Chantal Benelli

The Infrapatellar Fat Pad in Knee Osteoarthritis: An Important Source of Interleukin-6 and Its Soluble Receptor

OBJECTIVE Obesity is a potent risk factor in knee osteoarthritis (OA). It has been suggested that adipokines, secreted by adipose tissue (AT) and largely found in the synovial fluid of OA patients, derive in part from the infrapatellar fat pad (IFP), also known as Hoffas fat pad. The goal of this study was to characterize IFP tissue in obese OA patients and to compare its features with thigh subcutaneous AT to determine whether the IFP contributes to local inflammation in knee OA via production of specific cytokines. METHODS IFP and subcutaneous AT samples were obtained from 11 obese women (body mass index > or =30 kg/m2) with knee femorotibial OA. Gene expression was measured by real-time quantitative polymerase chain reaction. Cytokine concentrations in plasma and in conditioned media of cultured AT explants were determined by enzyme-linked immunosorbent assay or by Luminex xMAP technology. RESULTS In IFP tissue versus subcutaneous AT, there was a decrease in the expression of genes for key enzymes implicated in adipocyte lipid metabolism, whereas the expression levels of genes for AT markers remained similar. A 2-fold increase in the expression of the gene for interleukin-6 (IL-6), a 2-fold increase in the release of IL-6, and a 3.6-fold increase in the release of soluble IL-6 receptor (sIL-6R) were observed in IFP samples, compared with subcutaneous AT, but the rates of secretion of other cytokines in IFP samples were similar to the rates in subcutaneous AT. In addition, leptin secretion was decreased by 40%, whereas adiponectin secretion was increased by 70%, in IFP samples versus subcutaneous AT. CONCLUSION Our results indicate that the IFP cytokine profile typically found in OA patients could play a role in paracrine inflammation via the local production of IL-6/sIL-6R and that such a profile might contribute to damage in adjacent cartilage.

Expression of macrophage-selective markers in human and rodent adipocytes

CD14, CD68 and/or mouse F4/80 or human epidermal growth factor module‐containing mucin‐like receptor 1 (EMR1) are widely used as macrophage‐specific markers. Since macrophages infiltrate several tissues during inflammatory processes, CD14, CD68 and EMR1‐F4/80 have been employed to discriminate between tissue‐containing macrophages, like adipose tissue (AT), and other cells. Using real‐time PCR experiments, we show that isolated adipocytes from humans and mice AT express high levels of CD14 and CD68 mRNA, whereas EMR1‐F4/80 is mainly present in the macrophage‐containing stroma‐vascular fraction. Furthermore, fibroblasts‐like cells (adipoblasts), preadipocytes and adipocytes from the murine cell lines, 3T3‐F442A and BFC‐1, express CD14 and CD68 mRNA and protein as determined by fluorescence‐activated cell sorter, but not F4/80 which, as expected, is strongly expressed in the macrophage cell line RAW264.7. These results reinforce the view that EMR1‐F4/80 is the best macrophage marker to date and show that CD14 and CD68 are not macrophage‐specific proteins.

Inflammatory pathway genes belong to major targets of persistent organic pollutants in adipose cells.

Background: Epidemiological studies emphasize the possible role of persistent organic pollutants (POPs) in obesity and the metabolic syndrome. These pollutants are stored in adipose tissue (AT). Objectives: Our aim was to study the effects of POPs on human adipose cells and rodent AT. Methods: Using human multipotent adipose-derived stem cells, we carried out large-scale gene expression analysis to identify the major pathways modified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl (PCB) congener 126 (PCB-126), and PCB-153 and to evaluate their toxic effects. The effects of TCDD on gene expression and AT histology were also assessed in mice. Results: The most significantly regulated genes in both precursor cells and adipocytes were those involved in the inflammatory/immune response, cancer, and metabolism pathways. Interestingly, the fold induction and the number of modulated genes were higher in precursors than in adipocytes, suggesting that the former could be more sensitive to the effect of pollutants. When cells were treated with combinations of pollutants, the effects of the AhR ligands TCDD and PCB-126 were dominant compared with those of the non-dioxin-like PCB-153. The effects of AhR ligands were reduced by the AhR antagonist α-naphthoflavone. The regulation of inflammatory pathway was observed in wild-type AT but not in AhR-knockout mice. Conclusions: Both in vitro and in vivo studies showed that adipose cells were targets of AhR ligands and suggest that inflammation is one of the main regulated pathways. These observations suggest a possible contribution of pollutants to low-grade AT inflammation that accompanies the pathogenesis of metabolic diseases.

Pyruvate Dehydrogenase Kinase 4: Regulation by Thiazolidinediones and Implication in Glyceroneogenesis in Adipose Tissue

OBJECTIVE—Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled by PDK. RESEARCH DESIGN AND METHODS—Rosiglitazone was administered to Zucker fa/fa rats, and then PDK4 and PDK2 mRNAs were examined in subcutaneous, periepididymal, and retroperitoneal WAT, liver, and muscle by real-time RT-PCR. Cultured WAT explants from humans and rats and 3T3-F442A adipocytes were rosiglitazone-treated before analyses of PDK2 and PDK4 mRNA and protein. Small interfering RNA (siRNA) was transfected by electroporation. Glyceroneogenesis was determined using [1-14C]pyruvate incorporation into lipids. RESULTS—Rosiglitazone increased PDK4 mRNA in all WAT depots but not in liver and muscle. PDK2 transcript was not affected. This isoform selectivity was also found in ex vivo–treated explants. In 3T3-F442A adipocytes, Pdk4 expression was strongly and selectively induced by rosiglitazone in a direct and transcriptional manner, with a concentration required for half-maximal effect at 1 nmol/l. The use of dichloroacetic acid or leelamine, two PDK inhibitors, or a specific PDK4 siRNA demonstrated that PDK4 participated in glyceroneogenesis, therefore altering nonesterified fatty acid release in both basal and rosiglitazone-activated conditions. CONCLUSIONS—These data show that PDK4 upregulation in adipocytes participates in the hypolipidemic effect of thiazolidinediones through modulation of glyceroneogenesis.

Hyperinsulinism and Hyperammonemia Syndrome: Report of Twelve Unrelated Patients

Hyperinsulinism and hyperammonemia syndrome has been reported as a cause of moderately severe hyperinsulinism with diffuse involvement of the pancreas. The disorder is caused by gain of function mutations in the GLUD1 gene, resulting in a decreased inhibitory effect of guanosine triphosphate on the glutamate dehydrogenase (GDH) enzyme. Twelve unrelated patients (six males, six females) with hyperinsulinism and hyperammonemia syndrome have been investigated. The phenotypes were clinically heterogeneous, with neonatal and infancy-onset hypoglycemia and variable responsiveness to medical (diazoxide) and dietary (leucine-restricted diet) treatment. Hyperammonemia (90–200 μmol/L, normal <50 μmol/L) was constant and not influenced by oral protein, by protein- and leucine-restricted diet, or by sodium benzoate or N-carbamylglutamate administration. The patients had mean basal GDH activity (18.3 ± 0.9 nmol/min/mg protein) not different from controls (17.9 ± 1.8 nmol/min/mg protein) in cultured lymphoblasts. The sensitivity of GDH activity to inhibition by guanosine triphosphate was reduced in all patient lymphoblast cultures (IC50, or concentrations required for 50% inhibition of GDH activity, ranging from 140 to 580 nM, compared with control IC50 value of 83 ± 1.0 nmol/L). The allosteric effect of ADP was within the normal range. The activating effect of leucine on GDH activity varied among the patients, with a significant decrease of sensitivity that was correlated with the negative clinical response to a leucine-restricted diet in plasma glucose levels in four patients. Molecular studies were performed in 11 patients. Heterozygous mutations were localized in the antenna region (four patients in exon 11, two patients in exon 12) as well as in the guanosine triphosphate binding site (two patients in exon 6, two patients in exon 7) of the GLUD1 gene. No mutation has been found in one patient after sequencing the exons 5–13 of the gene.

Induction of an inflammatory and prodegradative phenotype in autologous fibroblast-like synoviocytes by the infrapatellar fat pad from patients with knee osteoarthritis.

The infrapatellar fat pad (IFP) of the knee joint has an inflammatory phenotype in osteoarthritis (OA). Its close proximity to the synovial membrane suggests that the IFP could be involved in the induction of OA synovitis. This study was undertaken to investigate the response of fibroblast‐like synoviocytes (FLS) to autologous IFP and subcutaneous adipose tissue (SCAT) from patients with severe knee OA.

Butyrate elicits a metabolic switch in human colon cancer cells by targeting the pyruvate dehydrogenase complex

Butyrate, a short‐chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO2, they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate‐stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC.

The pyruvate dehydrogenase complex in cancer: An old metabolic gatekeeper regulated by new pathways and pharmacological agents

Cancer cells exhibit an altered metabolism which is characterized by a preference for aerobic glycolysis more than mitochondrial oxidation of pyruvate. This provides anabolic support and selective growth advantage for cancer cells. Recently, a new concept has arisen suggesting that these metabolic changes may be due, in part, to an attenuated mitochondrial function which results from the inhibition of the pyruvate dehydrogenase complex (PDC). This mitochondrial complex links glycolysis to the Krebs cycle and the current understanding of its regulation involves the cyclic phosphorylation and dephosphorylation by specific pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs).

Xenobiotic-Metabolizing Cytochromes P450 in Human White Adipose Tissue: Expression and Induction

Lipophilic pollutants can accumulate in human white adipose tissue (WAT), and the consequences of this accumulation are still poorly understood. Cytochromes P450 (P450s) have recently been found in rat WAT and shown to be inducible through mechanisms similar to those in the liver. The aim of our study was to describe the cytochrome P450 pattern and their induction mechanisms in human WAT. Explants of subcutaneous and visceral WAT and primary culture of subcutaneous adipocytes were used as WAT models, and liver biopsies and primary culture of hepatocytes were used as liver models to characterize P450 expression in both tissues. The WAT and liver models were then treated with typical P450 inducers (rifampicin, phenobarbital, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and lipophilic pollutants (lindane, prochloraz, and chlorpyrifos), and the effects on P450 expression were studied. P450 expression was considerably lower in WAT than in the liver, except for CYP1B1 and CYP2U1, which were the most highly expressed adipose P450s in all individuals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and prochloraz induced CYP1A1 and CYP1B1 expression in both tissues. The aryl hydrocarbon receptor was also present in WAT. In contrast, neither phenobarbital nor rifampicin treatment induced CYP2 or CYP3 mRNA in WAT, and constitutive androstane receptor and pregnane X receptor were almost undetectable. These results suggest that the mechanisms by which P450s of family 1 are regulated in the liver are also functional in human WAT, but those regulating CYP2 and CYP3 expression are not.

E1 pyruvate dehydrogenase deficiency in a child with motor neuropathy.

ABSTRACT: We report the case of a boy who developed a motor neuropathy during infectious episodes at 18 mo and 3 y of age. When he was 7 y old, he suffered persistent weakness and areflexia; his resting lactate and pyruvate values were 3.65 mM and 398 μM, respectively (controls: 1.1 ± 0.3 mM and 90 ± 22 μM), and an exercise test demonstrated a lactic acidosis (13.6 mM; controls: 6.4 ± 1.3 mM) with a high pyruvate level (537 μM; controls: 176 ± 15 μM) and a low lactate/pyruvate ratio (24.2; controls: 35 ± 2). The results of polarographic studies on muscle mitochondria suggested a defect in pyruvate oxidation (pyruvate 17 ng atom O/min/mg protein; controls: 115 ± 42), whereas glutamate, palmitoylcarnitine, and succinate were good respiratory substrates. The activity of total pyruvate dehydrogenase complex (PDHC) in muscle mitochondria and in fresh mononuclear cells was markedly decreased (9.7 and 0.054 nmol 14CO2/min/mg protein, respectively; controls: 123 ± 4.5 and 0.733 ± 0.03, respectively). Immunochemical analysis in muscle mitochondria demonstrated an absence of the α and β El PDHC subunits. After 2 y of treatment with 500 mg/d thiamine, the patient was clinically improved. A genetic study of the main regions of mutations (exon 10 and 11) in the X chromosome encoding for the El α subunit of PDHC did not show any mutation. These data indicate that, although genetically different, this case enters in a very rare category of patients with PDHC deficiency without cerebral dysfunction and improved by thiamine + L-carnitine therapy.