Ponesimod Compared With Teriflunomide in Patients With Relapsing Multiple Sclerosis in the Active-Comparator Phase 3 OPTIMUM Study: A Randomized Clinical Trial.

Importance: To our knowledge, the Oral Ponesimod Versus Teriflunomide In Relapsing Multiple Sclerosis (OPTIMUM) trial is the first phase 3 study comparing 2 oral disease-modifying therapies for relapsing multiple sclerosis (RMS). Objective: To compare the efficacy of ponesimod, a selective sphingosine-1-phosphate receptor 1 (S1P1) modulator with teriflunomide, a pyrimidine synthesis inhibitor, approved for the treatment of patients with RMS. Design, Setting, and Participants: This multicenter, double-blind, active-comparator, superiority randomized clinical trial enrolled patients from April 27, 2015, to May 16, 2019, who were aged 18 to 55 years and had been diagnosed with multiple sclerosis per 2010 McDonald criteria, with a relapsing course from the onset, Expanded Disability Status Scale (EDSS) scores of 0 to 5.5, and recent clinical or magnetic resonance imaging disease activity. Interventions: Patients were randomized (1:1) to 20 mg of ponesimod or 14 mg of teriflunomide once daily and the placebo for 108 weeks, with a 14-day gradual up-titration of ponesimod starting at 2 mg to mitigate first-dose cardiac effects of S1P1 modulators and a follow-up period of 30 days. Main Outcomes and Measures: The primary end point was the annualized relapse rate. The secondary end points were the changes in symptom domain of Fatigue Symptom and Impact Questionnaire-Relapsing Multiple Sclerosis (FSIQ-RMS) at week 108, the number of combined unique active lesions per year on magnetic resonance imaging, and time to 12-week and 24-week confirmed disability accumulation. Safety and tolerability were assessed. Exploratory end points included the percentage change in brain volume and no evidence of disease activity (NEDA-3 and NEDA-4) status. Results: For 1133 patients (567 receiving ponesimod and 566 receiving teriflunomide; median [range], 37.0 [18-55] years; 735 women [64.9%]), the relative rate reduction for ponesimod vs teriflunomide in the annualized relapse rate was 30.5% (0.202 vs 0.290; P < .001); the mean difference in FSIQ-RMS, -3.57 (-0.01 vs 3.56; P < .001); the relative risk reduction in combined unique active lesions per year, 56% (1.405 vs 3.164; P < .001); and the reduction in time to 12-week and 24-week confirmed disability accumulation risk estimates, 17% (10.1% vs 12.4%; P = .29) and 16% (8.1% vs 9.9; P = .37), respectively. Brain volume loss at week 108 was lower by 0.34% (-0.91% vs -1.25%; P < .001); the odds ratio for NEDA-3 achievement was 1.70 (25.0% vs 16.4%; P < .001). Incidence of treatment-emergent adverse events (502 of 565 [88.8%] vs 499 of 566 [88.2%]) and serious treatment-emergent adverse events (49 [8.7%] vs 46 [8.1%]) was similar for both groups. Treatment discontinuations because of adverse events was more common in the ponesimod group (49 of 565 [8.7%] vs 34 of 566 [6.0%]). Conclusions and Relevance: In this study, ponesimod was superior to teriflunomide on annualized relapse rate reduction, fatigue, magnetic resonance imaging activity, brain volume loss, and no evidence of disease activity status, but not confirmed disability accumulation. The safety profile was in line with the previous safety observations with ponesimod and the known profile of other S1P receptor modulators. Trial Registration: ClinicalTrials.gov Identifier: NCT02425644.

Quantitative real-time PCR detection of insulin signalling-related genes in pancreatic islets isolated from healthy cats.

The cat has recently been proposed as a valuable model for type 2 diabetes mellitus (T2DM), because feline diabetes shares several similarities with the disease in humans. Impaired beta-cell function, decreased beta-cell mass, insulin resistance that is often related to obesity, and pancreatic amyloid deposition, are among these common features. In this study, and to further develop the cat as a model of T2DM, feline pancreatic islets were isolated and real-time PCR quantification of mRNA transcripts of genes central to beta-cell function and survival established. In particular, mRNA quantification systems were determined for insulin, the insulin enhancer pancreatic duodenal homeobox-1 (PDX-1), the insulin suppressor CCAAT/enhancer binding protein-beta (C/EBPbeta), glucose transporter isoform 2 (GLUT2), Fas receptor, the caspase-8 inhibitor FLIP (FLICE [caspase-8]-inhibitory protein) and two chemokines, interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Pancreatic islets were isolated by collagenase digestion from healthy cat donors. Partial feline mRNA sequences were determined for PDX-1, C/EBPbeta, GLUT2 and FLIP using primers identified from conserved regions of human, dog and rat mRNA. These novel and the previously available sequences (insulin, Fas receptor, IL-8 and MCP-1) were used to design feline-specific primers suitable for real-time PCR in isolated pancreatic islets. The adopted protocol of collagenase digestion yielded pancreatic islets that were frequently surrounded by acinar cells. Quantification of mRNA transcripts was simple and reproducible in healthy cats. Characterisation of genes related to insulin signalling in cats will prove useful to better understand the pathogenesis of feline diabetes and possibly of human T2DM.

Partial sequencing and expression of genes involved in glucose metabolism in adipose tissues and skeletal muscle of healthy cats.

Impaired insulin sensitivity is increasingly recognised in cats, but sequences of genes involved in insulin-signalling are largely undetermined in this species. In this study, extended feline mRNA sequences were determined for the adiponectin, glucose transporter-1 (GLUT1), GLUT4, peroxisome proliferative activated receptor-gamma1 (PPARgamma1), PPARgamma2, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1) and insulin receptor genes. Conserved dog-specific primers identified from human-dog mRNA alignments were used to amplify feline cDNA in the polymerase chain reaction (PCR). The feline sequences determined by this method were used to design feline-specific primers suitable for real-time PCR for quantification of gene expression in insulin sensitive tissues of healthy cats. Partial sequences of feline mRNAs had 86-95% identity with dog and human genes. Expression of adiponectin, GLUT1, GLUT4, PPARgamma1, PPARgamma2, PAI-1 and insulin receptor mRNA was detected and quantified in subcutaneous and visceral fat and skeletal muscle, whereas MCP-1 mRNA was detected in adipose tissue but not in skeletal muscle. Further characterisation of genes related to glucose metabolism in cats will provide additional insights into insulin-signalling mechanisms in this species.

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes.

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.

LPS induces interleukin-6 and interleukin-8 but not tumor necrosis factor-alpha in human adipocytes.

Adipose tissue-derived cytokines are presumably involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. Here we studied the lipopolysaccharide (LPS)-induced expression dynamics of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 in human adipose tissue biopsies, in preadipocyte-derived adipocytes, and in mesenchymal stem cell (MSC)-derived adipocytes. TNFalpha, IL-6, IL-8 and IL-10 secretions by adipose tissue explants were increased 5.5-, 19.5-, 3.5- and 12.5-fold, respectively, by LPS (1 microg/mL) administration. Concordantly, IL-6 and IL-8 release was dose-dependently induced in MSC-derived adipocytes by LPS (>10 pg/mL). In contrast, TNFalpha and IL-10 remained undetectable even at the highest LPS dose (1 microg/mL) after 24h. In MSC- and preadipocyte-derived adipocytes, respectively, exposure to LPS evoked a weak and transient induction of TNFalpha mRNA whereas induction of IL-6 and IL-8 mRNA were pronounced and sustained for at least 24h. Basal glucose uptake, lipolysis and IL-6 mRNA were induced by exogenous TNFalpha (10 ng/mL) but not by IL-6 (10 ng/mL), IL-8 (100 ng/mL) and IL-10 (20 ng/mL). In this adipocyte model TNFalpha induces well known metabolic effects, but together with previous reports these data suggest that inflammation-induced TNFalpha may derive from non-adipocyte sources in adipose tissue, likely to be macrophages.

Multipotential nestin and Isl-1 positive mesenchymal stem cells isolated from human pancreatic islets.

Mesenchymal cells in the developing pancreas express the neural stem cell marker nestin and the transcription factor islet-1 (Isl-1). Using defined culture conditions we isolated on a single cell basis nestin producing cells from human pancreatic islets. These cells were immortalized with lentiviral vectors coding for telomerase and mBmi. They are positive for Isl-1 and nestin and have the potential to adopt a pancreatic endocrine phenotype with expression of critical transcription factors including Ipf-1, Isl-1, Ngn-3, Pax4, Pax6, Nkx2.2, and Nkx6.1 as well as the islet hormones insulin, glucagon, and somatostatin. In addition, they can be differentiated into human albumin producing cells in vivo when grafted into a SCID mouse liver. In accordance with a mesenchymal phenotype, the cells were also able to adopt adipocytic or osteocytic phenotypes in vitro. In conclusion, cultured pancreatic islets contain nestin and Isl-1 positive mesenchymal stem cells with multipotential developmental capacity.

Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells.

Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin.

Cytokine-induced metabolic effects in human adipocytes are independent of endogenous nitric oxide.

Nitric oxide (NO) has been recognized as a potential mediator of inflammation-induced metabolic alterations, including insulin resistance. However, expression mechanisms and potential roles of endothelial and inducible NO synthases (eNOS and iNOS, respectively) in human adipocytes are poorly understood. In the present study, we aimed to analyze several aspects of NO-related gene expression and metabolite synthesis in basal and inflammation-activated human adipocyte models. eNOS mRNA was highly expressed in omental and to a lesser extent in human subcutaneous adipose tissue biopsies, but not in purified adipocytes, in mesenchymal stem cell (MSC)- and in preadipocyte-derived adipocytes, respectively. Trace amounts of iNOS mRNA were detected in adipose tissue samples of donors with abdominal infection, as opposed to noninfected subjects. Interferon-gamma, in combination with interleukin-1beta or lipopolysaccharide, evoked a transient (4 h < time < 24 h) iNOS mRNA expression in human MSC and preadipocyte-derived adipocytes, respectively. This induction was preceded by cytokine-specific mRNAs. In addition, it was accompanied by an activation of the tetrahydrobiopterin synthesis pathway and by inhibition of peroxisome proliferator-activated receptor-gamma2. In contrast to murine 3T3-L1-derived adipocytes, iNOS protein and NO oxidation products remained undetectable in iNOS mRNA-positive human adipocytes. Accordingly, coadministration of NOS inhibitors (i.e., Nomega-nitro-L-arginine methyl ester, Nomega-monomethyl-L-arginine, and 1400W) had no effects on insulin-mediated glucose uptake and lipolysis. We conclude that, in human adipocytes, endogenous NO is not involved in metabolic regulation during either basal or cytokine-activated conditions.

Autocrine/paracrine role of inflammation-mediated calcitonin gene-related peptide and adrenomedullin expression in human adipose tissue.

Human adipose tissue is a contributor to inflammation- and sepsis-induced elevation of serum procalcitonin (ProCT). Several calcitonin (CT) peptides, including ProCT, CT gene-related peptide (CGRP), and adrenomedullin (ADM) are suspected mediators in human inflammatory diseases. Therefore, we aimed to explore the expression, interactions, and potential roles of adipocyte-derived CT peptide production. Expression of CT peptide-specific transcripts was analyzed by RT-PCR and quantitative real-time PCR in human adipose tissue biopsies and three different inflammation-challenged human adipocyte models. ProCT, CGRP, and ADM secretions were assessed by immunological methods. Adipocyte transcriptional activity, glycerol release, and insulin-mediated glucose transport were studied after exogenous CGRP and ADM exposure. With the exception of amylin, CT peptides were expressed in adipose tissue biopsies from septic patients, inflammation-activated mature explanted adipocytes, and macrophage-activated preadipocyte-derived adipocytes. ProCT and CGRP productions were significantly augmented in IL-1beta and lipopolysaccharide-challenged mesenchymal stem cell-derived adipocytes but not in undifferentiated mesenchymal stem cells. In contrast, ADM expression occurred before and after adipogenic differentiation. Interferon-gamma coadministration inhibited IL-1beta-mediated ProCT and CGRP secretion by 78 and 34%, respectively but augmented IL-1beta-mediated ADM secretion by 50%. Exogenous CGRP and ADM administration induced CT, CGRP I, and CGRP II mRNAs and dose-dependently (10(-10) and 10(-6) m) enhanced glycerol release. In contrast, no CGRP- and ADM-mediated effects were noted on ADM, TNFalpha, and IL-1beta mRNA abundances. In summary, CGRP and ADM are two differentially regulated novel adipose tissue secretion factors exerting autocrine/paracrine roles. Their lipolytic effect (glycerol release) suggests a metabolic role in adipocytes during inflammation.

Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation.

Somatostatin (SRIF) is a well-known neuroendocrine secretion product. SRIF expression and secretion are induced after inflammation in murine macrophages and in endotoxin-injected sheep and pigs. Because adipocytes have been demonstrated to produce numerous cytokines and peptide hormones, we investigated the expression of SRIF and its receptors (SSTR1-5) in human adipose tissue after inflammatory stimulation in vitro and in tissues from patients with septic disease.Preadipocyte-derived adipocytes, mesenchymal stem cell-derived adipocytes, and mature explanted adipocytes expressed SRIF-mRNA after endotoxin [lipopolysaccharide (LPS)] or IL-1beta treatments. LPS- and IL-1beta-mediated SRIF-mRNA induction was blocked by pretreatment with dexamethasone. Using cocultures and quantitative real-time PCR, we demonstrate adipocyte SRIF induction by secretion factors from activated peripheral blood mononuclear cell-derived macrophages. In contrast to basal adipocytes, SRIF protein was detected in culture supernatants of LPS-treated and of combined TNFalpha/IL-1beta/LPS-treated adipocytes. SRIF protein was visualized by immunohistochemistry in explanted minced adipose tissue after overnight incubation in culture medium supplemented with combined IL-1beta and LPS. In septic patients, expression of SRIF-mRNA and SRIF protein was found in visceral, but not in sc, adipose tissue. Adipocyte mRNA abundance of SSTR 1-5 was differentially regulated by inflammatory treatments.Thus, human visceral adipose tissue secretes SRIF during inflammation and sepsis and expresses several SSTRs. It is tempting to speculate that visceral adipose tissue-derived SRIF plays a modulatory role in the immunological and metabolic response to inflammation.

Expression and secretion of procalcitonin and calcitonin gene-related peptide by adherent monocytes and by macrophage-activated adipocytes.

OBJECTIVE: To explore the roles of peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophages in sepsis-related increased procalcitonin and calcitonin gene-related peptide (CGRP) I production. DESIGN: Prospective, in vitro primary human cell culture study and human tissue samples gene expression analysis. SETTING: University hospital research laboratories. PATIENTS: Cells from healthy donors and septic patients. INTERVENTIONS: PBMCs were obtained from healthy donors. Isolation of pure monocyte cultures was performed by magnetic depletion of nonmonocyte cells from PBMCs. Adipose tissue biopsies and circulating leukocytes were collected from septic patients. Expressions of calcitonin messenger RNA and CGRP I messenger RNA were analyzed using reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction. Supernatant procalcitonin and CGRP protein content were determined by ultrasensitive chemiluminometric and radioimmunoassays, respectively. MEASUREMENTS AND MAIN RESULTS: PBMCs expressed and secreted procalcitonin and CGRP within 3-5 hrs after adherence to endothelial cells or plastic surfaces. This induction was transient, as it was not detectable after 18 hrs. No calcitonin or CGRP I messenger RNA was observed in leukocytes obtained from septic patients with markedly increased serum procalcitonin concentrations. Stimulation with cytokines, endotoxin, or Escherichia coli did not induce expression of calcitonin and CGRP I messenger RNA in PBMC-derived macrophages. However, inflammatory factors released from activated macrophages induced a marked expression of procalcitonin and CGRP in co-cultured human adipocytes. CONCLUSIONS: The adhesion-induced, transient expression and secretion of procalcitonin and CGRP in vitro may play an important role during monocyte adhesion and migration in vivo. PBMC-derived macrophages may contribute to the marked increase in circulating procalcitonin by recruiting parenchymal cells within the infected tissue, as exemplified with adipocytes.

In vitro and in vivo calcitonin I gene expression in parenchymal cells: a novel product of human adipose tissue.

Circulating levels of calcitonin precursors (CTpr), including procalcitonin (ProCT), increase up to several thousand-fold in human sepsis, and immunoneutralization improves survival in two animal models of this disease. Herein, we analyzed inflammation-mediated calcitonin I gene (CALC I) expression in human adipocyte primary cultures and in adipose tissue samples from infected and noninfected patients with different levels of serum ProCT. In ex vivo differentiated adipocytes, the expression of CT mRNA increased 24-fold (P < 0.05) after the administration of Escherichia coli endotoxin (lipopolysaccharide) and 37-fold (P < 0.05) after IL-1beta administration by 6 h. ProCT protein secretion into culture supernatant increased 13.5-fold (P < 0.01) with lipopolysaccharide treatment and 15.2-fold (P < 0.01) with IL-1beta after 48 h. In coculture experiments, adipocyte CT mRNA expression was evoked by E. coli-activated macrophages in which CT mRNA was undetectable. The marked IL-1beta-mediated ProCT release was inhibited by 89% during coadministration with interferon-gamma (IFNgamma). In patients with infection and markedly increased serum ProCT, CT mRNA was detected in adipose tissue biopsies. Hence, we demonstrate that ProCT, which is suspected to mediate deleterious effects in sepsis and inflammation, is a novel product of adipose tissue secretion. The inhibiting effect of IFNgamma on IL-1beta-induced CT mRNA expression and on ProCT secretion might explain previous observations that serum ProCT concentrations increase less in systemic viral compared with bacterial infections.

Diminished production of nitric oxide synthase cofactor tetrahydrobiopterin by rosiglitazone in adipocytes.

Increased nitric oxide (NO) synthesis has been proposed to participate in the generation of insulin resistance in adipose and muscle tissues. Therefore, we examined the potential rate-limiting role of tetrahydrobiopterin (BH4) in cytokine-induced NO synthesis, and the effect of peroxisome proliferator activated receptor-gamma (PPARgamma) activation using the insulin-sensitizer rosiglitazone on cytokine-induced BH4 synthesis in 3T3-L1 adipocytes. Our data indicate that modulated availability of the mandatory nitric oxide synthase (NOS) cofactor BH4 affected cytokine-induced NO generation. Semiquantitative linear range reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated that rosiglitazone not only reduced inducible nitric oxide synthase (iNOS) mRNA transcription, but also guanosine triphosphate cyclohydrolase (GTPCH), the rate-limiting and controlling step of BH4 synthesis. Accordingly, intracellular BH4 concentration was reduced by 45% following rosiglitazone treatment. Furthermore, we observed a transient inhibitory effect of natural PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PJ2) on cytokine-mediated iNOS and GTPCH induction. Thus, the inhibition of cytokine-induced NO synthesis by rosiglitazone is at least in part attributable to reduced availability of BH4, the synthesis of which might represent a potential new target in the treatment of type 2 diabetes and insulin resistance.