Role of inflammatory pathways in insulin resistance development in obese subjects without comorbidities

L’obésité est une pathologie multifactorielle qui contribue au développement de l’insulinorésistance (l’IR). De nos jours, même si de nombreux travaux ont permis de répondre à plusieurs questions relatives à la pathogenèse de l’IR, les mécanismes cellulaires et moléculaires qui sous-tendent la pathogenèse de l’IR ne sont pas encore complètement élucidés chez l’Homme. Deux hypothèses principales se sont dégagées de ces travaux.D’une part, certaines études suggèrent que le défaut primaire à l’origine de l’IR se trouverait au niveau du tissu adipeux, qui en libérant dans la circulation systémique des adipokines, des cytokines inflammatoires et des acides gras contribue au développement d’une inflammation et d’une hyperlipidémie systémique. Ces deux paramètres exercent des effets délétères sur la sensibilité à l’insuline des autres organes comme le muscle et le foie. D’autre part, le muscle étant l’organe gluco-régulateur par excellence en situation postprandiale, d’autres études le place au cœur de la physiopathologie de l’IR.Malgré ces controverses, aucune étude n’a à ce jour exploré simultanément l’inflammation et l’IR systémique /tissulaire dans le muscle et le tissu adipeux chez un même sujet. Dans ce contexte, l’objectif de ce travail de thèse était de mieux comprendre les mécanismes impliqués dans la mise en place de l’IR. Il s’inscrit dans le cadre d’un projet de recherche translationnel, qui compare dans une cohorte de femmes ménopausées, des sujets minces à des sujets obèses de grade I insulinosensibles (OIS) et insulinorésistants (OIR). Plusieurs paramètres systémiques et tissulaires (lipotoxicité, inflammation et activation du récepteur toll like receptor 4 (TLR4)) impliqués dans la physiopathologie de l’IR ont été analysés chez ces sujets.Les résultats de cette étude mettent en avant l’importance de l’activation des voies de l’immunité innée dans la régulation de la sensibilité à l’insuline. Ainsi, alors qu’aucune inflammation systémique n’est détectée, on observe une activation différentielle des voies de signalisation du récepteur TLR4 de l’immunité innée entre le tissu musculaire et le tissu adipeux des sujets OIR. La voie MyD88-dépendante qui est une voie pro-inflammatoire, est activée dans le muscle squelettique de ces sujets et est associée à une IR au sein de ce tissu. A l’inverse, la voie TRIF-dépendante qui est une voie anti-inflammatoire est activée au sein du tissu adipeux et permet le maintien de la sensibilité à l’insuline. Ce maintien de la réponse à l’insuline se fait grâce l’induction du système interféron et de l’enzyme anti-oxydante manganèse superoxyde dismutase (MnSOD). Dans ce travail, nous montrons d’une part, que le défaut de réponse à l’insuline musculaire se trouve au cœur de la physiopathologie de l’IR chez les sujets obèses de grade I. D’autre part, ces résultats mettent en exergue l’importance de la balance de régulation des voies de l’immunité innée « MyD88 » et « TRIF » dans le maintien de la sensibilité à l’insuline.Obesity is a multifactorial disease promoting the development of insulin resistance (IR). Nowadays, cellular and molecular mechanisms underlying IR pathogenesis in humans are not fully understood although many studies have been attempted to improve our knowledge. Tow hypotheses arose from these researches.On one hand, several studies have led to the idea that a chronic inflammatory state combined with adipose tissue (AT) dysfunction could be the so-called “central mechanism” leading to IR. AT dysfunction is associated with increase in the release of inflammatory adipokines/cytokines and free fatty acid (FFA), leading to systemic inflammation and lipid overload. These latter parameters would have deleterious effects on diverse organs such as muscles and liver by affecting their insulin sensitivityOn the other hand, skeletal muscle (SM) is responsible for 80% of glucose uptake and metabolism in postprandial state and muscle failure in this function is often considered as the first defect causing IR.Interestingly, despite these controversies, in human, insulin sensitivity and the onset of inflammation have so far never been investigated simultaneously at systemic level and locally in SM and AT in the same individual.In this context, the aim of this thesis was to better understand the mechanisms involved in IR development in grade I obese subject. It is based on a translational research project that compares in a cohort of postmenopausal women, lean subjects with grade I obese insulin-sensitive (OIS) and insulin-resistant (OIR) subjects. Several systemic and tissue parameters (lipotoxicity, inflammation and toll like receptor 4 (TLR4) activation), involved in the IR pathophysiology were analyzed in these subjects.Our results highlight the importance of innate immunity pathways activation in the regulation of insulin sensitivity. Thus, while no inflammation was detected at the systemic level, there is a differential activation of innate immune TLR4 signaling pathways between muscle and AT of OIR subjects. The MyD88-dependent pathway, a pro-inflammatory pathway, is activated in their SM and is associated with IR in this tissue. Conversely, the TRIF-dependent pathway which is an anti-inflammatory pathway is activated in the AT thus maintaining insulin sensitivity in this tissue. This is supported by the induction of interferon system and the antioxidant enzyme manganese superoxide dismutase (MnSOD).In this work, we show that SM defect is the central mechanism which determines IR in grade I obese subject. We have also highlighted the importance of regulation of the balance between the two innate immunity pathways "MyD88" and "TRIF" in maintaining insulin sensitivity

Decreased RNF41 expression leads to insulin resistance in skeletal muscle of obese women

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Skeletal Muscle Insulin Resistance and Absence of Inflammation Characterize Insulin-Resistant Grade I Obese Women

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Inflammation and fibrosis in skeletal muscle.

<p><b>Panel A:</b> Total macrophages (CD68⁺), and pro-inflammatory (CD68⁺/CD86⁺) and anti-inflammatory (CD68⁺/CD206⁺) macrophages were counted after staining in skeletal muscle. For CD68, and CD86 or CD206 markers, four and two non-consecutive entire sections per subject were respectively analyzed for 6 CT, 6 OIS and 5 OIR subjects. Data are expressed as mean ± SEM. <b>Panel B:</b> Relative quantification of Col5A, Col6A, TNFα, IL-1β, IL-6 and MCP-1, mRNA expression in skeletal muscle (n_CT = 10, n_OIS = 11, n_OIR = 9). Data are expressed as mean ± SEM and relatively to CT values, which were set at 1.0. <b>Panel C:</b> Western blot analysis of IκBα expression in skeletal muscle (n_CT = 10, n_OIS = 9, n_OIR = 9). The graph represents IκBα protein quantification after correction by α/β tubulin protein levels, used as an indicator of protein loading. Data are expressed as mean ± SEM and relatively to CT values, which were set at 1.0. $ P_{OIR <i>vs</i>. CT} = 0.04.</p

Insulin response in SAT.

<p><b>Panel A:</b> Western blot analysis of P-Akt/Akt <i>ratio</i> in SAT with and without incubation with insulin (n_CT = 9, n_OIS = 10, n_OIR = 9). The graph represents P-Akt protein quantification after correction by total Akt protein levels, used as an indicator of protein loading. Data are expressed as mean ± SEM and relatively to CT values, which were set at 1.0. § P_CT±insulin = 0.008; P_OIS±insulin = 0.002; P_OIR±insulin = 0.004. <b>Panel B:</b> Fold induction of P-Akt/Akt level in SAT after insulin stimulation. Data are expressed as mean ± SEM. <b>Panels C and D:</b> Correlations (Spearman analysis) between insulin-stimulated P-Akt/Akt fold induction in SAT and HOMA_IR (C) or GIR (D).</p

Insulin response in skeletal muscle.

<p><b>Panel A:</b> Western blot analysis of P-Akt/Akt <i>ratio</i> in skeletal muscle with and without incubation with insulin (n_CT = 10, n_OIS = 11, n_OIR = 8). The graph represents P-Akt protein quantification after correction by total Akt protein levels, used as an indicator of protein loading. Data are expressed as mean ± SEM and relatively to CT values, which were set at 1.0. § P_CT±insulin = 0.002; P_OIS±insulin = 0.002. P_OIR±insulin = 0.129. <b>Panel B:</b> Fold induction of P-Akt/Akt in skeletal muscle after insulin stimulation. Data are mean ± SEM. $ P_{OIR <i>vs</i>. CT} = 0.008 and # P_{OIR <i>vs</i>. OIS} = 0.0005. <b>Panels C and D:</b> Correlations (Spearman analysis) between insulin-stimulated P-Akt/Akt fold induction in skeletal muscle and HOMA_IR (C) or GIR (D).</p

Clinical and biological characteristics of the subjects.

<p>Clinical and biological characteristics of the subjects.</p

Levels of adipokines and markers of systemic inflammation.

<p>Levels of adipokines and markers of systemic inflammation.</p

Inflammation and fibrosis in SAT.

<p><b>Panel A:</b> Total macrophages (CD68⁺), pro-inflammatory (CD68⁺/CD86⁺), and anti-inflammatory (CD68⁺/CD206⁺) macrophages were counted after staining in SAT. For CD68, and CD86 or CD206 markers, four and two non-consecutive entire sections per subject were analyzed for 7 CT, 5 OIS and 6 OIR subjects. Data are expressed as mean ± SEM. $: P_{OIR <i>vs</i>. CT} = 0.008. <b>Panel B:</b> Adipocyte average size (μm²) in SAT; 10–15 sections per subject were respectively analyzed for 4 subjects per group. Data are expressed as mean ± SEM. <b>Panel C:</b> Relative quantification of Col5A, Col6A, TNFα, IL-1β, IL-6 and MCP-1 mRNA expression in SAT (n_CT = 10, n_OIS = 11, n_OIR = 9). Data are expressed as mean ± SEM and relatively to CT values, which were set at 1.0.$ P_{OIR <i>vs</i>. CT} = 0.004. <b>Panel D:</b> Western blot analysis of IκBα expression in SAT (n_CT = 9, n_OIS = 8, n_OIR = 8). The graph represents IκBα protein quantification after correction by α/β tubulin protein levels, used as an indicator of protein loading. Data are expressed as mean ± SEM and relatively to CT values, which were set at 1.0.</p