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1.
Tissue Eng Part A ; 27(5-6): 311-327, 2021 03.
Article in English | MEDLINE | ID: mdl-30734654

ABSTRACT

A stabilized cartilage construct without signs of hypertrophy in chondrocytes is still a challenge. Suspensions of adipose stem/stromal cells (ASCs) and cartilage progenitor cells (CPCs) were seeded into micromolded nonadhesive hydrogel to produce spheroids (scaffold- and serum-free method) characterized by size, immunohistochemistry, fusion, and biomechanical properties. After cell dissociation, they were characterized for mesenchymal cell surface markers, cell viability, and quantitative real-time polymerase chain reaction. Both targeted and nontargeted (shotgun mass spectrometry) analyses were conducted on the culture supernatants. Induced ASC spheroids (ø = 350 µm) showed high cell viability and CD73 downregulation contrasting to CD90. The transforming growth factor (TGF)-ß3/TGF-ß1 ratio and SOX9 increased (p < 0.05), whereas interleukin (IL)-6, IL-8, RUNX2, and ALPL decreased. Induced ASC spheroids were able to completely fuse and showed a higher force required to compression at day 14 (p < 0.0001). Strong collagen type II in situ was associated with gradual decrease of collagen type X and a lower COLXA1 gene expression at day 14 compared with day 7 (p = 0.0352). The comparison of the secretome content of induced and non-induced ASCs and CPCs identified 138 proteins directly relevant to chondrogenesis of 704 proteins in total. Although collagen X was absent, thrombospondin-1 (TSP-1), described as antiangiogenic and antihypertrophic, and cartilage oligomeric matrix protein (COMP), a biomarker of chondrogenesis, were upregulated in induced ASC spheroids. Our scaffold- and serum-free method mimics stable cartilage acting as a tool for biomarker discovery and for regenerative medicine protocols. Impact Statement Promising adult stem cell sources for cartilage regeneration include adipose stem/stromal cells (ASCs) from subcutaneous adipose tissue. Our main objective was the development of a reproducible and easy-to-handle scaffold- and serum-free method to obtain stable cartilage from induced ASC spheroids. In addition to targeted protein profiling and biomechanical analysis, we provide the first characterization of the secretome composition for ASC spheroids, providing a useful tool to monitor in vitro chondrogenesis and a noninvasive quality control of tissue-engineered constructs. Furthermore, our secretome analysis revealed a potential novel biomarker-thrombospondin-1 (TSP-1), known by its antiangiogenic properties and recently described as an antihypertrophic protein.


Subject(s)
Cartilage , Mesenchymal Stem Cells , Adipose Tissue , Cell Differentiation , Cells, Cultured , Chondrocytes , Chondrogenesis , Humans , Thrombospondin 1 , Tissue Engineering
2.
Mol Cell Biochem ; 447(1-2): 1-7, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29372531

ABSTRACT

The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.


Subject(s)
Amyloid/immunology , Antibodies/pharmacology , Insulin-Secreting Cells/immunology , Islet Amyloid Polypeptide/immunology , Islet Amyloid Polypeptide/toxicity , Protein Aggregation, Pathological/immunology , Animals , Antibodies/immunology , Humans , Insulin-Secreting Cells/pathology , Male , Mice , Protein Aggregation, Pathological/pathology
3.
Mol Cell Endocrinol ; 374(1-2): 56-64, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23623867

ABSTRACT

Peroxiredoxins are a family of six antioxidant enzymes (PRDX1-6), and may be an alternative system for the pancreatic beta cells to cope with oxidative stress. This study investigated whether the main diabetogenic pro-inflammatory cytokines or the anti-inflammatory cytokine IL-4 modulate PRDXs levels and putative intracellular pathways important for this process in the insulin-producing RINm5F cells. RINm5F cells expressed significant amounts of PRDX1, PRDX3 and PRDX6 enzymes. Only PRDX6 was modulated by cytokines, showing both mRNA and protein down-regulation following incubation of RINm5F cells with TNF-alpha and IFN-gamma but not with IL-1beta. Separately IFN-gamma or TNF-alpha decreased PRDX6 protein but not mRNA levels. The blockage of the JNK signalling and of the calpains and proteasome proteolysis systems restored PRDX6 protein levels. IL-4 alone did not modulate PRDXs levels. However, pre/co-incubation with IL-4 substantially prevented the decrease in PRDX6 induced by pro-inflammatory cytokines. Knockdown of PRDX6 increased susceptibility of RINm5F cells to the deleterious effects of pro-inflammatory cytokines and to oxidative stress. These results show that, from the PRDXs significantly expressed in RINm5F cells, only PRDX6 is modulated by the diabetogenic cytokines IFN-gamma and TNF-alpha. This PRDX6 down-regulation depends on the calpain and proteasome systems and JNK signalling. PRDX6 is an important enzyme for protection against oxidative stress and the interaction between pro- and anti-inflammatory cytokines might be important to determine the antioxidant capacity of the cells.


Subject(s)
Insulin-Secreting Cells/metabolism , Insulin/biosynthesis , Interferon-gamma/pharmacology , Peroxiredoxin VI/genetics , Tumor Necrosis Factor-alpha/pharmacology , Animals , Calpain/antagonists & inhibitors , Calpain/genetics , Calpain/metabolism , Cell Line , Gene Expression Regulation , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Interleukin-1beta/pharmacology , Interleukin-4/pharmacology , MAP Kinase Kinase 4/antagonists & inhibitors , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Oxidative Stress , Peroxiredoxin VI/antagonists & inhibitors , Peroxiredoxin VI/metabolism , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteolysis/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Signal Transduction
4.
Endocrinology ; 154(3): 1361-72, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23407453

ABSTRACT

Diabetes mellitus (DM) disrupts the pituitary-thyroid axis and leads to a higher prevalence of thyroid disease. However, the role of reactive oxygen species in DM thyroid disease pathogenesis is unknown. Dual oxidases (DUOX) is responsible for H(2)O(2) production, which is a cosubstrate for thyroperoxidase, but the accumulation of H(2)O(2) also causes cellular deleterious effects. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is another member of the nicotinamide adenine dinucleotide phosphate oxidase family expressed in the thyroid. Therefore, we aimed to evaluate the thyroid DUOX activity and expression in DM rats in addition to NOX4 expression. In the thyroids of the DM rats, we found increased H(2)O(2) generation due to higher DUOX protein content and DUOX1, DUOX2, and NOX4 mRNA expressions. In rat thyroid PCCL3 cells, both TSH and insulin decreased DUOX activity and DUOX1 mRNA levels, an effect partially reversed by protein kinase A inhibition. Most antioxidant enzymes remained unchanged or decreased in the thyroid of DM rats, whereas only glutathione peroxidase 3 was increased. DUOX1 and NOX4 expression and H(2)O(2) production were significantly higher in cells cultivated with high glucose, which was reversed by protein kinase C inhibition. We conclude that thyroid reactive oxygen species is elevated in experimental rat DM, which is a consequence of low-serum TSH and insulin but is also related to hyperglycemia per se.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Reactive Oxygen Species/metabolism , Thyroid Gland/metabolism , Animals , Base Sequence , Blood Glucose/metabolism , Cell Line , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/genetics , Dual Oxidases , Flavoproteins/genetics , Flavoproteins/metabolism , Gene Expression , Hydrogen Peroxide/metabolism , Insulin/blood , Insulin/metabolism , Insulin/pharmacology , Iodide Peroxidase/metabolism , Male , NADPH Oxidase 4 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Thyroid Diseases/etiology , Thyroid Diseases/genetics , Thyroid Diseases/metabolism , Thyroid Gland/drug effects , Thyrotropin/blood , Thyrotropin/metabolism
5.
Gen Physiol Biophys ; 31(1): 65-76, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22447832

ABSTRACT

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondrial apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax α protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.


Subject(s)
Dexamethasone/pharmacology , Gene Expression Regulation/physiology , Islets of Langerhans/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/physiology , Animals , Cells, Cultured , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Islets of Langerhans/drug effects , Mitochondria/drug effects , Rats , Rats, Wistar , Signal Transduction/drug effects
6.
Redox Rep ; 16(4): 173-80, 2011.
Article in English | MEDLINE | ID: mdl-21888768

ABSTRACT

OBJECTIVES: Reactive oxygen species (ROS) are involved in many physiological and pathological processes. In the present study, we analysed whether the synthetic glucocorticoid dexamethasone induces oxidative stress in cultured pancreatic islets and whether the effects of dexamethasone on insulin secretion, gene expression, and viability can be counteracted by concomitant incubation with N-acetylcysteine (NAC). METHODS: ROS production was measured by dichlorofluorescein (DCFH-DA) assay, insulin secretion by radioimmunoassay, intracellular calcium dynamics by fura-2-based fluorescence, gene expression by real-time polymerase chain reaction analyses and cell viability by the MTS assay. RESULTS: Dexamethasone (Dexa) increased ROS production and decreased glucose-stimulated insulin secretion after 72 hours incubation. Intracellular ROS levels were decreased and the insulin secretion capacity was recovered by concomitant treatment with Dexa+NAC. The total insulin content and intracellular Ca2+ levels were not modulated in either Dexa or Dexa+NAC groups. There was a decrease in the NAD(P)H production, used as an indicator of viability, after dexamethasone treatment. Concomitant incubation with NAC returned viability to control levels. Dexa also decreased synaptotagmin VII (SYT VII) gene expression. In contrast, the Dexa+NAC group demonstrated an increased expression of SYT VII compared to controls. Surprisingly, treatment with NAC decreased the gene expression of the antioxidant enzyme copper zinc superoxide dismutase soluble. DISCUSSION: Our results indicate that dexamethasone increases ROS production, decreases viability, and impairs insulin secretion in pancreatic rat islets. These effects can be counteracted by NAC, which not only decreases ROS levels but also modulates the expression of genes involved in the secretory pathway and those coding for antioxidant enzymes.


Subject(s)
Acetylcysteine/pharmacology , Dexamethasone/antagonists & inhibitors , Glucocorticoids/antagonists & inhibitors , Insulin/metabolism , Islets of Langerhans/drug effects , Protective Agents/pharmacology , Reactive Oxygen Species/metabolism , Animals , Cell Survival/drug effects , Cells, Cultured , Dexamethasone/toxicity , Free Radical Scavengers/pharmacology , Free Radicals/metabolism , Glucocorticoids/toxicity , Insulin Secretion , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Oxidative Stress/drug effects , Rats , Rats, Sprague-Dawley , Rats, Wistar , Synaptotagmins/drug effects , Synaptotagmins/metabolism
7.
Eur J Pharmacol ; 642(1-3): 37-46, 2010 Sep 10.
Article in English | MEDLINE | ID: mdl-20541544

ABSTRACT

The pentadecapeptide comprising the 104-118 amino acid sequence of the ilotropin-derived Reg3-related islet neogenesis-associated protein (INGAP-PP) has been implicated in beta cell neogenesis and enhancement of insulin secretion in pancreatic islets. The aim of this study was to investigate intracellular pathways by which INGAP-PP signals in insulin-producing cells. Treatment with INGAP-PP increased insulin secretion and intracellular calcium levels in MIN6 cells. INGAP-PP exposure activated c-Myc, serum and particularly nuclear factor-kappaB (NF-kappaB) response elements in insulin-producing cells (1.7+/-0.1, 1.8+/-0.1, 2.4+/-0.3 for RINm5F, and 1.3+/-0.1, 1.3+/-0.1 and 1.6+/-0.1 fold for MIN6 cells compared to controls, respectively). There was an increase in the proliferation rate of viable cells (162+/-17% for RINm5F and 155+/-13% for MIN6) that was accompanied by an increase in proliferating cell nuclear antigen (PCNA) protein expression (187+/-19% and 170+/-8% for RINm5F and MIN6 cells respectively) following INGAP-PP treatment. INGAP-PP increased the expression of the muscarinic M(3) receptor subtype (169+/-4% for RINm5F and 222+/-20% for MIN6 cells). Activation of multiple serum response elements by foetal calf serum also increased muscarinic M(3) receptor expression (173+/-9% for RINm5F and 140+/-7% for MIN6 cells). The blockade of NF-kappaB signalling pathway strongly decreased muscarinic M(3) receptor expression in response to both stimuli. In summary, a network of intracellular signals that includes activation of c-Myc signalling pathway and increased PCNA expression might be related to the increased proliferation rate of insulin-producing cells following incubation with INGAP-PP. NF-kappaB signalling plays an essential role in controlling the expression of the muscarinic M(3) receptor.


Subject(s)
Acetylcholine/metabolism , Cytokines/pharmacology , Gene Expression Regulation/drug effects , Insulin-Secreting Cells/cytology , Peptide Fragments/pharmacology , Receptor, Muscarinic M3/metabolism , Signal Transduction/drug effects , Transcription Factor RelA/metabolism , Active Transport, Cell Nucleus/drug effects , Amino Acid Sequence , Animals , Base Sequence , Carbachol/pharmacology , Catalytic Domain , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Cytokines/chemistry , Gene Knockdown Techniques , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Intracellular Space/drug effects , Intracellular Space/metabolism , Mice , Molecular Sequence Data , Pancreatitis-Associated Proteins , Peptide Fragments/chemistry , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Interfering/genetics , Rats , Reproducibility of Results , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Serum Response Element/genetics , Transcription Factor RelA/deficiency , Transcription Factor RelA/genetics , Up-Regulation/drug effects
8.
Free Radic Biol Med ; 47(10): 1386-93, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19698781

ABSTRACT

Pancreatic beta cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in beta cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic beta cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.


Subject(s)
Antioxidants/metabolism , Catalase/metabolism , Dexamethasone/toxicity , Insulin-Secreting Cells/drug effects , Animals , Caspase 3/metabolism , Catalase/biosynthesis , Catalase/genetics , Cell Death , Cells, Cultured , Gene Expression , Humans , Hydrogen Peroxide/metabolism , Insulin-Secreting Cells/metabolism , Oxidative Stress , Rats , Reactive Oxygen Species/metabolism
9.
Nutrition ; 25(7-8): 774-81, 2009.
Article in English | MEDLINE | ID: mdl-19251398

ABSTRACT

OBJECTIVE: The present study evaluated the effect of nutritional recovery with a soybean diet on the gene and protein expressions and protein phosphorylation of several enzymes and transcription factors involved in hepatic lipid metabolism. METHODS: Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with a 17% casein (CC and LC groups) or soybean (CS and LS groups) diet and with a 6% casein (LL group) diet until 90 d of life. RESULTS: The soybean diet enhanced serum insulin levels but decreased body and liver weights and hepatic lipid and glycogen concentrations. Liver peroxisome proliferator receptor-alpha mRNA abundance was higher in the LS and CS groups than in the LC and CC groups, but the protein content was similar in all groups. Hepatic acetyl-coenzyme A carboxylase (ACC)-alpha and ACCbeta mRNA expression was markedly lower in the LS and CS rats than in the LC and CC rats. ACC protein expression was lower in the CS group than in the CC, LC, and LS groups. Phospho-[Ser(79)]2-ACC content was similar in the CS, LC, and LS groups and lower than the CC group. In the CS rats this reduction paralleled the decrease in total ACC protein. Messenger RNA and protein expression of sterol regulatory element-binding protein 1c, adenosine monophosphate-activated protein kinase, and phospho-[Thr(172)]-adenosine monophosphate-activated protein kinase was not modified by the soybean diet. CONCLUSION: Thus, the soybean diet reduced the liver lipid concentration through downregulation of the ACC gene and protein expressions rather than by phosphorylation status, which possibly resulted in decreased lipogenesis and increased beta-oxidation.


Subject(s)
Acetyl-CoA Carboxylase/metabolism , Glycine max , Liver/enzymology , Malnutrition/diet therapy , Plant Preparations/pharmacology , Animals , Body Weight/drug effects , Caseins/pharmacology , Diet , Down-Regulation , Fatty Acids, Nonesterified/metabolism , Female , Glycogen/metabolism , Insulin/blood , Liver/metabolism , Male , Malnutrition/enzymology , Malnutrition/metabolism , Organ Size/drug effects , PPAR alpha/metabolism , Phosphorylation , Plant Preparations/administration & dosage , Pregnancy , RNA, Messenger/metabolism , Rats , Rats, Wistar
10.
Life Sci ; 82(9-10): 542-8, 2008 Feb 27.
Article in English | MEDLINE | ID: mdl-18234235

ABSTRACT

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), and p70S6K1 (S6K-1) proteins from pancreatic islets of rats fed a normal (17%; NP) or a low (6%; LP) protein diet for 8 weeks. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 16.7 mmol/L of glucose, or 2.8 mmol/L of glucose in the presence or absence of 20 mmol/L of leucine. Glucose- and leucine-induced insulin secretions were higher in NP than in LP islets. Western blotting analysis showed an increase in the expression of IR and PI3K protein levels whereas IRS1 and S6K-1 protein expression were lower in LP compared to NP islets. In addition, S6K-1 mRNA expression was also reduced in islets from LP rats. Our data indicate that a low protein diet modulates the levels of several proteins involved in the insulin secretion pathway. Particularly, the decrease in S6K-1 expression might be an important factor affecting either glucose- or leucine-induced insulin secretion.


Subject(s)
Diet, Protein-Restricted , Insulin/metabolism , Islets of Langerhans/metabolism , Ribosomal Protein S6 Kinases/metabolism , Animals , Animals, Newborn , Blotting, Western , Disease Models, Animal , Gene Expression Regulation, Enzymologic/drug effects , Glucose/pharmacology , In Vitro Techniques , Insulin Secretion , Leucine/pharmacology , Malnutrition/genetics , Malnutrition/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein S6 Kinases/genetics , Signal Transduction/drug effects
11.
J Endocrinol ; 195(1): 157-65, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17911407

ABSTRACT

Ciliary neurotrophic factor (CNTF) belongs to the cytokine family and increases neuron differentiation and/or survival. Pancreatic islets are richly innervated and express receptors for nerve growth factors (NGFs) and may undergo neurotypic responses. CNTF is found in pancreatic islets and exerts paracrine effects in neighboring cells. The aim of this study was to investigate possible effects of CNTF on neonatal rat pancreatic islet differentiation and/or survival. For this purpose, we isolated pancreatic islets from neonatal rats (1-2 days old) by the collagenase method and cultured for 3 days in RPMI medium with (CNTF) or without (CTL) 1 nM CNTF. Thereafter, glucose-stimulated insulin secretion (RIA), general metabolism by (NAD(P)H production; MTS), glucose metabolism ((14)CO(2) production), gene (RT-PCR), protein expression (western blotting), caspase-3 activity (Asp-Glu-Val-Asp (DEVD)), and apoptosis (DNA fragmentation) were analyzed. Our results showed that CNTF-treated islets demonstrated reduced glucose-induced insulin secretion. CNTF treatment did not affect glucose metabolism, as well as the expression of mRNAs and proteins that are crucial for the secretory process. Conversely, CNTF significantly increased mRNA and protein levels related to cell survival, such as Cx36, PAX4, and BCL-2, reduced caspase-3 activity, and islet cells apoptosis, suggesting that CNTF does not affect islet cell differentiation and, instead, acts as a survival factor reducing apoptosis by increasing the expression of the anti-apoptotic BCL-2 protein and decreasing caspase-3 activity.


Subject(s)
Ciliary Neurotrophic Factor/pharmacology , Islets of Langerhans/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western/methods , Caspase 3/genetics , Caspase 3/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Gene Expression , Glucose/metabolism , Islets of Langerhans/drug effects , NADP/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction/methods , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism
12.
Life Sci ; 74(26): 3211-22, 2004 May 14.
Article in English | MEDLINE | ID: mdl-15094322

ABSTRACT

The mechanisms underlying the onset of obesity are complex and not completely understood. An imbalance of autonomic nervous system has been proposed to be a major cause of great fat deposits accumulation in hypothalamic obesity models. In this work we therefore investigated the adrenal chromaffin cells in monosodium glutamate (MSG)-treated obese female mice. Newborn mice were injected daily with MSG (4 mg/g body weight) or saline (controls) during the first five days of life and studied at 90 days of age. The adrenal catecholamine content was 56.0% lower in the obese group when compared to lean controls (P < 0.0001). Using isolated adrenal medulla we observed no difference in basal catecholamine secretion percentile between obese and lean animals. However, the percentile of catecholamine secretion stimulated by high K+ concentration was lower in the obese group. There was a decrease in the tyrosine hydroxylase enzyme expression (57.3%, P < 0.004) in adrenal glands of obese mice. Interestingly, the expression of dopamine beta-hydroxylase was also reduced (47.0%, P < 0.005). Phenylethanolamine N-methyltransferase expression was not affected. Our results show that in the MSG model, obesity status is associated with a defective adrenal chromaffin cell function. We conclude that in MSG obesity the low total catecholamine content is directly related to a decrease of key catecholamine-synthesizing enzymes, which by its turn may lead to a defective catecholamine secretion.


Subject(s)
Adrenal Medulla/physiopathology , Catecholamines/metabolism , Hypothalamic Diseases/complications , Mixed Function Oxygenases/biosynthesis , Obesity/physiopathology , Phenylethanolamine N-Methyltransferase/biosynthesis , Adrenal Medulla/enzymology , Adrenal Medulla/metabolism , Animals , Disease Models, Animal , Dopamine beta-Hydroxylase/biosynthesis , Female , Hypothalamic Diseases/chemically induced , Mice , Obesity/enzymology , Obesity/etiology , Obesity/metabolism , Sodium Glutamate/toxicity , Tyrosine 3-Monooxygenase/biosynthesis
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