glucagon-like-peptide-1 and Neuroblastoma

Diabetes mellitus (DM) is one of the most common metabolic disorders characterized by hyperglycemia due to insufficiency of insulin and/or insulin resistance. Clinical studies have revealed a higher risk of neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease in diabetic patients. Recently, glucagon-like peptide-1 (GLP-1) is an attractive potential treatment modality for various neurodegenerative diseases. In our study, we aimed to investigate whether exenatide, a GLP-1 analogue, has neuroprotective effects against glucose and fructose-induced toxicity in human SH-SY5Y neuroblastoma cell line. Neurotoxicity was induced by incubating SH-SY5Y cells with different doses (25-100 mM) of glucose and fructose for 24, 48 and 72 hours. Following determination of the significant toxic doses of glucose and fructose, the cells were treated with various doses of exenatide (10-250 nM) in the presence or absence of glucose and fructose. Neurotoxicity was evaluated by MTT assay and Hoechst 33258 staining. Caspase-3 activity and the levels of advanced glycation end products (AGEs) were determined in the cytosolic fractions of treated cells. Our results demonstrated that both glucose and fructose treatments decreased cell viability in neuronal cells dose and time-dependently. Glucose and fructose-treated groups showed increased numbers of apoptotic cells, caspase-3 activity and AGEs levels. Treatment of the cells with exenatide significantly prevented cell death. The most prominent effect was observed at 100 nM exenatide-treated cultures. Our results suggest that high doses of glucose and fructose may lead to neurotoxicity, and exenatide may have protective effects against neuronal damage through its anti-apoptotic feature.

Topics: Cell Line, Tumor; Exenatide; Fructose; Glucagon-Like Peptide 1; Glucose; Humans; Hypoglycemic Agents; Neuroblastoma; Neuroprotective Agents

Several single incretin receptor agonists that are approved for the treatment of type 2 diabetes mellitus (T2DM) have been shown to be neuroprotective in cell and animal models of neurodegeneration. Recently, a synthetic dual incretin receptor agonist, nicknamed "twincretin," was shown to improve upon the metabolic benefits of single receptor agonists in mouse and monkey models of T2DM. In the current study, the neuroprotective effects of twincretin are probed in cell and mouse models of mild traumatic brain injury (mTBI), a prevalent cause of neurodegeneration in toddlers, teenagers and the elderly. Twincretin is herein shown to have activity at two different receptors, dose-dependently increase levels of intermediates in the neurotrophic CREB pathway and enhance viability of human neuroblastoma cells exposed to toxic concentrations of glutamate and hydrogen peroxide, insults mimicking the inflammatory conditions in the brain post-mTBI. Additionally, twincretin is shown to improve upon the neurotrophic effects of single incretin receptor agonists in these same cells. Finally, a clinically translatable dose of twincretin, when administered post-mTBI, is shown to fully restore the visual and spatial memory deficits induced by mTBI, as evaluated in a mouse model of weight drop close head injury. These results establish twincretin as a novel neuroprotective agent and suggest that it may improve upon the effects of the single incretin receptor agonists via dual agonism.

Topics: Animals; Body Temperature; Brain Injuries, Traumatic; Cell Line, Tumor; Cells, Cultured; CREB-Binding Protein; Disease Models, Animal; Embryo, Mammalian; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Incretins; Male; Maze Learning; Memory Disorders; Mice; Mice, Inbred ICR; Neuroblastoma; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Gastrointestinal Hormone; Recognition, Psychology; Signal Transduction

The "metabolic competition" for nutrients between cancer cells and the patient has emerged as an important research area. For pediatric oncology, it remains unclear whether the neuroendokrine regulation of appetite by gastrointestinal hormones such as ghrelin "eat", GLP-1 (glucagon-like peptide, "do not eat"), and PYY (peptide tyrosine-tyrosine, "do not eat") is influenced by tumor growth.. In a prospective randomized study, human hepatoblastoma (HB) and neuroblastoma (NB) cells (3 × 10(6)) were transplanted into the abdominal wall of immune-incompetent (nu/nu) rats (ethic committee approval: TVV43/11). Sham-operated animals received cell culture medium only. Tumor growth was allowed for 8 weeks. Then, all the animals underwent a 2-hour oGTT (oral glucose tolerance test) and were assessed for serum levels of glucose, insulin, ghrelin, GLP-1, and PYY. Finally, all tumor masses and adipose tissues were excised and calculated.. Total body weight (including tumor masses) differed for HB (329+31 g), but not for NB (358+22 g) compared with Sham (361+35 g). Subcutaneous adipose tissue was significantly decreased for both the tumor groups (HB=2.6 g, NB=2.1 g, and Sham=3.5 g). Only for NB, fasting glucose (3.4 + 0.6 mmol/L) and insulin (0.89+0.11 ng/mL) levels were significantly decreased compared with Sham (4.4+0.6 mmol/L; 1.19+0.36 ng/mL) only. During the oGTT (all data calculated as area under the curve, AUC) glucose levels were significantly increased for HB (104 ± 10) and NB (102 ± 13) compared with Sham (84 ± 3), but insulin levels remained similar for either group. Triglyceride levels were increased for HB (0.51 mmol/L) and especially NB (0.73 mmol/L) compared with Sham (0.34 mmol/L). Inflammatory parameters did not differ between the groups. Total ghrelin levels were significantly increased for NB (111 ± 10) and altered for HB (102 ± 15) compared with Sham (84 ± 8). Vice versa GLP-1 was statistically decreased in HB (92 ± 7) and NB (88 ± 12) compared with Sham (127 ± 13). Finally, PYY levels were nonsignificantly reduced for HB (117 ± 5) and NB (120 ± 4) compared with Sham (146 ± 12).

Topics: Animals; Biomarkers; Blood Glucose; Dipeptides; Ghrelin; Glucagon-Like Peptide 1; Glucose Tolerance Test; Hepatoblastoma; Homeostasis; Humans; Lipid Metabolism; Liver Neoplasms; Neoplasm Transplantation; Neuroblastoma; Prospective Studies; Random Allocation; Rats; Rats, Nude; Triglycerides

It is now clear that insulin signaling has important roles in regulation of neuronal functions in the brain. Dysregulation of brain insulin signaling has been linked to neurodegenerative disease, particularly Alzheimer's disease (AD). In this regard, there is evidence that improvement of neuronal insulin signaling has neuroprotective activity against amyloid β (Aβ)-induced neurotoxicity for patients with AD. Linagliptin is an inhibitor of dipeptidylpeptidase-4 (DPP-4), which improves impaired insulin secretion and insulin downstream signaling in the in peripheral tissues. However, whether the protective effects of linagliptin involved in Aβ-mediated neurotoxicity have not yet been investigated.. In the present study, we evaluated the mechanisms by which linagliptin protects against Aβ-induced impaired insulin signaling and cytotoxicity in cultured SK-N-MC human neuronal cells.. Our results showed that Aβ impairs insulin signaling and causes cell death. However, linagliptin significantly protected against Aβ-induced cytotoxicity, and prevented the activation of glycogen synthase kinase 3β (GSK3β) and tau hyperphosphorylation by restoring insulin downstream signaling. Furthermore, linagliptin alleviated Aβ-induced mitochondrial dysfunction and intracellular ROS generation, which may be due to the activation of 5' AMP-activated protein kinase (AMPK)-Sirt1 signaling. This upregulation of Sirt1 expression was also observed in diabetic patients with AD coadministration of linagliptin.. Taken together, our findings suggest linagliptin can restore the impaired insulin signaling caused by Aβ in neuronal cells, suggesting DPP-4 inhibitors may have therapeutic potential for reducing Aβ-induced impairment of insulin signaling and neurotoxicity in AD pathogenesis.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; AMP-Activated Protein Kinases; Amyloid beta-Peptides; Cell Line, Tumor; Cell Survival; Chromones; Diabetes Mellitus; Dipeptidyl-Peptidase IV Inhibitors; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Glucagon-Like Peptide 1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Insulin-Like Growth Factor I; Linagliptin; Male; Membrane Potential, Mitochondrial; Morpholines; Neuroblastoma; Peptide Fragments; Reactive Oxygen Species; RNA, Messenger; Sirtuin 1; Superoxide Dismutase; Superoxide Dismutase-1

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

Topics: Animals; Carboxypeptidase H; Cell Line; Cells, Cultured; Enteroendocrine Cells; Glucagon-Like Peptide 1; Glucagon-Like Peptide 2; Mice; Neuroblastoma; Pancreas; Proglucagon; RNA, Small Interfering; Secretory Vesicles; Signal Transduction

PAS kinase (PASK) is a nutrient sensor that is highly conserved throughout evolution. PASK-deficient mice reveal a metabolic phenotype similar to that described in S6 kinase-1 S6K1-deficient mice that are protected against obesity. Hypothalamic metabolic sensors, such as AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR), play an important role in feeding behavior, the homeostasis of body weight, and energy balance. These sensors respond to changes in nutrient levels in the hypothalamic areas involved in feeding behavior and in neuroblastoma N2A cells, and we have recently reported that those effects are modulated by the anorexigenic peptide glucagon-like peptide-1 (GLP-1). Here, we identified PASK in both N2A cells and rat VMH and LH areas and found that its expression is regulated by glucose and GLP-1. High levels of glucose decreased Pask gene expression. Furthermore, PASK-silenced N2A cells record an impaired response by the AMPK and mTOR/S6K1 pathways to changes in glucose levels. Likewise, GLP-1 effect on the activity of AMPK, S6K1, and other intermediaries of both pathways and the regulatory role at the level of gene expression were also blocked in PASK-silenced cells. The absence of response to low glucose concentrations in PASK-silenced cells correlates with increased ATP content, low expression of mRNA coding for AMPK upstream kinase LKB1, and enhanced activation of S6K1. Our findings indicate that, at least in N2A cells, PASK is a key kinase in GLP-1 actions and exerts a coordinated response with the other metabolic sensors, suggesting that PASK might play an important role in feeding behavior.

Topics: Acetyl-CoA Carboxylase; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Energy Metabolism; Gene Expression Regulation, Enzymologic; Gene Knockdown Techniques; Gene Silencing; Glucagon-Like Peptide 1; Glucose; Hypothalamus; Male; Mice; Neuroblastoma; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases; RNA, Messenger

Increasing evidence suggests that glucagon-like peptide-1 (GLP-1), an incretin hormone of current interest in type 2 diabetes, is neuroprotective in both cell culture and animal models. To characterize the neuroprotective properties of GLP-1 and associated underlying mechanisms, we over-expressed the GLP-1 receptor (GLP-1R) on human neuroblastoma SH-SY5Y cells to generate a neuronal culture system featuring enhanced GLP-1R signaling. In GLP-1R over-expressing SH-SY5Y (SH-hGLP-1R#9) cells, GLP-1 and the long-acting agonist exendin-4 stimulated cell proliferation and increased cell viability by 2-fold at 24 h at physiologically relevant concentrations. This GLP-1R-dependent action was mediated via the protein kinase A and phosphoinositide 3-kinase signaling pathways, with the MAPK pathway playing a minor role. GLP-1 and exendin-4 pretreatment dose-dependently protected SH-hGLP-1R#9 cells from hydrogen peroxide (H(2)O(2))- and 6-hydroxydopamine-induced cell death. This involved amelioration of elevated caspase 3 activity, down-regulation of pro-apoptotic Bax and up-regulation of anti-apoptotic Bcl-2 protein. In the presence of 6-hydroxydopamine, GLP-1's ability to lower caspase-3 activity was abolished with the phosphoinositide 3-kinase inhibitor, LY2940002, and partly reduced with the protein kinase A inhibitor, H89. Hence, GLP-1R mediated neurotrophic and anti-apoptotic actions co-contribute to the neuroprotective property of GLP-1 in neuronal cell cultures, and reinforce the potential therapeutic value of GLP-1R agonists in neurodegenerative disorders involving oxidative stress.

Topics: Activating Transcription Factor 4; Adrenergic Agents; Apoptosis; bcl-2-Associated X Protein; Bromodeoxyuridine; Butadienes; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Hydrogen Peroxide; Hypoglycemic Agents; Neuroblastoma; Neuroprotective Agents; Nitriles; Oxidants; Oxidopamine; Peptides; Proto-Oncogene Proteins c-bcl-2; Receptors, Glucagon; Signal Transduction; Time Factors; Transfection; Venoms