Biphasic Effect of Sildenafil on Energy Sensing is Mediated by Phosphodiesterases 2 and 3 in Adipocytes and Hepatocytes.

Sirt1 (Sirtuin 1), AMPK (AMP-activated protein kinase), and eNOS (endothelial nitric oxide synthase) modulate hepatic energy metabolism and inflammation and play a major role in the development of NASH. Cyclic nucleotide phosphodiesterases (PDEs) play an important role in signal transduction by modulating intracellular levels of cyclic nucleotides. We previously found the PDE5 inhibitor sildenafil to synergize with leucine and leucine-metformin combinations in preclinical studies of NASH and obesity. However, efficacy is diminished at higher sildenafil concentrations. Herein, we have successfully modeled the U-shaped sildenafil dose-response in vitro and utilized this model to assess potential mechanisms of this dose-response relationship. Adipocytes and liver cells were treated with leucine (0.5 mM) and different concentrations of sildenafil (1 nM to 100 µM). cAMP, cGMP, and P-AMPK protein expression were used to demonstrate the biphasic response for increasing concentrations of sildenafil. The reversal with higher sildenafil levels was blunted by PDE2 inhibition. These data indicate that sildenafil-mediated increases in cGMP inhibits PDE3 at lower concentrations, which increases cAMP. However, further increases in cGMP from higher sildenafil concentrations activate PDE2 and consequently decrease cAMP, which demonstrates crosstalk between cAMP and cGMP via PDE2, PDE3, and PDE5. These changes in cAMP concentration are further reflected in downstream effects, including AMPK activation.

Erratum: Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice.

[This corrects the article on p. 33 in vol. 7, PMID: 28533928.].

Leucine-nicotinic acid synergy stimulates AMPK/Sirt1 signaling and regulates lipid metabolism and lifespan in Caenorhabditis elegans, and hyperlipidemia and atherosclerosis in mice.

BACKGROUND/AIMS: Nicotinic acid (NA), a lipid-lowering drug, serves as a source of NAD+, the cofactor for Sirt1. Leucine (Leu) stimulates the AMPK/Sirt1 axis and amplifies the effects of other AMPK/Sirt1 activating compounds. Therefore, we tested the interactive effects of leucine and low dose NA on AMPK/Sirt1 signaling and downstream effects of lipid metabolism in cell culture, C. elegans and mice. METHODS: LDL-receptor knockout mice were fed an atherogenic Western diet supplemented with leucine (24 g/kg diet) and sub-therapeutic NA combinations (50 mg/kg diet and 250 mg/kg diet) or low therapeutic NA (1000 mg/kg diet) for 8 weeks to evaluate markers of hyperlipidemia and atherosclerosis. RESULTS: NA-Leu increased P-AMPK and Sirt1 in adipocytes and myotubes. In C. elegans, NA-Leu increased P-AMPK and DAF-16 (FOXO), reduced lipid accumulation and increased median survival under mild oxidative stress conditions. In the mice, NA-Leu reduced total cholesterol, cholesterol esters, plasma triglycerides, atherosclerotic lesion size, lipid area, and aortic macrophage infiltration, similar to the therapeutic NA dose. CONCLUSION: Leu amplifies the effects of NA on lipid metabolism, hyperlipidemia and atherosclerosis in mice, at least in part by activation of the AMPK/Sirt1 axis. This combination may be a potential therapeutic alternative for hyperlipidemia and atherosclerosis.

Activation of the AMPK/Sirt1 pathway by a leucine-metformin combination increases insulin sensitivity in skeletal muscle, and stimulates glucose and lipid metabolism and increases life span in Caenorhabditis elegans.

BACKGROUND: We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for NAD+, thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction. METHODS: Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met-Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. RESULTS: Leu (0.5mmol/L)+Met (50-100µmol/L) synergistically activated Sirt1 (p<0.001) at low (≤100µmol/L) NAD+ levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sirt inhibition or silencing. Met-Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (~50%, p<0.002) evident within 30 min as well as a 60% reduction in insulin EC50. In addition, in HepG2 liver cells nuclear CREB regulated transcription coactivator 2 (CRTC2) protein expression and phosphorylation of glycogen synthase was decreased, while glycogen synthase kinase phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by ~25%, while addition of Leu+Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p=0.023), restored normal glucose utilization and increased fat oxidation ~two-fold (p<0.005), while metformin exerted no independent effect at any concentration (0.1-0.5mmol/L). CONCLUSION: Thus, Leu and Met synergize to enable Sirt1 activation at low NAD+ concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met-Leu's full action, which result in improvements in energy metabolism and insulin sensitivity.

Prevention of pancreatic cancer in a hamster model by cAMP decrease.

Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). We have previously shown that these cancers overexpressed stress neurotransmitters and cyclic adenosine monophosphate (cAMP) while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) was suppressed. Using a hamster model, the current study has tested the hypothesis that cAMP decrease by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cAMP and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers and phosphorylated signaling proteins, which stimulate cell proliferation, and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP decrease by GABA supplementation inhibits multiple cancer stimulating pathways in cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism.

A Combination of Leucine, Metformin, and Sildenafil Treats Nonalcoholic Fatty Liver Disease and Steatohepatitis in Mice.

Sirt1, AMPK, and eNOS modulate hepatic energy metabolism and inflammation and are key players in the development of NASH. L-leucine, an allosteric Sirt1 activator, synergizes with low doses of metformin or sildenafil on the AMPK-eNOS-Sirt1 pathway to reverse mild NAFLD in preclinical mouse models. Here we tested a possible multicomponent synergy to yield greater therapeutic efficacy in NAFLD/NASH. Liver cells and macrophages or an atherogenic diet induced NASH mouse model was treated with two-way and three-way combinations. The three-way combination Sild-Met-Leu increased hepatic fatty acid oxidation and reduced lipogenic gene expression and inflammatory marker in vitro. In mice, Sild-Met-Leu reduced the diet induced increases of ALT, TGFß, PAI-1, IL1ß, and TNFα, hepatic collagen expression, and nearly completely reversed hepatocyte ballooning and triglyceride accumulation, while all two-way combinations had only modest effects. Therefore, these data provide preclinical evidence for therapeutic efficacy of Sild-Met-Leu in the treatment of NAFLD and NASH.

Nicotine induces self-renewal of pancreatic cancer stem cells via neurotransmitter-driven activation of sonic hedgehog signalling.

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

Regulation of nonsmall-cell lung cancer stem cell like cells by neurotransmitters and opioid peptides.

Nonsmall-cell lung cancer (NSCLC) is the leading type of lung cancer and has a poor prognosis. We have shown that chronic stress promoted NSCLC xenografts in mice via stress neurotransmitter-activated cAMP signaling downstream of beta-adrenergic receptors and incidental beta-blocker therapy was reported to improve clinical outcomes in NSCLC patients. These findings suggest that psychological stress promotes NSCLC whereas pharmacologically or psychologically induced decreases in cAMP may inhibit NSCLC. Cancer stem cells are thought to drive the development, progression and resistance to therapy of NSCLC. However, their potential regulation by stress neurotransmitters has not been investigated. In the current study, epinephrine increased the number of cancer stem cell like cells (CSCs) from three NSCLC cell lines in spheroid formation assays while enhancing intracellular cAMP and the stem cell markers sonic hedgehog (SHH), aldehyde dehydrogenase-1 (ALDH-1) and Gli1, effects reversed by GABA or dynorphin B via Gαi -mediated inhibition of cAMP formation. The growth of NSCLC xenografts in a mouse model of stress reduction was significantly reduced as compared with mice maintained under standard conditions. Stress reduction reduced serum levels of corticosterone, norepinephrine and epinephrine while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and opioid peptides increased. Stress reduction significantly reduced cAMP, VEGF, p-ERK, p-AKT, p-CREB, p-SRc, SHH, ALDH-1 and Gli1 in xenograft tissues whereas cleaved caspase-3 and p53 were induced. We conclude that stress neurotransmitters activate CSCs in NSCLC via multiple cAMP-mediated pathways and that pharmacologically or psychologically induced decreases in cAMP signaling may improve clinical outcomes in NSCLC patients.

Differential modulation of nicotine-induced gemcitabine resistance by GABA receptor agonists in pancreatic cancer cell xenografts and in vitro.

BACKGROUND: Pancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. Based on our report that low dose nicotine within the range of NRT causes gemcitabine resistance in pancreatic cancer, our current study has tested the hypothesis that GABA or the selective GABA-B-R agonist baclofen used to treat alcohol dependence reverse nicotine-induced gemcitabine resistance in pancreatic cancer. METHODS: Using mouse xenografts from the gemcitabine--sensitive pancreatic cancer cell line BXPC-3, we tested the effects of GABA and baclofen on nicotine-induced gemcitabine resistance. The levels of cAMP, p-SRC, p-ERK, p-AKT, p-CREB and cleaved caspase-3 in xenograft tissues were determined by ELISA assays. Expression of the two GABA-B receptors, metalloproteinase-2 and 9 and EGR-1 in xenograft tissues was monitored by Western blotting. Mechanistic studies were conducted in vitro, using cell lines BXPC-3 and PANC-1 and included analyses of cAMP production by ELISA assay and Western blots to determine protein expression of GABA-B receptors, metalloproteinase-2 and 9 and EGR-1. RESULTS: Our data show that GABA was as effective as gemcitabine and significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. These effects of GABA were accompanied by decreases in cAMP, p-CREB, p-AKT, p-Src, p-ERK metalloproteinases-9 and -2 and EGR-1 and increases in cleaved caspase-3 in xenografts whereas baclofen had the opposite effects. In vitro exposure of cells to single doses or seven days of nicotine induced the protein expression of MMP-2, MMP-9 and EGR-1 and these responses were blocked by GABA. Baclofen downregulated the protein expression of GABA-B-Rs in xenograft tissues and in cells exposed to baclofen for seven days in vitro. This response was accompanied by inversed baclofen effects from inhibition of cAMP formation after single dose exposures to stimulation of cAMP formation in cells pretreated for seven days. CONCLUSIONS: These findings suggest GABA as a promising single agent for the therapy of pancreatic cancer and to overcome nicotine-induced gemcitabine resistance whereas treatment with baclofen may increase gemcitabine resistance.

A linguistically informed autosomal STR survey of human populations residing in the greater Himalayan region.

The greater Himalayan region demarcates two of the most prominent linguistic phyla in Asia: Tibeto-Burman and Indo-European. Previous genetic surveys, mainly using Y-chromosome polymorphisms and/or mitochondrial DNA polymorphisms suggested a substantially reduced geneflow between populations belonging to these two phyla. These studies, however, have mainly focussed on populations residing far to the north and/or south of this mountain range, and have not been able to study geneflow patterns within the greater Himalayan region itself. We now report a detailed, linguistically informed, genetic survey of Tibeto-Burman and Indo-European speakers from the Himalayan countries Nepal and Bhutan based on autosomal microsatellite markers and compare these populations with surrounding regions. The genetic differentiation between populations within the Himalayas seems to be much higher than between populations in the neighbouring countries. We also observe a remarkable genetic differentiation between the Tibeto-Burman speaking populations on the one hand and Indo-European speaking populations on the other, suggesting that language and geography have played an equally large role in defining the genetic composition of present-day populations within the Himalayas.

Chronic nicotine inhibits the therapeutic effects of gemcitabine on pancreatic cancer in vitro and in mouse xenografts.

AIM OF STUDY: Smoking is an established risk factor for pancreatic cancer and nicotine replacement therapy (NRT) often accompanies chemotherapy. The current study has tested the hypothesis that chronic exposure to low dose nicotine reduces the responsiveness of pancreatic cancer to the leading therapeutic for this cancer, gemcitabine. METHODS: The effects of chronic nicotine (1 µm/L) on two pancreatic cancer cell lines in vitro and in a xenograft model were assessed by immunoassays, Western blots and cell proliferation assays. RESULTS: Exposure in vitro to nicotine for 7 days inhibited the gemcitabine-induced reduction in viable cells, gemcitabine-induced apoptosis as indicated by reduced expression of cleaved caspase-3 while inducing the phosphorylation of signalling proteins extracellular signal-regulated kinase (ERK), v-akt thymoma viral oncogene homolog (protein kinase B, AKT) and Src. Nicotine (1 µm/L) in the drinking water for 4 weeks significantly reduced the therapeutic response of mouse xenografts to gemcitabine while reducing the induction of cleaved caspase-3 and the inhibition of phosphorylated forms of multiple signalling proteins by gemcitabine in xenograft tissues. CONCLUSIONS: Our experimental data suggest that continued moderate smoking and NRT may negatively impact therapeutic outcomes of gemcitabine on pancreatic cancer and that clinical studies in cancer patients are now warranted.

Role for stromal heterogeneity in prostate tumorigenesis.

Prostate cancer develops through a stochastic mechanism whereby precancerous lesions on occasion progress to multifocal adenocarcinoma. Analysis of human benign and cancer prostate tissues revealed heterogeneous loss of TGF-ß signaling in the cancer-associated stromal fibroblastic cell compartment. To test the hypothesis that prostate cancer progression is dependent on the heterogeneous TGF-ß responsive microenvironment, a tissue recombination experiment was designed in which the ratio of TGF-ß responsive and nonresponsive stromal cells was varied. Although 100% TGF-ß responsive stromal cells supported benign prostate growth and 100% TGF-ß nonresponsive stromal cells resulted in precancerous lesions, only the mixture of TGF-ß responsive and nonresponsive stromal cells resulted in adenocarcinoma. A computational model was used to resolve a mechanism of tumorigenic progression in which proliferation and invasion occur in two independent steps mediated by distinct stromally derived paracrine signals produced by TGF-ß nonresponsive and responsive stromal cells. Complex spatial relationships of stromal and epithelial cells were incorporated into the model on the basis of experimental data. Informed by incorporation of experimentally derived spatial parameters for complex stromal-epithelial relationships, the computational model indicated ranges for the relative production of paracrine factors by each cell type and provided bounds for the diffusive range of the molecules. Because SDF-1 satisfied model predictions for an invasion-promoting paracrine factor, a more focused computational model was subsequently used to investigate whether SDF-1 was the invasion signal. Simulations replicating SDF-1 expression data revealed the requirement for cooperative SDF-1 expression, a prediction supported biologically by heterotypic stromal interleukin-1ß signaling between fibroblastic cell populations. The cancer stromal field effect supports a functional role for the unaltered fibroblasts as a cooperative mediator of cancer progression.

Molecular insights into the origins of the Shompen, a declining population of the Nicobar archipelago.

The Shompen, one of the most isolated and poorly understood contemporary hunter-gatherer populations, inhabit Great Nicobar Island, the southernmost island of the Nicobar archipelago. Morphological imprints in the Shompen were interpreted to favour a mixed Indo-Chinese, Malay, Negrito and Dravidian origin. Analyses of the mitochondrial, Y-chromosomal and autosomal gene pool of contemporary Shompen have revealed low diversity, illustrating a founder effect in the island population. Mitochondrial sequence analyses revealed the presence of two haplogroups of R lineage: B5a, and a newly defined clade, R12. Y-chromosomal analyses demonstrated the occurrence of a single lineage found predominantly in Austro-Asiatic speakers across Asia. With the different types of genetic markers analysed, the Shompen exhibit varying levels of genetic relatedness with the Nicobarese, and Austro-Asiatic speakers of mainland India and Southeast Asia. These genetic analyses provide evidence that the Shompen, an offshoot of the Nicobarese, are descendants of Mesolithic hunter-gatherers of Southeast Asian origin, deriving from at least two source populations.

A prehistory of Indian Y chromosomes: evaluating demic diffusion scenarios.

Understanding the genetic origins and demographic history of Indian populations is important both for questions concerning the early settlement of Eurasia and more recent events, including the appearance of Indo-Aryan languages and settled agriculture in the subcontinent. Although there is general agreement that Indian caste and tribal populations share a common late Pleistocene maternal ancestry in India, some studies of the Y-chromosome markers have suggested a recent, substantial incursion from Central or West Eurasia. To investigate the origin of paternal lineages of Indian populations, 936 Y chromosomes, representing 32 tribal and 45 caste groups from all four major linguistic groups of India, were analyzed for 38 single-nucleotide polymorphic markers. Phylogeography of the major Y-chromosomal haplogroups in India, genetic distance, and admixture analyses all indicate that the recent external contribution to Dravidian- and Hindi-speaking caste groups has been low. The sharing of some Y-chromosomal haplogroups between Indian and Central Asian populations is most parsimoniously explained by a deep, common ancestry between the two regions, with diffusion of some Indian-specific lineages northward. The Y-chromosomal data consistently suggest a largely South Asian origin for Indian caste communities and therefore argue against any major influx, from regions north and west of India, of people associated either with the development of agriculture or the spread of the Indo-Aryan language family. The dyadic Y-chromosome composition of Tibeto-Burman speakers of India, however, can be attributed to a recent demographic process, which appears to have absorbed and overlain populations who previously spoke Austro-Asiatic languages.

Phylogeny and antiquity of M macrohaplogroup inferred from complete mt DNA sequence of Indian specific lineages.

BACKGROUND: Analysis of human complete mitochondrial DNA sequences has largely contributed to resolve phylogenies and antiquity of different lineages belonging to the majorhaplogroups L, N and M (East-Asian lineages). In the absence of whole mtDNA sequence information of M lineages reported in India that exhibits highest diversity within the sub-continent, the present study was undertaken to provide a detailed analysis of this macrohaplogroup to precisely characterize and unravel the intricate phylogeny of the lineages and to establish the antiquity of M lineages in India. RESULTS: The phylogenetic tree constructed from sequencing information of twenty-four whole mtDNA genome revealed novel substitutions in the previously defined M2a and M6 lineages. The most striking feature of this phylogenetic tree is the recognition of two new lineages, M30 and M31, distinguished by transitions at 12007 and 5319, respectively. M30 comprises of M18 and identifies a potential new sub-lineage possessing substitution at 16223 and 16300. It further branches into M30a sub-lineage, defined by 15431 and 195A substitution. The age of M30 lineage was estimated at 33,042 YBP, indicating a more recent expansion time than M2 (49,686 YBP). The M31 branch encompasses the M6 lineage along with the previously defined M3 and M4 lineages. Contradictory to earlier reports, the M5 lineage does not always include a 12477 substitution, and is more appropriately defined by a transversion at 10986A. The phylogenetic tree also identifies a potential new lineage in the M* branch with HVSI sequence as 16223,16325. Substitutions in M25 were in concordance with previous reports. CONCLUSION: This study describes five new basal mutations and recognizes two new lineages, M30 and M31 that substantially contribute to the present understanding of macrohaplogroup M. These two newly erected lineages include the previously independent lineages M18 and M6 as sub-lineages within them, respectively, suggesting that most mt DNA genomes might arise as limited offshoots of M trunk. Furthermore, this study supports the non existence of lineages such as M3 and M4 that are solely defined on the basis of fast mutating control region motifs and hence, establishes the importance of coding region markers for an accurate understanding of the phylogeny. The deep roots of M phylogeny clearly establish the antiquity of Indian lineages, especially M2, as compared to Ethiopian M1 lineage and hence, support an Asian origin of M majorhaplogroup.