[State of the art on the pathophysiology, diagnosis and treatment of non-alcoholic steatohepatitis (NASH)]. / État des lieux sur la physiopathologie, le diagnostic et les traitements de la stéato-hépatite non alcoolique (NASH).

NAFLD or non-alcoholic fatty liver disease is one of the complications of obesity and diabetes, the prevalence of which is increasing. The causes of the pathology and its development towards its severe form, NASH or non-alcoholic steatohepatitis, are multiple and still poorly understood. Many different pharmacological classes are being tested in clinical trials to treat NASH, but no pharmaceutical treatment is currently on the market. Moreover, the diagnosis of certainty is only possible by liver biopsy and histological analysis, an invasive procedure with high risk for the patient. It is therefore necessary to better understand the natural history of the disease in order to identify therapeutic targets, but also to identify markers for the diagnosis and monitoring of the disease using a blood sample, which will allow an improvement in patient management.

Role of bile acid receptor FXR in development and function of brown adipose tissue.

Bile acids act as signalling molecules that contribute to maintenance of energy homeostasis in mice and humans. Activation of G-protein-coupled bile acid receptor TGR5 induces energy expenditure in brown adipose tissue (BAT). However, a role for the nuclear bile acid receptor Farnesoid X receptor (FXR) in BAT has remained ambiguous. We aimed to study the potential role of FXR in BAT development and functioning. Here we demonstrate low yet detectable expression of the α1/2 isoforms of FXR in murine BAT that markedly decreases upon cold exposure. Moderate adipose tissue-specific FXR overexpression in mice induces pronounced BAT whitening, presenting with large intracellular lipid droplets and extracellular collagen deposition. Expression of thermogenic marker genes including the target of Tgr5, Dio2, was significantly lower in BAT of chow-fed aP2-hFXR mice compared to wild-type controls. Transcriptomic analysis revealed marked up-regulation of extracellular matrix formation and down-regulation of mitochondrial functions in BAT from aP2-hFXR mice. In addition, markers of cell type lineages deriving from the dermomyotome, such as myocytes, as well as markers of cellular senescence were strongly induced. The response to cold and ß3-adrenergic receptor agonism was blunted in these mice, yet resolved BAT whitening. Newborn cholestatic Cyp2c70-/- mice with a human-like bile acid profile also showed distinct BAT whitening and upregulation of myocyte-specific genes, while thermogenic markers were down-regulated. Ucp1 expression inversely correlated with plasma bile acid levels. Therefore, bile acid signalling via FXR has a role in BAT function already early in tissue development. Functionally, FXR activation appears to oppose TGR5-mediated thermogenesis.

Association of 1-deoxy-sphingolipids with steatosis but not steatohepatitis nor fibrosis in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most important cause of chronic liver disease in the western world. Steatosis can be accompanied by inflammation and cell damage (non-alcoholic steatohepatitis, NASH), and even liver fibrosis. Sphingolipids are a heterogeneous class of lipids and essential components of the plasma membrane and plasma lipoproteins. The atypical class of deoxy-sphingolipids has been implicated in the metabolic syndrome and type 2 diabetes. AIM: To determine if circulating (deoxy)sphingolipids are associated with NAFLD and its different entities, steatosis, inflammatory changes (inflammation and ballooning) and fibrosis. METHODS: Sphingolipids were analysed by LC-MS after hydrolysing the N-acyl and O-linked headgroups in plasma of obese adults who underwent a liver biopsy in suspicion of NAFLD. RESULTS: Two-hundred and eighty-eight patients were included. There was no association between typical sphingolipids and NAFLD and its different entities. There was a significant association between the presence of steatosis and the concentrations of deoxy-sphinganine [exp(B) 11.163 with CI (3.432, 36.306) and p < 0.001] and deoxy-sphingosine [exp(B) 8.486 with CI (3.437, 20.949) and p < 0.001]. There was no association between these deoxy-sphingolipids and activity of the steatohepatitis, nor was there any association with fibrosis. Differences in deoxy-sphingolipids also correlated independently with the presence of the metabolic syndrome, but not diabetes. CONCLUSION: Deoxy-sphingolipids are elevated in patients with steatosis compared to those without fatty liver, but not different between the different NAFLD subtypes, suggesting that deoxy-sphingolipid bases might be involved in steatogenesis, but not in the further progression of NAFLD to NASH nor in fibrogenesis.

Roux-en-Y gastric bypass increases systemic but not portal bile acid concentrations by decreasing hepatic bile acid uptake in minipigs.

Roux-en-Y gastric bypass (RYGB) surgery is widely used in the management of morbid obesity. RYGB improves metabolism independently of weight loss by still unknown mechanisms. Bile acids (BAs) are good candidates to explain this benefit, since they regulate metabolic homeostasis and their systemic concentrations increase upon RYGB. Here we analyzed the mechanisms underlying the increase in systemic BA concentrations after RYGB and the role of the liver therein. To this aim, we used the Göttingen-like minipig, a human-size mammalian model, which allows continuous sampling and simultaneous analysis of pre-hepatic portal and systemic venous blood. BA concentrations and pool composition were measured in portal blood, containing intestinal reabsorbed BAs and compared to systemic blood during a standardized meal test before and after RYGB. Systemic total BA concentrations increased after RYGB, due to an increase in conjugated BAs. Interestingly, the ratio of portal:systemic conjugated BAs decreased after RYGB, indicating a role for the liver in systemic BA concentrations changes. In line, hepatic expression of BA transporter genes decreased after RYGB. Our results show that the increase in systemic BAs after surgery is due to decreased selective hepatic recapture. Thus, alterations in hepatic function contribute to the increase in systemic BAs after RYGB.

Topical ivermectin improves allergic skin inflammation.

BACKGROUND: Ivermectin (IVM) is widely used in both human and veterinary medicine to treat parasitic infections. Recent reports have suggested that IVM could also have anti-inflammatory properties. METHODS: Here, we investigated the activity of IVM in a murine model of atopic dermatitis (AD) induced by repeated exposure to the allergen Dermatophagoides farinae, and in standard cellular immunological assays. RESULTS: Our results show that topical IVM improved allergic skin inflammation by reducing the priming and activation of allergen-specific T cells, as well as the production of inflammatory cytokines. While IVM had no major impact on the functions of dendritic cells in vivo and in vitro, IVM impaired T-cell activation, proliferation, and cytokine production following polyclonal and antigen-specific stimulation. CONCLUSION: Altogether, our results show that IVM is endowed with topical anti-inflammatory properties that could have important applications for the treatment of T-cell-mediated skin inflammatory diseases.

PPARß in macrophages and atherosclerosis.

Macrophages are central cells in the genesis and development of atherosclerosis, one of the major causes of cardiovascular diseases. Macrophages take up lipids (mainly cholesterol and triglycerides) from lipoproteins thus transforming into foam cells. Moreover, through the efflux pathway, macrophages are the main actors of the elimination of excessive tissue cholesterol toward extra-cellular acceptors. Macrophages participate in the control of inflammation by displaying different functional phenotypes, from the M1 pro-inflammatory to the M2 anti-inflammatory state. The nuclear receptor Peroxisome Proliferator-Activated Receptor (PPAR)ß (also called PPARδ or PPARß/δ) is expressed in macrophages where it plays a different role in the control of lipid metabolism, inflammation and phagocytosis of apoptotic cells. This review will summarize our current understanding of how PPARß regulates macrophage biology and its impact on atherosclerosis. Differences between studies and species-specific macrophage gene regulation will be discussed.

Influence of Roux-en-Y gastric bypass on plasma bile acid profiles: a comparative study between rats, pigs and humans.

BACKGROUND: Roux-en-Y gastric bypass (RYGBP) is the most widely used bariatric surgery procedure, which induces profound metabolic and physiological effects, such as substantial improvements in obesity, type 2 diabetes and their comorbidities. Increasing evidence identifies bile acids (BAs) as signaling molecules that contribute to the metabolic improvement after RYGBP. However, how and to what extent BAs mediate the metabolic effects of RYGBP still remains unclear and requires mechanism of action studies using preclinical models. In this study, we compared plasma BA profiles before and after RYGBP in two animal models, rats and pigs, with humans to evaluate their translational potential. METHODS: Plasma BAs were profiled in rats, pigs and humans by liquid chromatography coupled with tandem mass spectrometry before and after RYGBP. RESULTS: RYGBP increased baseline plasma total BA concentrations in humans and in the two animal models to a similar extent (∼3-fold increase), despite differences in presurgery BA levels and profiles between the models. However, qualitatively, RYGBP differently affected individual plasma BA species, with similar increases in some free species (cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)), different increases in glyco-conjugated species depending on the model and globally no increase in tauro-conjugated species whatever the model. CONCLUSIONS: The tested animal models share similar quantitative RYGBP-induced increases in peripheral blood BAs as humans, which render them useful for mechanistic studies. However, they also present qualitative differences in BA profiles, which may result in different signaling responses. Such differences need to be taken into account when translating results to humans.

Temporal changes in bile acid levels and 12α-hydroxylation after Roux-en-Y gastric bypass surgery in type 2 diabetes.

Since the publication of the above article it has been noted that the author S O'Brien should have been listed as CS O'Brien. The authors should therefore appear as follows: R Dutia, M Embrey, CS O'Brien, RA Haeusler, KK Agénor, P Homel, J McGinty, RP Vincent, J Alaghband-Zadeh, B Staels, CW le Roux, J Yu and B Laferrère The corrected article html and online pdf versions have been amended. The authors wish to apologise for any inconvenience caused.

Peroxisome Proliferator-Activated Receptor γ Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1) in Human Macrophages.

Tissue factor (TF) is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa). Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that, together with PPARα and PPARß/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARß/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway.

Skeletal muscle functions around the clock.

In mammals, the central clock localized in the central nervous system imposes a circadian rhythmicity to all organs. This is achieved thanks to a well-conserved molecular clockwork, involving interactions between several transcription factors, whose pace is conveyed to peripheral tissues through neuronal and humoral signals. The molecular clock plays a key role in the control of numerous physiological processes and takes part in the regulation of metabolism and energy balance. Skeletal muscle is one of the peripheral organs whose function is under the control of the molecular clock. However, although skeletal muscle metabolism and performances display circadian rhythmicity, the role of the molecular clock in the skeletal muscle has remained unappreciated for years. Peripheral organs such as skeletal muscle, and the liver, among others, can be desynchronized from the central clock by external stimuli, such as feeding or exercise, which impose a new rhythm at the organism level. In this review, we discuss our current understanding of the clock in skeletal muscle circadian physiology, focusing on the control of myogenesis and skeletal muscle metabolism.

The neuron-derived orphan receptor 1 (NOR1) is induced upon human alternative macrophage polarization and stimulates the expression of markers of the M2 phenotype.

BACKGROUND: Atherosclerosis is an inflammatory disease in which macrophages play a crucial role. Macrophages are present in different phenotypes, with at the extremes of the spectrum the classical M1 pro-inflammatory and the alternative M2 anti-inflammatory macrophages. The neuron-derived orphan receptor 1 (NOR1), together with Nur77 and Nurr1, are members of the NR4A orphan nuclear receptor family, expressed in human atherosclerotic lesion macrophages. However, the role of NOR1 in human macrophages has not been studied yet. OBJECTIVES: To determine the expression and the functions of NOR1 in human alternative macrophages. METHODS AND RESULTS: In vitro IL-4 polarization of primary monocytes into alternative M2 macrophages enhances NOR1 expression in human but not in mouse macrophages. Moreover, NOR1 expression is most abundant in CD68+MR+ alternative macrophage-enriched areas of human atherosclerotic plaques in vivo. Silencing NOR1 in human alternative macrophages decreases the expression of several M2 markers such as the Mannose Receptor (MR), Interleukin-1 Receptor antagonist (IL-1Ra), CD200 Receptor (CD200R), coagulation factor XIII A1 polypeptide (F13A1), Interleukin 10 (IL-10) and the Peroxisome Proliferator-Activated Receptor (PPAR)γ. Bioinformatical analysis identified F13A1, IL-1Ra, IL-10 and the Matrix Metalloproteinase-9 (MMP9) as potential target genes of NOR1 in human alternative macrophages. Moreover, expression and enzymatic activity of MMP9 are induced by silencing and repressed by NOR1 overexpression in M2 macrophages. CONCLUSIONS: These data identify NOR1 as a transcription factor induced during alternative differentiation of human macrophages and demonstrate that NOR1 modifies the alternative macrophage phenotype.

The coronary artery disease-associated gene C6ORF105 is expressed in human macrophages under the transcriptional control of PPARγ.

Coronary artery disease (CAD) is a major cause of morbidity and mortality. Mutations in C6ORF105, associated with decreased gene expression, positively correlate with the risk of CAD in Chinese populations. Moreover, the C6ORF105-encoded protein may play a role in coagulation. Here, we report that C6ORF105 gene expression is lower in circulating mononuclear cells from obese diabetic than lean subjects. Moreover, C6ORF105 is expressed in human macrophages and atherosclerotic lesions, where its expression positively correlates with expression of the transcription factor Peroxisome Proliferator-Activated Receptor (PPAR)γ. Activation of PPARγ increases, in a PPARγ-dependent manner, the expression of C6ORF105 in human macrophages and atherosclerotic lesions.

Temporal changes in bile acid levels and 12α-hydroxylation after Roux-en-Y gastric bypass surgery in type 2 diabetes.

INTRODUCTION: Gastric bypass surgery (GBP) leads to sustained weight loss and significant improvement in type 2 diabetes (T2DM). Bile acids (BAs), signaling molecules which influence glucose metabolism, are a potential mediator for the improvement in T2DM after GBP. This study sought to investigate the effect of GBP on BA levels and composition in individuals with T2DM. METHODS: Plasma BA levels and composition and fibroblast growth factor (FGF)-19 levels were measured during fasting and in response to an oral glucose load before and at 1 month and 2 years post GBP in 13 severely obese women with T2DM. RESULTS: A striking temporal change in BA levels and composition was observed after GBP. During the fasted state, BA concentrations were generally reduced at 1 month, but increased 2 years post GBP. Postprandial BA levels were unchanged 1 month post GBP, but an exaggerated postprandial peak was observed 2 years after the surgery. A significant increase in the 12α-hydroxylated/non12α-hydroxylated BA ratio during fasting and postprandially at 2 years, but not 1 month, post GBP was observed. Significant correlations between BAs vs FGF-19, body weight, the incretin effect and peptide YY (PYY) were also found. CONCLUSIONS: This study provides evidence that GBP temporally modifies the concentration and composition of circulating BAs in individuals with T2DM, and suggests that BAs may be linked to the improvement in T2DM after GBP.

Efficacy and safety of initial combination treatment with sitagliptin and pioglitazone--a factorial study.

AIM: To evaluate the efficacy and safety of initial combination therapy of sitagliptin 100 mg/day coadministered with all marketed doses of pioglitazone in patients with type 2 diabetes. METHODS: Patients with A1c ≥7.5 and ≤11.0% were randomized among seven arms that received, once daily, 100 mg sitagliptin alone; 15, 30 or 45 mg pioglitazone alone, or 100 mg sitagliptin plus 15, 30 or 45 mg pioglitazone for 54 weeks. The primary endpoint was change from baseline in A1c at week 24. Protocol-specified analyses compared combination therapies with monotherapies at respective dose-strengths and combination of sitagliptin plus pioglitazone 30 mg with pioglitazone 45 mg monotherapy. Post-hoc analyses compared sitagliptin plus pioglitazone 15 mg with pioglitazone monotherapy at the two higher doses. RESULTS: Initial combination therapy with sitagliptin and pioglitazone provided significantly greater reductions in A1c (0.4-0.7% differences) and other glycaemic endpoints than either monotherapy at the same doses. Combining sitagliptin with low-dose pioglitazone generally produced greater glycaemic improvements than higher doses of pioglitazone monotherapy (0.3-0.4% differences in A1c). Combination therapy was generally well tolerated; adverse events (AEs) of hypoglycaemia were reported with similar incidence (7.8-11.1%) in all treatment groups over the 54 weeks of study; oedema was reported in 0.5% of patients in the sitagliptin monotherapy group and 2.7-5.3% among pioglitazone-treated groups. Significant weight gain was observed in all combination-treated groups compared with the sitagliptin monotherapy group. CONCLUSIONS: Initial combination therapy with sitagliptin and pioglitazone provided better glycaemic control than either monotherapy and was generally well tolerated.

Profiling serum bile acid glucuronides in humans: gender divergences, genetic determinants, and response to fenofibrate.

Glucuronidation, catalyzed by uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic bile acids (BAs). We aimed to (i) characterize the circulating BA-glucuronide (BA-G) pool composition in humans, (ii) determine how sex and UGT polymorphisms influence this composition, and (iii) analyze the effects of the lipid-lowering drug fenofibrate on the circulating BA-G profile in 300 volunteers and 5 cholestatic patients. Eleven BA-Gs were determined in pre- and postfenofibrate samples. Men exhibited higher BA-G concentrations, and various genotype/BA-G associations were discovered in relevant UGT genes. The chenodeoxycholic acid-3G (CDCA-3G) concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA-3G formation. Fenofibrate exposure increased the serum levels of five BA-G species, including CDCA-3G, and upregulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrated that fenofibrate stimulates BA glucuronidation in humans and thus reduces BA toxicity in the liver.

A gene variant of PNPLA3, but not of APOC3, is associated with histological parameters of NAFLD in an obese population.

OBJECTIVE: Mechanisms explaining the relationship in non-alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance are poorly understood. A genetic basis has been suggested. We studied the association between the genes patatin-like phospholipase domain-containing protein 3 (PNPLA3) and apolipoprotein C3 (APOC3) and metabolic and histological parameters of NAFLD in obese patients. DESIGN AND METHODS: Overweight and obese patients underwent a metabolic and liver assessment. If NAFLD was suspected, liver biopsy was proposed. APOC3 variant rs2854117 and PNPLA3 variant rs738409 were genotyped. RESULTS: Four hundred seventy patients were included (61.1% had liver biopsy). The percentage of patients with non-alcoholic steatohepatitis (NASH) was significantly different according to the PNPLA3 variant. After adjustment for age and body mass index, the PNPLA3 variant was associated with alanine aminotransferase (P < 0.001) and aspartate aminotransferase (P < 0.001). The PNPLA3 variant was associated with more severe features of steatohepatitis: steatosis (P < 0.001), lobular inflammation (P < 0.001), and ballooning (P = 0.002), but not with liver fibrosis, anthropometry, or insulin resistance. No significant difference in liver histology, anthropometric, or metabolic parameters was found between carriers and non-carriers of the APOC3 variant. CONCLUSIONS: PNPLA3 polymorphism rs738409 was associated with NASH and the severity of necroinflammatory changes independently of metabolic factors. No association between APOC3 gene variant rs2854117 and histological or metabolic parameters of NAFLD was found.

Downregulation of the tumour suppressor p16INK4A contributes to the polarisation of human macrophages toward an adipose tissue macrophage (ATM)-like phenotype.

AIMS/HYPOTHESIS: Human adipose tissue macrophages (ATMs) display an alternatively activated (M2) phenotype, but are still able to produce excessive inflammatory mediators. However, the processes driving this particular ATM phenotype are not understood. Genome-wide association studies associated the CDKN2A locus, encoding the tumour suppressor p16(INK4A), with the development of type 2 diabetes. In the present study, p16(INK4A) levels in human ATMs and the role of p16(INK4A) in acquiring the ATM phenotype were assessed. METHODS: Gene expression of p16 ( INK4A ) in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16(INK4A) levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16(INK4A) in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16(INK4A). RESULTS: Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16 ( INK4A ). In vitro, IL-4-induced M2 polarisation resulted in lower p16(INK4A) protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16(INK4A) in MDMs mediated by siRNA increased the expression of M2 marker genes and enhanced the response to lipopolysaccharide (LPS), to give a phenotype resembling that of ATM. By contrast, adenovirus-mediated overproduction of p16(INK4A) in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16(INK4A) overproduction inhibits LPS- and palmitate-induced Toll-like receptor 4 (TLR4)-nuclear factor of κ light polypeptide gene enhancer in B cells (NF-κB) signalling. CONCLUSIONS/INTERPRETATION: These results show that p16(INK4A) inhibits the acquisition of the ATM phenotype. The age-related increase in p16(INK4A) level may thus influence normal ATM function and contribute to type 2 diabetes risk.

Generation and characterization of a humanized PPARδ mouse model.

BACKGROUND AND PURPOSE: Humanized mice for the nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ), termed PPARδ knock-in (PPARδ KI) mice, were generated for the investigation of functional differences between mouse and human PPARδ and as tools for early drug efficacy assessment. EXPERIMENTAL APPROACH: Human PPARδ function in lipid metabolism was assessed at baseline, after fasting or when challenged with the GW0742 compound in mice fed a chow diet or high-fat diet (HFD). KEY RESULTS: Analysis of PPARδ mRNA levels revealed a hypomorph expression of human PPARδ in liver, macrophages, small intestine and heart, but not in soleus and quadriceps muscles, white adipose tissue and skin. PPARδ KI mice displayed a small decrease of high-density lipoprotein-cholesterol whereas other lipid parameters were unaltered. Plasma metabolic parameters were similar in wild-type and PPARδ KI mice when fed chow or HFD, and following physiological (fasting) and pharmacological (GW0742 compound) activation of PPARδ. Gene expression profiling in liver, soleus muscle and macrophages showed similar gene patterns regulated by mouse and human PPARδ. The anti-inflammatory potential of human PPARδ was also similar to mouse PPARδ in liver and isolated macrophages. CONCLUSIONS AND IMPLICATIONS: These data indicate that human PPARδ can compensate for mouse PPARδ in the regulation of lipid metabolism and inflammation. Overall, this novel PPARδ KI mouse model shows full responsiveness to pharmacological challenge and represents a useful tool for the preclinical assessment of PPARδ activators with species-specific activity.

Which is the eligible patient to be treated with pioglitazone? The expert view.

Pioglitazone has an important role in the treatment of patients with Type 2 diabetes. The drug can help patients to achieve sustained glycemic control and may delay the requirement for insulin. Pioglitazone may provide benefits beyond its effects on glycemia, with data suggesting it may confer anti-atherosclerotic and cardioprotective properties. Attention should be given to possible side effects relating to class effects of TZD, and selection of appropriate patients to be prescribed pioglitazone will enable optimum benefits to be derived from pioglitazone treatment.