Molecular approaches to examine the phosphorylation state of the C type natriuretic peptide receptor.

The intracellular domain of the C type natriuretic peptide receptor (NPRC) contains one threonine and several serine residues where phosphorylation is thought to occur. Several phosphorylation consensus sequences for various kinases have been identified within the intracellular domain of NPRC, but the exact residues that are phosphorylated and the specific kinases responsible for their phosphorylation have not been thoroughly defined. Here we introduce a recombinant GST fusion protein and a rat gastric mucosa (RGM1) cell line as molecular tools to study the phosphorylation state of NPRC in vitro and in vivo, respectively. We utilize a previously characterized polyclonal antibody against NPRC to probe for total NPRC protein and various phosphospecific and substrate motif antibodies to probe for phosphorylation of NPRC. Phosphoprotein staining reagents were used with a phosphoprotein control set to detect phosphorylation of NPRC at serine and threonine residues. Recombinant GST-NPRC fusion protein was phosphorylated in vitro by RGM1 lysate in the presence of adenosine-5'-triphosphate (ATP). Western blot analysis using a monoclonal phospho-Thr antibody, which exclusively detects phosphorylated threonine residues, and does not cross-react with phosphorylated serine residues revealed NPRC immunoprecipitated from RGM1 lysate is phosphorylated on a threonine residue. Global analysis of the entire rat NPRC sequence using a protein kinase A (PKA) prediction algorithm, identified five putative PKA phosphorylation sites containing a serine residue and one containing a threonine residue, Thr 505. Taken together, the data presented here suggest that rat NPRC is a substrate for PKA and Thr 505 located within the intracellular domain of NPRC is a likely candidate site for the phosphorylation.

Muscle-specific knockout of PKC-lambda impairs glucose transport and induces metabolic and diabetic syndromes.

Obesity, the metabolic syndrome, and type 2 diabetes mellitus (T2DM) are major global health problems. Insulin resistance is frequently present in these disorders, but the causes and effects of such resistance are unknown. Here, we generated mice with muscle-specific knockout of the major murine atypical PKC (aPKC), PKC-lambda, a postulated mediator for insulin-stimulated glucose transport. Glucose transport and translocation of glucose transporter 4 (GLUT4) to the plasma membrane were diminished in muscles of both homozygous and heterozygous PKC-lambda knockout mice and were accompanied by systemic insulin resistance; impaired glucose tolerance or diabetes; islet beta cell hyperplasia; abdominal adiposity; hepatosteatosis; elevated serum triglycerides, FFAs, and LDL-cholesterol; and diminished HDL-cholesterol. In contrast to the defective activation of muscle aPKC, insulin signaling and actions were intact in muscle, liver, and adipocytes. These findings demonstrate the importance of aPKC in insulin-stimulated glucose transport in muscles of intact mice and show that insulin resistance and resultant hyperinsulinemia owing to a specific defect in muscle aPKC is sufficient to induce abdominal obesity and other lipid abnormalities of the metabolic syndrome and T2DM. These findings are particularly relevant because humans who have obesity, impaired glucose tolerance, and T2DM reportedly have defective activation and/or diminished levels of muscle aPKC.

The C type natriuretic peptide receptor tethers AHNAK1 at the plasma membrane to potentiate arachidonic acid-induced calcium mobilization.

Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C gamma1 (PLCgamma1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLCgamma1 at the plasma membrane. Here, we identify the C-type natriuretic peptide receptor (NPR-C), an atypical G protein-coupled receptor, as a protein binding partner for AHNAK1 in various cell types. Mass spectrometry and MASCOT analysis of excised bands from NPR-C immunoprecipitation studies revealed multiple signature peptides corresponding to AHNAK1. Glutathione S-transferase (GST) pulldown assays using GST- AHNAK1 fusion proteins corresponding to each of the distinct domains of AHNAK1 showed the C1 domain of AHNAK1 associates with NPR-C. The role of NPR-C in mediating AA-dependent AHNAK1 calcium signaling was explored in various cell types, including 3T3-L1 preadipocytes during the early stages of differentiation. Sucrose density gradient centrifugation studies showed AHNAK1 resides in the nucleus, cytoplasm, and at the plasma membrane, but small interfering RNA (siRNA)-mediated knockdown of NPR-C resulted in AHNAK1 accumulation in the nucleus. Overexpression of a portion of AHNAK1 resulted in augmentation of intracellular calcium mobilization, whereas siRNA-mediated knockdown of NPR-C or AHNAK1 protein resulted in attenuation of intracellular calcium mobilization in response to phorbol 12-myristate 13-acetate. We characterize the novel association between AHNAK1 and NPR-C and provide evidence that this association potentiates the AA-induced mobilization of intracellular calcium. We address the role of intracellular calcium in the various cell types that AHNAK1 and NPR-C were found to associate.

C-type natriuretic peptide receptor expression in pancreatic alpha cells.

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 +/- 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4-23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.

B-type natriuretic peptide decreases gastric emptying and absorption.

Natriuretic peptides have been shown to decrease contractility of isolated gastric smooth muscle cells. However there is a paucity of research showing whether this effect has functional significance in the whole animal. The objective of this study was to test whether intravenously administered B-type Natriuretic Peptide (BNP) has an effect on gastric emptying and/or absorption in a whole animal mouse model. C57BL/6-Wild-type (WT) and Natriuretic Peptide Receptor type A (NPR-A) knockout (KO) mice were used in these studies. Gastric contractility was examined in anesthetized mice before and after BNP vs. vehicle injection. Gastric emptying of gavage fed 70 Kilo Dalton (kDa) FITC-dextran and absorption of 4 kDa FITC-dextran were compared in BNP vs. vehicle treated conscious WT and KO mice. BNP decreased gastric contractility (measured in change in intragastric pressure) from 2.26 +/- 0.29 to 1.44 +/- 0.11 mmHg (P < 0.05), pressure returned to 2.08 +/- 0.17 after 5 BNP half-lives (P < 0.05). There was no significant change in the vehicle or KO. BNP also decreased gastric emptying in WT mice compared to vehicle, 87.8 +/- 0.8% vs. 97.3 +/- 1.04% (P < 0.05) and this effect showed a dose-response relationship. In KO mice emptying was 95.8 +/- 0.5% (BNP) vs. 91.7 +/- 0.7% (Vehicle) (P > 0.05). The absorption in WT mice was 28.2 +/- 7.8 (relative fluorescence units) for BNP vs. 91 +/- 25.9 for vehicle (P < 0.05). For KO mice absorption was 64.3 +/- 14.9 for BNP vs. 60.6 +/- 17.4 for vehicle (P > 0.05). The results show that BNP decreases intragastric pressure, emptying and absorption by acting via the NPR-A receptor. We postulate that this effect is aimed at decreasing preload through decreased water and electrolyte absorption from the GI tract and may also be responsible for the symptoms of impaired gastrointestinal function observed in heart failure patients.

Four cardiac hormones eliminate 4-fold more human glioblastoma cells than the green mamba snake peptide.

UNLABELLED: Within 24h four cardiac hormones, i.e., vessel dilator, kaliuretic peptide, atrial natriuretic peptide, and long acting natriuretic peptide decrease the number of human glioblastoma cells 75%, 68%, 67%, and 65% while Dendroaspis (green mamba) peptide caused a 17% decrease when each were utilized at 100 microM. The four cardiac hormones decreased DNA synthesis 65-87% and increased cyclic GMP 1.3- to 3.8-fold in the glioblastoma cells. Natriuretic peptide receptors (NPR)-A and -C were present. CONCLUSION: four cardiac hormones eliminate up to 75% of glioblastoma cells via cyclic GMP-mediated up to 87% decrease in DNA synthesis.

Four cardiac hormones cause cell death of melanoma cells and inhibit their DNA synthesis.

BACKGROUND: There will be an estimated 59,940 new cases of melanoma and 8,110 deaths from melanoma in the United States in 2007. There has been no improvement in survival with melanomas in the last 22 years, with current treatment indicating that new treatment(s) of melanoma are drastically needed. Four cardiac hormones ie, atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide, have significant anticancer effects in adenocarcinomas. METHODS: Dose-response curves evaluated the effects of these cardiovascular hormones on cell death and DNA synthesis in several melanoma cell lines in culture for 96 hours. Receptors to mediate these peptide hormones effects were examined in the melanoma cells with Western blots. Their intracellular mediator-analog 8-bromo-cyclic GMP was used to determine if it could mimic their effects on decreasing melanoma cell number and DNA synthesis. RESULTS: The four cardiac hormones caused cell death in up to 71% (P < 0.001) of the melanoma cells within 24 hours. Cardiac hormone receptors (NPR-A, -B, -C) were present on the melanoma cells, and each of the peptide hormones decreased DNA synthesis within the melanoma cells up to 73% (P < 0.001) at their 1-microM concentrations. 8-Bromo-cyclic GMP mimicked their effects, decreasing the number of melanoma cells up to 67% and their DNA synthesis by 58% (both at P < 0.01). CONCLUSIONS: These results indicate that 4 cardiac hormones have potent beneficial effects by increasing cell death in up to 71% of melanoma cells within 24 hours mediated in part by a 73% decrease in their DNA synthesis.

Four cardiac hormones eliminate up to two-thirds of human breast cancers in athymic mice.

BACKGROUND: Four cardiac hormones i.e. atrial natriuretic peptide (ANP), vessel dilator, long acting natriuretic peptide (LANP) and kaliuretic peptide have anticancer effects in vitro. MATERIALS AND METHODS: These four cardiac hormones were infused subcutaneously for 28 days with weekly fresh hormones at 3 nM min(-1) kg(-1) body weight in athymic mice bearing human breast adenocarcinomas. RESULTS: Vessel dilator, LANP, kaliuretic peptide and ANP eliminated 67%, 50%, 67% and 33% of the HTB-132 human breast adenocarcinomas. LANP eliminated 100% and vessel dilator 1/3 of CRL-2327 breast adenocarcinomas. There was no recurrence of the breast cancers in the primary site and no metastasis except in the ANP-treated group in one year post-treatment. The natriuretic peptide receptors-A and -C were decreased 50% and 31%, respectively, in metastatic versus primary ANP-treated breast adenocarcinomas. CONCLUSION: Four cardiac hormones eliminate up to two-thirds of human breast adenocarcinomas in athymic mice.

Elimination of up to 80% of human pancreatic adenocarcinomas in athymic mice by cardiac hormones.

BACKGROUND: Four cardiac hormones have anticancer effects in vitro: i) atrial natriuretic peptide (ANP), ii) vessel dilator, iii) long acting natriuretic peptide (LANP), and iv) kaliuretic peptide. MATERIALS AND METHODS: These cardiac hormones were infused subcutaneously for 28 days with weekly fresh hormones at 3 nM min(-1) kg(-1) body weight in athymic mice bearing human pancreatic adenocarcinomas. RESULTS: ANP, vessel dilator, LANP and kaliuretic peptide eliminated 80%, 33%, 20% and 14% of the pancreatic adenocarcinomas. Even in the treated animals which did not have a total cure, their tumor volume decreased to less than 10% (and with vessel dilator to 2%) of that of the untreated animals. The natriuretic peptide receptor (NPR)-A receptor was decreased 33% to 55% in the metastatic lesions compared to the primary pancreatic adenocarcinoma. CONCLUSION: Four cardiac hormones eliminated up to 80% of human pancreatic adenocarcinomas in athymic mice.

Vessel dilator: most potent of the atrial natriuretic peptides in decreasing the number and DNA synthesis of human squamous lung cancer cells.

Within 24 h four peptide hormones, i.e. vessel dilator, long acting natriuretic peptide, kaliuretic peptide, and atrial natriuretic peptide and their intracellular mediator cyclic GMP decreased the number of human squamous lung cancer cells 51, 22, 25, 21, and 30%, respectively. There was not any proliferation in the 3 days following this decrease in cell number. Vessel dilator decreased DNA synthesis 85% in the squamous lung cancer cells. Thus, vessel dilator significantly decreased the number of human squamous lung cancer cells and their DNA synthesis, mediated in part by cyclic GMP, more than other peptide hormones.

Immunocytochemical localization of atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide in human pancreatic adenocarcinomas.

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo.

Four peptides decrease human colon adenocarcinoma cell number and DNA synthesis via cyclic GMP.

BACKGROUND: Mortality from colon cancer is significant with an expected 30,350 colon cancer deaths in 2005 with current treatment(s). Long-acting natriuretic peptide, vessel dilator, kaliuretic peptide, and atrial natriuretic peptide have significant anticancer effects in breast and pancreatic adenocarcinomas. AIM OF STUDY: Whether these peptide hormones have anticancer effects in colon adenocarcinoma cells and whether these effects are specifically mediated by cyclic GMP has not been determined. METHODS: These peptide hormones were evaluated for anticancer effects in human colon adenocarcinoma cells and to determine whether their anticancer effects are specifically mediated by cyclic GMP. RESULTS: There was a 89-97% decrease (p <0.001 for each) in colon adenocarcinoma cells within 24 h with 1 mM of these peptide hormones. There was a significant (p <0.05) decrease in human colon cancer cell number with each 10-fold increase in concentration from 1 to 1,000 microM (i.e., 1 mM) of these four peptide hormones without any proliferation in the 3 d following this decrease. These same hormones decreased DNA synthesis 65-83% (p <0.001). Cyclic GMP antibody inhibited 75- 80% of these peptides' ability to decrease colon adenocarcinoma cell number and inhibited 92-96% of their DNA synthesis effects and 97% of cyclic GMP's effects. Western blots revealed that for the first time natriuretic peptide receptors (NPR) A and C were present in colon adenocarcinoma cells. CONCLUSIONS: Four peptide hormones eliminate up to 97% of colon cancer cells within 24 h with their DNA effects specifically mediated by cyclic GMP.

Effects of cardiac hormones on arterial pressure and sodium excretion in NPRA knockout mice.

These studies were designed to determine if the atria contains natriuretic substances that act through a non-natriuretic peptide type A (NPRA) receptor mechanism. C57BL/6 mice, either wild-type NPRA++ (WT) or NPRA-- knockout (KO), were anesthetized with pentobarbital. Catheters were placed in the trachea, carotid artery, jugular vein, and bladder. Urine was collected for six 30-min periods. Both groups received an iv injection of 100 ng of rat atrial natriuretic peptide (rANP) in 200 microl of saline after the first period (30 mins) and 200 microl of rat atrial extract after the fourth period (120 mins). ANP injection increased urine flow (UF) to 2.7 +/- 0.5 microl/min in the WT versus 1.9 +/- 0.2 in KO. Extract increased UF to 7.9 +/- 1.5 microl/min in WT versus 2.7 +/- 0.4 in KO (P < 0.01). ANP increased sodium excretion (ENa) to 0.47 +/- 0.10 micromoles/min in WT versus 0.27 +/- 0.04 in KO (P < 0.05). Extract increased ENa to 1.44 +/- 0.47 micromoles/min in WT versus 0.26 +/- 0.06 in KO (P < 0.05). Extract decreased mean arterial pressure (MAP) to 62 +/- 3 mm Hg in the WT versus 81 +/- 5 in KO (P < 0.01). ENa and MAP responses to extract in KO were not different from responses to 200 microl of saline. A constant 150-min infusion of rat atrial extract increased urine flow by 3-fold and ENa by 5-fold (both P < 0.05) in the WT mice but had no significant effect in the KO mice. Thus, acute renal and MAP responses to atrial extracts require the NPRA receptor.

Regulation of Kupffer cell TNF gene expression during experimental acute pancreatitis: the role of p38-MAPK, ERK1/2, SAPK/JNK, and NF-kappaB.

We have demonstrated that Kupffer cell-derived tumor necrosis factor (TNF) mediates pancreatitis-associated liver injury. The aim of this study was to determine the role of p38 mitogen-activated protein kinase (MAPK), extracellular stress-related kinase 1/2 (ERK1/2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and nuclear factor-kappaB (NF-kappaB) in TNF gene expression within Kupffer cells. TNF and TNF-mRNA were measured in rat livers perfused with elastase. TNF, TNF-mRNA, NF-kappaB activation, and phosphorylated p38-MAPK, SAPK/JNK, and ERK1/2 were determined in Kupffer cells treated with elastase. Elastase increased TNF and upregulated TNF-mRNA in livers (P<0.03) and Kupffer cells (P<0.001). Phosphorylated p38-MAPK, SAPK/JNK, and ERK1/2 and activated NF-kappaB were detected in Kupffer cells at 7 minutes; at 60 minutes, TNF-mRNA peaked and NF-kappaB returned to baseline, whereas all three kinases remained activated. Gadolinium inhibited elastase-induced upregulation of TNF-mRNA (P < 0.001), TNF production (P<0.001), and attenuated SAPK/JNK, as well as ERK1/2, but not p38-MAPK. Both UO126 and SB203580 significantly inhibited elastase-induced upregulation of TNF-mRNA and TNF production (P<0.001), but only UO126 inhibited activation of NF-kappaB. It was concluded that pretranscriptional regulation of TNF gene expression in Kupffer cells follows an orderly activation of p38-MAPK, ERK1/2, and SAPK/JNK that may not converge on NF-kappaB. The seemingly limited duration of NF-kappaB activation may be important in "switching off" the cytokine cascade during acute pancreatitis.

Kupffer cell-derived Fas ligand plays a role in liver injury and hepatocyte death.

Liver injury is an important prognostic indicator during acute pancreatitis. The aim of this study was to determine the role of Fas ligand (FasL) in hepatocyte injury. Liver parenchymal enzymes were measured in cocultures of hepatocytes and Kupffer cells treated with elastase. FasL and FasL mRNA were measured in elastase-treated Kupffer cells. Hepatocytes were treated with FasL and their viability was assessed by monotetrazolium (MTT), apoptosis by flow cytometry, as well as caspase-3 and p38-mitogen-activated protein kinase (MAPK) by immunoblotting. Elastase increased aspartate aminotransferase and lactate dehydrogenase in cocultures of hepatocyte and Kupffer cells (P<0.040). Elastase increased FasL production from Kupffer cells (P=0.02) and upregulated FasL mRNA (FasL/beta-2 microglobulin (BMG): 0.23+/-0.03 vs. 0.11+/-0.003; P=0.04). FasL increased alanine aminotransferase and lactate dehydrogenase (P<0.03) and reduced hepatocyte viability by 45% (P=0.01). FasL increased the number of dually labeled cells with AnnexinV/7AAD (P=0.03) while upregulating cleavage of caspase-3 and the phosphorylation of p38-MAPK. FasL antibody attenuated the FasL-related increase in dually labeled cells (P=0.02), the cleavage of caspase-3, and phosphorylation of p38-MAPK. Pancreatic elastase upregulates FasL within Kupffer cells. FasL induces hepatocyte injury and death and upregulates p38-MAPK and caspase-3 within hepatocytes. The ability to manipulate interactions between Kupffer cells and hepatocytes may have important therapeutic implications.

Heart failure mice exhibit decreased gastric emptying and intestinal absorption.

Our recent study showed that intravenously administered B-type natriuretic peptide (BNP) decreases gastric emptying and intestinal absorption in mice. We aimed to test whether acute myocardial injury and heart failure have similar effects. Wild-type (WT) and natriuretic peptide receptor type A (NPR-A) knockout (KO) mice underwent cryoinfarction (myocardial infarction [MI]) of the left ventricle (LV) versus sham. LV dysfunction was confirmed by echocardiography. Percent gastric emptying and intestinal absorption were measured and analyzed one and two weeks after infarction, by gavage feeding the mice with fluorescein-isothiocyanate-dextran. Ejection fraction was 48 ± 3% versus 64 ± 2% (P < 0.05) and fractional shortening was 24 ± 2% versus 35 ± 2% (P < 0.01), MI versus sham, respectively. BNP levels (pg/mL) were 4292 ± 276 one week after MI versus 105 ± 11 in sham (n = 5, P < 0.05) and 1964 ± 755 two weeks after MI (n = 5, P < 0.05). Gastric emptying was significantly decreased, 68 ± 6% in MI versus 89 ± 3% in sham (n = 5, P < 0.05) one week after MI and 82 ± 0.5% versus 98 ± 0.4%, MI versus sham (n = 5, P < 0.05), two weeks post-MI. Absorption, measured in relative plasma fluorescence units in WT mice, was 350 ± 79 in MI versus 632 ± 121 in sham (n = 6, P < 0.05). KO mice did not show a significant difference in emptying or absorption compared with sham. These findings suggest that MI and LV dysfunction decrease gastric emptying and absorption in mice through a mechanism that involves NPR-A.

Roux-en-Y gastric bypass alters tumor necrosis factor-α but not adiponectin signaling in immediate postoperative period in obese rats.

BACKGROUND: Adiponectin has anti-inflammatory properties and is increased with weight loss. Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that negatively regulates adiponectin. Previously, we have demonstrated that Roux-en-Y gastric bypass (RYGB) induces weight loss and improves steatosis in obese rats. We hypothesized that RYGB would alter the interplay of TNF-α and adiponectin signaling in the postoperative period. METHODS: Obese Sprague-Dawley male rats that had undergone RYGB (n = 5) or sham (n = 4) were euthanatized at 9 weeks postoperatively. The adiponectin levels from serial serum samples were measured by enzyme-linked immunosorbent assay. Adiponectin, adiponectin receptor 2, and TNF-α mRNA from adipose and liver samples were quantified by reverse transcriptase-polymerase chain reaction. Data are presented as mean ± standard deviation; using a t test, P <.05 was significant. RESULTS: RYGB did not change the serum adiponectin, adipose tissue adiponectin mRNA, or hepatic adiponectin receptor 2 levels compared with the levels in the sham-operated rats (P >.05). However, the TNF-α mRNA levels had decreased in the adipose tissue (P >.05) but remained unchanged in the liver compared with the sham controls (P >.05). CONCLUSION: Surgically-induced weight loss in a rat model of RYGB did not increase adiponectin signaling in the immediate postoperative period but was associated with decreased pro-inflammatory signaling in the adipose tissue. During this period, pro-inflammatory signaling might play a more important role than adiponectin. Additional studies with longer follow-up are necessary to determine whether adiponectin plays a role in weight loss and improvement of steatosis after RYGB.

Does LKB1 mediate activation of hepatic AMP-protein kinase (AMPK) and sirtuin1 (SIRT1) after Roux-en-Y gastric bypass in obese rats?

INTRODUCTION: Roux-en-Y gastric bypass (RYGB) improves steatosis and reduces liver triglycerides in obese rats. Sirtuin1 (SIRT1) and AMP-activated protein kinase (AMPK) are key metabolic regulators that reduce lipogenesis and increase fatty acid oxidation. LKB1 phosphorylates AMPK and may activate SIRT1. We hypothesize that RYGB in obese rats is associated with an upregulation of the LKB1-AMPK-SIRT1 signaling pathway. METHODS: Obese Sprague-Dawley male rats underwent RYGB or sham. Liver tissue was obtained at 9 weeks postoperatively. Protein levels of SIRT1, LKB1, p-LKB1, AMPKalpha, p-AMPKalpha, and p-protein kinase C-zeta (PKC-zeta ) were determined. Protein associations of LKB1 with each of SIRT1, AMPKalpha, and PKC-zeta were determined by coimmunoprecipitation.Data are mean +/- SD; for t test, p<0.05 was significant. RESULTS: RYGB increased protein levels of hepatic AMPKalpha, p-AMPKalpha, and SIRT1 (all p<0.001 vs. sham); p-LKB1 but not LKB1 increased after RYGB (p<0.001 vs. sham). Physical interactions of LKB1-AMPK and LKB1-SIRT1 increased after RYGB (p<0.001 vs. sham). Although PKC-zeta mRNA and p-PKC-zeta did not change, interactions between LKB1 and PKC-zeta increased after RYGB (p<0.001 vs. sham). CONCLUSION: RYGB increases hepatic levels of SIRT1, AMPK, and p-AMPK as well as increasing interactions of LKB1 with AMPK or SIRT1. p-PKC-zeta may play an intermediary role in the interaction between AMPK and SIRT. These findings demonstrate key signaling changes in powerful metabolic regulators that may account for the resolution of steatosis after RYGB.

Identification, regulation and anti-proliferative role of the NPR-C receptor in gastric epithelial cells.

Evidence suggests that functional atrial natriuretic peptide (ANP) receptors occur in surface gastric mucosal epithelial cells. To evaluate functional aspects of ANP in a model of these cells we examined the expression of natriuretic peptide receptors (NPR) subtypes A and C in the non-transformed rat gastric mucosal epithelial cell line RGM1. Transcripts for NPR-A and NPR-C were detected in RGM1 cells by RT-PCR. However, only NPR-C protein was detected by Western blot and immunohistochemical analyses. Specific saturable binding of (125)I-ANP to RGM1 cells revealed a single class of high affinity binding sites (K (d) = 208 +/- 71pM, B (max) = 110,000 +/- 14,000 sites/cell, Hill coefficient = 0.97 +/- 0.05). ANP (IC(50) 130 +/- 47pM), BNP (IC(50) 716 +/- 26 pM), CNP (IC(50) 356 +/- 85pM) and C-ANP (IC(50) 134 +/- 13pM), a specific ligand for NPR-C, effectively displaced (125)I-ANP binding. Cross-linking of (125)I-ANP to cells labeled predominantly a protein of 66,000 Da. These data suggest that (125)I-ANP binding was primarily to NPR-C. ANP and C-ANP inhibited forskolin- and prostaglandin E(2) (PGE(2))-stimulated cAMP in a PTx-sensitive fashion. PGE(2), transforming growth factor-+/-1 (TGF-+/-1), forskolin, 8-bromo-cyclic AMP, and phorbol-12-myristate-13-acetate (PMA) caused a dose-dependent decrease in specific (125)I-ANP binding, whereas epidermal growth factor (EGF), 8-bromo-cyclic GMP and 4+/--phorbol didecanoate had no effect. PGE(2), forskolin, TGF-+/-1 and PMA significantly decreased (125)I-ANP B (max) values, NPR-C protein and steady-state NPR-C transcript levels. H89, a protein kinase A inhibitor, blocked the reduction of NPR-C mRNA produced by both forskolin and PGE(2.) GF109203X, a protein kinase C inhibitor, abolished the PMA-induced decrease in NPR-C transcripts but only partially blocked that produced by TGF-+/-1. RGM1 cells exhibited a dose-dependent decrease in both DNA synthesis and cell proliferation when cultured in the presence of ANP or C-ANP. These findings indicate that RGM1 cells express functional NPR-C receptors that can influence RGM1 cell proliferation and are down-regulated by PGE(2) and TGF-+/-1.

Four cardiac hormones eliminate up to 82% of human medullary thyroid carcinoma cells within 24 hours.

Four cardiac hormones, i.e., atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide, which have anticancer effects, were evaluated for the first time on any endocrine cancer to determine if they have anticancer effects in an endocrine cancer. These four cardiac hormones were evaluated for their anticancer, DNA synthesis, and receptor status in human medullary thyroid cancer cells. There was a significant (p < 0.001) decrease in human medullary thyroid cancer cells with each 10-fold increase from 1 to 100 microM of the four cardiac hormones. There was an 81%, 68%, 71%, and 66% elimination within 24 h of medullary thyroid cancer cells secondary to vessel dilator, kaliuretic peptide, atrial natriuretic peptide, and long-acting natriuretic peptide, respectively (p < 0.0001). Three days after treatment with these peptide hormones, there was no proliferation of the medullary thyroid cancer cells. These cardiac hormones decreased DNA synthesis in the medullary thyroid cells from 65% to 84% (p < 0.0001). Western blots revealed natriuretic peptide receptors-A and -C were present in human medullary thyroid cancer cells. These results indicate the four cardiac hormones have potent anticancer effects by eliminating up to 82% of human medullary thyroid carcinoma cells within 24 h of treatment.