P2Y13 receptor deficiency favors adipose tissue lipolysis and worsens insulin resistance and fatty liver disease.

Excessive lipolysis in white adipose tissue (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In humans, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocyte lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicated that mice lacking P2Y13-R showed a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared with their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R-knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.

Plasma Level of ATPase Inhibitory Factor 1 and Intrinsic Capacity in Community-Dwelling Older Adults: Prospective Data From the MAPT Study.

BACKGROUND: Intrinsic capacity (IC) is a concept related to functionality that reflects healthy aging. ATPase inhibitory factor 1 (IF1) is a multifaceted protein that regulates mitochondrial oxidative phosphorylation (OXPHOS), and may be involved in IC. The objective of this study is to investigate the association between plasma levels of IF1 and IC changes in community-dwelling older adults. METHODS: Community-dwelling older adults from the Multidomain Alzheimer Preventive Trial (MAPT Study) were enrolled in this study. A composite IC score was calculated based on 4 IC domains: locomotion, psychological dimension, cognition, and vitality (with data available annually over 4 years of follow-up). Secondary analyses were conducted on the sensory domain (with data available only for 1 year of follow-up). Mixed-model linear regression adjusted for confounders was conducted. RESULTS: A total of 1 090 participants with usable IF1 values were included in the study (75.3 ± 4.4 years; 64% females). Compared to the lowest quartile, both the low- and high-intermediate IF1 quartiles were found to be cross-sectionally associated with greater composite IC scores across 4 domains (ßlow-intermediate, 1.33; 95% confidence interval [CI] 0.06-2.60 and ßhigh-intermediate, 1.78; 95% CI 0.49-3.06). In the secondary analyses, the highest quartile was found to be associated with a slower decline in composite IC scores across 5 domains over 1 year (ßhigh 1.60; 95% CI 0.06-3.15). The low- and high-intermediate IF1 quartiles were also found to be cross-sectionally associated with greater locomotion (ßlow-intermediate, 2.72; 95% CI 0.36-5.08) and vitality scores (ßhigh-intermediate, 1.59; 95% CI 0.06-3.12), respectively. CONCLUSIONS: This study is the first to demonstrate that levels of circulating IF1, a mitochondrial-related biomarker, are associated with IC composite scores in both cross-sectional and prospective analyses among community-dwelling older adults. However, further research is needed to confirm these findings and elucidate the potential underlying mechanisms that may explain these associations.

Intermittent Fasting Resolves Dyslipidemia and Atherogenesis in Apolipoprotein E-Deficient Mice in a Diet-Dependent Manner, Irrespective of Sex.

In humans and animal models, intermittent fasting (IF) interventions promote body weight loss, improve metabolic health, and are thought to lower cardiovascular disease risk. However, there is a paucity of reports on the relevance of such nutritional interventions in the context of dyslipidemia and atherosclerotic cardiovascular diseases. The present study assessed the metabolic and atheroprotective effects of intermittent fasting intervention (IF) in atherosclerosis-prone apolipoprotein E-deficient (Apoe-/-) mice. Groups of male and female Apoe-/- mice were fed a regular (chow) or atherogenic (high-fat, high-cholesterol, HFCD) diet for 4 months, either ad libitum or in an alternate-day fasting manner. The results show that IF intervention improved glucose and lipid metabolism independently of sex. However, IF only decreased body weight gain in males fed chow diet and differentially modulated adipose tissue parameters and liver steatosis in a diet composition-dependent manner. Finally, IF prevented spontaneous aortic atherosclerotic lesion formation in mice fed chow diet, irrespective of sex, but failed to reduce HFCD-diet-induced atherosclerosis. Overall, the current work indicates that IF interventions can efficiently improve glucose homeostasis and treat atherogenic dyslipidemia, but a degree of caution is warranted with regard to the individual sex and the composition of the dietary regimen.

The Multifaceted ATPase Inhibitory Factor 1 (IF1) in Energy Metabolism Reprogramming and Mitochondrial Dysfunction: A New Player in Age-Associated Disorders?

Significance: The mitochondrial oxidative phosphorylation (OXPHOS) system, comprising the electron transport chain and ATP synthase, generates membrane potential, drives ATP synthesis, governs energy metabolism, and maintains redox balance. OXPHOS dysfunction is associated with a plethora of diseases ranging from rare inherited disorders to common conditions, including diabetes, cancer, neurodegenerative diseases, as well as aging. There has been great interest in studying regulators of OXPHOS. Among these, ATPase inhibitory factor 1 (IF1) is an endogenous inhibitor of ATP synthase that has long been thought to avoid the consumption of cellular ATP when ATP synthase acts as an ATP hydrolysis enzyme. Recent Advances: Recent data indicate that IF1 inhibits ATP synthesis and is involved in a multitude of mitochondrial-related functions, such as mitochondrial quality control, energy metabolism, redox balance, and cell fate. IF1 also inhibits the ATPase activity of cell-surface ATP synthase, and it is used as a cardiovascular disease biomarker. Critical Issues: Although recent data have led to a paradigm shift regarding IF1 functions, these have been poorly studied in entire organisms and in different organs. The understanding of the cellular biology of IF1 is, therefore, still limited. The aim of this review was to provide an overview of the current understanding of the role of IF1 in mitochondrial functions, health, and diseases. Future Directions: Further investigations of IF1 functions at the cell, organ, and whole-organism levels and in different pathophysiological conditions will help decipher the controversies surrounding its involvement in mitochondrial function and could unveil therapeutic strategies in human pathology. Antioxid. Redox Signal. 37, 370-393.

Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis.

BACKGROUND: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and 2 lysosomal proteins, the NPC1 (Niemann-Pick C1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. METHODS: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells. To establish whether Wnt5a also protects against cholesterol accumulation in human vascular smooth muscle cells, we used a CRISPR/Cas9 guided nuclease approach to generate human vascular smooth muscle cells knockout for Wnt5a. RESULTS: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 (mechanistic target of rapamycin complex 1) kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum. Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the endoplasmic reticulum, and promoted atherosclerosis. CONCLUSIONS: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

A reference measurement of circulating ATPase inhibitory factor 1 (IF1) in humans by LC-MS/MS: Comparison with conventional ELISA.

ATPase inhibitory factor 1 (IF1) is a 9.5 kDa protein that binds to mitochondrial and plasma membrane ATP synthase and selectively inhibits ATP hydrolysis. Recently, IF1 was identified in systemic circulation in humans. IF1 appeared as an independent determinant of HDL-cholesterol with lower levels in coronary heart disease (CHD) patients. Moreover, IF1 was also found to negatively associate with mortality in these patients, supporting the notion that circulating IF1 could be a promising biomarker of cardiovascular disease. However, in previous studies, IF1 was quantified by a non-standardized competitive enzyme-linked immunosorbent assay (ELISA). Herein, we have validated a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) enabling the accurate quantification of IF1 in human plasma. Plasma IF1 was trypsin-digested through an optimized procedure before LC-MS/MS analysis. The method was successfully validated over 4 independent experiments into the range of 100-1500 ng/mL. Intra- and inter-assay variation coefficients had never exceeded 14.2% and accuracy ranged between 95% and 102% for the selected EAGGAFGK peptide marker. Subsequently, the results of the LC-MS/MS method were compared with those obtained using ELISA in 204 individuals from the GENES study. We found that IF1 plasma levels obtained using both techniques were strongly correlated (r = 0.89, p < 0.0001), while the Bland-Altman plot did not indicate any major statistically significant differences. To clinically validate LC-MS/MS, we confirmed the positive correlation between IF1 plasma levels and HDL-cholesterol (r = 0.38, p < 0.0001). Besides, we found lower IF1 plasma levels in CHD patients compared to controls (431 ± 132 ng/mL and 555 ± 173 ng/mL, respectively; p < 0.0001). Hence, it can be concluded that the presented LC-MS/MS analytical method provides a highly specific strategy for IF1 quantification in human plasma and could be proposed as a reference method.

Serum level of HDL particles are independently associated with long-term prognosis in patients with coronary artery disease: The GENES study.

HDL-Cholesterol (HDL-C) is not an accurate surrogate marker to measure the cardioprotective functions of HDL in coronary artery diseases (CAD) patients. Hence, measurement of other HDL-related parameters may have prognostic superiority over HDL-C. In this work, we examined the predictive value of HDL particles profile for long-term mortality in CAD patients and to compare its informative value to that of HDL-C and apoA-I. HDL particles profiles were measured by nuclear magnetic resonance (NMR) spectroscopy in 214 male participants with stable CAD (45-74 years). Median follow up was 12.5 years with a 36.4% mortality rate. Cardiovascular mortality accounted for 64.5%. Mean concentrations of total HDL particles (HDL-P), small-sized HDL (SHDL-P) and apoA-I were lower in deceased than in surviving patients whereas no difference was observed according to HDL-C and large HDL particles. All NMR-HDL measures were correlated between themselves and with other HDL markers (HDL-C, apoA-I and LpA-I). In a multivariate model adjusted for cardiovascular risk factors and bioclinical variables, HDL-P and SHDL-P displayed the strongest inverse association with all-cause and cardiovascular mortality. Weaker associations were recorded for apoA-I. Based on our results, we conclude that HDL particle profile measured by NMR spectroscopy should be considered to better stratify risk in population at high risk or in the setting of pharmacotherapy.

Paclitaxel induces lymphatic endothelial cells autophagy to promote metastasis.

Cytotoxic therapy for breast cancer inhibits the growth of primary tumors, but promotes metastasis to the sentinel lymph nodes through the lymphatic system. However, the effect of first-line chemotherapy on the lymphatic endothelium has been poorly investigated. In this study, we determined that paclitaxel, the anti-cancer drug approved for the treatment of metastatic or locally advanced breast cancer, induces lymphatic endothelial cell (LEC) autophagy to increase metastases. While paclitaxel treatment was largely efficacious in inhibiting LEC adhesion, it had no effect on cell survival. Paclitaxel inhibited LEC migration and branch point formation by inducing an autophagy mechanism independent of Akt phosphorylation. In vivo, paclitaxel mediated a higher permeability of lymphatic endothelium to tumor cells and this effect was reversed by chloroquine, an autophagy-lysosome inhibitor. Despite a strong effect on reducing tumor size, paclitaxel significantly increased metastasis to the sentinel lymph nodes. This effect was restricted to a lymphatic dissemination, as chemotherapy did not affect the blood endothelium. Taken together, our findings suggest that the lymphatic system resists to chemotherapy through an autophagy mechanism to promote malignant progression and metastatic lesions. This study paves the way for new combinative therapies aimed at reducing the number of metastases.

Liraglutide improves hepatic steatosis and metabolic dysfunctions in a 3-week dietary mouse model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an emerging health problem worldwide. However, efficacious pharmacological treatment for NASH is lacking. A major issue for preclinical evaluation of potential therapeutics for NASH is the limited number of appropriate animal models, i.e., models that do not require long-term dietary intervention and adequately mimic disease progression in humans. The present study aimed to evaluate a 3-wk dietary mouse model of NASH and validate it by studying the effects of liraglutide, a compound in advanced clinical development for NASH. C57BL6/J mice were fed a diet high in fat (60%), cholesterol (1.25%), and cholic acid (0.5%), along with 2% hydroxypropyl-ß-cyclodextrin in drinking water (HFCC-CDX diet). Histological and biological parameters were measured at 1 and 3 wk. After 1-wk diet induction, liraglutide was administrated daily for 2 wk and then NASH-associated phenotypic aspects were evaluated in comparison with control mice. Prior to treatment with liraglutide, mice fed the HFCC-CDX diet for 1 wk developed liver steatosis and had increased levels of oxidative-stress markers and hepatic and systemic inflammation. For mice not treated with liraglutide, these aspects were even more pronounced after 3 wk of the dietary period, with additional liver insulin resistance and fibrosis. Liraglutide treatment corrected the diet-induced alterations in glucose metabolism and significantly reduced hepatic steatosis and inflammation. This study provides a novel 3-wk dietary model of mice that rapidly develop NASH features, and this model will be suitable for evaluating the therapeutic efficacy of compounds in preclinical drug development for NASH.NEW & NOTEWORTHY We propose a diet high in fat (60%), cholesterol (1.25%), and cholic acid (0.5%) along with 2% hydroxypropyl-ß-cyclodextrin in drinking water (HFCC-CDX diet) as a new dietary model of nonalcoholic steatohepatitis. We used the HFCC-CDX model to reproduce the main features of disease development in humans for the purpose of facilitating the rapid screening of drug candidates and prioritizing the more promising candidates for advanced preclinical assessment and subsequent clinical trials.

Characterization of immortalized human dermal microvascular endothelial cells (HMEC-1) for the study of HDL functionality.

BACKGROUND: Primary cultures endothelial cells have been used as models of endothelial related diseases such atherosclerosis. Biological behavior of primary cultures is donor-dependent and data could not be easily reproducible; endothelial cell lines are emerging options, particularly, human dermal microvascular endothelial cells (HMEC-1), that should be validated to substitute primary cultures for the study of HDL functions. METHODS: Morphology, size and granularity of cells were assessed by phase contrast microscopy and flow cytometry of HMEC-1. The adhesion molecules, ICAM-1and VCAM-1 after TNF-α stimulation, and endothelial markers CD105 endoglin, as well as HDL receptor SR-BI were determined by flow cytometry. Internalization of HDL protein was demonstrated by confocal microscopy using HDL labeled with Alexa Fluor 488. HUVECs were used as reference to compared the characteristics with HMEC-1. RESULTS: HMEC-1 and HUVEC had similar morphologies, size and granularity. HMEC-1 expressed endothelial markers as HUVECs, as well as functional SR-B1 receptor since the cell line was able to internalize HDL particles. HMEC-1 effectively increased ICAM-1 and VCAM-1 expression after TNF-α stimulation. HUVECs showed more sensibility to TNF-α stimulus but the range of ICAM-1 and VCAM-1 expression was less homogeneous than in HMEC-1, probably due to biological variation of the former. Finally, the expression of adhesion molecules in HMEC-1 was attenuated by co-incubation with HDL. CONCLUSION: HMEC-1 possess characteristics of endothelial cells, similar to HUVECs, being a cell line suitable to evaluate the functionality of HDL vis-à-vis the endothelium.

Ecto-F1-ATPase/P2Y pathways in metabolic and vascular functions of high density lipoproteins.

The atheroprotective property of High Density Lipoprotein (HDL) is supported by many epidemiological studies and cellular and in vivo approaches on animal models. While the anti-atherogenic effects of HDL are thought to derive primarily from its role in reverse cholesterol transport, together with anti-inflammatory, anti-oxidant, anti-thrombotic and cytoprotective properties, the mechanisms that support these effects are still not completely understood. However, many advances in identifying the cellular partners involved in HDL functions have been made over the last two decades. This review highlights the diverse roles of the HDL receptor ecto-F1-ATPase coupled to purinergic P2Y receptors in the modulation of important metabolic and vascular functions of HDL. On hepatocytes, the ecto-F1-ATPase is coupled to P2Y13 receptor and contributes to HDL holoparticle endocytosis. On endothelial cells, ecto-F1-ATPase/P2Ys pathway is involved in HDL-mediated endothelial protection and HDL transcytosis. The clinical relevance of this F1-ATPase/P2Ys axis in humans has recently been supported by the identification of serum F1-ATPase inhibitor (IF1) as an independent determinant of HDL-Cholesterol (HDL-C) and coronary heart disease risk. Therapeutic strategies targeting F1-ATPase/P2Y pathways for the treatment of atherosclerosis are currently being explored.

Identification of the F1-ATPase at the cell surface of colonic epithelial cells: role in mediating cell proliferation.

F1 domain of F(1)F(o)-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F(1)-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F(1)-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F(1)-ATPase and the amino acids in the F(1)-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F(1)-ATPase and the loss of its biological activity. In addition we demonstrated that F(1)-ATPase mediates the growth effects of the peptide. Indeed, blocking F(1)-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F(1)-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F(1)-ATPase in the pro-proliferative action of this gastrointestinal peptide.

A switch of G protein-coupled receptor binding preference from phosphoinositide 3-kinase (PI3K)-p85 to filamin A negatively controls the PI3K pathway.

Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K.

Leptin receptor activation increases Sam68 tyrosine phosphorylation and expression in human trophoblastic cells.

Leptin is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy, promoting antiapoptotic and trophic effects. Leptin receptor is present in trophoblastic cells and leptin may fully activate signaling. We have previously implicated the RNA-binding protein Sam68 in leptin signal transduction in immune cells. In the present work, we have studied the possible role of Sam68 in leptin receptor signaling in trophoblastic cells (JEG-3 cells). Leptin dose-dependently stimulated Sam68 phosphorylation in JEG-3 cells, as assessed by immunoprecipitation and immunoblot with anti-phosphotyrosine antibodies. As previously observed in other systems, tyrosine phosphorylation of Sam68 in response to leptin inhibits its RNA binding capacity. Besides, leptin stimulation dose-dependently increases Sam68 expression in JEG-3 cells, as assessed by quantitative PCR. Consistently, the amount of Sam68 protein is increased after 24h of leptin stimulation of trophoblastic cells. In order to study the possible role of Sam68 on leptin receptor synthesis, we employed antisense strategy to knockdown the expression of Sam68. We have found that a decrease in Sam68 expression leads to a decrease in leptin receptor amount in JEG-3 cells, as assessed both by quantitative PCR and immunoblot. These results strongly suggest the participation of Sam68 in leptin receptor signaling in human trophoblastic cells, and therefore, Sam68 may mediate some of the leptin effects in placenta.

Reprint of: Metabolic effects and mechanism of action of the chromogranin A-derived peptide pancreastatin.

Pancreastatin is one of the regulatory peptides derived from intracellular and/or extracellular processing of chromogranin A, the soluble acidic protein present in the secretory granules of the neuroendocrine system. While the intracellular functions of chromogranin A include formation and maturation of the secretory granule, the major extracellular functions are generation of biologically active peptides with demonstrated autocrine, paracrine or endocrine activities. In this review, we will focus on the metabolic function of one of these peptides, pancreastatin, and the mechanisms underlying its effects. Many different reported effects have implicated PST in the modulation of energy metabolism, with a general counterregulatory effect to that of insulin. Pancreastatin induces glycogenolysis in liver and lipolysis in adipocytes. Metabolic effects have been confirmed in humans. Moreover, naturally occurring human variants have been found, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptide's potency to inhibit cellular glucose uptake. Thus, qualitative hereditary alterations in pancreastatin's primary structure may give rise to interindividual differences in glucose and lipid metabolism. Pancreastatin activates a receptor signaling system that belongs to the seven-spanning transmembrane receptor coupled to a Gq-PLCß-calcium-PKC signaling pathway. Increased pancreastatin plasma levels, correlating with catecholamines levels, have been found in insulin resistance states, such as gestational diabetes or essential hypertension. Pancreastatin plays important physiological role in potentiating the metabolic effects of catecholamines, and may also play a pathophysiological role in insulin resistance states with increased sympathetic activity.

Role of leptin in the activation of immune cells.

Adipose tissue is an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that may be present in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Leptin is one of the most important hormones secreted by adipocytes, with a variety of physiological roles related to the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response, innate and adaptive response, both in normal and pathological conditions. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response.

Metabolic effects and mechanism of action of the chromogranin A-derived peptide pancreastatin.

Pancreastatin is one of the regulatory peptides derived from intracellular and/or extracellular processing of chromogranin A, the soluble acidic protein present in the secretory granules of the neuroendocrine system. While the intracellular functions of chromogranin A include formation and maturation of the secretory granule, the major extracellular functions are generation of biologically active peptides with demonstrated autocrine, paracrine or endocrine activities. In this review, we will focus on the metabolic function of one of these peptides, pancreastatin, and the mechanisms underlying its effects. Many different reported effects have implicated PST in the modulation of energy metabolism, with a general counterregulatory effect to that of insulin. Pancreastatin induces glycogenolysis in liver and lipolysis in adipocytes. Metabolic effects have been confirmed in humans. Moreover, naturally occurring human variants have been found, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptide's potency to inhibit cellular glucose uptake. Thus, qualitative hereditary alterations in pancreastatin's primary structure may give rise to interindividual differences in glucose and lipid metabolism. Pancreastatin activates a receptor signaling system that belongs to the seven-spanning transmembrane receptor coupled to a Gq-PLCbeta-calcium-PKC signaling pathway. Increased pancreastatin plasma levels, correlating with catecholamines levels, have been found in insulin resistance states, such as gestational diabetes or essential hypertension. Pancreastatin plays important physiological role in potentiating the metabolic effects of catecholamines, and may also play a pathophysiological role in insulin resistance states with increased sympathetic activity.

A gastrin precursor, gastrin-gly, upregulates VEGF expression in colonic epithelial cells through an HIF-1-independent mechanism.

One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.

Antitumor effects of somatostatin.

Since its discovery three decades ago as an inhibitor of GH release from the pituitary gland, somatostatin has attracted much attention because of its functional role in the regulation of a wide variety of physiological functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. In addition to its negative role in the control of endocrine and exocrine secretions, somatostatin and analogs also exert inhibitory effects on the proliferation and survival of normal and tumor cells. Over the past 15 years, studies have begun to reveal some of the molecular mechanisms underlying the antitumor activity of somatostatin. This review covers the present knowledge in the antitumor effect of somatostatin and analogs and discusses the perspectives of novel clinical strategies based on somatostatin receptor sst2 gene transfer therapy.

Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation.

Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.