Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (beta-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPgammaS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPbetaS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK(A) receptor antagonist L-364,718 (K(d) = 0.24 nM) and tritiated CCK(B) receptor antagonist L-365,260 (K(d) = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in beta-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK(A) receptor-selective) and CR 2945 (CCK(B) receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase-polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK(B) receptors in beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein alpha-subunits demonstrated inhibition of CCK-stimulated PLC activity in beta-TC3 cells by G(q/11)alpha antiserum (70%), in Rin 1027-B2 cells by G(q/11)alpha antiserum (70%) and G(i)-3alpha antiserum (23%), and in INR1-G9 cells by G(q/11)alpha antiserum (60%) and G(o)alpha antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.
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PMID:Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines. 1093 May 42

Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.
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PMID:Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells. 1093 83

Clinical and experimental studies have implicated high circulating levels of the cytokine tumour necrosis factor-alpha (TNF-alpha) in the pathogenesis of insulin resistance, not only in obesity and diabetes, but also in clinical conditions associated with cachexia and sepsis. TNF-alpha impairs insulin-mediated glucose uptake in adipocytes, but because of lipolytic effects the interpretation of clinical studies and the extent to which TNF-alpha affects muscle insulin sensitivity are unclear. In addition, protein kinase C (PKC) has recently been implicated in the mechanism of TNF-alpha-induced insulin resistance. The present study investigated the effects of TNF-alpha and a PKC inhibitor (RO-318220) on basal and insulin-stimulated 2-[(3)H]deoxyglucose uptake in cultured L6 myoblasts. Reverse transcriptase-PCR analysis confirmed that L6 myoblasts express TNF-alpha receptors I and II (p60 and p80). Dose-response curves for glucose uptake were fitted to a quadratic function to derive C(I-150) values (concentration of insulin required to increase glucose uptake by 50%). Incubation with TNF-alpha at 1 or 10 ng/ml for 24 h had no significant effect on basal glucose uptake, insulin sensitivity or maximal insulin responsiveness. C(I-150) values (means+/-S.E.M.) were as follows: basal, 91.2+/-13 nM; 1 ng/ml TNF-alpha, 102+/-12 nM; and basal, 70.8+/-13 nM; 10 ng/ml TNF-alpha, 43.7+/-40 nM. PKC inhibition markedly attenuated glucose uptake, but there was no difference in insulin sensitivity with RO-318220 alone compared with RO-318220+TNF-alpha. In conclusion, although increased TNF-alpha expression and plasma concentrations have been implicated in the pathogenesis of insulin resistance in various clinical states, there is no evidence that TNF-alpha impairs insulin-stimulated glucose uptake in a skeletal-muscle-derived cell line.
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PMID:Effects of tumour necrosis factor-alpha and inhibition of protein kinase C on glucose uptake in L6 myoblasts. 1099 95

Age-related bone loss is thought to be due to impaired osteoblast functions. Insulin-like growth factors (IGFs) have been shown to be important stimulators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated with changes in components of the IGF-system including IGF-I, IGF-II, IGF-binding proteins (IGFBPs) and IGFBP-specific proteases. We employed a human diploid osteoblast cell line obtained from trabecular bone explants and that exhibit typical characteristics of in vitro senescence during serial subculturing. Using a non-competitive reverse-transcriptase polymerase-chain reaction (RT-PCR) assay, we found that the constitutive level of IGF-I mRNA decreased progressively to 49.9 +/- 4.9% in old osteoblasts as compared to the levels found in the young cells. No age-related change was found in IGF-II steady-state mRNA levels. Changes in IGFBPs gene expression and protein production were assessed using Northern blot analysis and Western ligand blotting (WLB), respectively. IGFBP-3 mRNA levels decreased to 30% and protein production to 16% in aged osteoblasts as compared to levels found in young cells. We also found age-related decreases in mRNA levels of both IGFBP-4 and IGFBP-5 to 70% and 60% in aged osteoblasts, respectively, compared to young cells. While IGFBP-5 protein was not detected by WLB, IGFBP-4 protein production showed a biphasic change with 50% decrease in middle-aged cells and a subsequent increase in aged osteoblasts to levels similar to those in young osteoblasts. We found an age-related increase in the immunoreactive levels of IGFBP-4 protease, however, no detectable IGFBP-4 or IGFBP-3 protease activities in conditioned media from osteoblast cultures were observed. Our findings demonstrate that osteoblast aging is associated with impaired production of the stimulatory components of the IGF-system, that may be a mechanism contributing to age-related decline in osteoblast functions.
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PMID:Changes in the insulin-like growth factor-system may contribute to in vitro age-related impaired osteoblast functions. 1112 90

Mitochondrial adenosine triphosphate (ATP) generation plays a major role in insulin secretion in pancreatic islet beta cells. The relationship between age and nutritional status of the islet and mitochondrial gene messenger RNA (mRNA) expression was investigated. Three animal groups were studied: infant (12-day-old) rats fed either mother's milk or a high carbohydrate (HC) diet; young (2 to 4-month-old) rats; and old (12 to 14-month-old) rats. The expression of mitochondrial cytochrome oxidase (CYO) (subunits I, II, and III), beta-nicotinamide adenine dinucleotide, reduced form dehydrogenase subunit 4 (NADH-DH4), and ATP synthase (subunit 6) (ATP-SYN6) mRNAs was characterized by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The mitochondrial gene mRNAs were identified in each of the groups of rat islets and in RINm5F cells. CYO-II mRNA expression in young and old rat pancreatic islets was 12.7- and 8.2-fold higher, respectively, compared with the level in infant rat islets. The expression of NADH-DH4 and ATP-SYN6 mRNAs was 47% and 40% lower, respectively, in young rat islets compared with the level in infant rat islets. CYO-I, CYO-III, and cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GPDH) mRNA expression did not differ between experimental groups. Artificial rearing of infant rat pups on a HC diet for 8 days lead to a 3.3-fold increase in islet CYO-II mRNA expression compared with mother-fed pups. However, glucose (11 mmol/L) stimulation of cultured isolated islets from young and old rats for 4 days failed to affect the expression level of mitochondrial gene mRNAs. Thus, aging affected the differential expression of CYO-II, NADH-DH4, and ATP-SYN6 mRNAs in rat islets. CYO-II mRNA expression was modulated only in infant rat islets after in vivo administration of carbohydrate.
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PMID:Mitochondrial-encoded gene regulation in rat pancreatic islets. 1122 30

ABSTRACT Because prenatal and perinatal undernutrition are associated with type 2 diabetes later in life, we posed the question whether nutrient deprivation during puberty would also result in a decreased ability to secrete insulin. Chronically catheterized, unstressed Sprague Dawley rats, fed ad libitum, were studied before puberty (Pre, n = 14) and after puberty (Post, n = 8). Moderately caloric-restricted rats (fed 70% of the control diet, n = 9), were studied after puberty. Insulin secretion was assessed using a hyperglycemic clamp at a glucose concentration of 300 mg/dL, or with a primed continuous infusion of intralipid (plasma FFA levels approximately 1.5 mM) at a plasma glucose concentration of 200 mg/dL. Stimulated insulin levels increased in Post rats by 3- to 4-fold compared with Pre rats (from 4.6 +/- 0.4 ng/mL Pre to 12.8 +/- 0.7 ng/mL Post, and from 4.5 +/- 0.4 ng/mL Pre to 15.8 +/- 0.7 ng/mL Post, respectively, p < 0.001, at a glucose concentration of 300 mg/dL, and 200 mg/dL with intralipid). Caloric restriction prevented any rise in insulin secretion (3.8 +/- 0.5 and 4.6 +/- 0.5 ng/mL in the caloric-restricted rats at glucose concentrations of 300 mg/dL and 200 mg/dL with intralipid, respectively). A semiquantitative reverse-transcriptase PCR procedure was used to assess basal and stimulated insulin mRNA levels. Caloric restriction did not compensate by enhancing insulin mRNA levels in response to glucose stimulation. Moderate food deprivation during puberty reduced the capacity of the pancreas to secrete insulin in response to different nutrient stimuli. We hypothesize that puberty has an important role in beta-cell maturation and any major nutrient modification may have deleterious consequences later in life.
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PMID:Food deprivation limits insulin secretory capacity in postpubertal rats. 1126 28

Insulin-secreting pancreatic islet beta-cells possess anion-permeable Cl- channels (I(Cl,islet)) that are swelling-activated, but the role of these channels in the cells is unclear. The Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and niflumic acid were evaluated for their ability to inhibit I(Cl,islet) in clonal beta-cells (HIT cells). Both drugs blocked the channel, but the blockade due to niflumic acid was less voltage-dependent than the blockade due to DIDS. HIT cell volume initially increased in hypotonic solution and was followed by a regulatory volume decrease (RVD). The addition of niflumic acid and, to a lesser extent, DIDS to the hypotonic solution potentiated swelling and blocked the RVD. In isotonic solution, niflumic acid produced swelling, suggesting that islet Cl- channels are activated under basal conditions. The channel blockers glyburide, gadolinium, or tetraethylammonium-Cl did not alter hypotonic-induced swelling or volume regulation. The Na/K/2Cl transport blocker furosemide produced cell shrinkage in isotonic solution and blocked cell swelling normally induced by hypotonic solution. Perifused HIT cells secreted insulin when challenged with hypotonic solutions. However, this could not be completely attributed to I(Cl,islet)-mediated depolarization, because secretion persisted even when Cl- channels were fully blocked. To test whether blocker-resistant secretion occurred via a distal pathway, distal secretion was isolated using 50 mmol/l potassium and diazoxide. Under these conditions, glucose-dependent secretion was blunted, but hypotonically induced secretion persisted, even with Cl- channel blockers present. These results suggest that beta-cell swelling stimulates insulin secretion primarily via a distal I(Cl,islet)-independent mechanism, as has been proposed for K(ATP)-independent glucose- and sulfonylurea-stimulated insulin secretion. Reverse transcriptase-polymerase chain reaction of HIT cell mRNA identified a CLC-3 transcript in HIT cells. In other systems, CLC-3 is believed to mediate swelling-induced outwardly rectifying Cl- channels. This suggests that the proximal effects of swelling to regulate cell volume may be mediated by CLC-3 or a closely related Cl- channel.
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PMID:Chloride channels regulate HIT cell volume but cannot fully account for swelling-induced insulin secretion. 1133 43

In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.
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PMID:Insulin-like growth factor type 1 upregulates uncoupling protein 3. 1158 36

Members of the TGF-beta superfamily of cytokines have been implicated in pancreatic cancer, pancreatitis and in regulation and differentiation of pancreatic endocrine and exocrine cells. Different TGF-beta members signal through phosphorylation of different signal transduction proteins, which eventually form oligomers with SMAD 4 and translocate to the nucleus. Reverse transcriptase-polymerase chain reaction showed that SMADs 1, 2 and 4 are expressed in pancreatic islets. Immunostaining revealed that SMAD 1 and 4 predominantly were expressed by islet insulin and glucagon cells. Since SMAD 1 is known to transduce signals from receptors binding bone morphogenetic protein (BMP) these results indicate a previously unknown role of BMP-like ligands in islet function.
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PMID:Expression of SMAD signal transduction molecules in the pancreas. 1168 56

Effective therapies are now available that can stop the progression of HIV infection and significantly delay the onset of AIDS. The "highly active antiretroviral therapy" (HAART) is a combination of potent antiretroviral drugs such as viral protease inhibitors or nucleoside-analogue reverse-transcriptase inhibitors, that has a variety of serious side effects, including lipodystrophy, a pathology characterized by accumulation of visceral fat, breast adiposity, cervical fat-pads, hyperlipidemia, insulin resistance as well as fat wasting in face and limbs. There is still an open debate that concerns the precise responsibility of HAART as well as metabolic pathways and mechanisms that are involved in the onset of lipodystrophy. The similarities with multiple symmetric lipomatosis (MSL), in which mitochondria impairment plays a crucial role, lead to the hypothesis that drug-induced damages to mitochondrial DNA are able to alter mitochondria functionality to an extent that is similar to what occurs in MSL. In addition, several evidences indicate that HAART is also linked to a deregulated production of tumour necrosis factor-alpha, which uses mitochondria as intracellular targets. In this paper, we review data concerning the role of mitochondria in the pathogenesis of lipodystrophy, and advance a unifying hypothesis involving either direct or indirect effects of the drugs employed during HAART.
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PMID:Mitochondria in the pathogenesis of lipodystrophy induced by anti-HIV antiretroviral drugs: actors or bystanders? 1174 23


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