Glucagon-like Peptide-1 Receptor Agonist Exendin-4 Research Articles

Insulin Secretion Induced by Glucose-dependent Insulinotropic Polypeptide Requires Phosphatidylinositol 3-Kinase γ in Rodent and Human β-Cells

PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in β-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and β-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 μmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and β-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.

GLP-1 receptor agonism ameliorates hepatic VLDL overproduction and de novo lipogenesis in insulin resistance.

Background/objectivesFasting dyslipidemia is commonly observed in insulin resistant states and mechanistically linked to hepatic overproduction of very low density lipoprotein (VLDL). Recently, the incretin hormone glucagon-like peptide-1 (GLP-1) has been implicated in ameliorating dyslipidemia associated with insulin resistance and reducing hepatic lipid stores. Given that hepatic VLDL production is a key determinant of circulating lipid levels, we investigated the role of both peripheral and central GLP-1 receptor (GLP-1R) agonism in regulation of VLDL production.MethodsThe fructose-fed Syrian golden hamster was employed as a model of diet-induced insulin resistance and VLDL overproduction. Hamsters were treated with the GLP-1R agonist exendin-4 by intraperitoneal (ip) injection for peripheral studies or by intracerebroventricular (ICV) administration into the 3rd ventricle for central studies. Peripheral studies were repeated in vagotomised hamsters.ResultsShort term (7–10 day) peripheral exendin-4 enhanced satiety and also prevented fructose-induced fasting dyslipidemia and hyperinsulinemia. These changes were accompanied by decreased fasting plasma glucose levels, reduced hepatic lipid content and decreased levels of VLDL-TG and -apoB100 in plasma. The observed changes in fasting dyslipidemia could be partially explained by reduced respiratory exchange ratio (RER) thereby indicating a switch in energy utilization from carbohydrate to lipid. Additionally, exendin-4 reduced mRNA markers associated with hepatic de novo lipogenesis and inflammation. Despite these observations, GLP-1R activity could not be detected in primary hamster hepatocytes, thus leading to the investigation of a potential brain–liver axis functioning to regulate lipid metabolism. Short term (4 day) central administration of exendin-4 decreased body weight and food consumption and further prevented fructose-induced hypertriglyceridemia. Additionally, the peripheral lipid-lowering effects of exendin-4 were negated in vagotomised hamsters implicating the involvement of parasympathetic signaling.ConclusionExendin-4 prevents fructose-induced dyslipidemia and hepatic VLDL overproduction in insulin resistance through an indirect mechanism involving altered energy utilization, decreased hepatic lipid synthesis and also requires an intact parasympathetic signaling pathway.

Exendin-4 improves cardiac function in mice overexpressing monocyte chemoattractant protein-1 in cardiomyocytes

The incretin hormone glucagon-like peptide-1 (Glp1) is cardioprotective in models of ischemia-reperfusion injury, myocardial infarction and gluco/lipotoxicity. Inflammation is a factor in these models, yet it is unknown whether Glp1 receptor (Glp1r) agonists are protective against cardiac inflammation. We tested the hypothesis that the Glp1r agonist Exendin-4 (Ex4) is cardioprotective in mice with cardiac-specific monocyte chemoattractant protein-1 overexpression. These MHC-MCP1 mice exhibit increased cardiac monocyte infiltration, endoplasmic reticulum (ER) stress, apoptosis, fibrosis and left ventricular dysfunction. Ex4 treatment for 8weeks improved cardiac function and reduced monocyte infiltration, fibrosis and apoptosis in MHC-MCP1 mice. Ex4 enhanced expression of the ER chaperone glucose-regulated protein-78 (GRP78), decreased expression of the pro-apoptotic ER stress marker CCAAT/-enhancer-binding protein homologous protein (CHOP) and increased expression of the ER calcium regulator Sarco/Endoplasmic Reticulum Calcium ATPase-2a (SERCA2a). These findings suggest that the Glp1r is a viable target for treating cardiomyopathies associated with stimulation of pro-inflammatory factors.

Abstract 205: Mitochondrial Sirt3 Is Upregulated By Glucagon-like Peptide-1 Receptor Activation And Contributes To Reversal Of Cardiac Mitochondrial Remodeling Induced By Type 2 Diabetes.

RATIONALE: Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that maintains basal ATP yield and its expression level is increased by fasting, exercise, and some NAD+ intermediates. We recently reported that the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 (Ex-4) ameliorated cardiac mitochondrial remodeling in diabetic cardiomyopathy via increase in cardiac cyclic AMP (AJP2013). Because changes in cyclic AMP level is regulated by adenylyl cyclase which is one of the downstream target of Ex-4, we hypothesized that SIRT3 may involve in the Ex-4-mediated myocardial reverse remodeling of mitochondria in diabetic mice. METHODS: Type 2 diabetic Mice (16-week old male) were allocated into experimental groups as follows: Ex-4 (24 nmole/kg/day, subcutaneously administrated by osmotic pump for 40 days, DIO/Ex-4) and vehicle control (DIO/CON). Heart samples and cultured rat neonatal cardiomyocytes were subjected to mitochondrial fractionation using density gradient. RESULTS: Cardiac cyclic AMP concentration and phosphorylation of CREB were elevated in DIO-ex4, suggesting successful administration of Ex-4. Electron microscopic analysis revealed that Ex-4 reversed destroyed cristae structure and defragmented mitochondria of DIO/CON heart. The ratio of expression levels of Mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2) was consistently suppressed by Ex-4 treatment, suggesting normalization of mitochondrial morphology. Of note, DIO-CON exhibited marked decrease in cardiac SIRT3 level compared to lean/nondiabetic counterpart, which was reversed by exendin-4 treatment. Pharmacological production of intracellular cAMP levels (Isoprotelenol (10 microM) and 8-bromo-cyclic AMP (1 mM)) increased SIRT3 mRNA in cultured primary cardiomyocytes. The AMP-activated protein kinase (AMPK) inhibitition blocked the increase in SIRT3 mRNA, indicating that the SIRT3 expression was regulated by AMPK-dependent manner. CONCLUSIONS: Our results indicate that Ex-4 reversed abnormal suppression of SIRT3 in mitochondria via activating the PKA/AMPK-dependent pathway.

Glucagon-like peptide-1 receptor activation in the nucleus accumbens core suppresses feeding by increasing glutamatergic AMPA/kainate signaling.

Glucagon-like peptide-1 receptor (GLP-1R) activation in the nucleus accumbens (NAc) core is pharmacologically and physiologically relevant for regulating palatable food intake. Here, we assess whether GLP-1R signaling in the NAc core of rats modulates GABAergic medium spiny neurons (MSNs) through presynaptic-glutamatergic and/or presynaptic-dopaminergic signaling to control feeding. First, ex vivo fast-scan cyclic voltammetry showed that the GLP-1R agonist exendin-4 (Ex-4) does not alter dopamine release in the NAc core. Instead, support for a glutamatergic mechanism was provided by ex vivo electrophysiological analyses showing that Ex-4 activates presynaptic GLP-1Rs in the NAc core to increase the activity of MSNs via a glutamatergic, AMPA/kainate receptor-mediated mechanism, indicated by increased mEPSC frequency and decreased paired pulse ratio in core MSNs. Only a small, direct excitatory effect on MSNs by Ex-4 was observed, suggesting that the contribution of postsynaptic GLP-1R to MSN activity is minimal. The behavioral relevance of the electrophysiological data was confirmed by the finding that intracore injection of the AMPA/kainate receptor antagonist CNQX attenuated the ability of NAc core GLP-1R activation by Ex-4 microinjection to suppress food intake and body weight gain; in contrast, intracore NMDA receptor blockade by AP-5 did not inhibit the energy balance effects of NAc core Ex-4. Together, these data provide evidence for a novel glutamatergic, but not dopaminergic, mechanism by which NAc core GLP-1Rs promote negative energy balance.

Abstract 429: Central Glucagon-Like Peptide-1 Reduces Intestinal Chylomicron Production via Melanocortin-4-Receptor Signaling

Insulin resistance and the metabolic syndrome are associated with intestinal overproduction of chylomicrons, resulting in postprandial dyslipidemia and atherogenic remnants. It is increasingly clear that a reduction in chylomicrons is achieved by the action of glucagon-like peptide-1 (GLP-1) in the periphery. However, this peptide is also generated by central neurons and regulates activity in brain regions that affect sympathetic signaling. Indeed, through sympathetic pathways GLP-1 can modulate peripheral lipogenesis. Therefore, we examined the potential role of peripheral and central GLP-1 in regulating chylomicron production. Healthy hamsters received an oral fat load followed by an intraperitoneal (IP) or intracerebroventricular (ICV) injection of the GLP-1 receptor (GLP-1R) agonist exendin-4. Postprandial triglyceride (TG) and apolipoprotein B48 (apoB48) levels from plasma TG-rich lipoproteins (TRL) were found to be reduced over 6 h. The effects of central exendin-4 were mirrored by ICV MK-0626, an inhibitor of GLP-1 degradation, and reversed by the GLP-1R antagonist exendin9-39. Given a potential link between central GLP-1 and the melanocortin-4-receptor (MC4R) system, hamsters received an MC4R antagonist ICV alongside exendin-4 and this prevented a lowering in TRL-TG levels. To determine whether subsequent communication with the gut was through sympathetic pathways, α- and β-adrenergic receptor blockers were infused intravenously prior to ICV exendin-4 treatment and prevented exendin-4 from reducing TRL-TG. Finally, to examine the interplay between central and peripheral GLP-1R signaling, exendin-4 was given IP while exendin9-39 was given ICV and vice-versa. IP exendin-4 diminished TRL-TG levels during central antagonism, while IP exendin9-39 prevented the action of ICV exendin-4. This suggests that peripheral GLP-1Rs may be potentiated by central stimulation, but can also act independently. Overall, we demonstrate a GLP-1-sensitive brain-gut axis for regulating chylomicron production, involving the MC4R system and sympathetic pathways. Future studies will investigate whether central GLP-1 signals via pro-opiomelanocortin neurons and sympathetic neurotransmitters to affect enterocyte chylomicron output.

Abstract 438: Peripheral but Not Central Glucagon-Like Peptide-1 Receptor Agonism Regulates Fasting Dyslipidemia by Reducing VLDL Production

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces plasma glucose levels and thus acts as an important drug target in the treatment of type 2 diabetes (T2D). Our laboratory has further shown that GLP-1 regulates intestinal lipoprotein metabolism and may also play a similar role in the liver. Hepatic lipids are packaged with apolipoprotein B-100 (apoB100) into very low density lipoproteins (VLDL) and secreted into the plasma. Dysregulation of VLDL production results in the fasting dyslipidemia that is observed in T2D. We postulate that GLP-1 receptor (GLP-1R) agonism regulates fasting dyslipidemia by decreasing VLDL production in insulin resistance through a peripheral or central pathway. To test this, experiments were conducted in fructose-fed insulin resistant Syrian golden hamsters. Hamsters were given twice daily intraperitoneal(i.p) injections of the GLP-1R agonist exendin-4 (ex4; 5nM/kg) for 7 days and placed into metabolic cages. Plasma was collected for lipid analysis following i.p poloxamer to prevent lipoprotein uptake and livers were excised. Peripheral ex4 prevented fructose-induced fasting dyslipidemia and decreased fasting plasma- and VLDL-triglyceride (TG), -cholesterol and -apoB100 accumulation. Ex4 also reduced food intake and body weight and thus pair-fed controls were added. Pair-feeding did not account for the full ex4 lipid-lowering effect. In comparison to both controls, ex4 treated hamsters had reduced respiratory exchange ratio and CO 2 production indicating a switch from glucose to fat metabolism as the main source of energy, and also decreased hepatic mRNA markers for de novo lipogenesis. To determine involvement of a central pathway, hamsters received a 4 day intracerebroventricular administration of ex4 (250ng) into the third ventricle. Central ex4 reduced body weight and food intake but did not modulate fasting plasma and VLDL-lipid or apoB100 accumulation, further suggesting that decreases in food intake cannot solely explain the changes in VLDL production. Our studies suggest that GLP-1R agonism reduces fasting dyslipidemia in insulin resistance by decreasing VLDL production through a peripheral pathway. GLP-1R agonism may be a potential therapy in the treatment of fasting dyslipidemia.

Uncoupling Protein-2 Mediates DPP-4 Inhibitor-Induced Restoration of Endothelial Function in Hypertension Through Reducing Oxidative Stress

Although uncoupling protein 2 (UCP2) negatively regulates intracellular reactive oxygen species (ROS) production and protects vascular function, its participation in vascular benefits of drugs used to treat cardiometabolic diseases is largely unknown. This study investigated whether UCP2 and associated oxidative stress reduction contribute to the improvement of endothelial function by a dipeptidyl peptidase-4 inhibitor, sitagliptin, in hypertension. Pharmacological inhibition of cyclooxygenase-2 (COX-2) but not COX-1 prevented endothelial dysfunction, and ROS scavengers reduced COX-2 mRNA and protein expression in spontaneously hypertensive rats (SHR) renal arteries. Angiotensin II (Ang II) evoked endothelium-dependent contractions (EDCs) in C57BL/6 and UCP2 knockout (UCP2KO) mouse aortae. Chronic sitagliptin administration attenuated EDCs in SHR arteries and Ang II-infused C57BL/6 mouse aortae and eliminated ROS overproduction in SHR arteries, which were reversed by glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin 9-39, AMP-activated protein kinase (AMPK)α inhibitor compound C, and UCP2 inhibitor genipin. By contrast, sitagliptin unaffected EDCs in Ang II-infused UCP2KO mice. Sitagliptin increased AMPKα phosphorylation, upregulated UCP2, and downregulated COX-2 expression in arteries from SHR and Ang II-infused C57BL/6 mice. Importantly, exendin 9-39, compound C, and genipin reversed the inhibitory effect of GLP-1R agonist exendin-4 on Ang II-stimulated mitochondrial ROS rises in SHR endothelial cells. Moreover, exendin-4 improved the endothelial function of renal arteries from SHR and hypertensive patients. We elucidate for the first time that UCP2 serves as an important signal molecule in endothelial protection conferred by GLP-1-related agents. UCP2 could be a useful target in treating hypertension-related vascular events. UCP2 inhibits oxidative stress and downregulates COX-2 expression through GLP-1/GLP-1R/AMPKα cascade.

The therapeutic potential of glucagon-like peptide-1 analogs in the treatment of Alzheimer’s disease

Alzheimer’s disease (AD) affects more than 25% of individuals over 80 years of age and its prevalence is expected to rise with the increase of life expectancy. Existing drugs only provide symptomatic benefits but there are no currently available diseasemodifying therapies. However, evidence from preclinical studies indicates that several drugs approved for other indications could have beneficial effects on Alzheimer’s pathology, and some of them are currently being evaluated in clinical trials [1]. In the last decades an increase in the incidence of both type 2 diabetes (T2D) and neurodegenerative disorders has been observed, posing a significant health and social challenge due to increased morbidity and mortality. T2D increases the risk of AD and there is an increased incidence of T2D in AD patients [2], probably due to common pathophysiological mechanisms shared between these diseases, and one that can be identified is insulin desensitization. Indeed, in AD brain, insulin signaling is desensitized, showing a molecular profile similar to that found in peripheral tissues of diabetic subjects [3]. Glucagon-like peptide-1 (GLP-1) is an incretin hormone with several effects on glycemic homeostasis, stimulating insulin and decreasing glucagon secretion. Currently, the GLP-1 receptor agonists exendin-4, liraglutide and lixisenatide are approved for treatment of T2D [4]. Besides its metabolic effects, GLP-1 has also been shown to act as a growth factor in the brain, inducing neurite growth and protecting against oxidative injury; GLP-1 receptor knock-out mice present with impaired hippocampal long-term potentiation of synaptic transmission [5]. The GLP-1 receptor is found in neurons throughout the nervous system, is maintained during aging and disease, and GLP-1 and its analogs effectively cross the blood–brain barrier. In the context of AD-related brain amyloid pathology, the preclinical results of GLP-1 analogs have shown promising potential. Exendin-4 has been shown to reduce endogenous levels of β-amyloid in the mouse brain [6]. Liraglutide has also been shown to protect synapses from the detrimental effects of amyloid [7,8] and it was able to significantly reduce the β-amyloid plaque load and the total amount of β-amyloid in the brain of a transgenic mouse model of AD. Importantly, in a late-stage animal model of AD, liraglutide demonstrated restorative effects in both brain structure and function, reducing both β-amyloid plaques and soluble amyloid oligomers [9], suggesting that liraglutide could potentially influence the amyloid production. Moreover, GLP-1 analogs have been demonstrated to promote cell proliferation in the rat hippocampus and increase the number of neuronal progenitor cells in mouse models of diabetes. GLP-1R activators induce the differentiation of neuronal stem cell and stimulate neurite outgrowth in a manner similar to nerve “…preclinical evidence strongly supports the hypothesis that GLP-1 analogs can represent promising candidates for Alzheimer’s disease treatment.”

Glucagon-like Peptide-1 Suppresses the Proliferation and Migration of Vascular Smooth Muscle Cells: Implications for Preventive Effects on Atherosclerosis

Our group previously demonstrated the suppressive effect of glucagon-like peptide-1(GLP-1)on macrophage-driven atherosclerosis in apolipoprotein E-de�- cient (apoE ­/­ )mice. In the present study we investigated the suppressive effect of GLP-1 on the atherogenic phenotype of vascular smooth muscle cells(VSMCs) in vivo using apoE ­/­ mice, and the proliferation and migration of human VSMCs in vitro. A 4-week infusion of GLP-1 in 17-week-old apoE ­/­ mice significantly reduced the proliferative VSMC phenotype stained with SMemb. Platelet-derived growth factor(PDGF) -BB signicantly stimulated the proliferation of human aortic VSMCs by three fold. Both 0.1 and 1 nmol / l GLP-1 signicantly suppressed the PDGF-induced VSMC proliferation, and this suppressive effect was significantly abolished by the GLP-1 receptor antagonist exendin(9−39)(50 nmol / l) . The GLP-1 receptor agonists liraglutide(100 nmol / l)and exendin-4(100 nmol / l)mim- icked GLP-1, signicantly suppressing PDGF-induced VSMC proliferation. PDGF- BB signicantly stimulated the migration of human aortic VSMCs by 1.7 -fold, and this effect was signicantly suppressed by 1 nmol / l GLP-1. Thesendings suggest that GLP-1-related treatments may prevent the progression of atherosclerotic lesions by suppressing the proliferation and migration of VSMCs, which are characteristic features of atherosclerosis.

Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage migration in vitro via the NF-κB pathway

To investigate the effects of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 on oxidized low-density lipoprotein (ox-LDL)-induced inhibition of macrophage migration and the mechanisms underlying the effects of exendin-4. Primary peritoneal macrophages were extracted from the peritoneal cavity of mice treated with 3% thioglycollate (2 mL, ip). Migration of the macrophages was examined using a cell migration assay. Macrophage migration-related factors including leptin-like ox-LDL receptor (LOX-1), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin-1 (IL-1)β, matrix metalloproteinase-2 (MMP-2), intercellular adhesion molecule (ICAM)-1 and macrophage migration inhibitory factor (MIF) were measured using semi-quantitative RT-PCR. Expression of MIF and ICAM-1 proteins was examined with ELISA. Gelatin zymography was used to evaluate the activity of MMP-9. Activation of the NF-κB pathway was determined by confocal laser scanning microscopy. Treatment of the macrophages with ox-LDL (50 μg/mL) markedly suppressed the macrophage migration. Furthermore, ox-LDL treatment substantially increased the expression of the macrophage migration-related factors, the activity of MMP-9 and the translocation of the NF-κB p65 subunit. These effects of ox-LDL were significantly ameliorated by pretreatment with the specific NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (100 μmol/L). These effects of ox-LDL were also significantly ameliorated by pretreatment with exendin-4 (25 and 50 nmol/L). Exendin-4 ameliorates the inhibition of ox-LDL on macrophage migration in vitro, via suppressing ox-LDL-induced expression of ICAM-1 and MIF, which is probably mediated by the NF-κB pathway.

Design, Synthesis, and Biological Activity of Novel Dicoumarol Glucagon-like Peptide 1 Conjugates

Twelve novel dicoumarol glucagon-like peptide 1 (GLP-1) conjugates were designed, synthesized, and tested for biological activity. All derivatives retained receptor activation efficacy, and exhibited improved albumin affinity and in vitro stability in rat plasma. The in vivo elimination half-lives of 13c and 13 l (22.07 and 18.78 h, respectively) were much longer than those of the GLP-1 receptor agonists exendin-4 (2.82 h) and liraglutide (12.53 h). The prolonged in vivo antidiabetic effects of 13c and 13 l on db/db mice were confirmed by the hypoglycemic efficacy test and the multiple intraperitoneal glucose tolerance test. Importantly, a once daily administration of 13c to db/db mice for 7 weeks provided long-term beneficial effects by lowering glycated hemoglobin (HbA1c) levels to 5.05%, which was lower than with liraglutide treatment (5.41%). These results suggest that 13c is a promising long-lasting GLP-1 mimetic that may be suitable for clinical use following further research.

Indirect Effects of Glucagon-Like Peptide-1 Receptor Agonist Exendin-4 on the Peripheral Circadian Clocks in Mice

Circadian clocks in peripheral tissues are powerfully entrained by feeding. The mechanisms underlying this food entrainment remain unclear, although various humoral and neural factors have been reported to affect peripheral clocks. Because glucagon-like peptide-1 (GLP-1), which is rapidly secreted in response to food ingestion, influences multiple humoral and neural signaling pathways, we suggest that GLP-1 plays a role in the food entrainment of peripheral clocks. To test this, we compared the effects of exendin-4, a GLP-1 receptor agonist, on mRNA expression of the clock genes (Clock, Bmal1, Nr1d1, Per1, Per2, and Cry1) with those of refeeding. In addition, we investigated whether exendin-4 could affect the rhythms of the peripheral clocks. In male C57BL/6J mice, although refeeding rapidly (within 2 h) altered mRNA levels of Per1 and Per2 in the liver and that of Per1 in adipose tissue, a single i.p. injection of exendin-4 did not cause such changes. However, unlike the GLP-1 receptor antagonist exendin-(9–39), exendin-4 significantly influenced Per1 mRNA levels in the liver at 12 h after injection. Moreover, pretreatment with exendin-4 affected the rapid-feeding-induced change in Per1 not only in the liver, but also in adipose tissue, without effect on food intake. Furthermore, during light-phase restricted feeding, repeated dosing of exendin-4 at the beginning of the dark phase profoundly influenced both the food intake and daily rhythms of clock gene expression in peripheral tissues. Thus, these results suggest that exendin-4 modulates peripheral clocks via multiple mechanisms different from those of refeeding.

The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors

Glucagon-like peptide-1 receptor (GLP-1R) activation in the ventral tegmental area (VTA) is physiologically relevant for the control of palatable food intake. Here, we tested whether the food intake-suppressive effects of VTA GLP-1R activation are mediated by glutamatergic signaling within the VTA. Intra-VTA injections of the GLP-1R agonist exendin-4 (Ex-4) reduced palatable high-fat food intake in rats primarily by reducing meal size; these effects were mediated in part via glutamatergic AMPA/kainate but not NMDA receptor signaling. Additional behavioral data indicated that GLP-1R expressed specifically within the VTA can partially mediate the intake- and body weight-suppressive effects of systemically administered Ex-4, offering the intriguing possibility that this receptor population may be clinically relevant for food intake control. Intra-VTA Ex-4 rapidly increased tyrosine hydroxylase levels within the VTA, suggesting that GLP-1R activation modulates VTA dopaminergic signaling. Further evidence for this hypothesis was provided by electrophysiological data showing that Ex-4 increased the frequency of AMPA-mediated currents and reduced the paired/pulse ratio in VTA dopamine neurons. Together, these data provide novel mechanisms by which GLP-1R agonists in the mesolimbic reward system control for palatable food intake.

Protection of Glucagon-Like Peptide-1 in Cisplatin-Induced Renal Injury Elucidates Gut-Kidney Connection

Accumulating evidence of the beyond-glucose lowering effects of a gut-released hormone, glucagon-like peptide-1 (GLP-1), has been reported in the context of remote organ connections of the cardiovascular system. Specifically, GLP-1 appears to prevent apoptosis, and inhibition of dipeptidyl peptidase-4 (DPP-4), which cleaves GLP-1, is renoprotective in rodent ischemia-reperfusion injury models. Whether this renoprotection involves enhanced GLP-1 signaling is unclear, however, because DPP-4 cleaves other molecules as well. Thus, we investigated whether modulation of GLP-1 signaling attenuates cisplatin (CP)-induced AKI. Mice injected with 15 mg/kg CP had increased BUN and serum creatinine and CP caused remarkable pathologic renal injury, including tubular necrosis. Apoptosis was also detected in the tubular epithelial cells of CP-treated mice using immunoassays for single-stranded DNA and activated caspase-3. Treatment with a DPP-4 inhibitor, alogliptin (AG), significantly reduced CP-induced renal injury and reduced the renal mRNA expression ratios of Bax/Bcl-2 and Bim/Bcl-2. AG treatment increased the blood levels of GLP-1, but reversed the CP-induced increase in the levels of other DPP-4 substrates such as stromal cell-derived factor-1 and neuropeptide Y. Furthermore, the GLP-1 receptor agonist exendin-4 reduced CP-induced renal injury and apoptosis, and suppression of renal GLP-1 receptor expression in vivo by small interfering RNA reversed the renoprotective effects of AG. These data suggest that enhancing GLP-1 signaling ameliorates CP-induced AKI via antiapoptotic effects and that this gut-kidney axis could be a new therapeutic target in AKI.

Glucagon‐Like Peptide‐1 Receptor Agonist Administration Suppresses Both Water and Saline Intake in Rats

Glucagon-like peptide-1 (GLP-1) plays an important role in energy homeostasis. Injections of GLP-1 receptor (GLP-1R) agonists suppress food intake, and endogenous GLP-1 is released when nutrients enter the gut. There is also growing evidence that the GLP-1 system is involved in the regulation of body fluid homeostasis. GLP-1R agonists suppress water intake independent of their effects on food intake. It is unknown, however, whether this suppressive effect of GLP-1R agonists extends to saline intake. Accordingly, we tested the effect of the GLP-1R agonists liraglutide (0.05μg) and exendin-4 (0.05μg) on water and saline intake, as stimulated either by angiotensin II (AngII) or by water deprivation with partial rehydration (WD-PR). Each agonist suppressed AngII-induced water intake; however, only exendin-4 suppressed saline intake. WD-PR-induced water and saline intakes were both attenuated by each agonist. Analysis of drinking microstructure after WD-PR found a reliable effect of the agonists on burst number. Furthermore, exendin-4 conditioned a robust taste avoidance to saccharine; however, there was no similar effect of liraglutide. To evaluate the relevance of the conditioned taste avoidance, we tested whether inducing visceral malaise by injection of lithium chloride (LiCl) suppressed fluid intake. Injection of LiCl did not suppress water or saline intakes. Overall, these results indicate that the fluid intake suppression by GLP-1R activation is not selective to water intake, is a function of post-ingestive feedback, and is not secondary to visceral malaise.

Mitochondrial SIRT3 is upregulated by Glucagon-like peptide-1 receptor activation and contributes to reversal of cardiac mitochondrial remodeling induced by type 2 diabetes

Rationale: Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates mitochondrial function and maintains basal ATP yield, whereas the role in myocardium remain uncertain. We reported that the GLP-1 receptor agonist exendin-4 (Ex-4) ameliorated cardiac mitochondrial remodeling in diabetic cardiomyopathy via cyclic AMP axis (ESC 2012, AHA 2012). Changes in intracellular ATP levels are associated with cyclic AMP, which is generated by adenylyl cyclase utilizing ATP, we thus hypothesized that SIRT3 may involve in Ex-4-mediated myocardial reverse remodeling in diabetes. Methods: Type 2 diabetic mice were allocated into Ex-4 (24 nmole/kg/day, DIO/Ex-4) and vehicle (DIO/CON). Heart samples and cultured cardiomyocytes were subjected to mitochondrial fractionation followed by mRNA and protein analyses. Results: Cardiac cyclic AMP concentration and phosphorylation of CREB were elevated in DIO/Ex-4, suggesting successful administration of Ex-4. Electron microscopy revealed that DIO/CON exhibited mitochondria with destroyed cristae structure and defragmented, which was reversed by Ex-4 (Fig. A). The ratio of Mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2), which modulates mitochondria morphology, was consistently suppressed by Ex-4 (Fig. B), suggesting normalization of mitochondrial morphology. DIO-CON exhibited decrease in cardiac SIRT3 level compared to nondiabetic counterpart, which was reversed by Ex-4 (Fig. C). Increased cAMP levels (Isoprotelenol and 8-bromo-cyclic AMP) enhanced SIRT3 mRNA and protein levels in cultured cardiomyocytesm which was blocked by AMP-activated protein kinase (AMPK) inhibitor (compound C). ![Figure][1] Conclusions: Our results indicate that Ex-4 reversed abnormal suppression of SIRT3 in mitochondria via activating the PKA/AMPK-dependent pathway. [1]: pending:yes

Abstract 146: Glucagon-like Peptide-1 Receptor Activation Promotes Dual Benefits For Reversing Microvasculopathy And Mitochondrial Damage In Diabetic Heart.

PURPOSE: Glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) is a remedy for type 2 diabetes mellitus (T2DM). Ex4 ameliorates cardiac dysfunction in preclinical and clinical settings. However, it remains unclear whether the impact of Ex4 on cardiac remodeling in diabetic cardiomyopathy (DMC), of which primary characteristics are microvasculopathy and mitochondrial damage. Methods and Results: Diet-induced T2DM (DIO) mice and age- and gender-matched lean control mice were allocated into EX4 (24 nmole/kg/day for 40 days; DIO-Ex4 and LEAN-Ex4) and vehicle groups (DIO-veh and LEAN-veh). We first confirmed the GLP-1R expression in every single chamber of mouse heart by immunoblotting and PCR. Ex4 treatment ameliorated both systemic and cardiac insulin resistance without affecting body weight in DIO. Cardiac capillary density of DIO-veh was reduced compared to those LEAN-veh, which were reversed by Ex4 treatment. Tube formation assay and immunoblot analysis using culture endothelial cells revealed that Ex4 directly enhanced in vitro angiogenesis in a PKA/eNOS-dependent fashion. Systolic and diastolic left-ventricular (LV) dysfunctions observed in DIO-veh were restored by Ex4 with decline in LV wall thickening. Myocardial fibrosis detected using sirius-red staining and tissue oxidative stress detected by a fluorescence indicator DHE were attenuated in DIO-Ex4. Of note, analyses using transmission electron microscopy and a fluorescence indicator for damaged mitochondria (mitotracker red) revealed that Ex4 treatment reversed cardiac mitochondrial remodeling and increased healthy mitochondria. Ex4 treatment modulated cardiac oxidative stress balance by upregulating antioxidative molecules (SOD, thioredoxin, glutathione peroxidase) and reduction of NOX4 level; whereas it had no influence on NOX2 level. Conclusions: Ex4 enhances cardiac angiogenesis via GLP-1R-mediated activation of PKA/eNOS axis and accelerates reverses remodeling of myocardial mitochondria, at least in part, via its facilitating effects on antioxidative defense.

Abstract 147: Glucagon-like Peptide-1 Receptor Activation Reverses Cardiac Mitochondrial Remodeling In Advanced Type 2 Diabetes Via Suppression Of Nox4.

PURPOSE: Damaged cardiac mitochondria are one of the critical features of diabetic cardiomyopathy. We indeed demonstrated that diet-induced type 2 diabetes mellitus (T2DM) exhibited cardiac mitochondria remodeling, which were reversed by glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) (submitted as another abstract to BCVS 2013); however, it remains unclear whether the mitochondrial remodeling induced by diabetes may be reversible even at an advanced disease stage and Ex4 may be strong enough to exhibit the cardioprotective effects on such a progressive T2DM. Methods and Results: Genetic T2DM model (KKAy mice of which casual blood glucose levels is more than 30 mmol/L) were allocated into exendin-4 (Ex4, 24 nmole/kg/day for 40 days; KK-Ex4) and vehicle groups (KK-v). In KK-v, their systolic left-ventricular (LV) function was normal range, whereas they exhibited diastolic LV dysfunction and myocardial hypertrophy, which were ameliorated by Ex4. Myocardial fibrosis and cardiac steatosis (detected by Oil-red-O-staining and anti-ceramide antibody) were reduced in KK-ex4. Transmission electron microscopy revealed that Ex-4 normalized cardiac mitochondrial remodeling in terms of morphology, size, and number observed in KK-v. Ex4 attenuated mitochondrial damage which was detected by its specific dye mitotracker-red and alternatively monitored by mitofusin (Mfn)1/Mfn2 ratio. DHE staining revealed that Ex-4 reduced cardiac oxidative stress observed in KK-v. Ex4 modulated cardiac oxidative stress balance by reduction of NOX4 level; whereas it had no influence on NOX2 level and antioxidative molecules (SOD, thioredoxin, glutathione peroxidase), contrarily to those cases observed in diet-induced T2DM heart. Conclusions: Mitochondrial remodeling induced by T2DM was found to be reversible even at an advanced disease stage and Ex4 was strong enough to exhibit the cardioprotective effects on such a progressive T2DM by attenuating cardiac oxidative stress, at least in part, by suppression of NOX4.

Hindbrain GLP-1 receptor-mediated suppression of food intake requires a PI3K-dependent decrease in phosphorylation of membrane-bound Akt

Glucagon-like peptide-1 (GLP-1) receptors (GLP-1R) expressed in the nucleus tractus solitarius (NTS) are physiologically required for the control of feeding. Recently, NTS GLP-1R-mediated suppression of feeding was shown to occur via a rapid PKA-induced suppression of AMPK and activation of MAPK signaling. Unknown are the additional intracellular signaling pathways that account for the long-term hypophagic effects of GLP-1R activation. Because cAMP/PKA activity can promote PI3K/PIP3-dependent translocation of Akt to the plasma membrane, we hypothesize that hindbrain GLP-1R-mediated control of feeding involves a PI3K-Akt-dependent pathway. Importantly, the novel evidence presented here challenges the dogmatic view that PI3K phosphorylation results in an obligatory activation of Akt and instead supports a growing body of literature showing that activation of cAMP/PKA can inhibit Akt phosphorylation at the plasma membrane. Behavioral data show that inhibition of hindbrain PI3K activity by a fourth icv administration of LY-294002 (3.07 μg) attenuated the food intake- and body weight-suppressive effects of a fourth icv administration of the GLP-1R agonist exendin-4 (0.3 μg) in rats. Hindbrain administration of triciribine (10 μg), an inhibitor of PIP3-dependent translocation of Akt to the cell membrane, also attenuated the intake-suppressive effects of a fourth icv injection of exendin-4. Immunoblot analyses of ex vivo NTS tissue lysates and in vitro GLP-1R-expressing neurons (GT1-7) support the behavioral findings and show that GLP-1R activation decreases phosphorylation of Akt in a time-dependent fashion. Current data reveal the requirement of PI3K activation, PIP3-dependent translocation of Akt to the plasma membrane, and suppression in phosphorylation of membrane-bound Akt to mediate the food intake-suppressive effects of hindbrain GLP-1R activation.