Monocarboxylate transporter 1 deficiency impacts CD8+ T lymphocytes proliferation and recruitment to adipose tissue during obesity.

Lactate sits at the crossroad of metabolism, immunity, and inflammation. The expression of cellular lactate transporter MCT1 (known as Slc16a1) increases during immune cell activation to cope with the metabolic reprogramming. We investigated the impact of MCT1 deficiency on CD8+ T cell function during obesity-related inflammatory conditions. The absence of MCT1 impaired CD8+ T cell proliferation with a shift of ATP production to mitochondrial oxidative phosphorylation. In Slc16a1 f/f Tcell cre mice fed a high-fat diet, a reduction in the number of CD8+ T cells, which infiltrated epididymal visceral adipose tissue (epiWAT) or subcutaneous adipose tissue, was observed. Adipose tissue weight and adipocyte area were significantly reduced together with downregulation of adipogenic genes only in the epiWAT. Our findings highlight a distinct effect of MCT1 deficiency in CD8+ T cells in the crosstalk with adipocytes and reinforce the concept that targeting immunometabolic reprogramming in lymphocyte could impact the immune-adipose tissue axis in obesity.

Regulatory mechanisms of the early phase of white adipocyte differentiation: an overview.

The adipose organ comprises two main fat depots termed white and brown adipose tissues. Adipogenesis is a process leading to newly differentiated adipocytes starting from precursor cells, which requires the contribution of many cellular activities at the genome, transcriptome, proteome, and metabolome levels. The adipogenic program is accomplished through two sequential phases; the first includes events favoring the commitment of adipose tissue stem cells/precursors to preadipocytes, while the second involves mechanisms that allow the achievement of full adipocyte differentiation. While there is a very large literature about the mechanisms involved in terminal adipogenesis, little is known about the first stage of this process. Growing interest in this field is due to the recent identification of adipose tissue precursors, which include a heterogenous cell population within different types of adipose tissue as well as within the same fat depot. In addition, the alteration of the heterogeneity of adipose tissue stem cells and of the mechanisms involved in their commitment have been linked to adipose tissue development defects and hence to the onset/progression of metabolic diseases, such as obesity. For this reason, the characterization of early adipogenic events is crucial to understand the etiology and the evolution of adipogenesis-related pathologies, and to explore the adipose tissue precursors' potential as future tools for precision medicine.

The untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci.

This observational study determined the lipidome of cow milk during subclinical intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted approach. Among the pathogens causing bovine IMI, NAS have become the most frequently isolated bacteria from milk samples. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, peptidome, and metabolome, the milk lipidome during mammary gland inflammation remains undisclosed. To cover this gap, we determined the milk lipidome of 17 dairy cows with IMI caused by NAS (NAS-IMI), and we compared the results with those of healthy quarter milk from 11 cows. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,556 measured lipids, the abundance of 597 changed more than 10-fold in quarter milk with NAS-IMI compared with healthy quarters. The results demonstrate the influence of NAS-IMI on the milk lipidome, implying significant changes in lipid species belonging to the family of triacylglycerols and sphingomyelins, and contribute to the understanding of inflammatory processes in the bovine udder, highlighting potential novel biomarkers for improving mastitis diagnostics.

Neuroactive steroids and diabetic complications in the nervous system.

Important complications of diabetes mellitus in the nervous system are represented by diabetic peripheral neuropathy and diabetic encephalopathy. In this context, an important link is represented by neuroactive steroids (i.e., steroids coming from peripheral glands and affecting nervous functionality as well as directly synthesized in the nervous system). Indeed, diabetes does not only affect the reproductive axis and consequently the levels of sex steroid hormones, but also those of neuroactive steroids. Indeed, as will be here summarized, the levels of these neuromodulators present in the central and peripheral nervous system are affected by the pathology in a sex-dimorphic way. In addition, some of these neuroactive steroids, such as the metabolites of progesterone or testosterone, as well as pharmacological tools able to increase their levels have been demonstrated, in experimental models, to be promising protective agents against diabetic peripheral neuropathy and diabetic encephalopathy.

Non-insulin anti-diabetic drugs: An update on pharmacological interactions.

Nowadays, the goal in the management of type 2 diabetes mellitus (T2DM) remains personalized control of glucose. Since less than 50% of patients with T2DM achieve glycemic treatment goal and most of them take medications for comorbidities associated to T2DM, drug interactions, namely pharmacokinetic and pharmacodynamic interactions, may enhance or reduce the effect of compounds involved in hyperglycemia. Hence, clinicians should be aware of the severe complications in T2DM patients in case of a concomitant use of these medications. It is within this context that this review aims to evaluate the effect of a second drug on the pharmacokinetic of these compounds which may lead, along with several pharmacodynamic interactions, to severe clinical complications, i.e., hypoglycemia. Available drugs already approved in Europe, USA and Japan have been included.

Attenuation of diet-induced obesity and induction of white fat browning with a chemical inhibitor of histone deacetylases.

BACKGROUND/OBJECTIVES: In the last decade, a strict link between epigenetics and metabolism has been demonstrated. Histone deacetylases (HDACs) have emerged as key epigenetic regulators involved in metabolic homeostasis in normal and pathologic conditions. Here we investigated the effect of the class I HDAC inhibitor MS-275 in a model of obesity induced by a high-fat diet (HFD). METHODS: C57BL6/J male mice were fed HFD for 17 weeks and then randomized in two groups, treated intraperitoneally with vehicle dimethylsulfoxide (DMSO) or with the class I selective HDAC inhibitor MS-275 every other day for 22 days. Glucose tolerance test and measurement of body temperature during cold exposure were performed. Adipose tissues and liver were phenotypically characterized through histological analysis. Gene and protein expression analysis of brown and white adipose tissues (WATs) were performed. RESULTS: MS-275 treated mice showed 10% reduction of body weight, lower adipocyte size and improved glucose tolerance. Inhibition of class I HDAC determined reduction of adipocyte size and of fat mass, paralleled by higher expression of adipose functionality markers and by increased rate of lipolysis and fatty acid ß-oxidation. MS-275 also promoted thermogenic capacity, related to 'browning' of visceral and subcutaneous WAT, showing increased expression of uncoupling protein 1. In brown adipose tissue, we observed limited effects on gene expression and only reduction of brown adipocyte size. CONCLUSIONS: This study provides evidence that class I HDAC inhibition stimulated functionality and oxidative potential of adipose tissue, improving glucose tolerance and ameliorating the metabolic profile in diet-induced obese mice.

Liver X receptors, nervous system, and lipid metabolism.

Lipids in the nervous system are represented by cholesterol and phospholipids as constituents of cell membranes and, in particular, of myelin. Therefore, lipids are finely regulated to guarantee physiological functions. In the central nervous system, cholesterol is locally synthesized due to the presence of the blood brain barrier. In the peripheral nervous system cholesterol is either up-taken by lipoproteins and/or produced by de novo biosynthesis. Defects in lipid homeostasis in these tissues lead to structural and functional changes that often result in different pathological conditions depending on the affected pathways (i.e. cholesterol biosynthesis, cholesterol efflux, fatty acid biosynthesis etc.). Alterations in cholesterol metabolism in the central nervous system are linked to several disorders such as Alzheimer's disease, Huntington disease, Parkinson disease, Multiple sclerosis, Smith-Lemli-Opitz syndrome, Niemann-Pick type C disease, and glioblastoma. In the peripheral nervous system changes in lipid metabolism are associated with the development of peripheral neuropathy that may be caused by metabolic disorders, injuries, therapeutics, and autoimmune diseases. Transcription factors, such as the Liver X receptors (LXR), regulate both cholesterol and fatty acid metabolism in several tissues including the nervous system. In the last few years several studies elucidated the biology of LXR in the nervous system due to the availability of knock-out mice and the development of synthetic ligands. Here, we review a survey of the literature focused on the central and peripheral nervous system and in physiological and pathological settings with particular attention to the roles played by LXR in both districts.

Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Bile acids and their signaling pathways: eclectic regulators of diverse cellular functions.

The field of bile acids has witnessed an impulse in the last two decades. This has been the result of cloning the genes encoding enzymes of bile acid synthesis and their transporters. There is no doubt that the identification of Farnesoid X Receptor (FXR, NR1H4) as the bile acid receptor has contributed substantially to attract the interest of scientists in this area. When FXR was cloned by Forman et al. [1], farnesol metabolites were initially considered the physiological ligands. After identifying FXR and other nuclear receptors as bile acid sensors [2-4], it has become clear that bile acids are involved in the regulation of lipid and glucose metabolism and that these molecules are eclectic regulators of diverse cellular functions. In this review, we will summarize the current knowledge of the functions regulated by bile acids and how their physiological receptors mediate the signaling underlying numerous cellular responses.

Age-related changes in bile acid synthesis and hepatic nuclear receptor expression.

BACKGROUND: Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7alpha-hydroxylase and related nuclear receptor expression in human livers. DESIGN: Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7alpha-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. RESULTS: Ageing was inversely correlated with serum 7alpha-hydroxy-4-cholesten-3-one and with cholesterol 7alpha-hydroxylase mRNA levels (r = -0.44 and r = -0.45 on a semi-log scale, respectively, P < 0.05). Among different nuclear factors, cholesterol 7alpha-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 (r = 0.55 on a log scale, P < 0.05); hepatocyte nuclear factor-4 levels were also inversely correlated with age (r = -0.64 on a semi-log scale, P < 0.05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7alpha-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7alpha-hydroxylase was observed. CONCLUSIONS: Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7alpha-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism.

Decreased hepatic expression of PPAR-gamma coactivator-1 in cholesterol cholelithiasis.

BACKGROUND: Cholesterol cholelithiasis (gallstone disease) is a common disease in the Western world. The aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholesterol gallstone disease. MATERIALS AND METHODS: Surgical liver biopsies were obtained from 11 patients with untreated cholesterol cholelithiasis and nine gallstone-free subjects; mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and related nuclear receptors and coactivators were assayed by quantitative real-time RT-PCR. RESULTS: No differences between the two groups were detected in mRNA levels of CYP7A1 and related nuclear receptors, with the exception of peroxysome proliferator-activated receptor-gamma coactivator 1 (PGC-1), which was significantly (P < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with farnesoid X receptor (FXR) in gallstone patients (r = 0.87 on a log scale, P < 0.01), but not in control subjects; in gallstone patients PGC-1 expression was also correlated with hepatocyte nuclear factor 4 (HNF-4) (r = 0.78, P < 0.01). CONCLUSION: These findings suggest that PGC-1 can play a role in the prevention of cholesterol gallstone disease in humans; this might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models and other coactivators. The present data might help to understand the pathophysiology and possibly focus on new therapeutical targets in cholesterol gallstone disease.

High pressure liquid chromatography and electrospray ionization mass spectrometry are advantageously integrated into a two-levels approach to detection and identification of haemoglobin variants.

Detecting and correctly identifying haemoglobin (Hb) variants is typically achieved by a two-levels laboratory approach. We report our experience in dealing with 91 Hb variants, including a number of frequent and a few rare variants. Screening included akaline agarose gel electrophoresis (AGE), ion-exchange automated high-performance liquid chromatography (HPLC) and a test for deoxyhaemoglobin solubility. Identification was based on electrospray ionization-mass spectrometry (ESI-MS). Our results confirmed the advantages of HPLC over AGE for screening, because of the occurrence of some electrophoretically 'silent' variants. ESI-MS permitted the definitive identification of 90 of the 91 variants included in the study, in some cases (e.g. HbS) through the application of a simple protocol (direct injection of the sample), in other cases requiring the application of more demanding procedures (purification of the variant chain and peptide analysis after enzymatic or chemical cleavage). In an additional case (Hb J-Oxford), ESI-MS assay did not lead to definitive identification, but gave indications for designing the appropriate primers to focus DNA sequence analysis on the specific region of the gene. Deoxyhaemoglobin solubility test was positive only in the presence of HbS. We conclude that HPLC and ESI-MS are advantageously integrated into a two-level analytical system for the detection and confirmation of variant Hbs.

LXR (liver X receptor) and HNF-4 (hepatocyte nuclear factor-4): key regulators in reverse cholesterol transport.

Cholesterol homoeostasis is the result of the fine tuning between intake and disposal of this molecule. High levels of cholesterol in the blood are detrimental as they may lead to excessive accumulation in vessel walls, a condition predisposing to the development of atherosclerotic lesions. Cholesterol is removed from the vessel wall and transported to the liver through a process called reverse cholesterol transport. Nuclear receptors are among the most important transcription factors regulating genes involved in different steps of reverse cholesterol transport. Here, we discuss the role of the nuclear receptors LXR (liver X receptor) and HNF-4alpha (hepatocyte nuclear factor-4alpha) in different steps of reverse cholesterol transport. LXR controls the transcription of crucial genes in cholesterol efflux from macrophages and its transport to the liver, such as ABCA1 (ATP binding cassette A1), CYP27A1 (sterol 27-hydroxylase), CLA-1 (scavenger receptor type B1) and apolipoprotein E. Some oxysterols present in oxidized low-density lipoproteins and proinflammatory cytokines modulate the activity of LXR by antagonizing the effect of activators of this receptor, thus contributing to cholesterol accumulation in macrophages. Bile acid synthesis, which represents the final step of reverse cholesterol transport, is transcriptionally regulated by several nuclear receptors at the level of the liver-specific cytochrome P450 cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme of this metabolic pathway. Bile acids returning to the liver through the enterohepatic circulation down-regulate CYP7A1 transcription via the bile acid sensors farnesoid X receptor and HNF-4alpha. Based on this evidence, these nuclear receptors are candidate targets of new drugs for the treatment and prevention of atherosclerotic disease.

Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae).

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions, and thus inhibition of MMP activity might have therapeutic potential. The methanolic extract and the identified compounds from the bark of Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) were tested on the activity, production, and gene expression of MMP-9. The extract produced a concentration-dependent inhibition (50-95% at 10-50 microg/ml) of MMP-9 activity. The inhibitory activity was retained in the ethyl acetate-soluble fraction (50-95% inhibition at 10-50 microg/ml) which also reduced the release of MMP-9 by mouse peritoneal macrophages up to 80%. In the ethyl acetate-soluble fraction, two active fractions, 5A and 5B were identified. HPLC-MS and NMR analyses of these fractions indicated the presence of gallocatechin, ellagic acid, and its glycoside derivatives. Since the absolute configuration of gallocatechin was not determined, in the next experiments both (+)-gallocatechin (2R,3S) and (-)-gallocatechin (2S,3R) were tested, and (-)-epigallocatechin (2R,3R) was included for comparison. 5A and 5B inhibited MMP-9 secretion, an observation which correlated with the decrease of MMP-9 promoter activity and the downregulation of mRNA levels. All compounds decreased MMP-9 mRNA levels and secretion. Ellagic acid, (+)-gallocatechin and (-)-epigallocatechin, but not (-)gallocatechin inhibited promoter-driven transcription. Thus configuration at C2 (R) of the flavanol seem to be critical for the interaction with the promoter.

The negative effects of bile acids and tumor necrosis factor-alpha on the transcription of cholesterol 7alpha-hydroxylase gene (CYP7A1) converge to hepatic nuclear factor-4: a novel mechanism of feedback regulation of bile acid synthesis mediated by nuclear receptors.

Bile acids regulate the cholesterol 7alpha-hydroxylase gene (CYP7A1), which encodes the rate-limiting enzyme in the classical pathway of bile acid synthesis. Here we report a novel mechanism whereby bile acid feedback regulates CYP7A1 transcription through the nuclear receptor hepatocyte nuclear factor-4 (HNF-4), which binds to the bile acid response element (BARE) at nt -149/-118 relative to the transcription start site. Using transient transfection assays of HepG2 cells with Gal4-HNF-4 fusion proteins, we show that chenodeoxycholic acid (CDCA) dampened the transactivation potential of HNF-4. Overexpression of a constitutive active form of MEKK1, an upstream mitogen-activated protein kinase (MAPK) module triggered by stress signals, strongly repressed the promoter activity of CYP7A1 via the consensus sequence for HNF-4 embedded in the BARE. Similarly, MEKK1 inhibited the activity of HNF-4 in the Gal4-based assay. The involvement of the MEKK1-dependent pathway in the bile acid-mediated repression of CYP7A1 was confirmed by co-transfecting a dominant negative form of the stress-activated protein kinase kinase, SEK, which abolished the effect of CDCA upon CYP7A1 transcription. Treatment of transfected HepG2 cells with tumor necrosis factor alpha (TNF-alpha), an activator of the MEKK1 pathway, led to the repression of CYP7A1 via the HNF-4 site in the BARE. TNF-alpha also inhibited the transactivation potential of HNF-4. Collectively, our results demonstrate for the first time that HNF-4, in combination with a MAPK signaling pathway, acts as a bile acid sensor in the liver. Furthermore, the effects of CDCA and TNF-alpha converge to HNF-4, which binds to the BARE of CYP7A1, suggesting a link between the cascades elicited by bile acids and pro-inflammatory stimuli in the liver.