MUSCARINIC M1 AND M3 RECEPTOR GENE EXPRESSION DURING PANCREATIC REGENERATION AND INSULIN SECRETION IN RATS

The present work is an attempt to understand the role of acetylcholine muscarinic M1 and M3 receptors during pancreatic regeneration and insulin secretion. The work focuses on the changes in the muscarinic M1 and M3 receptors in brain and pancreas during pancreatic regeneration. The effect of these receptor subtypes on insulin secretion and pancreatic P-cell proliferation were studied in vitro using rat primary pancreatic islet culture. Muscarinic Ml and M3 receptor kinetics and gene expression studies during pancreatic regeneration and insulin secretion will help to elucidate the role of acetylcholine functional regulation of pancreatic u-cell proliferation and insulin secretion.The cholinergic system through muscarinic M1 and M3 receptors play an important role in the regulation of pancreatic (3-cell proliferation and insulin secretion . Cholinergic activity as indicated by acetylcholine esterase, a marker for cholinergic system, decreased in the brain regions - hypothalamus, brain stem, corpus striatum, cerebral cortex and cerebellum during pancreatic regeneration. Pancreatic muscarinic M1 and M3 receptor activity increased during proliferation indicating that both receptors are stimulatory to (3-cell division. Acetylcholine dose dependently increase EGF induced DNA synthesis in pancreatic islets in vitro, which is inhibited by muscarinic antagonist atropine confirming the role of muscarinic receptors. Muscarinic M1 and M3 receptor antagonists also block acetycholine induced DNA synthesis suggesting the importance of these receptors in regeneration. Acetylcholine also stimulated glucose induced insulin secretion in vitro which is inhibited by muscarinic M1 and M3 receptor antagonists. The muscarinic receptors activity and their functional balance in the brain and pancreas exert a profound influence in the insulin secretion and also regeneration of pancreas

Dopamine Receptor Gene Expression In Streptozotocin Induced Diabetic Rats And Its Role In Insulin Secretion

Diabetes Mellitus is a metabolic disorder associated with insulin deficiency, which not.only affects the carbohydrate metabolism but also is associated with various central and peripheral complications. Chronic hyperglycemia during diabetes mellitus is a major initiator of diabetic microvascular complications like retinopathy, neuropathy, The central nervous system (CNS) neurotransmitters play an important role in the regulation of glucose homeostasis. These neurotransmitters mediate rapid intracellular communications not only within the central nervous system but also in the peripheral tissues. They exert their function through receptors present in both neuronal and non neuronal cell surface that trigger second messenger signaling pathways. Dopamine is a neurotransmitter that has been implicated in various central neuronal degenerative disorders like Parkinson's disease and behavioral diseases like Schizophrenia. Dopamine is synthesised from tyrosine, stored in vesicles in axon terminals and released when the neuron is depolarised. Dopamine interacts with specific membrane receptors to produce its effect. Dopamine plays an important role both centrally and peripherally. The recent identification of five dopamine receptor subtypes provides a basis for understanding dopamine's central and peripheral actions . Dopamine receptors are classified into two major groups : DA D1 like and DA D2 like. Dopamine D1 like receptors consists of DA D1 and DA D5 receptors . Dopamine D2 like receptors consists of DA D2, DA D3 and DA D4 receptors. Stimulation of the DA D1 receptor gives rise to increased production of cAMP. Dopamine D2 receptors inhibit cAMP production, but activate the inositol phosphate second messenger system . Impairment of central dopamine neurotransmission causes muscle rigidity, hormonal regulation , thought disorder and cocaine addiction. Peripheral dopamine receptors mediate changes in blood flow, glomerular filtration rate, sodium excretion and catecholamine release. The dopamine D2 receptors increased in the corpus striatum and cerebral cortex but decreased in the hypothalamus and brain stem indicating their involvement in regulating insulin secretion. Dopamine D2 receptor which has a stimulatory effecton insulin secretion decreased in the pancreatic islets during diabetes. Our in vitro studies confirmed the stimulatory role of dopamine D2 receptors in stimulation of glucose induced insulin secretion. A detailed study at the molecular level on the mechanisms involved in the role of dopamine in insulin secretion, its functional modification could lead to therapeutic interventions that will have immense clinical importance.

GABAA and GABAB Receptor Gene Expression and Functional Regulation During Pancreatic Regeneration and Insulin Secretion in Rats

The present study demonstrate the functional alterations of the GABAA and GABAB receptors and the gene expression during the regeneration of pancreas following partial pancreatectomy. The role of these receptors in insulin secretion and pancreatic DNA synthesis using the specific agonists and antagonists also are studied in vitro. The alterations of GABAA and GABAR receptor function and gene expression in the brain stem, crebellum and hypothalamus play an important role in the sympathetic regulation of insulin secretion during pancreatic regeneration. Previous studies have given much information linking functional interaction between GABA and the peripheral nervous system. The involvement of specific receptor subtypes functional regulation during pancreatic regeneration has not given emphasis and research in this area seems to be scarce. We have observed a decreased GABA content, down regulation of GABAA receptors and an up regulation of GABAB receptors in the cerebral cortex, brain stem and hypothalamus. Real Time-PCR analysis confirmed the receptor data in the brain regions. These alterations in the GABAA and GABAB receptors of the brain are suggested to govern the regenerative response and growth regulation of the pancreas through sympathetic innervation. In addition, receptor binding studies and Real Time-PCR analysis revealed that during pancreatic regeneration GABAA receptors were down regulated and GABAB receptors were up regulated in pancreatic islets. This suggests an inhibitory role for GABAA receptors in islet cell proliferation i.e., the down regulation of this receptor facilitates proliferation. Insulin secretion study during 1 hour showed GABA has inhibited the insulin secretion in a dose dependent manner in normal and hyperglycaemic conditions. Bicuculline did not antagonize this effect. GABAA agonist, muscimol inhibited glucose stimulated insulin secretion from pancreatic islets except in the lowest concentration of 1O-9M in presence of 4mM glucose.Musclmol enhanced insulin secretion at 10-7 and 10-4M muscimol in presence of 20mM glucose- 4mM glucose represents normal and 20mM represent hyperglycaemic conditions. GABAB agonist, baclofen also inhibited glucose induced insulin secretion and enhanced at the concentration of 1O-5M at 4mM glucose and at 10-9M baclofen in presence of 20mM glucose. This shows a differential control of the GABAA and GABAB receptors over insulin release from the pancreatic islets. During 24 hours in vitro insulin secretion study it showed that low concentration of GABA has inhibited glucose stimulated insulin secretion from pancreatic islets. Muscimol, the GABAA agonist, inhibited the insulin secretion but, gave an enhanced secretion of insulin in presence of 4mM glucose at 10-7 , 10-5 and 1O-4M muscimol. But in presence of 20mM glucose muscimol significantly inhibited the insulin secretion. GABAB agonist, baclofen also inhibited glucose induced insulin secretion in presence of both 4mM and 20mM glucose. This shows the inhibitory role of GABA and its specific receptor subtypes over insulin synthesis from pancreatic bete-islets. In vitro DNA synthesis studies showed that activation of GABAA receptor by adding muscimol, a specific agonist, inhibited islet DNA synthesis. Also, the addition of baclofen, a specific agonist of GABAB receptor resulted in the stimulation of DNA synthesis.Thus the brain and pancreatic GABAA and GABAB receptor gene expression differentially regulates pancreatic insulin secretion and islet cell proliferation during pancreatic regeneration. This will have immense clinical significance in therapeutic applications in the management of Diabetes mellitus.

5-HT1A and -HT2c Receptor Gene Expression: Their Functional Role in Pancreatic Regeneration and Insulin Secretion in Rats

Department of Biotechnology, Cochin University of Science and Technology

Muscarinic M1 Receptor Gene Expression in Streptozotocin Induced Diabetic Rats: Regulation of Insulin Secretion By Aegle marmelose And Costus pictus Leaf Extracts

The present work is to understand the alterations of total Muscarinic and Muscarinic MI receptors in brain and pancreatic islets of Streptozotocin induced diabetic rats. The work focuses on the evaluation of the antihyperglycemic activity of aqueous extracts of Aegle marmelose and Costus pictus leaves in vivo and the changes in the total Muscarinic and Muscarinic MI receptors during diabetes and after the treatment with insulin. The insulin secretory activity of Aegle marmelose and Costus pictus leaf extracts and the effect of cholinergic receptor agonist were investigated in vitro using rat primary pancreatic islet culture. Muscarinic MI receptor kinetics and gene expression during diabetes and regulation of insulin secretion by Aegle marmelose and Costus pie/us leaf extracts will help us to elucidate the role of Muscarinic and Muscarinic MI receptors in hyperglycemia and the regulatory activity of these plant extracts on insulin secretion through Muscarinic receptors.

“Adrenergic receptors and monoamines in the brain and pancreatic islets of streptozotoein diabetic rats: Role in insulin secretion as a function of age”

I) To study the changes in the content of brain rrrorroamirres in streptozotocirr-irrduced tliabetes as a lirnction of age and to lirrd the role oliadrenal lrornroncs in diabetic state. 2) To assess the adrenergic receptor function in the brain stem ofstreptozotocin-induced diabetic rats ofdillerent ages. 3) To study the changes in the basal levels of second messenger cAMP in the brain stenr ofstreptozotocin-induced diabetic rats as a function of age. 4) To study the changes occurring in the content ofmorroamines and their metabolites in whole pancreas and isolated pancreatic islets of streptozotocin-diabetic rats as a function ofage and the effect of adrenal hormones. 5) To study the adrenergic receptors and basal levels of cAMP in isolated pancreatic islets in young and old streptozotoein-diabetic rats. 6) The in virro study of CAMP content in pancreatic islets of young and old rats and its ellect on glucose induced insulin secretion. 7) 'lhe in vitro study on the involvement of dopamine and corticosteroids in glucose induced insulin secretion in pancreatic islets as a function of age.

Amygdala and ventromedial prefrontal cortex are inversely coupled during regulation of negative affect and predict the diurnal pattern of cortisol secretion among older adults

Among younger adults, the ability to willfully regulate negative affect, enabling effective responses to stressful experiences, engages regions of prefrontal cortex (PFC) and the amygdala. Because regions of PFC and the amygdala are known to influence the hypothalamic-pituitary-adrenal axis, here we test whether PFC and amygdala responses during emotion regulation predict the diurnal pattern of salivary cortisol secretion. We also test whether PFC and amygdala regions are engaged during emotion regulation in older (62- to 64-year-old) rather than younger individuals. We measured brain activity using functional magnetic resonance imaging as participants regulated (increased or decreased) their affective responses or attended to negative picture stimuli. We also collected saliva samples for 1 week at home for cortisol assay. Consistent with previous work in younger samples, increasing negative affect resulted in ventral lateral, dorsolateral, and dorsomedial regions of PFC and amygdala activation. In contrast to previous work, decreasing negative affect did not produce the predicted robust pattern of higher PFC and lower amygdala activation. Individuals demonstrating the predicted effect (decrease s attend in the amygdala), however, exhibited higher signal in ventromedial prefrontal cortex (VMPFC) for the same contrast. Furthermore, participants displaying higher VMPFC and lower amygdala signal when decreasing compared with the attention control condition evidenced steeper, more normative declines in cortisol over the course of the day. Individual differences yielded the predicted link between brain function while reducing negative affect in the laboratory and diurnal regulation of endocrine activity in the home environment.

Triacylglycerol-rich lipoprotein-gene interactions in endothelial cells

Lipoproteins such as LDL (low-density lipoprotein) and oxidized LDL have potentially adverse effects on endothelial cells due to their ability to activate pro-inflammatory pathways regulated via the transcription factor NF-kappaB (nuclear factor kappaB). Triacylglycerol-rich lipoproteins (the chylomicrons, very-low-density lipoprotein and their respective remnant particles) have also been implicated in the induction of a pro-inflammatory phenotype and up-regulation of adhesion molecule expression. Although early studies supported the proposal that LPL (lipoprotein lipase)-mediated hydrolysis of TRLs (triglyceride-rich lipoproteins) at the endothelium could activate the NFkappaB pathway, more recent studies provide evidence of pro-and anti-inflammatory responses when cells are exposed to fatty acids of TRL particles. A large number of genes are up- and down-regulated when cells are exposed to TRL, with the net effect reflecting receptor- and nonreceptor-mediated pathways that are activated or inhibited depending on fatty acid type, the lipid and apolipoprotein composition of the TRL and the presence or absence of LPL. Early concepts of TRL particles as essentially pro-inflammatory stimuli to the endothelium provide an overly simplistic view of their impact on the vascular compartment.

Pathogenicity of the bacterium Pseudomonas putida from the entomopathogenic nematode Steinernema abbasi and its secretion against Galleria mellonella larvae

The Entomopathogenic bacterium Pseudomonas putida from Steinernema abbasi and its metabolic secretions were lethal to the Galleria mellonella larvae. Different laboratory experiments on time interval, substrate, moisture, temperature, dose, penetration of cells, stored and dried metabolites were conducted in sand and filter paper bioassays. It was concluded that death was probably due to the toxic metabolites. This bacterium and its metabolites were found very effective at 30 degree C. Penetration of bacterium was rapid after application on G. mellonella larvae. P. putida cells were recovered from the haemocoele when suspensions containing bacterial cells were applied to the G. mellonella indicating that bacterial symbionts do have a free-living existence and can enter the haemocoele in the absence of nematode vector. Stored metabolite and dried metabolites were found persistent for long time. This bacterium or its toxic secretions can be used for insect control that can be important component of integrated pest management against different insect pests. P. putida and its secretions are suggested as the most appropriate suspension to apply against insect pest control program in tropical ecological regions.

Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion

The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are co-ordinated by the outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane beta-barrel. Here we report isolation and structural/functional characterization of a novel middle domain of the Caf1A usher from Yersinia pestis. The isolated UMD (usher middle domain) is a highly soluble monomeric protein capable of autonomous folding. A 2.8 angstrom (1 angstrom = 0.1 nm) resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor-strand-complemented Caf1 fibre subunit. Moreover, these proteins displayed significant structural similarity. Although UMD is in the middle of the predicted amphipathic beta-barrel of Caf1A, the usher still assembled in the membrane in the absence of this domain. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1 fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Comparison of isolated UMD with the recent strcture of the corresponding domain of PapC usher revealed high similarity of the core structures, suggesting a universal structural adaptation of FGL (F(1)G(1) long) and FGS (F(1)G(1) short) chaperone/usher pathways for the secretion of different types of fibres. The functional role of two topologically different states of this plug domain suggested by structural and biochemical results is discussed.

A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with beta 1- and beta 3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLC gamma 2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in alpha-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for alpha-granule secretion and therefore may play a central role in the regulation of platelet function. (Blood. 2008; 112: 4523-4531)

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of rnRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from prehierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-1A, -1B and -II consistent with tissue responsiveness. Preovulatory granulosa cells F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. RMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. RMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-Subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' OB mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intra-ovarian OMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles

Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins.

Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: Possible role of transforming growth factor alpha

The objective was to investigate the potential role of the oocyte in modulating proliferation and basal, FSH-induced and insulin-like growth factor (IGF)-induced secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E-2), and progesterone (P-4) by mural bovine granulosa cells. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin and androstenedione, and the effects of ovine FSH and IGF analogue (LR3-IGF-1) were tested alone and in the presence of denuded bovine oocytes (2, 8, or 20 per well). Medium was changed every 48 h, cultures were terminated after 144 h, and viable cell number was determined. Results are based on combined data from four independent cultures and are presented for the last time period only when responses were maximal. Both FSH and IGF increased (P < 0.001) secretion of inh A, act A, FS, E-2, and P-4 and raised cell number. In the absence of FSH or IGF, coculture with oocytes had no effect on any of the measured hormones, although cell number was increased up to 1.8-fold (P < 0.0001). Addition of oocytes to FSH-stimulated cells dose-dependently suppressed (P < 0.0001) inh A (6-fold maximum suppression), act A (5.5-fold), FS (3.6-fold), E-2 (4.6-fold), and P-4 (2.4-fold), with suppression increasing with FSH dose. Likewise, oocytes suppressed (P < 0.001) IGF-induced secretion of inh A, act A, FS, and E-2 (P < 0.05) but enhanced IGF-induced P-4 secretion (1.7-fold; P < 0.05). Given the similarity of these oocyte-mediated actions to those we observed previously following epidermal growth factor (EGF) treatment, we used immunocytochemistry to determine whether bovine oocytes express EGF or transforming growth factor (TGF) alpha. Intense staining with TGFalpha antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGFalpha to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGFalpha but not EGF, and TGFalpha is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.