Levels In Peripheral Tissues Research Articles

Frataxin levels in peripheral tissue in Friedreich ataxia.

ObjectiveFriedreich ataxia (FRDA) is an autosomal recessive ataxia resulting from mutations in the frataxin gene (FXN). Such mutations, usually expanded guanine–adenine–adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues. The goal was to understand the relationship of frataxin levels in peripheral tissues to disease status.MethodsFrataxin levels were measured in buccal cells and blood, and analyzed in relation to disease features. Site-directed mutant frataxin was also transfected into human embryonic kidney cells to model results from specific point mutations.ResultsThere was no evidence for change in frataxin levels over time with repeated measures analysis, although linear regression analysis of cross-sectional data predicted a small increase over decades. GAA repeat length predicted frataxin levels in both tissues, and frataxin levels themselves predicted neurological ratings (accounting for age). Compound heterozygous patients for a GAA expansion and a point mutation in FXN generally had lower levels of frataxin than those homozygous for the presence of two GAA repeat expansions, though levels varied dramatically between tissues in some compound heterozygotes for point mutations. The G130V mutation led to decreased levels of frataxin in vitro as well as in vivo, while the R165C mutation produced normal immunoreactive levels of frataxin both in vitro and in vivo. Start codon mutations led to low levels of frataxin in buccal cells but preserved immunoreactive frataxin levels in blood.InterpretationThe present data show that peripheral frataxin levels reflect disease features in FRDA, but emphasize the need for interpretation of such levels in the context of specific mutations.

Plasma BDNF levels associate with Pittsburgh Compound B binding in the brain

BackgroundBrain-derived neurotrophic factor (BDNF) plays an important role in Alzheimer's disease (AD) and other neurodegenerative disorders. BDNF function is adversely affected by amyloid beta in AD. BDNF levels in brain and peripheral tissues are lower in patients with AD and mild cognitive impairment (MCI) than in controls. Here we examined the association between plasma levels of BDNF and amyloid deposition in the brain measured with Pittsburgh Compound B (PiB). MethodOur data set consisted of 18 AD, 56 MCI, and 3 normal control Alzheimer's Disease Neuroimaging Initiative-1 (ADNI1) subjects with available [11C] PiB and peripheral blood protein data. Magnetic resonance imaging (MRI)-coregistered positron emission tomography data were smoothed with a 15 mm kernel and mapped onto three-dimensional (3D) hemispheric models using the warping deformations computed in cortical pattern matching of the associated MRI scans. We applied linear regression to examine in 3D the associations between BDNF and PiB standard uptake value ratio, while adjusting for age and sex. We used permutation statistics thresholded at P < .01 for multiple comparisons correction. ResultsPlasma BDNF levels showed significant negative associations with left greater than right amyloid burden in the lateral temporal, inferior parietal, inferior frontal, anterior and posterior cingulate, and orbitofrontal regions (left Pcorrected = .03). ConclusionsAs hypothesized, lower plasma levels of BDNF were significantly associated with widespread brain amyloidosis.

Comparative biochemical characterization of the monoacylglycerol lipase inhibitor KML29 in brain, spinal cord, liver, spleen, fat and muscle tissue

Monoacylglycerol lipase (MAGL) is part of the endocannabinoid and the prostaglandin signaling system. MAGL degrades the endocannabinoid 2-arachidonoylglycerol (2-AG) into glycerol and arachidonic acid. MAGL-induced arachidonic acid is the primary source for prostaglandin synthesis in the brain. 2-AG mainly induces neuroprotective and anti-inflammatory effects, whereas prostaglandins are related to pro-inflammatory effects inducing neurotoxicity. Therefore, inhibition of MAGL represents a promising target for neurological diseases characterized by inflammation. However, as 2-AG is an agonist for the cannabinoid receptor 1 (CB1), inhibition of MAGL might be associated with unwanted cannabimimetic effects. Here, we show that oral administration of KML29, a highly selective inhibitor of MAGL, induced large and dose-dependent changes in 2-AG levels in vivo in brain and spinal cord of mice. Of note, MAGL inhibition by KML29 induced a decrease in prostaglandin levels in brain and most peripheral tissues but not in the spinal cord. MAGL expression was highest in fat, liver and brain, whereas the cytosolic phospholipase A2 (cPLA2), a further enzyme responsible for arachidonic acid production, was highly expressed in spinal cord, muscle and spleen. In addition, high doses (10 mg/kg) of KML29 induced some cannabimimetic effects in vivo in the tetrad test, including hypothermia, analgesia and hypomotility without induction of cataleptic behavior. In summary, inhibition of MAGL by KML29 represents a promising strategy for targeting the cannabinoid and prostaglandin system of the brain with only a moderate induction of cannabimimetic effects.

Hyperhomocysteinemia potentiates hyperglycemia-induced inflammatory monocyte differentiation and atherosclerosis.

Hyperhomocysteinemia (HHcy) is associated with increased diabetic cardiovascular diseases. However, the role of HHcy in atherogenesis associated with hyperglycemia (HG) remains unknown. To examine the role and mechanisms by which HHcy accelerates HG-induced atherosclerosis, we established an atherosclerosis-susceptible HHcy and HG mouse model. HHcy was established in mice deficient in cystathionine β-synthase (Cbs) in which the homocysteine (Hcy) level could be lowered by inducing transgenic human CBS (Tg-hCBS) using Zn supplementation. HG was induced by streptozotocin injection. Atherosclerosis was induced by crossing Tg-hCBS Cbs mice with apolipoprotein E-deficient (ApoE−/−) mice and feeding them a high-fat diet for 2 weeks. We demonstrated that HHcy and HG accelerated atherosclerosis and increased lesion monocytes (MCs) and macrophages (MØs) and further increased inflammatory MC and MØ levels in peripheral tissues. Furthermore, Hcy-lowering reversed circulating mononuclear cells, MC, and inflammatory MC and MC-derived MØ levels. In addition, inflammatory MC correlated positively with plasma Hcy levels and negatively with plasma s-adenosylmethionine–to–s-adenosylhomocysteine ratios. Finally, l-Hcy and d-glucose promoted inflammatory MC differentiation in primary mouse splenocytes, which was reversed by adenoviral DNA methyltransferase-1. HHcy and HG, individually and synergistically, accelerated atherosclerosis and inflammatory MC and MØ differentiation, at least in part, via DNA hypomethylation.

Lipolysis, and not hepatic lipogenesis, is the primary modulator of triglyceride levels in streptozotocin-induced diabetic mice.

Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels. To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin-deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin-induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signaling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial triglycerides clearance, which was associated with decreased lipoprotein lipase and peroxisome proliferator-activated receptor α mRNA levels in peripheral tissues and decreased lipoprotein lipase activity in skeletal muscle, heart, and brown adipose tissue. Diabetic heterozygous lipoprotein lipase knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial triglycerides clearance. Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic triglycerides production and secretion.

The LXR–Idol Axis Differentially Regulates Plasma LDL Levels in Primates and Mice

The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR receptor stability independent of SREBP and PCSK9, but its relevance to plasma lipid levels is unknown. Here we demonstrate that the effects of the LXR-IDOL axis are both tissue and species specific. In mice, LXR agonist induces Idol transcript levels inperipheral tissues but not in liver, and does not change plasma LDL levels. Accordingly, Idol-deficient mice exhibit elevated LDLR protein levels in peripheral tissues, but not in the liver. By contrast, LXR activation in cynomolgus monkeys induces hepatic IDOL expression, reduces LDLR protein levels, and raises plasma LDL levels. Knockdown of IDOL in monkeys with an antisense oligonucleotide blunts the effect of LXR agonist on LDL levels. These results implicate IDOL as a modulator of plasma lipid levels in primates and support further investigation into IDOL inhibition as a potential strategy for LDL lowering in humans.

Effect of Sitagliptin and Glimepiride on Glucose Homeostasis and cAMP Levels in Peripheral Tissues of HFD/STZ Diabetic Rats

Introduction: T2DM is a group of metabolic disorders manifested by hyperglycemia as a result of insulin insufficiency and/or resistance. The main goal of antidiabetic therapies is to lower glucose levels, and therefore prevent development of diabetes complications. DPP-4 inhibitors (e.g. sitagliptin) are relatively new antidiabetic drugs which inhibit the activity of DPP-4 enzyme and therefore prevent rapid degradation of incretin hormones. Objective: We investigated effects of sitagliptin on glucose homeostasis, lipid profile, and insulin signaling by determination of cAMP levels in peripheral tissues of HFD/STZ diabetic rats, compared to glimepiride. Methods: The experimental rats were divided into five groups, each group comprising 10 rats. Group (1) served as the normal control rats and administered DMSO (without treatments) as the vehicle. The rest of the groups were rendered diabetic by feeding HFD containing 40% fats for 4 weeks, followed by a single I.P. injection of STZ (45 mg/kg of body weight). One week after STZ injection, the rats with FBG level of ≥ 200 mg/dl were considered diabetic. Group (2) served as the diabetic untreated rats and administered DMSO (without treatments) as the vehicle. Group (3) served as diabetic rats treated with glimepiride (0.1 mg/kg of body weight). Group (4) and group (5) served as diabetic rats treated with sitagliptin (10 and 30 mg/kg of body weight, respectively). Treatments were dissolved in DMSO and were given orally for 4 weeks. At the end of the treatment period, the blood, liver and adipose tissues (White and brown) were collected for biochemical analysis. Results: In normal control rats, the highest content of cAMP was observed in BAT. Diabetic rats showed an elevation in cAMP levels of liver and WAT to be 1.3 and 3.9 fold control values, respectively, while in BAT, cAMP level decreased to be 0.4 fold control value. Sitagliptin and glimepiride significantly decreased cAMP levels in liver and WAT. Conclusion: We conclude that sitagliptin and glimepiride have comparable effects on glucose homeostasis. Both drugs have cAMP-lowering effect which may suggest their potential protecting effect against vascular complications of diabetes.

Characterization of proopiomelanocortin in the snakeskin gourami (Trichopodus pectoralis) and its expression in relation to food intake

Proopiomelanocortin (POMC) is the precursor of several hormones involved in physiological systems including feed intake. The snakeskin gourami (Trichopodus pectoralis) POMC complementary DNA (TpPOMC) was cloned and characterized. Phylogenetic analysis showed that TpPOMC was clustered in a major POMC lineage in fish. Analysis of the Ka to Ks ratios for the entire POMC sequence and for each hormonal segment suggested that different POMC-derived peptide segments were subject to different evolutionary pressures. High expression level of TpPOMC was observed in all brain regions, with the highest levels in the diencephalon and pituitary gland. In situ hybridization also revealed that TpPOMC-expressing cells were distributed in discrete brain regions. The transcription level of TpPOMC was also found at moderate levels in several peripheral tissues, including gills, liver, head kidney, trunk kidney, stomach, intestine, spleen, ovary and testis, and at a low level in muscle. The expression level of TpPOMC was evaluated in each brain region (telencephalon, mesencephalon, metencephalon, and diencephalon together with the pituitary gland) at 1 h before the first and the last meals of the day and compared with expression levels at a time interval between the first and the last meals of the day. Low expression levels of TpPOMC were found at 1 h before the last meal of the day (P < 0.05). These finding suggest that decreased POMC expression level may lead to reduced melanocyte-stimulating hormones, which may in part be responsible for stimulating food intake. The effect of short-term fasting (24 h) on TpPOMC expression level in each brain region was also investigated. In telencephalon and diencephalon together with the pituitary gland, TpPOMC messenger RNA reached a nadir at 12 h of fasting, whereas TpPOMC transcript showed a nadir at 6 h of fasting in metencephalon and mesencephalon. A peak of TpPOMC level was observed at 18 h of fasting in metencephalon and diencephalon together with the pituitary gland. These findings suggest that TpPOMC expression is affected by nutritional status.

New Lmna knock-in mice provide a molecular mechanism for the ‘segmental aging’ in Hutchinson–Gilford progeria syndrome†

Lamins A and C (products of the LMNA gene) are found in roughly equal amounts in peripheral tissues, but the brain produces mainly lamin C and little lamin A. In HeLa cells and fibroblasts, the expression of prelamin A (the precursor to lamin A) can be reduced by miR-9, but the relevance of those cell culture studies to lamin A regulation in the brain was unclear. To address this issue, we created two new Lmna knock-in alleles, one (Lmna(PLAO-5NT)) with a 5-bp mutation in a predicted miR-9 binding site in prelamin A's 3' UTR, and a second (Lmna(PLAO-UTR)) in which prelamin A's 3' UTR was replaced with lamin C's 3' UTR. Neither allele had significant effects on lamin A levels in peripheral tissues; however, both substantially increased prelamin A transcript levels and lamin A protein levels in the cerebral cortex and the cerebellum. The increase in lamin A expression in the brain was more pronounced with the Lmna(PLAO-UTR) allele than with the Lmna(PLAO-5NT) allele. With both alleles, the increased expression of prelamin A transcripts and lamin A protein was greater in the cerebral cortex than in the cerebellum. Our studies demonstrate the in vivo importance of prelamin A's 3' UTR and its miR-9 binding site in regulating lamin A expression in the brain. The reduced expression of prelamin A in the brain likely explains why children with Hutchinson-Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease.

Abstract 1183: Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside.

Abstract Purpose: DNA adduct formation is a critical step in platinum(Pt)–induced cell death. The goal of this project is to measure carboplatin-DNA adducts after a single non-toxic microdose of [14C]carboplatin, use this information to predict efficacy before cancer patients receive toxic chemotherapy and assess the underlying chemoresistance mechanisms, thus enhancing personalized therapy. Methods: Six non-small cell lung cancer (NSCLC) and six bladder cancer cell lines were treated with 1 μM (microdose) or 100 μM (therapeutic dose) carboplatin, but with equal amounts of [14C]carboplatin. Carboplatin-DNA monoadduct levels were determined by measuring 14C content in genomic DNA with accelerator mass spectrometry (AMS). Cellular sensitivity to carboplatin (IC50 values) was correlated with drug-DNA adduct levels. To validate the in vitro findings, a pilot Phase 0 trial with microdosing [14C]carboplatin was initiated to determine the recommended Phase II dose (RP2D) of [14C]carboplatin for optimal measurement of DNA monoadducts and pharmacokinetics (PK) in lung and bladder cancer. PK and DNA monoadduct levels in peripheral blood mononuclear cells and tumor tissues were determined by liquid scintillation counting and AMS, respectively, validated with inductively coupled plasma mass spectrometry (ICP-MS), and correlated with tumor response to chemotherapy. Results: DNA monoadduct levels in cell lines induced by microdose and therapeutic concentrations of carboplatin were highly linearly proportional (r2 = 0.95, p &amp;lt; 0.001) as measured by AMS, and correlated to the total Pt-DNA adduct levels induced by therapeutic carboplatin (r2 = 0.80, p &amp;lt; 0.001) as measured by ICP-MS. DNA adduct levels were superior in predicting resistance to carboplatin when compared to the mRNA expression of ERCC1, a DNA repair gene commonly used as a resistant biomarker of Pt-based chemotherapy. Nine patients were recruited at the pilot Phase 0 trial. The Level I dose of [14C]carboplatin at 107 dpm/Kg with total carboplatin at 1% of therapeutic dose (area under curve of 6) was determined as the RP2D. No clinical toxicity was observed as a result of microdosing. The radiation exposure from 14C was less than a chest X-ray per patient. DNA monoadduct levels and PK parameters of carboplatin at the microdose and therapeutic doses were highly correlated (p&amp;lt;0.001), suggesting that this non-toxic microdosing approach can be used to predict DNA adduct levels and PK of therapeutic doses. Patients with the highest DNA monoadduct levels responded best to carboplatin-based chemotherapy, although more data is needed to achieve statistical significance in the clinical data. Conclusions: This novel microdosing approach predicts DNA monoadduct levels and PK of therapeutic carboplatin dosing; preliminary data suggest the feasibility of predicting chemosensitivity and the possibility of guiding personalized dosing. Citation Format: Chong-xian Pan, Sisi Wang, Miaoling He, Tao Li, Tzu-yin Lin, Hongyong Zhang, Michael Malfatti, David Gandara, Tianhong Li, Ken Yoneda, Joyce Lee, Marc Dall'Era, George Cimino, Ralph de Vere White, Kenneth Turteltaub, Paul Henderson. Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2013-1183

Host B7x Promotes Pulmonary Metastasis of Breast Cancer

B7x (B7-H4 or B7S1) is an inhibitory member of the B7 family of T cell costimulation. It is expressed in low levels in healthy peripheral tissues, such as the lung epithelium, but is overexpressed in a variety of human cancers with negative clinical associations, including metastasis. However, the function of B7x in the context of cancer, whether expressed on cancer cells or on surrounding "host" tissues, has not been elucidated in vivo. We used the 4T1 metastatic breast cancer model and B7x knockout (B7x (-/-)) mice to investigate the effect of host tissue-expressed B7x on cancer. We found that 4T1 cells were B7x negative in vitro and in vivo, and B7x(-/-) mice had significantly fewer lung 4T1 tumor nodules than did wild-type mice. Furthermore, B7x(-/-) mice showed significantly enhanced survival and a memory response to tumor rechallenge. Mechanistic studies revealed that the presence of B7x correlated with reduced general and tumor-specific T cell cytokine responses, as well as with an increased infiltration of immunosuppressive cells, including tumor-associated neutrophils, macrophages, and regulatory T cells, into tumor-bearing lungs. Importantly, tumor-associated neutrophils strongly bound B7x protein and inhibited the proliferation of both CD4 and CD8 T cells. These results suggest that host B7x may enable metastasizing cancer cells to escape local antitumor immune responses through interactions with the innate and adaptive immune systems. Thus, targeting the B7x pathway holds much promise for improving the efficacy of immunotherapy for metastatic cancer.

The involvement of microRNAs in neurodegenerative diseases

Neurodegenerative diseases (NDDs) originate from a loss of neurons in the central nervous system and are severely debilitating. The incidence of NDDs increases with age, and they are expected to become more common due to extended life expectancy. Because no cure is available, these diseases have become a major challenge in neurobiology. The increasing relevance of microRNAs (miRNAs) in biology has prompted investigation into their possible involvement in neurodegeneration in order to identify new therapeutic targets. The idea of using miRNAs as therapeutic targets is not far from realization, but important issues need to be addressed before moving into the clinics. Here, we review what is known about the involvement of miRNAs in the pathogenesis of NDDs. We also report the miRNA expression levels in peripheral tissues of patients affected by NDDs in order to evaluate their application as biomarkers of disease. Finally, discrepancies, innovations, and the effectiveness of collected data will be elucidated and discussed.

Radiosynthesis and preclinical evaluation of [11C]prucalopride as a potential agonist PET ligand for the 5-HT4 receptor

BackgroundSerotonin 5-HT4 receptor (5-HT4-R) agonists are potential therapeutic agents for enterokinetic and cognitive disorders and are marketed for treatment of constipation. The aim of this study was to develop an agonist positron emission tomography (PET) ligand in order to label the active G-protein coupled 5-HT4-R in peripheral and central tissues. For this purpose prucalopride, a high-affinity selective 5-HT4-R agonist, was selected.Methods[11C]Prucalopride was synthesized from [11C]methyl triflate and desmethyl prucalopride, and its LogDoct,pH7.4 was determined. Three distinct studies were performed with administration of IV [11C]prucalopride in male rats: (1) The biodistribution of radioactivity was measured ex vivo; (2) the kinetics of radioactivity levels in brain regions and peripheral organs was assessed in vivo under baseline conditions and following pre-treatment with tariquidar, a P-glycoprotein efflux pump inhibitor; and (3) in vivo stability of [11C]prucalopride was checked ex vivo in plasma and brain extracts using high-performance liquid chromatography.Results[11C]Prucalopride was synthesized in optimised conditions with a yield of 21% ± 4% (decay corrected) and a radiochemical purity (>99%), its LogDoct,pH7.4 was 0.87. Ex vivo biodistribution studies with [11C]prucalopride in rats showed very low levels of radioactivity in brain (maximal 0.13% ID·g−1) and ten times higher levels in certain peripheral tissues. The PET studies confirmed very low brain levels of radioactivity under baseline conditions; however, it was increased three times after pre-treatment with tariquidar. [11C]Prucalopride was found to be very rapidly metabolised in rats, with no parent compound detectable in plasma and brain extracts at 5 and 30 min following IV administration. Analysis of levels of radioactivity in peripheral tissues revealed a distinct PET signal in the caecum, which was reduced following tariquidar pre-treatment. The latter is in line with the role of the P-glycoprotein pump in the gut.Conclusion[11C]Prucalopride demonstrated low radioactivity levels in rat brain; a combination of reasons may include rapid metabolism in the rat in particular, low passive diffusion and potential P-glycoprotein substrate. In humans, further investigation of [11C]prucalopride for imaging the active state of 5-HT4-R is worthwhile, in view of the therapeutic applications of 5-HT4 agonists for treatment of gastrointestinal motility disorders.

Influence of Pre-analytical Variables on VEGF Gene Expression and Circulating Protein Concentrations

The extended role of vascular endothelial growth factor (VEGF) in human pathophysiology led us to evaluate pre-analytical parameters possibly influencing its levels in peripheral blood and tissues. The effects on VEGF protein levels and mRNA expression were measured after storage delay (blood and tissue), use of different types of anticoagulants (blood), and after different numbers of freeze-thaw cycles (blood). Blood from healthy donors was sampled simultaneously in ethylene diamine tetraacetic acid (EDTA), acid citrate dextrose (ACD-A), hirudin, and serum separation tubes. For each anticoagulant, VEGF was measured by enzyme-linked immunosorbent assay (ELISA) with different conditions of delay at 4°C before centrifugation (2 h, 4 h, or 48 h) and of different numbers of freeze-thaw cycles (1, 2, and 10). The transcripts coding for the VEGF165 isoform were quantified in peripheral blood mononuclear cells by RT-PCR. Muscle biopsy samples were frozen with delays of 15, 30, or 60 min after surgery. VEGF expression was quantified on immunofluorescence stained slides. The period of storage and the number of freeze-thaw cycles correlated with an increase in the levels of circulating VEGF (for each anticoagulant but not for serum) and its expression in PBMCs. VEGF expression measured from muscle biopsy sections was higher with freezing delays, with a peak at 30 and 60 min as compared to 15 min. The most reliable conditions for measuring both circulating VEGF and its gene expression are to reduce time between blood collection and centrifugation, and to avoid multiple freeze-thaw cycles. Serum collection tubes with no additive and no separator were less sensitive to the pre-analytical variations analyzed in this study. Freezing delay had a significant influence on VEGF protein expression in tissue samples.

Short chain fatty acids may elicit an innate immune response from preadipocytes: A potential link between bacterial infection and inflammatory diseases

Short chain fatty acids (SCFAs) such as acetate, propionate and butyrate are produced by bacterial fermentation of dietary fiber. The highest concentrations of SCFAs in the body are found in the colon. Elevated dietary acetate has been shown to have anti-inflammatory effects in mouse models of colitis and inflammatory diseases in peripheral tissues. The details of how dietary SCFAs stimulate reduced inflammation in peripheral tissues have not been determined. I suggest that SCFA concentrations in peripheral tissues are generally not sufficient to locally produce a significant anti-inflammatory effect from immune cells. Moreover it is possible that elevated SCFA levels in peripheral tissues may actually stimulate an inflammatory response. The hypothesis is presented that preadipocytes and other cells with immune function such as fibroblasts in peripheral tissues elicit an inflammatory innate immune response when exposed to SCFAs at millimolar concentrations. A role for SCFAs in activating an immune response in preadipocytes is possible given the expression of a SCFA receptor in these cells, the demonstration that adipocytes and preadipocytes have immunity related functions, the observation that 2 mM SCFAs stimulated the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA from 3T3-L1 preadipocytes and that concentrations of SCFAs can reach elevated levels at sites of bacterial infection. A SCFA-induced inflammatory response from preadipocytes and other cells with immune function, such as fibroblasts, may provide a further contributing factor linking bacterial infection to the development of insulin resistance and the severity of inflammatory diseases such as atherosclerosis.

Prognostic significance of matrix metalloproteinase-1 levels in peripheral plasma and tumour tissues of lung cancer patients

Matrix metalloproteinase-1 (MMP-1) participates in a variety of physiological and pathological processes. We previously found that MMP-1 was one of the lung cancer-related proteins detectable in peripheral blood. To validate our preliminary observations and explore the clinical significance of MMP-1 for lung cancer further, we carried out the present study. The concentrations of MMP-1 in circulating plasma specimens of 170 lung cancer patients and 70 healthy individuals were measured by an enzyme-linked immunosorbance assay. The expression status of the MMP-1 in archival tissue samples from 122 lung cancer patients was examined by immunohistochemical analysis. The correlation between the MMP-1 levels and prognosis of the lung cancer patients was then assessed statistically. Protein levels of MMP-1 were considerably raised in the plasma from lung cancer patients relative to those in healthy controls. The high plasma MMP-1 levels were associated with advanced-stage of the disease and significantly lower overall survival rate of the patients. Coincidently, MMP-1 protein extraordinarily overexpressed in the tumour tissues of lung cancer; and the up-regulated MMP-1 was associated with the progression (including tumour size, staging and lymphatic invasion), especially with decreased survival rate of the patients. Statistic analysis revealed that MMP-1 protein levels had an independent influence on survival. MMP-1 levels were elevated in both tumour tissue and blood; the latter may serve as an independent predictor for survival of lung cancer patients. MMP-1 protein levels in plasma/serum thus represent a potential and clinically relevant biomarker for the prognosis of patients with lung cancers.

Impact of Growth Hormone Receptor Blockade on Substrate Metabolism during Fasting in Healthy Subjects

Experimental studies in GH-deficient patients and in healthy subjects receiving somatostatin-infusion suggest that GH is an important regulator of substrate metabolism during fasting. These models may not adequately reflect the selective effects of GH, and GH receptor (GHR) blockade offers a new model to define the metabolic role of GH. The aim of this study was to investigate the impact of GHR blockade on substrate metabolism and insulin sensitivity during fasting. We conducted a randomized, placebo-controlled, crossover study in 10 healthy young men. After 36 h of fasting with saline or pegvisomant (GHR blockade), the subjects were studied during a 4-h basal period and 2.5-h hyperinsulinemic euglycemic clamp. We measured whole-body and forearm glucose, lipid, and protein metabolism, peripheral insulin sensitivity, and acyl and desacyl ghrelin. GHR blockade significantly suppressed circulating free fatty acids (1226 +/- 83 vs. 1074 +/- 65 micromol/liter; P = 0.03) and ketone bodies (3080 +/- 271 vs. 2015 +/- 235 micromol/liter; P <or= 0.01), as well as forearm uptake of free fatty acids (0.341 +/- 0.150 vs. 0.004 +/- 0.119 micromol/100 ml x min; P < 0.01) and lipid oxidation (1.3 +/- 0.1 vs. 1.2 +/- 0.1 mg/kg x min; P = 0.03) in the basal period. By contrast, IGF-I levels in either serum or peripheral tissues were not impacted by GHR blockade, and protein metabolism was also unaffected. Basal glucose levels were elevated by GHR blockade, but insulin sensitivity was similar; this was associated with an increased acyl/desacyl ghrelin ratio. GHR blockade, without changes in circulating or tissue IGF-I levels, selectively suppresses lipid mobilization and oxidation after short-term fasting. This supports the notion that stimulation of lipolysis is a primary and important effect of GH.

Peripheral endocannabinoid dysregulation in obesity: relation to intestinal motility and energy processing induced by food deprivation and re‐feeding

Endocannabinoids in tissues controlling energy homeostasis are altered in obesity, thus contributing to metabolic disorders. Here we evaluate endocannabinoid dysregulation in the small intestine of mice with diet-induced obesity (DIO) and in peripheral tissues of Zucker and lean rats following food deprivation and re-feeding. Intestinal transit, evaluated using rhodamine-B-labelled dextran, and small intestinal endocannabinoid levels, measured by liquid chromatography mass spectrometry, were measured in mice fed normal or high-fat diets (HFDs). Endocannabinoid levels were measured also in various tissues of lean and Zucker rats fed ad libitum or following overnight food deprivation with and without subsequent re-feeding. After 8 weeks of HFD, baseline intestinal transit was increased in DIO mice and enhanced by cannabinoid CB(1) receptor antagonism less efficaciously than in lean mice. Small intestinal anandamide and 2-arachidonoylglycerol levels were reduced and increased respectively. In Zucker rats, endocannabinoids levels were higher in the pancreas, liver and duodenum, and lower in the subcutaneous adipose tissue. Food deprivation increased endocannabinoid levels in the duodenum and liver of both rat strains, in the pancreas of lean rats and in adipose tissues of Zucker rats. Reduced anandamide levels might account for increased intestinal motility in DIO mice. Regulation of endocannabinoid levels in rat peripheral tissues, induced by food deprivation and re-feeding, might participate in food intake and energy processing and was altered in Zucker rats. These data, together with previous observations, provide further evidence for dysregulation of peripheral endocannabinoids in obesity.

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass ( Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36‰), isotonic water (15‰), brackish water (4‰) and freshwater (0‰). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.

CB1 Cannabinoid Receptor Inhibition: Promising Approach for Heart Failure?

Endocannabinoids are part of a novel signaling system, along with their cellular receptors and several proteins implicated in their synthesis, release, transport, and degradation. The endocannabinoids are endogenous lipid mediators generated by almost all cell types both in the brain and in the peripheral tissues, which have a wide range of biological effects similar to those of marijuana. 2-Arachidonoylglycerol and arachidonoyl ethanolamide (anandamide) are the 2 best characterized endocannabinoids. Their effects are mediated by 2 G protein–coupled receptors, CB1 and CB2, as well as additional, as-yet unidentified ones.1 2-Arachidonoylglycerol binds to both CB2 and CB1 receptors, whereas arachidonoyl ethanolamide has higher affinity for CB1 receptors and may also bind to vanilloid VR1 receptors. The tissue levels of endocannabinoids are determined by the balance between their biosynthesis (involving phospholipase D– and diacyl-glycerol lipase–dependent and other pathways), cellular uptake, and degradation by fatty acid amide hydrolase and/or monoacylglycerol lipases.1,2 Both CB2 and CB1 receptors are negatively coupled to adenylate cyclase through Gi/o proteins but can also signal via protein kinases A and C, mitogen-activated protein kinases, and cyclooxygenase 2 pathways, among others.1,3 The CB1 receptor is widely distributed in the central nervous system1 and at lower levels in various peripheral tissues (eg, myocardium,4–6 human coronary artery endothelial and smooth muscle cells,7,8 adipose tissue,9,10 and the liver10–12). Initially it was thought that CB2 receptors were expressed only in immune and hematopoietic cells, but recent studies have also established their presence in the myocardium,6 human coronary endothelial and smooth muscle cells,7,8 brain,13 and liver.12 Modulation of the endocannabinoid system (ECS) may be therapeutically exploited in various cardiovascular (CV) disorders ranging from circulatory shock, stroke, atherosclerosis and restenosis, and hypertension, to cirrhotic cardiomyopathy, myocardial infarction, and chronic heart failure.1,14,15