Postprandial leptin and adiponectin in response to sugar and fat in obese and normal weight individuals.

PURPOSE: Adipokines produced by white adipose tissue are central in the development of lifestyle diseases. Individuals in industrialized countries spend a substantial part of life in the non-fasting, postprandial state, which is associated with increased oxidation and inflammation. The aim was to study postprandial adiponectin and leptin levels after an oral fat tolerance test (OFTT) and an oral glucose tolerance test (OGTT) in obese (OB) and healthy, normal weight individuals (NW). METHODS: Fifty adults with obesity (BMI ≥ 30) and 17 healthy, NW were included. Postprandial triglyceride (TG), adiponectin, and leptin levels were measured every second hour during an 8 h OFTT, and every half hour during a 2 h OGTT. RESULTS: Compared with the basal level, postprandial levels of adiponectin following OFTT showed a slight initial peak, followed by a significant decrease at 8 h, in the NW. In the OB these changes were abolished. Postprandial levels of leptin decreased significantly from basal levels in the OFTT, in the NW, whereas in the OB, leptin was unchanged except for a slight increase from 2 to 8 h. During the OGTT both adiponectin and leptin levels remained unchanged in the NW, but decreased significantly in the OB. In addition, the OB had delayed TG clearance at 6 h. CONCLUSIONS: A fatty meal gives postprandial changes in the secretion of adiponectin and leptin in NW, but not in OB. Our observations indicate that a potential postprandial regulatory role of adiponectin and leptin is impaired in OB, and of importance in a more comprehensive understanding of the delayed postprandial TG clearance in obese individuals.

Screening for hepatitis C in a general adult population in a low-prevalence area: the Tromsø study.

BACKGROUND: Chronic hepatitis C virus (HCV) infection can progress to cirrhosis and end-stage liver disease in a substantial proportion of patients. The infection is frequently asymptomatic, leaving many infected individuals unaware of the diagnosis until complications occur. This advocates the screening of healthy individuals. The aim of this study was to estimate the prevalence of HCV infection in the general adult population of the municipality of Tromsø, Norway, and to evaluate the efficiency of such an approach in a presumed low-prevalence area. METHODS: The study was part of the seventh survey of the Tromsø Study (Tromsø 7) in 2015-2016. Sera from 20,946 individuals aged 40 years and older were analysed for antibodies to HCV (anti-HCV). A positive anti-HCV test was followed up with a new blood test for HCV RNA, and the result of any previous laboratory HCV data were recorded. Samples positive for anti-HCV and negative for HCV RNA were tested with a recombinant immunoblot assay. All HCV RNA positive individuals were offered clinical evaluation. RESULTS: Among 20,946 participants, HCV RNA was detected in 33 (0.2%; 95% CI: 0.1-0.3), of whom 13 (39.4%; 95% CI: 22.7-56.1) were unaware of their infection. The anti-HCV test was confirmed positive in 134 individuals (0.6%; 95% CI: 0.5-0.7) with the highest prevalence in the age group 50-59 years. Current or treatment-recovered chronic HCV-infection was found in 85 individuals (0.4%; 95% CI: 0.3-0.5) and was associated with an unfavorable psychosocial profile. CONCLUSION: In this population-based study, the prevalence of viraemic HCV infection was 0.2%. A substantial proportion (39%) of persons with viraemic disease was not aware of their infectious status, which suggests that the current screening strategy of individuals with high risk of infection may be an inadequate approach to identify chronic HCV infection hidden in the general population.

Leptin to adiponectin ratio - A surrogate biomarker for early detection of metabolic disturbances in obesity.

AIM: To study if the leptin to adiponectin (L:A) ratio, can be a potential biomarker for postprandial triglyceride clearance, insulin resistance (IR) or leptin resistance (LR) in apparently healthy obese, and obese individuals with established metabolic disease. METHODS AND RESULTS: Fifty adult subjects with obesity (BMI ≥30); of which 36 metabolic healthy obese (MHO), and 14 metabolic dysregulated obese (MDO), with clinical and/or biochemical signs of metabolic disease were included. Seventeen healthy, normal weight subjects represented the control group. Postprandial triglyceride (TG) levels were measured in an 8 h oral fat tolerance test (OFTT). IR by HOMA-IR, L:A ratio and indirect LR were measured. In the MHO group, 71.4%, 69.4% and 86.1%, had delayed TG clearance, IR and LR, respectively; whereas in the MDO group this was detected in 85.7%, 71.4% and 91.7%, respectively. A combination of all three metabolic risk factors was found in 39.8% of the MHO and in 42.9% of the MDO patients. Receiver operating characteristics (ROC) analysis revealed that a cut-off value for the L:A ratio of >1.65 for the control group (PPV 1.0, NPV 0.91) and >3.65 for the obese subjects (PPV 0.86, NPV 0.48) predicted the delayed TG clearance with a good specificity and sensitivity. Detecting a combined risk with at least 2/3 metabolic risk factors, the ROC yielded the most suitable L:A ratio cut-off at >1.88. CONCLUSION: L:A ratio was able to detect early metabolic disturbances in obese individuals, and may be a potential useful clinical surrogate biomarker of metabolic disorders.

Future complications of chronic hepatitis C in a low-risk area: projections from the hepatitis c study in Northern Norway.

BACKGROUND: Hepatitis C (HCV) infection causes an asymptomatic chronic hepatitis in most affected individuals, which often remains undetected until cirrhosis and cirrhosis-related complications occur. Screening of high-risk subjects in Northern Norway has revealed a relatively low prevalence in the general population (0.24%). Despite this, late complications of HCV infection are increasing. Our object was to estimate the future prevalence and complications of chronic HCV infection in the period 2013-2050 in a low-risk area. METHODS: We have entered available data into a prognostic Markov model to project future complications to HCV infection. RESULTS: The model extrapolates the prevalence in the present cohort of HCV-infected individuals, and assumes a stable low incidence in the projection period. We predict an almost three-fold increase in the incidence of cirrhosis (68 per 100,000), of decompensated cirrhosis (21 per 100,000) and of hepatocellular carcinoma (4 per 100,000) by 2050, as well as a six-fold increase in the cumulated number of deaths from HCV-related liver disease (170 per 100,000 inhabitants). All estimates are made assuming an unchanged treatment coverage of approximately 15%. The estimated numbers can be reduced by approximately 50% for cirrhosis, and by approximately one third for the other endpoints if treatment coverage is raised to 50%. CONCLUSION: These projections from a low-prevalence area indicate a substantial rise in HCV-related morbidity and mortality in the coming years. The global HCV epidemic is of great concern and increased treatment coverage is necessary to reduce the burden of the disease.

A rapid chemokine response of macrophage inflammatory protein (MIP)-1α, MIP-1ß and the regulated on activation, normal T expressed and secreted chemokine is associated with a sustained virological response in the treatment of chronic hepatitis C.

The role of chemokines in chronic hepatitis C virus (HCV) infection is not fully understood. The present study aimed to characterize the baseline serum concentrations and the initial ß-chemokine response to treatment with interferon-α and ribavirin with respect to the final clinical outcome of virological response to treatment. Serum concentrations of alanine aminotransferase (ALT) and of the CC subfamily chemokines [macrophage inflammatory protein (MIP)-1α, MIP-1ß, monocyte chemoattractant protein (MCP)-1 and the regulated on activation, normal T expressed and secreted (RANTES) chemokine] were measured in patients with chronic HCV infection and in healthy individuals. Necroinflammation and fibrosis were scored in liver biopsies. Treatment outcomes were classified as with or without a sustained virological response after a full-course treatment according to the genotypes. The main treatment group consisted of 72 patients with chronic hepatitis C, whereas 24-h blood samples were available for 42 patients. Increased baseline levels of all CC chemokines were found in the two responder groups compared to the healthy controls, although significant levels were reached only for MIP-1α and MCP-1. No correlation was observed between chemokine levels and serum ALT levels, any histological necroinflammatory parameters, or the fibrosis grade. After 24 h of treatment, increases in MIP-1α, MIP-1ß and RANTES levels were exclusively observed in the group with sustained virological response. MCP-1 was also significantly increased after 24 h in both responder groups, although no differences were observed between the two responder groups. In conclusion, an early MIP-1α, MIP-1ß, and RANTES response may predict a sustained response to virological treatment.

Accuracy of a monoclonal antibody-based stool antigen test in the diagnosis of Helicobacter pylori infection.

BACKGROUND: Recent availability of tests for Helicobacter pylori antigens in stool samples has provided potentially useful tools for epidemiological studies and clinical settings. The aim of this study was to evaluate a monoclonal antibody-based H. pylori antigen stool test in the primary diagnosis of H. pylori infection, and to study the test performance after patients were treated with lanzoprazole, and after eradication therapy. METHODS: The study included 122 dyspeptic patients. At gastroscopy, biopsy specimens were obtained for culture and histology. Stool antigen and [14C]-urea breath tests were performed concurrently. Positive culture alone or a positive [14C]-urea breath test in combination with positive histology defined the reference standard. Forty-three Hp +ve patients were treated with lanzoprazole for 2 to 4 weeks, and stool antigen tests were performed on days 1 and 7 post-treatment. After eradication therapy, 32 patients were re-examined for H. pylori infection. RESULTS: Prevalence of H. pylori was 44.3%. Sensitivity and specificity for the stool antigen test in the primary diagnosis of H. pylori infection were 98% and 94%, with positive and negative likelihood ratios of 16.7 and 0.02, respectively. All patients had positive stool tests immediately after lanzoprazole treatment, whereas 2 patients had negative stool tests after 7 days. Triple therapy rendered all patients stool test negative. CONCLUSIONS: The monoclonal antibody-based stool antigen test is an accurate tool in the primary diagnosis of H. pylori infection and after eradication therapy. Lanzoprazole treatment does not influence the clinical performance of the test.

Phospholipase C activation in rat pituitary adenoma (GH) cells.

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (C-proteins) G(q) alpha and/or G(11) alpha has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G(q) and/or G(11) confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses showing that anti G(q)/11 alpha-sera coprecipitated PL-C activity. In essence, only G(q)/11 (but neither G(12) G(13) nor G(o)) seems to mediate the TRH-sensitive PL-C activity, while G(o) may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B gamma-complex also, to some extent, may stimulate GH(3) pituitary cell line PL-C activity. Finally, the steady state levels of G(q)/(11) alpha mRNA and protein were down regulated upon long term exposure of the GH(3) cells to TRH (but not to vasoactive intestinal peptide = VIP).

Protein domains involved in both in vivo and in vitro interactions between human T-cell leukemia virus type I tax and CREB.

Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-bp repeats and the transactivator protein Tax. The 21-bp repeats can be subdivided into three motifs known as A, B, and C, each of which is important for maximal gene expression in response to Tax. The B motif contains nucleotide sequences known as a cyclic AMP response element (CRE) or tax-response element which binds members of the ATF/CREB family of transcription factors. Though mutations of this element in the HTLV-I LTR eliminate tax activation, Tax will not activate most other promoters containing these CRE sites. In this study, we investigated the mechanism by which Tax activates gene expression in conjunction with members of the ATF/CREB family. We found that Tax enhanced the binding of one member of the ATF/CREB family, CREB 1, to each of the three HTLV-I LTR 21-bp repeats but not another member designated CRE-BP1 or CREB2. Tax enhanced the binding of CREB1 to nonpalindromic CRE binding sites such as those found in the HTLV-I LTR, but Tax did not enhance the binding of CREB1 to palindromic CRE binding sites such as found in the somatostatin promoter. This finding may help explain the failure of Tax to activate promoters containing consensus CRE sites. These studies were extended by use of the mammalian two-hybrid system. Tax was demonstrated to interact directly with CREB1 but not with other bZIP proteins, including CREB2 and Jun. Site-directed mutagenesis of both Tax and CREB1 demonstrated that the amino terminus of Tax and both the basic and the leucine zipper regions of CREB1 were required for direct interactions between these proteins both in vivo and in vitro. This interaction occurred in vivo and thus did not require the presence of the HTLV-I 21-bp repeats, as previously suggested. These results define the domains required for interaction between Tax and CREB that are likely critical for the activation of HTLV-I gene expression.

G-proteins: implications for pathophysiology and disease.

This article focuses on the involvement of G-proteins in neuroendocrine secretion, cell growth and phenotype alterations. The current concept of hormonal activation of the GTPase cycle, as well as the molecular diversity of G-proteins families and receptor*G-protein*effector coupling, are described. Also described are certain G-proteins as possible proto-oncogenes and how point mutations and frame shift mutations alter G-protein function and determine the characteristics of various endocrine diseases. The article outlines in detail how receptors and G-proteins interact in prolactin and growth-hormone-secreting pituicytes, how G-proteins are involved in the growth and differentiation of preadipocytes and osteoblasts. All in all, it seems that hormonal activation through G-proteins is modulated through direct intra- and inter-signalling system cross-talk at the plasma membrane level (short-term) and through interactions on the level of transcription (HREs) from tyrosine kinases, steroid-like hormones and metabolic pathways. Pharmacological intervention to treat diseases where G-proteins are involved should take both long and short-term regulatory phenomena into consideration.

Role of flanking E box motifs in human immunodeficiency virus type 1 TATA element function.

Human immunodeficiency virus type 1 (HIV-1) gene expression is dependent on a number of cis-acting DNA elements present in the HIV-1 long terminal repeat. Previous studies have demonstrated that the TATA element is critical for basal and Tat-induced HIV-1 gene expression. The HIV-1 TATA region has an unusual structure in that the TATA sequence is flanked by two palindromic sequence motifs (CANNTG) known as E boxes which can serve as binding sites for the basic helix-loop-helix (bHLH) class of DNA-binding proteins. In this study, we performed site-directed mutagenesis of both the TATA and the flanking E box sequences of HIV-1. We also substituted the sequences flanking the adenovirus E3 promoter TATA sequence for those flanking the HIV-1 TATA sequence. Constructs were assayed for their levels of basal and Tat-induced gene expression by both in vitro transcription and transient expression assays. Both the TATA box and flanking sequences including the E box motifs were found to be important in modulating both basal gene expression and Tat-induced HIV-1 gene expression. Gel retardation analysis demonstrated that binding of both the recombinant TATA-binding protein (TBP) and the TFIID fraction which contains both TBP and TBP-associated factors was dependent primarily on the TATA element. However, competition analysis suggested that the E boxes may play a role in stabilizing the binding of TFIID but not recombinant TBP. Two proteins representing different classes of bHLH proteins, E47 and AP-4, were assayed for their ability to bind to the flanking E box motifs. We isolated a cDNA clone encoding the complete AP-4 protein and demonstrated that both AP-4 and E47 bound specifically to the 3' E box motif, which contains sequences that correspond to the consensus binding site (CAGCTG). Gel retardation analysis indicated that the binding of AP-4 to the E boxes excluded the binding of TBP to the TATA box. These studies are consistent with a model in which different classes of cellular bHLH proteins may be involved in regulating HIV-1 TATA element function by either inhibiting or promoting the assembly of different preinitiation transcriptional complexes.

Cell-specific expression and function of adenylyl cyclases in rat pituitary tumour cell lines.

The present study demonstrates cell-specific distribution and describes distinct functional regulation of different adenylyl cyclases (AC, types I-VI) in rat pituitary cell tumor cell lines (GH12C1, GH3 and GH4C1 cells) and pituitary tissue. Northern-blot analysis revealed a distinct pattern of cell-specific expression of the different AC types; Ca2+/calmodulin (CaM)-insensitive AC type II was found in all cell lines tested except GH(1)2C1 cells. The Ca(2+)-inhibitable AC type VI was found in all cell types tested. We observed a lack of the Ca2+/CaM-sensitive AC type I in GH3 and GH4C1 cells. GH(1)2C1 cells exclusively contained both Ca2+/CaM-sensitive AC types I and III, the latter previously believed to be specific for olfactory tissue. An additional transcript of AC type III was found in rat brain and rat liver tissue. AC type IV, which is Ca2+/CaM insensitive, could be detected in the prolactin-producing GH3 and GH4C1 cells and pituitary tissue but not in growth-hormone-producing GH(1)2C1 cells. Basal and vasoactive-intestinal-peptide-(VIP)-releasing-hormone, somatostatin (SRIF) and thyrotropin-releasing-hormone (TRH)-modulation of AC activity was measured in the presence of 100 microM EGTA, anti-CaM serum (dilution 1:2000) or 10 microM trifluoroperazine. Antisera against guanine-nucleotide-binding protein (G-protein) alpha subunits (G(i)-2 alpha, Gs alpha) and beta subunits (G beta 35/36) and CaM were added for functional studies of the SRIF and VIP-modulated AC in GH(1)2C1 and GH3 cells. These experiments indicate that the VIP and the SRIF receptors are coupled to a Ca2+/CaM-sensitive AC in GH(1)2C1 cells, different from the AC involved in the regulation of cAMP levels in GH3 and GH4C1 cells. In addition, the beta gamma-complex is possibly able to modulate SRIF-inhibited AC activity by potentiating the inhibitory effect. The TRH receptor in GH3 and GH4C1 cells is coupled to a Ca2+/CaM-sensitive AC which is different from the already cloned forms of AC types I and III. We, therefore, conclude that hormone regulation of pituitary tumour cell functions differs between the GH cell lines, due to specific utilisation of AC types.

Induction of peroxisomal acyl-CoA oxidase by 3-thia fatty acid, in hepatoma cells and hepatocytes in culture is modified by dexamethasone and insulin.

The effects of tetradecylthioacetic acid (TTA) (50 microM), dexamethasone (0.25 microM) and insulin (0.4 microM) on induction of peroxisomal acyl-CoA oxidase activity and mRNA levels were studied in short term cultures of Morris 7800C1 and MH1C1 hepatoma cells and of rat hepatocytes. Dexamethasone and TTA resulted in parallel increases in the enzyme activity and the steady state mRNA content in the hepatoma cells. Combination of dexamethasone and TTA resulted in a synergistic and parallel stimulation of both the enzyme activity and the mRNA levels up to 11-12-fold and maximal changes were observed after 14 days of treatment. Semiquantitative immunoblot analyses of acyl-CoA oxidase were in concordance with enzyme and mRNA results. Insulin counteracted the inductive effects of dexamethasone and TTA on all parameters. The half-life of the acyl-CoA oxidase mRNA increased after treatment with the 3-thia fatty acid (t1/2 = 10.0 h +/- 0.4) compared to control (t1/2 = 5.9 h +/- 0.3). However, in combination with dexamethasone there was no further increase in the mRNA stability (t1/2 = 8.0 h +/- 0.3). Southern blot analysis did not reveal any changes on the oxidase gene level in any treatment group. TTA alone or in combination with dexamethasone did not affect the expression of either the glucocorticoid receptor or the peroxisomal proliferator acting receptor (PPAR) steady state mRNA levels. In cultured hepatocytes the acyl-CoA oxidase was modified in similar manner by these treatments, but the changes were less marked. We suggest that the changes in peroxisomal acyl-CoA oxidase activity in hepatoma cells are due to a major effect on the level of mRNA, involving both transcriptional effects and message stabilization.

Modulation of G proteins and second messenger responsiveness by steroid hormones in GH3 rat pituitary tumour cells.

We have investigated the modulation of different G protein alpha- and beta-subunit levels in prolactin (PRL) and growth hormone producing rat pituitary adenoma cells (GH3 cells) in culture after prolonged exposure (6-48 h) to the steroid hormones 17 beta-oestradiol and dexamethasone. Gi-3 alpha- and G beta-subunits were the only G protein subunits which increased in response to 10(-6) M oestradiol (to approximately 150 and 200% of controls, respectively), while the other alpha-subunits investigated (Gs alpha, Gi-2 alpha and G(o) alpha) remained relatively unchanged. Thyroliberin (TRH)--and guanosine 5'-[beta gamma-imido]trisphosphate (Gpp(NH)p)-elicited adenylyl cyclase (AC) activities were reduced during 6-12 h of oestradiol treatment (by 60 and 20%, respectively), while the inhibitory effect of somatostatin (SRIF) increased by approximately 100%. Dexamethasone (10(-6) M) increased levels of the stimulatory G protein Gs alpha (to approximately 340%) and decreased levels of Gi-3 alpha (to 25%). After 48 h, the AC response to TRH was reduced by approximately 70%, whereas the effect of the other modulators remained close to controls. We conclude that G protein subunits in GH3 cells are subject to specific regulation by steroid hormones and that this may be important in the tuning of the responsiveness of PRL secretion to hormones in the in vivo situation.

'Cross-talk' between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells.

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the alpha-subunits of the stimulatory and inhibitory G-proteins of AC (Gs alpha and G(i)-2 alpha) in cultured prolactin-producing rat pituitary adenoma cells (GH3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6-48 h) with ionomycin (1 microM) or 1-oleoyl-2-acetylglycerol (OAG; 1 microM) showed that ionomycin regulated Gs alpha levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered Gs alpha levels by more than 50% at all time-points. G(i)-2 alpha levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane Gs protein alpha-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of 'cross-talk' between the PLC- and AC-dependent signalling pathways.

Hypothalamic hormones modulate G protein levels and second messenger responsiveness in GH3 rat pituitary tumour cells.

Thyroliberin (TRH), vasoactive intestinal peptide (VIP) and somatostatin (SRIF) act through receptors that are coupled to guanine nucleotide-binding regulatory proteins (G proteins). Regulation of hormone action may occur at the level of G protein coupling to the receptor or effector systems. In this study we demonstrate that prolonged exposure (for up to 48 hr) of cultured rat pituitary adenoma GH3 cells to these hormones caused homologous and to some extent heterologous attenuation of the adenylyl cyclase (AC) (EC 4.6.1.1) responsiveness. In addition, TRH and SRIF diminished both TRH- and guanosine 5'-[beta gamma-imido]-triphosphate-enhanced phospholipase C (PLC) (EC 3.1.4.3) activity within the same time-course. Measurements of cells membrane levels of Gs protein alpha-subunit (Gs alpha), G(i)-1 alpha/G(i)-2 alpha, G(i)-3 alpha, G(o) alpha and G beta by immunoblotting were performed. TRH and VIP upregulated levels of all G proteins except G(o) alpha and G beta. In contrast, SRIF caused a marked reduction of G beta levels. Thus, TRH and VIP, both acting through Gs, both modulated the alpha-subunit levels of this signal transducer, whereas SRIF, which possibly acts through G(i)-2, did not change the steady state level of G(i)-2 alpha. The actions of TRH, VIP and SRIF are multifaceted at the G protein level, where modulations of subtypes not directly involved in their actions may occur. These findings emphasize the complexity expected to be found in the in vivo situation.

The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein.

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.

Regulation of G protein mRNA levels by thyroliberin, vasoactive intestinal peptide and somatostatin in prolactin-producing rat pituitary adenoma cells.

We have investigated the regulation of mRNA levels of alpha- and beta-subunits of guanine nucleotide-binding regulatory proteins (G proteins) by peptide hormones in prolactin producing rat pituitary adenoma cells (GH3 cells) in culture. The cells were treated with thyroliberin (1 microM), vasoactive intestinal peptide (1 microM) or somatostatin (10 microM) for 6 to 48 hours. Thyroliberin and vasoactive intestinal peptide increased the levels of Gs alpha Go alpha, Gi-2 alpha, Gi-3 alpha, Gx alpha, G beta 36 and mRNAs. The effect of vasoactive intestinal peptide was however earlier and more pronounced. Gi-2 alpha mRNA levels showed the quantitatively largest alterations. Somatostatin upregulated Gs alpha and downregulated Go alpha and Gi-2 mRNAs. G protein mRNAs for Gi-2 alpha and Go alpha were increased by exposure of the cells to a medium devoid of serum. We conclude that G protein mRNA levels are subjected to alterations by hormones that act through the corresponding G proteins in the regulation of prolactin synthesis and secretion.

Cell-specific expression of guanine nucleotide-binding proteins in rat testicular cells.

Guanine nucleotide-binding proteins (G proteins) are key components in membrane signal transduction that may play an important role in testis function. The present study is the first description of cell-specific differences in the contents of G protein alpha-subunits and their mRNAs in isolated rat testicular cells (pachytene spermatocytes, round spermatids, Sertoli cells, peritubular cells). By using Western blot techniques G1-3 alpha was shown to be the only pertussis toxin (PTX) substrate present in all the testicular cells examined. Surprisingly, we observed a lack of immunoreactive Gi-1 alpha/Gi-2 alpha protein in pachytene spermatocytes and round spermatids in spite of significant levels of the corresponding mRNAs as revealed by Northern analysis. No immunoreactive Gs alpha was detected in germ cells, in agreement with previous findings that the hormone-sensitive adenylyl cyclase is absent in these cell types. Peritubular cells and Sertoli cells contained no Go alpha, whereas high levels of both immunoreactive protein and mRNA were found in pachytene spermatocytes. This indicates that the Go protein may play a role at this stage of spermatogenesis. The stimulation of phospholipase C (PLC) in germ cell membranes by 5'-guanylyl imidophosphate indicates that PTX-sensitive PLC activation may be mediated by Go alpha or Gi-3 alpha.

Signal transduction alterations in GH(1)2C1 rat pituitary tumour cells following treatment with 5-azacytidine.

In GH(1)2C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of G alpha s RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of G alpha i and/or G alpha o. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH(1)2C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of G alpha i/G alpha s protein levels.