Marjorie Poggi

Metabolic endotoxemia initiates obesity and insulin resistance

Diabetes and obesity are two metabolic diseases characterized by insulin resistance and a low-grade inflammation. Seeking an inflammatory factor causative of the onset of insulin resistance, obesity, and diabetes, we have identified bacterial lipopolysaccharide (LPS) as a triggering factor. We found that normal endotoxemia increased or decreased during the fed or fasted state, respectively, on a nutritional basis and that a 4-week high-fat diet chronically increased plasma LPS concentration two to three times, a threshold that we have defined as metabolic endotoxemia. Importantly, a high-fat diet increased the proportion of an LPS-containing microbiota in the gut. When metabolic endotoxemia was induced for 4 weeks in mice through continuous subcutaneous infusion of LPS, fasted glycemia and insulinemia and whole-body, liver, and adipose tissue weight gain were increased to a similar extent as in high-fat–fed mice. In addition, adipose tissue F4/80-positive cells and markers of inflammation, and liver triglyceride content, were increased. Furthermore, liver, but not whole-body, insulin resistance was detected in LPS-infused mice. CD14 mutant mice resisted most of the LPS and high-fat diet–induced features of metabolic diseases. This new finding demonstrates that metabolic endotoxemia dysregulates the inflammatory tone and triggers body weight gain and diabetes. We conclude that the LPS/CD14 system sets the tone of insulin sensitivity and the onset of diabetes and obesity. Lowering plasma LPS concentration could be a potent strategy for the control of metabolic diseases.

C3H/HeJ mice carrying a toll-like receptor 4 mutation are protected against the development of insulin resistance in white adipose tissue in response to a high-fat diet

Aims/hypothesisInflammation is associated with obesity and has been implicated in the development of diabetes and atherosclerosis. During gram-negative bacterial infection, lipopolysaccharide causes an inflammatory reaction via toll-like receptor 4 (TLR4), which has an essential function in the induction of innate and adaptative immunity. Our aim was to determine what role TLR4 plays in the development of metabolic phenotypes during high-fat feeding.Materials and methodsWe evaluated metabolic consequences of a high-fat diet in TLR4 mutant mice (C3H/HeJ) and their respective controls.ResultsTLR4 inactivation reduced food intake without significant modification of body weight, but with higher epididymal adipose tissue mass and adipocyte hypertrophy. It also attenuated the inflammatory response and increased glucose transport and the expression levels of adiponectin and lipogenic markers in white adipose tissue. In addition, TLR4 inactivation blunted insulin resistance induced by lipopolysaccharide in differentiated adipocytes. Increased feeding efficiency in TLR4 mutant mice was associated with lower mass and lower expression of uncoupling protein 1 gene in brown adipose tissue. Finally, TLR4 inactivation slowed the development of hepatic steatosis, reducing the liver triacylglycerol content and also expression levels of lipogenic and fibrosis markers.Conclusions/interpretationTLR4 influences white adipose tissue inflammation and insulin sensitivity, as well as liver fat storage, and is important in the regulation of metabolic phenotype during a fat-enriched diet.

Plasminogen activator inhibitor-1, adipose tissue and insulin resistance

Purpose of review Plasminogen activator inhibitor (PAI)-1 is a physiological inhibitor of plasminogen activators (urokinase and tissue types) and vitronectin. It is synthesized by adipose tissue, and its levels in plasma are increased in obesity and reduced with weight loss. Circulating PAI-1 level predicts development of type 2 diabetes, suggesting that it may be causally related to development of obesity. A role for PAI-1 in development of obesity has only partially been established, however. This review summarizes current knowledge, gives context to developments thus far and discusses controversies. Recent findings In addition to its role in atherothrombosis, PAI-1 might be involved in adipose tissue development. PAI-1 is produced by ectopic fat depots under the influence of inducers. Among the most recently described inducers are inflammation, oxidative stress and circadian clock protein. PAI-1 may play several roles in contributing to obesity: through indirect effects on insulin signalling, by influencing adipocyte differentiation and by regulating recruitment of inflammatory cells within adipose tissue. Summary These recent findings emphasize the involvement of PAI-1 in controlling the biology of adipose tissue; PAI-1 is an attractive new therapeutic target to retard the metabolic complications that accompany obesity.

Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

BackgroundIt is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.ResultsA proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.ConclusionGiven its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

The inflammatory receptor CD40 is expressed on human adipocytes: contribution to crosstalk between lymphocytes and adipocytes.

Aims/hypothesisObesity is associated with adipose tissue inflammation. The CD40 molecule, TNF receptor superfamily member 5 (CD40)/CD40 ligand (CD40L) pathway plays a role in the onset and maintenance of the inflammatory reaction, but has not been studied in human adipose tissue. Our aim was to examine CD40 expression by human adipocytes and its participation in adipose tissue inflammation.MethodsCD40 expression was investigated in human whole adipose tissue and during adipocyte differentiation by real-time PCR, Western blot and immunohistochemistry. The CD40/CD40L pathway was studied using recombinant CD40L (rCD40L) in adipocyte culture and neutralising antibodies in lymphocyte/adipocyte co-culture.ResultsCD40 mRNA levels in subcutaneous adipose tissue were higher in the adipocyte than in the stromal–vascular fraction. CD40 expression was upregulated during adipocyte differentiation. Addition of rCD40L to adipocytes induced mitogen activated protein kinase (MAPK) activation, stimulated inflammatory adipocytokine production, and decreased insulin-induced glucose transport in parallel with a downregulation of IRS1 and GLUT4 (also known as SCL2A4). rCD40L decreased the expression of lipogenic genes and increased lipolysis. CD40 mRNA levels were significantly higher in subcutaneous adipose tissue than in visceral adipose tissue of obese patients and were positively correlated with BMI, and with IL6 and leptin mRNA levels. Lymphocyte/adipocyte co-culture led to an upregulation of proinflammatory adipocytokines and a downregulation of leptin and adiponectin. Physical separation of the two cell types attenuated these effects, suggesting the involvement of a cell–cell contact. Blocking the CD40/CD40L interaction with neutralising antibodies reduced IL-6 secretion from adipocytes.Conclusions/interpretationAdipocyte CD40 may contribute to obesity-related inflammation and insulin resistance. T lymphocytes regulate adipocytokine production through both the release of soluble factor(s) and heterotypic contact with adipocytes involving CD40.

Plasmacytoid Dendritic Cells Protect Against Atherosclerosis by Tuning T-Cell Proliferation and Activity

Rationale: Unlike conventional dendritic cells, plasmacytoid DCs (PDC) are poor in antigen presentation and critical for type I interferon response. Though proposed to be present in human atherosclerotic lesions, their role in atherosclerosis remains elusive. Objective: To investigate the role of PDC in atherosclerosis. Methods and Results: We show that PDC are scarcely present in human atherosclerotic lesions and almost absent in mouse plaques. Surprisingly, PDC depletion by 120G8 mAb administration was seen to promote plaque T-cell accumulation and exacerbate lesion development and progression in LDLr−/− mice. PDC depletion was accompanied by increased CD4+ T-cell proliferation, interferon-&ggr; expression by splenic T cells, and plasma interferon-&ggr; levels. Lymphoid tissue PDC from atherosclerotic mice showed increased indoleamine 2,3-dioxygenase (IDO) expression and IDO blockage abrogated the PDC suppressive effect on T-cell proliferation. Conclusions: Our data reveal a protective role for PDC in atherosclerosis, possibly by dampening T-cell proliferation and activity in peripheral lymphoid tissue, rendering PDC an interesting target for future therapeutic interventions.

Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance.

Significance Inflammation is a critical contributor to the pathogenesis of metabolic disorders associated with obesity. A group of molecules crucial in regulating the immune system are costimulatory molecules, including CD40. Our current study shows that CD40 acts as a double-edged sword in the metabolic syndrome through the initiation of differential signaling cascades. The CD40-TNF receptor-associated factor (TRAF) 2/3/5 signaling pathway protects against metabolic dysfunction and inflammation associated with obesity; conversely, the CD40-TRAF6 pathway contributes to the detrimental consequences of obesity. In the present study, we therefore designed, validated, and used a small-molecule inhibitor that blocks CD40-TRAF6 interactions. The improvement of insulin resistance by this specific CD40-TRAF6 inhibitor could represent a therapeutic breakthrough in the field of immunometabolism. The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40−/− mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40−/− mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance.

CD40L Deficiency Ameliorates Adipose Tissue Inflammation and Metabolic Manifestations of Obesity in Mice

Objective—Obese adipose tissue shows hallmarks of chronic inflammation, which promotes the development of metabolic disorders. The mechanisms by which immune cells interact with each other or with metabolism-associated cell types, and the players involved, are still unclear. The CD40-CD40L costimulatory dyad plays a pivotal role in immune responses and in diseases such as atherosclerosis and may therefore be a mediator of obesity. Here we investigated whether CD40L is involved in adipose tissue inflammation and its associated metabolic changes. Methods and Results—To assess a putative role of CD40L in obesity in vivo, we evaluated metabolic and inflammatory consequences of 18 weeks of high-fat feeding in CD40L+/+ and CD40L−/− mice. In addition, C57Bl6 mice were injected with neutralizing anti-CD40L (&agr;CD40L) antibody for 12 weeks while being fed a high-fat diet. Genetic deficiency of CD40L attenuated the development of diet-induced obesity, hepatic steatosis, and increased systemic insulin sensitivity. In adipose tissue, it impaired obesity-induced immune cell infiltration and the associated deterioration of glucose and lipid metabolism. Accordingly, &agr;CD40L treatment improved systemic insulin sensitivity, glucose tolerance, and CD4+ T-cell infiltration in adipose tissue with limited effects on adipose tissue weight. Conclusion—CD40L plays a crucial role in the development of obesity-induced inflammation and metabolic complications.

HDLs activate ADAM17‐dependent shedding

The tumor necrosis factor‐alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high‐density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs‐stimulated TNF release occurred also on cell‐free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1‐dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17‐dependent cleavage of TNF in the non‐raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17‐dependent processing of transmembrane substrates. J. Cell. Physiol. 214: 687–693, 2008.

Down regulation of tissue inhibitor of metalloproteinase-3 (timp-3) expression is necessary for adipocyte differentiation

Matrix metalloproteinase activity is essential for proper extracellular matrix remodeling that takes place during adipose tissue formation. Four tissue inhibitors of matrix metalloproteinases (TIMPs) regulate their activity. However, the role of TIMPs in adipocyte differentiation is poorly understood. We found that the expression of all TIMPs was modified during adipocyte differentiation, but that of TIMP-3 was distinguished by its extreme down-regulation. TIMP-3 expression was closely linked to the differentiation process. Indeed, it remained low during the adipocyte differentiation but increased when cell differentiation was prevented. We identified the transcription factor Sp1 as being responsible for the regulation of TIMP-3 expression during adipocyte differentiation. Overexpression of TIMP-3 reduced adipocyte differentiation, underlining its active role in this process. TIMP-3 overexpression decreased the expression of the early and obligate key inductors of adipogenesis Krüppel-like factor 4 (Klf4), early growth response 2 (Egr2/Krox20), and CAAT/enhancer-binding protein β (C/EBPβ). Our results indicate that during preadipocyte differentiation, the Sp1-dependent decrease in TIMP-3 expression is required for the successful implementation of the adipocyte differentiation program.