Machine Learning based histology phenotyping to investigate the epidemiologic and genetic basis of adipocyte morphology and cardiometabolic traits

Genetic studies have recently highlighted the importance of fat distribution, as well as overall adiposity, in the pathogenesis of obesity-associated diseases. Using a large study (n = 1,288) from 4 independent cohorts, we aimed to investigate the relationship between mean adipocyte area and obesity-related traits, and identify genetic factors associated with adipocyte cell size. To perform the first large-scale study of automatic adipocyte phenotyping using both histological and genetic data, we developed a deep learning-based method, the Adipocyte U-Net, to rapidly derive mean adipocyte area estimates from histology images. We validate our method using three state-of-the-art approaches; CellProfiler, Adiposoft and floating adipocytes fractions, all run blindly on two external cohorts. We observe high concordance between our method and the state-of-the-art approaches (Adipocyte U-net vs. CellProfiler: R2visceral = 0.94, P < 2.2 × 10-16, R2subcutaneous = 0.91, P < 2.2 × 10-16), and faster run times (10,000 images: 6mins vs 3.5hrs). We applied the Adipocyte U-Net to 4 cohorts with histology, genetic, and phenotypic data (total N = 820). After meta-analysis, we found that mean adipocyte area positively correlated with body mass index (BMI) (Psubq = 8.13 × 10-69, βsubq = 0.45; Pvisc = 2.5 × 10-55, βvisc = 0.49; average R2 across cohorts = 0.49) and that adipocytes in subcutaneous depots are larger than their visceral counterparts (Pmeta = 9.8 × 10-7). Lastly, we performed the largest GWAS and subsequent meta-analysis of mean adipocyte area and intra-individual adipocyte variation (N = 820). Despite having twice the number of samples than any similar study, we found no genome-wide significant associations, suggesting that larger sample sizes and a homogenous collection of adipose tissue are likely needed to identify robust genetic associations.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.C.A.G received a pump priming grant from Novo Nordisk to carry out this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.published version, accepted versio

Rôles des aldose réductases dans l'homéostasie des tissus adipeux blancs humains et murins

Aldose reductases are NADPH-dependent oxydoreductases described for their involvement in cellular detoxification and glucose reduction. The discovery of Akr1b7 expression in murine adipose tissue together with the prostaglandin F2α Synthase (PGFS) activity of some isoforms suggest unreleased biological roles for these enzymes. Prostaglandin F2α (PGF2α) inhibiting adipogenesis, this PGFS function highlights AKR1B potential involvement in white adipose tissue (WAT) physiology. This work aimed at characterising the expression of all AKR1B in both murine and human WAT and understanding their impact on adipose tissue homeostasis and especially on adipogenesis and lipolysis. We showed that all AKR1B were expressed in murine WAT. Akr1b3, Akr1b8 and Akr1b16 were both expressed in the stromal vascular fraction (containing immune cells, vascular cells, progenitors…) and in the adipose fraction. In contrast, Akr1b7 was not expressed in adipocytes. In vitro analyses indicated that, except for Akr1b16, murine AKR1B isoform expression increased early and transiently during adipogenesis. In human, AKR1B1 was expressed in human subcutaneous WAT from obese patients whereas AKR1B10 was hardly detectable (western blot, RT‑qPCR). In vitro, AKR1B1 expression increased throughout adipocyte differentiation unlike AKR1B10, which was preferentially expressed in undifferentiated cells. Using an AKR1B specific inhibitor, we demonstrated that AKR1B1 PGFS activity was a dampen to adipogenesis. We also showed that mechanisms regulating PGF2α action differed according to the species. In human cells, the expression of FP receptor was time-regulated whereas, in murine cells, PGFS expression and thus, PGF2α synthesis, limited PGF2α activity during adipogenesis. Akr1b7 knockout mice have decreased PGF2α intratissular levels associated with an expansion of adipose tissue resulting from an increase of adipogenesis and an adipocyte hypertrophia without any modification of lipogenic enzymes expression (Volat et al., 2012). These data, in agreement with PGF2α anti-adipogenic action, suggest an impact on lipolysis. We demonstrated that loss of Akr1b7 led to a decrease of WAT lipolytic activity. The use of murine (3T3‑L1) and human (hMADS) differentiated cells allowed us to show that the stimulation of lipolysis in response to FP activation was, in part, due to an increase of HSL phosphorylation (active form) and an increase of ATGL accumulation. The third part of this work consisted in characterizing the phenotype of transgenic mice overexpressing AKR1B1 in WAT (aP2‑AKR1B1 mice) in order to study the biological role of this human isoform.Les aldose réductases (AKR1B) sont des oxydoréductases dépendantes du NADPH initialement décrites pour leurs fonctions de détoxication cellulaire et de réduction du glucose. La découverte de l’expression d’Akr1b7 dans le tissu adipeux murin ainsi que l’activité prostaglandine F2α synthase (PGFS) spécifique de certaines isoformes suggèrent des rôles biologiques inédits pour ces enzymes. La prostaglandine F2α (PGF2α) inhibant l’adipogenèse, cette fonction PGFS met en avant l’implication des AKR1B dans la physiologie du tissu adipeux blanc (TAB). L’objectif de ces travaux était de caractériser l’expression de l’ensemble des AKR1B au sein des TAB murins et humains et de comprendre leur impact sur l’homéostasie du tissu adipeux et en particulier sur l’adipogenèse et la lipolyse. Nous avons montré que l’ensemble des AKR1B était exprimé dans le TAB murin. Akr1b3, Akr1b8 et Akr1b16 sont exprimées à la fois dans les fractions stroma‑vasculaires (contenant des cellules immunitaires, vasculaires, progénitrices…) et adipocytaires. A l’inverse, Akr1b7 n’est pas exprimé par les adipocytes. Les analyses réalisées in vitro indiquent qu’à l’exception d’Akr1b16, les isoformes murines des AKR1B voient leur expression augmenter précocement et transitoirement au cours de l’adipogenèse. Chez l’homme, l’isoforme AKR1B1 est exprimée dans le TAB sous‑cutané de patients obèses alors qu’AKR1B10 est difficilement détectable (western blot, RT‑qPCR). In vitro, l’expression d’AKR1B1 augmente tout au long de la différenciation adipocytaire contrairement à AKR1B10 qui est préférentiellement exprimé dans les cellules indifférenciées. L’utilisation d’un inhibiteur spécifique des AKR1B montre que l’activité PGFS d’AKR1B1 constitue un frein à l’adipogenèse. Nous montrons aussi que les mécanismes régulant l’action de la PGF2α diffèrent en fonction des espèces. Chez l’homme, l’expression du récepteur FP est régulée dans le temps alors que dans les cellules murines, c’est l’expression des PGFS et donc la synthèse de PGF2α qui définit, au cours de l’adipogenèse, la fenêtre d’action de cette prostaglandine. Les souris invalidées pour la PGFS Akr1b7 présentent une diminution des quantités intra‑tissulaires en PGF2α associée à une expansion accrue de leurs tissus adipeux due à une augmentation de l’adipogenèse et à une hypertrophie adipocytaire sans modification de l’expression des enzymes impliquées dans la lipogenèse (Volat et al., 2012). Ces données en accord avec le rôle anti‑adipogénique de la PGF2α suggèrent aussi une action sur la lipolyse. Nous démontrons ici que la perte d’Akr1b7 entraîne une diminution de l’activité lipolytique du TAB. L’utilisation de cellules murines (3T3‑L1) et humaines (hMADS) différenciées en adipocytes, nous a permis de montrer que la stimulation de l’activité lipolytique suite à l’activation du récepteur FP résultait en partie d’une augmentation de la phosphorylation de HSL (forme active) et de l’accumulation de la lipase ATGL. Le troisième volet de ce travail de thèse a consisté à caractériser un modèle de souris transgénique surexprimant AKR1B1 dans le TAB (souris aP2‑AKR1B1) afin d’étudier le rôle biologique de cette isoforme humaine

Roles of aldose reductases in homeostasis of human and murine white adipose tissues

Les aldose réductases (AKR1B) sont des oxydoréductases dépendantes du NADPH initialement décrites pour leurs fonctions de détoxication cellulaire et de réduction du glucose. La découverte de l’expression d’Akr1b7 dans le tissu adipeux murin ainsi que l’activité prostaglandine F2α synthase (PGFS) spécifique de certaines isoformes suggèrent des rôles biologiques inédits pour ces enzymes. La prostaglandine F2α (PGF2α) inhibant l’adipogenèse, cette fonction PGFS met en avant l’implication des AKR1B dans la physiologie du tissu adipeux blanc (TAB). L’objectif de ces travaux était de caractériser l’expression de l’ensemble des AKR1B au sein des TAB murins et humains et de comprendre leur impact sur l’homéostasie du tissu adipeux et en particulier sur l’adipogenèse et la lipolyse. Nous avons montré que l’ensemble des AKR1B était exprimé dans le TAB murin. Akr1b3, Akr1b8 et Akr1b16 sont exprimées à la fois dans les fractions stroma‑vasculaires (contenant des cellules immunitaires, vasculaires, progénitrices…) et adipocytaires. A l’inverse, Akr1b7 n’est pas exprimé par les adipocytes. Les analyses réalisées in vitro indiquent qu’à l’exception d’Akr1b16, les isoformes murines des AKR1B voient leur expression augmenter précocement et transitoirement au cours de l’adipogenèse. Chez l’homme, l’isoforme AKR1B1 est exprimée dans le TAB sous‑cutané de patients obèses alors qu’AKR1B10 est difficilement détectable (western blot, RT‑qPCR). In vitro, l’expression d’AKR1B1 augmente tout au long de la différenciation adipocytaire contrairement à AKR1B10 qui est préférentiellement exprimé dans les cellules indifférenciées. L’utilisation d’un inhibiteur spécifique des AKR1B montre que l’activité PGFS d’AKR1B1 constitue un frein à l’adipogenèse. Nous montrons aussi que les mécanismes régulant l’action de la PGF2α diffèrent en fonction des espèces. Chez l’homme, l’expression du récepteur FP est régulée dans le temps alors que dans les cellules murines, c’est l’expression des PGFS et donc la synthèse de PGF2α qui définit, au cours de l’adipogenèse, la fenêtre d’action de cette prostaglandine. Les souris invalidées pour la PGFS Akr1b7 présentent une diminution des quantités intra‑tissulaires en PGF2α associée à une expansion accrue de leurs tissus adipeux due à une augmentation de l’adipogenèse et à une hypertrophie adipocytaire sans modification de l’expression des enzymes impliquées dans la lipogenèse (Volat et al., 2012). Ces données en accord avec le rôle anti‑adipogénique de la PGF2α suggèrent aussi une action sur la lipolyse. Nous démontrons ici que la perte d’Akr1b7 entraîne une diminution de l’activité lipolytique du TAB. L’utilisation de cellules murines (3T3‑L1) et humaines (hMADS) différenciées en adipocytes, nous a permis de montrer que la stimulation de l’activité lipolytique suite à l’activation du récepteur FP résultait en partie d’une augmentation de la phosphorylation de HSL (forme active) et de l’accumulation de la lipase ATGL. Le troisième volet de ce travail de thèse a consisté à caractériser un modèle de souris transgénique surexprimant AKR1B1 dans le TAB (souris aP2‑AKR1B1) afin d’étudier le rôle biologique de cette isoforme humaine.Aldose reductases are NADPH-dependent oxydoreductases described for their involvement in cellular detoxification and glucose reduction. The discovery of Akr1b7 expression in murine adipose tissue together with the prostaglandin F2α Synthase (PGFS) activity of some isoforms suggest unreleased biological roles for these enzymes. Prostaglandin F2α (PGF2α) inhibiting adipogenesis, this PGFS function highlights AKR1B potential involvement in white adipose tissue (WAT) physiology. This work aimed at characterising the expression of all AKR1B in both murine and human WAT and understanding their impact on adipose tissue homeostasis and especially on adipogenesis and lipolysis. We showed that all AKR1B were expressed in murine WAT. Akr1b3, Akr1b8 and Akr1b16 were both expressed in the stromal vascular fraction (containing immune cells, vascular cells, progenitors…) and in the adipose fraction. In contrast, Akr1b7 was not expressed in adipocytes. In vitro analyses indicated that, except for Akr1b16, murine AKR1B isoform expression increased early and transiently during adipogenesis. In human, AKR1B1 was expressed in human subcutaneous WAT from obese patients whereas AKR1B10 was hardly detectable (western blot, RT‑qPCR). In vitro, AKR1B1 expression increased throughout adipocyte differentiation unlike AKR1B10, which was preferentially expressed in undifferentiated cells. Using an AKR1B specific inhibitor, we demonstrated that AKR1B1 PGFS activity was a dampen to adipogenesis. We also showed that mechanisms regulating PGF2α action differed according to the species. In human cells, the expression of FP receptor was time-regulated whereas, in murine cells, PGFS expression and thus, PGF2α synthesis, limited PGF2α activity during adipogenesis. Akr1b7 knockout mice have decreased PGF2α intratissular levels associated with an expansion of adipose tissue resulting from an increase of adipogenesis and an adipocyte hypertrophia without any modification of lipogenic enzymes expression (Volat et al., 2012). These data, in agreement with PGF2α anti-adipogenic action, suggest an impact on lipolysis. We demonstrated that loss of Akr1b7 led to a decrease of WAT lipolytic activity. The use of murine (3T3‑L1) and human (hMADS) differentiated cells allowed us to show that the stimulation of lipolysis in response to FP activation was, in part, due to an increase of HSL phosphorylation (active form) and an increase of ATGL accumulation. The third part of this work consisted in characterizing the phenotype of transgenic mice overexpressing AKR1B1 in WAT (aP2‑AKR1B1 mice) in order to study the biological role of this human isoform

Aldo-Keto Reductases 1B in Endocrinology and Metabolism

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Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

International audienceAldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions

Aldose Reductases Influence Prostaglandin F 2α Levels and Adipocyte Differentiation in Male Mouse and Human Species

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GLP-1 analogue-induced weight loss does not improve obesity-induced AT dysfunction

Glucagon-like peptide-1 (GLP-1) analogues aid weight loss which improves obesity-associated adipose tissue (AT) dysfunction. GLP-1 treatment may however also directly influence AT which expresses GLP-1 receptors. This study aimed to assess the impact of GLP-1 analogue treatment on subcutaneous AT inflammatory and fibrotic responses, compared to weight loss by calorie reduction (control). Among the 39 participants with type 2 diabetes recruited, 30 age-matched participants were randomised to 4 months treatment with Liraglutide (n=22) or calorie restriction-based on dietetic counselling (n=8). Assessments included clinical characteristics and repeated subcutaneous abdominal AT biopsies. Liraglutide resulted in weight loss in most participants (-3.12±1.72kg, p=0.007) and significant reduction in visceral AT. It was more effective in lowering fasting glucose, in comparison to weight loss by dieting. However TNFA AT-expression (p=0.0005), MCP-1 expression (p=0.027) and its serum levels (p=0.048) increased with Liraglutide, suggestive of an inflammatory response unlike in the diet arm in which a trend of lower CD14 expression (p=0.09) was found. Liraglutide treatment also increased expression of factors involved in ECM deposition, TGFbeta and Collagen-1 ( TGFB1 : before 0.73±0.09AU, after 1.00±0.13AU, p=0.006; COL1A1 : 0.84±0.09AU versus 1.49±0.26AU, p=0.026). Liraglutide thus appears to induce an inflammatory response in AT and influences ECM remodelling. Despite its superior effect on glycaemia, Liraglutide does not improve obesity-associated AT dysfunction in subcutaneous tissue. It is yet unclear whether this limits AT storage capacity for lipids. This may be of importance in patients being re-exposed to positive energy balance such as post GLP-1 discontinuation