (Glp1rflox/floxxCdh5cre) GLP-1R knockout mice. CONCLUSIONS: GLP-1R activation attenuates cardiovascular complications of arterial hypertension by reduction of vascular inflammation through selective actions requiring the endothelial but not the myeloid cell GLP-1R.

Does glucagon-like peptide-1 induce diuresis and natriuresis by modulating afferent renal nerve activity?

Glucagon-like peptide-1 (GLP-1), an incretin hormone, has diuretic and natriuretic effects. The present study was designed to explore the possible underlying mechanisms for the diuretic and natriuretic effects of GLP-1 via renal nerves in rats. Immunohistochemistry revealed that GLP-1 receptors were avidly expressed in the pelvic wall, the wall being adjacent to afferent renal nerves immunoreactive to calcitonin gene-related peptide, which is the dominant neurotransmitter for renal afferents. GLP-1 (3 µM) infused into the left renal pelvis increased ipsilateral afferent renal nerve activity (110.0 ± 15.6% of basal value). Intravenous infusion of GLP-1 (1 µg·kg-1·min-1) for 30 min increased renal sympathetic nerve activity (RSNA). After the distal end of the renal nerve was cut to eliminate the afferent signal, the increase in efferent renal nerve activity during intravenous infusion of GLP-1 was diminished compared with the increase in total RSNA (17.0 ± 9.0% vs. 68.1 ± 20.0% of the basal value). Diuretic and natriuretic responses to intravenous infusion of GLP-1 were enhanced by total renal denervation (T-RDN) with acute surgical cutting of the renal nerves. Selective afferent renal nerve denervation (A-RDN) was performed by bilateral perivascular application of capsaicin on the renal nerves. Similar to T-RDN, A-RDN enhanced diuretic and natriuretic responses to GLP-1. Urine flow and Na+ excretion responses to GLP-1 were not significantly different between T-RDN and A-RDN groups. These results indicate that the diuretic and natriuretic effects of GLP-1 are partly governed via activation of afferent renal nerves by GLP-1 acting on sensory nerve fibers within the pelvis of the kidney.

The effect of body mass index on glucagon-like peptide receptor gene expression in the post mortem brain from individuals with mood and psychotic disorders.

There is an increasing interest in the putative role of glucagon-like peptide 1 receptor (GLP-1R) agonists as novel therapeutic agents for mental disorders. Herein, we investigated the expressions of GLP-1R and GLP-2R genes, and its relationship with body mass index (BMI), in the post-mortem brain tissue of patients with mood (MD) and psychotic disorders. Brain samples were localized to the dorsolateral prefrontal cortex (dlPFC) (n = 459) and hippocampus (n = 378). After adjustment for age, sex, ethnicity, post-mortem interval (PMI) and BMI, we observed significant differences, between healthy controls and MD subjects, in GLP-1R and GLP-2R gene expression in the dlPFC (ß = 1.504, p = 0.004; and ß = 1.305, p = 0.011, respectively); whereas in the hippocampus, only GLP-1R expression was significantly associated with MD (ß = -1.28, p = 0.029). No significant differences were found in relation to schizophrenia. In addition, we observed a moderating effect of MD diagnosis on the associations between BMI, GLP-1R and GLP-2R expression values in the dlPFC (ß = -0.05, p = 0.003; and ß = -0.04, p = 0.004, respectively). There was a similar moderating effect for GLP-1R in the hippocampus (ß = 0.043, 95% CI 0.003; 0.08 p = 0.03), but in an opposite direction than observed in the dlPFC. This is the first evidence of abnormal gene expression of GLP-1R and GLP-2R in postmortem brain of individuals with MD, providing a rationale for further inquiry and proof of principle interventional studies.

Age-related change of GLP-1R expression in rats can be detected by [18F]AlF-NOTA-MAL-Cys39-exendin-4.

The glucagon-like peptide-1 receptor (GLP-1R) has been demonstrated as a potential therapeutic target for some neurological diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and stroke. Besides, its distribution and density in brain regions are closely associated with cognition, motor function, learning and memory. Given the relationship between age and these neurological diseases, we firstly examined the influences of age on GLP-1R expression using [18F]AlF-NOTA-MAL-Cys39-exendin-4 microPET imaging. The image showed that GLP-1R expression in nearly all regions of the brain of aged rats was evidently lower than that of normal rats. Significant differences were found in olfactory, striatum, hypothalamus, substantial nigra, and hippocampus, which have inseparable relations with some mental and neurological diseases such as PD and AD. Data obtained from biodistribution and immunohistochemistry staining also confirmed the image results. Taken together, these results illustrated decreased expression of GLP-1R in the brain of aged rats can be detected by [18F]AlF-NOTA-MAL-Cys39-exendin-4, which implied GLP-1R as a reliable target and GLP-1R PET imaging could be a promising technology in the field of neurological diseases.

Exendin-4 enhances radiation response of prostate cancer.

BACKGROUND: Exendin-4, one of the most widely used antidiabetic drugs, has recently been reported to have potential antitumor effects in cancers. Prostate cancer (PC) is one of the most common cancers in male patients with type 2 diabetes mellitus, and radiotherapy plays a vital role in the therapy of PC. Whether exendin-4 has the potential to enhance PC response to ionizing radiation (IR) remains unknown. We aimed to explore whether exendin-4 radiosensitizes PC cells. METHODS: GLP-1 receptor (GLP-1R) expression in PC tissue samples and cell lines were analyzed, Human prostate cancer cells (PC3 and LNCap) were treated with IR and exendin-4, and subjected to proliferation, clone formation, cell cycle, immunoblotting, and immunohistochemical analysis. An in situ prostate tumor of animal model was established. RESULTS: We found that GLP-1R was expressed in human PC tissues and cell lines. 1-100 nM exendin-4 promoted the anti-proliferation effects of IR in vitro and in vivo, and enhanced radiation-induced G2/M cycle arrest in PC cells in a dose-dependent manner. Furthermore, Ex-4 increased AMPK phosphorylation, decrease the levels of p-mTOR, cyclin B, and p34cdc2 . CONCLUSIONS: Our study suggested exendin-4 radiosensitizes PC cells via activation of AMPK A and subsequent inhibition of p-mTOR, cyclin B, and p34cdc2 activation.

GLP-1 neurons form a local synaptic circuit within the rodent nucleus of the solitary tract.

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (GLP-1R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In this study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse crossbreeding strategy revealed that <1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not use GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Expression of the glucagon-like peptide-1 receptor and its role in regulating autophagy in endometrial cancer.

BACKGROUND: A previous report showed that a glucagon-like peptide-1 receptor (GLP-1R) agonist (exenatide) induced apoptosis in endometrial cancer cells. However, the pathophysiological role of GLP-1R in endometrial cancer has not been fully elucidated. Here, we investigated the effects of the GLP-1R agonist liraglutide in endometrial cancer cells and examined the association between GLP-1R expression and clinicopathological characteristics in endometrial cancer patients. METHODS: Human Ishikawa endometrial cancer cells were treated with different concentrations of liraglutide. To assess the effects of liraglutide, cell viability, colony formation, flow cytometry, Western blotting, and immunofluorescence assays were performed. Autophagy induction was examined by analyzing LC3 and p62 expression and autophagosome accumulation. Moreover, using a tissue microarray, we analyzed GLP-1R expression in 154 endometrial cancer tissue samples by immunohistochemistry. RESULTS: In accordance with the previous report, liraglutide inhibited Ishikawa cell growth in a dose-dependent manner. Liraglutide significantly induced autophagy, and phosphorylated AMPK expression was elevated. Immunohistochemical analysis revealed that GLP-1R expression was associated with positive estrogen receptor and progesterone receptor status, and higher GLP-1R expression was significantly correlated with better progression-free survival. CONCLUSIONS: The use of liraglutide to target autophagy in endometrial cancer cells may be a novel potential treatment for endometrial cancer. Furthermore, higher GLP-1R expression may be associated with better prognosis in endometrial cancer patients.

Glucagon-like peptide-1 mediates effects of oral galactose in streptozotocin-induced rat model of sporadic Alzheimer's disease.

Insulin resistance and metabolic dysfunction in the brain are considered to be the pathophysiological core of sporadic Alzheimer's disease (sAD). In line with that fact, nutrients that could have therapeutic effects at this level have been investigated as possible targets in AD therapy. Galactose, an epimer of glucose, may serve as an alternative source of energy, and given orally may stimulate secretion of the incretin hormone glucagon-like peptide-1 (GLP-1). Our preliminary research indicated that oral galactose might prevent development of memory impairment in a rat model of sAD generated by intracerebroventricular administration of streptozotocin (STZ-icv). Here, we explored whether chronic oral galactose treatment could have beneficial effects on cognitive deficits already manifested at the time of initiation of galactose treatment in adult STZ-icv rats (treatment initiated 1 month after STZ-icv injection). The results clearly show that a 2-month exposure to oral galactose (200 mg/kg/day administered in a drink ad libitum) normalises impaired learning and memory functions. Memory improvement was accompanied by an improvement in brain glucose hypometabolism measured by 18fluorodeoxyglucose-positron emission tomography neuroimaging and by increments in active GLP-1 plasma levels as well as by an increased expression of GLP-1 receptors in the hippocampus and hypothalamus. Our findings provide strong evidence of beneficial effects of oral galactose treatment in the STZ-icv rat model of sAD and present possible underlying mechanisms including both direct effects of galactose within the brain and indirect GLP-1-induced neuroprotective effects that might open a new, dietary-based strategy in sAD treatment.

Autocrine Interleukin-10 Mediates Glucagon-Like Peptide-1 Receptor-Induced Spinal Microglial ß-Endorphin Expression.

The glucagon-like peptide-1 (GLP-1) receptor agonist exenatide stimulates microglial ß-endorphin expression and subsequently produces neuroprotection and antinociception. This study illustrated an unrecognized autocrine role of IL-10 in mediation of exenatide-induced ß-endorphin expression. Treatment with exenatide in cultured primary spinal microglia concentration dependently stimulated the expression of the M2 microglial markers IL-10, IL-4, Arg 1, and CD206, but not the M1 microglial markers TNF-α, IL-1ß, IL-6, or CD68. Intrathecal exenatide injection also significantly upregulated spinal microglial expression of IL-10, IL-4, Arg 1, and CD206, but not TNF-α, IL-1ß, IL-6, or CD68. Intrathecal injection of exenatide stimulated spinal microglial expression of IL-10 and ß-endorphin in neuropathic rats. Furthermore, treatment with IL-10 (but not IL-4) stimulated ß-endorphin expression in cultured primary microglia, whereas treatment with ß-endorphin failed to increase IL-10 expression. The IL-10-neutralizing antibody entirely blocked exenatide-induced spinal microglial expression of ß-endorphin in vitro and in vivo and fully blocked exenatide mechanical antiallodynia in neuropathic rats. Moreover, specific cAMP/PKA/p38 signal inhibitors and siRNA/p38ß, but not siRNA/p38α, completely blocked exenatide-induced IL-10 expression in cultured primary microglia. Knock-down of IL-10 receptor-α mRNA using siRNA fully inhibited exenatide-induced spinal microglial ß-endorphin expression and mechanical antiallodynia in neuropathy. Exenatide also markedly stimulated phosphorylation of the transcription factor STAT3 in cultured primary microglia and ß-endorphin stimulation was completely inhibited by the specific STAT3 activation inhibitor. These results revealed that IL-10 in microglia mediated ß-endorphin expression after GLP-1 receptor activation through the autocrine cAMP/PKA/p38ß/CREB and subsequent IL-10 receptor/STAT3 signal pathways.SIGNIFICANCE STATEMENT Activation of GLP-1 receptors specifically and simultaneously stimulates the expression of anti-inflammatory cytokines IL-10 and IL-4, as well as the neuroprotective factor ß-endorphin from microglia. GLP-1 receptor agonism induces ß-endorphin expression and antinociception through autocrine release of IL-10. Activation of GLP-1 receptors stimulates IL-10 and ß-endorphin expression subsequently through the Gs-cAMP/PKA/p38ß/CREB and IL-10/IL-10 receptor-α/STAT3 signal transduction pathways.

Human Epicardial Fat Expresses Glucagon-Like Peptide 1 and 2 Receptors Genes.

Epicardial adipose tissue (EAT) is an easily measurable visceral fat of the heart with unique anatomy, functionality, and transcriptome. EAT can serve as a therapeutic target for pharmaceutical agents targeting the fat. Glucagon-like peptide-1 (GLP-1) and GLP-2 analogues are newer drugs showing beneficial cardiovascular and metabolic effects. Whether EAT expresses GLP- 1 and 2 receptors (GLP-1R and GLP-2R) is unknown. RNA-seq analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate the presence of GLP-1R and GLP-2R in EAT and subcutaneous fat (SAT) obtained from 8 subjects with coronary artery disease and type 2 diabetes mellitus undergoing elective cardiac surgery. Immunofluorescence was also performed on EAT and SAT samples using Mab3f52 against GLP-1R. Our RNA-sequencing (RNA-seq) analysis showed that EAT expresses both GLP-1R and GLP-2R genes. qRT-PCR analysis confirmed that GLP-1R expression was low but detected by 2 different sets of intron-spanning primers. GLP-2R expression was detected in all patients and was found to be 5-fold higher than GLP-1R. The combination of accurately spliced reads from RNA-seq and successful amplification using intron-spanning primers indicates that both GLP-1R and GLP-2R are expressed in EAT. Immunofluorescence clearly showed that GLP-1R is present and more abundant in EAT than SAT. This is the first time that human EAT is found to express both GLP-1R and GLP-2R genes. Pharmacologically targeting EAT may induce beneficial cardiovascular and metabolic effects.

Endogenous Glucagon-like Peptide-1 Receptor Signaling in the Nucleus Tractus Solitarius is Required for Food Intake Control.

Alhough the glucagon-like peptide-1 (GLP-1) system is critical to energy balance control and is a target for obesity pharmacotherapies, the receptor-population-mediating effects of endogenous GLP-1 signaling are not fully understood. To address this, we developed a novel adeno-associated virus (AAV-GLP-1R) that utilizes short hairpin RNA to chronically knock down GLP-1 receptors (GLP-1R) in rats. As pharmacological studies highlight the hindbrain nucleus tractus solitarius (NTS) as a brain region important for GLP-1R-mediated effects on energy balance, AAV-GLP-1R was injected into the NTS to examine the role of endogenous NTS GLP-1R signaling in energy balance control. Chow intake and meal size were significantly increased following chronic NTS GLP-1R knockdown. In addition, NTS GLP-1R knockdown significantly increased self-administration of palatable food under both fixed and progressive ratio schedules of reinforcement. Collectively, these data demonstrate that endogenous NTS GLP-1R signaling is required for the control of food intake and motivation to feed, and provide a new strategy to investigate the importance of distinct GLP-1R populations in the control of a variety of functions.

Liraglutide prevents microvascular insulin resistance and preserves muscle capillary density in high-fat diet-fed rats.

Muscle microvasculature critically regulates endothelial exchange surface area to facilitate transendothelial delivery of insulin, nutrients, and oxygen to myocytes. Insulin resistance blunts insulin-mediated microvascular recruitment and decreases muscle capillary density; both contribute to lower microvascular blood volume. Glucagon-like peptide 1 (GLP-1) and its analogs are able to dilate blood vessels and stimulate endothelial cell proliferation. In this study, we aim to determine the effects of sustained stimulation of the GLP-1 receptors on insulin-mediated capillary recruitment and metabolic insulin responses, small arterial endothelial function, and muscle capillary density. Rats were fed a high-fat diet (HFD) for 4 wk with or without simultaneous administration of liraglutide and subjected to a euglycemic hyperinsulinemic clamp for 120 min after an overnight fast. Insulin-mediated muscle microvascular recruitment and muscle oxygenation were determined before and during insulin infusion. Muscle capillary density was determined and distal saphenous artery used for determination of endothelial function and insulin-mediated vasodilation. HFD induced muscle microvascular insulin resistance and small arterial vessel endothelial dysfunction and decreased muscle capillary density. Simultaneous treatment of HFD-fed rats with liraglutide prevented all of these changes and improved insulin-stimulated glucose disposal. These were associated with a significantly increased AMPK phosphorylation and the expressions of VEGF and its receptors. We conclude that GLP-1 receptor agonists may exert their salutary glycemic effect via improving microvascular insulin sensitivity and muscle capillary density during the development of insulin resistance, and early use of GLP-1 receptor agonists may attenuate metabolic insulin resistance as well as prevent cardiovascular complications of diabetes.

Gastric Bypass-Related Effects on Glucose Control, ß Cell Function and Morphology in the Obese Zucker Rat.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) may improve beta cell function by mechanisms other than caloric restriction and body weight loss. We aimed to assess the impact of anatomical and hormonal alterations specific to RYGB on glucose homeostasis, ß cell function and morphology. METHODS: Male Zucker(fa/fa) rats underwent either RYGB (n = 11) or sham surgeries (n = 10). Five of the shams were then food restricted and body weight matched (BWM) to the RYGB rats. Six male Zucker(fa/+) rats underwent sham surgery and served as additional lean controls. Twenty-seven days after surgery, an oral glucose tolerance test (OGTT) was performed and plasma levels of glucose, insulin and glucagon-like peptide-1 (GLP-1) were measured. Immunohistological analysis of pancreatic islets was performed, and GLP-1 receptor and PDX-1 mRNA content were quantified. RESULTS: Shams consumed more food and gained more weight compared to both RYGB and BWM (p < 0.001). Hyperglycaemia was evident in ad libitum-fed shams, whilst postprandial glucose levels were lower in RYGB compared to the BWM sham group (p < 0.05). During the OGTT, RYGB rats responded with >2.5-fold increase of GLP-1. Histology revealed signs of islet degeneration in ad libitum-fed shams, but not in RYGB and sham BWM controls (p < 0.001). GLP-1 receptor and PDX-1 mRNA content was similar between the RYGB and BWM shams but higher compared to ad libitum shams (p < 0.05). CONCLUSIONS: Combined molecular, cellular and histological analyses of pancreatic function suggest that weight loss alone, and not the enhancement of GLP-1 responses, is predominant for the short-term ß cell protective effects of RYGB.

Metformin ameliorates lipotoxicity-induced mesangial cell apoptosis partly via upregulation of glucagon like peptide-1 receptor (GLP-1R).

Glucagon like peptide-1 receptor (GLP-1R), known to be expressed in pancreatic beta cells, is also expressed in glomerular mesangial cells and its agonist has protective effects in diabetic nephropathy. However, its regulatory mechanisms by lipotoxicity in glomerular mesangial cells are not understood. We found that palmitate-mediated lipotoxicity increased apoptosis and decreased GLP-1R expression in a rat mesangial cell line. Silencing GLP-1R expression also increased mesangial cell apoptosis. Interestingly, metformin, one of the biguanide drugs that has anti-diabetic effects, attenuated lipotoxicity-induced mesangial cell apoptosis and restored GLP-1R expression. Moreover, this treatment alleviated GLP-1R knockdown-induced mesangial cell apoptosis. To further evaluate in vivo, diabetic obese db/db mice were administered metformin. Glomerular GLP-1R expression was diminished in db/db mice, as compared with db/m control mice. However, this decrease significantly recovered on metformin administration. Together, these data provide novel evidence that lipotoxicity decreases the mesangial GLP-1R expression in intact cells and in vivo. The decrease induced mesangial cell apoptosis. Furthermore, we provided the evidence that metformin treatment has a renal protective effect partly via increased mesangial GLP-1R expression. Our data suggested that regulation of GLP-1R expression could be a promising approach to treat diabetic nephropathy and the novel mechanism of metformin mediated GLP-1R regulation.