An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Conjugating uncoupler compounds with hydrophobic hydrocarbon chains to achieve adipose tissue selective drug accumulation.

One potential approach for treating obesity is to increase energy expenditure in brown and white adipose tissue. Here we aimed to achieve this outcome by targeting mitochondrial uncoupler compounds selectively to adipose tissue, thus avoiding side effects from uncoupling in other tissues. Selective drug accumulation in adipose tissue has been observed with many lipophilic compounds and dyes. Hence, we explored the feasibility of conjugating uncoupler compounds with a lipophilic C8-hydrocarbon chain via an ether bond. We found that substituting the trifluoromethoxy group in the uncoupler FCCP with a C8-hydrocarbon chain resulted in potent uncoupling activity. Nonetheless, the compound did not elicit therapeutic effects in mice, likely as a consequence of metabolic instability resulting from rapid ether bond cleavage. A lipophilic analog of the uncoupler compound 2,6-dinitrophenol, in which a C8-hydrocarbon chain was conjugated via an ether bond in the para-position (2,6-dinitro-4-(octyloxy)phenol), exhibited increased uncoupling activity compared to the parent compound. However, in vivo pharmacokinetics studies suggested that 2,6-dinitro-4-(octyloxy)phenol was also metabolically unstable. In conclusion, conjugation of a hydrophobic hydrocarbon chain to uncoupler compounds resulted in sustained or improved uncoupling activity. However, an ether bond linkage led to metabolic instability, indicating the need to conjugate lipophilic groups via other chemical bonds.

Defective extracellular matrix remodeling in brown adipose tissue is associated with fibro-inflammation and reduced diet-induced thermogenesis.

The relevance of extracellular matrix (ECM) remodeling is reported in white adipose tissue (AT) and obesity-related dysfunctions, but little is known about the importance of ECM remodeling in brown AT (BAT) function. Here, we show that a time course of high-fat diet (HFD) feeding progressively impairs diet-induced thermogenesis concomitantly with the development of fibro-inflammation in BAT. Higher markers of fibro-inflammation are associated with lower cold-induced BAT activity in humans. Similarly, when mice are housed at thermoneutrality, inactivated BAT features fibro-inflammation. We validate the pathophysiological relevance of BAT ECM remodeling in response to temperature challenges and HFD using a model of a primary defect in the collagen turnover mediated by partial ablation of the Pepd prolidase. Pepd-heterozygous mice display exacerbated dysfunction and BAT fibro-inflammation at thermoneutrality and in HFD. Our findings show the relevance of ECM remodeling in BAT activation and provide a mechanism for BAT dysfunction in obesity.

CBL/CAP Is Essential for Mitochondria Respiration Complex I Assembly and Bioenergetics Efficiency in Muscle Cells.

CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.

Allostatic hypermetabolic response in PGC1α/ß heterozygote mouse despite mitochondrial defects.

Aging, obesity, and insulin resistance are associated with low levels of PGC1α and PGC1ß coactivators and defective mitochondrial function. We studied mice deficient for PGC1α and PGC1ß [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro-thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1α4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria-rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ-specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice.

Publisher Correction: GDF15 mediates the effects of metformin on body weight and energy balance.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

GDF15 mediates the effects of metformin on body weight and energy balance.

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.

Diet-Induced Obese Mice and Leptin-Deficient Lepob/ob Mice Exhibit Increased Circulating GIP Levels Produced by Different Mechanisms.

As glucose-dependent insulinotropic polypeptide (GIP) possesses pro-adipogenic action, the suppression of the GIP hypersecretion seen in obesity might represent a novel therapeutic approach to the treatment of obesity. However, the mechanism of GIP hypersecretion remains largely unknown. In the present study, we investigated GIP secretion in two mouse models of obesity: High-fat diet-induced obese (DIO) mice and leptin-deficient Lepob/ob mice. In DIO mice, plasma GIP was increased along with an increase in GIP mRNA expression in the lower small intestine. Despite the robust alteration in the gut microbiome in DIO mice, co-administration of maltose and the α-glucosidase inhibitor (α-GI) miglitol induced the microbiome-mediated suppression of GIP secretion. The plasma GIP levels of Lepob/ob mice were also elevated and were suppressed by fat transplantation. The GIP mRNA expression in fat tissue was not increased in Lepob/ob mice, while the expression of an interleukin-1 receptor antagonist (IL-1Ra) was increased. Fat transplantation suppressed the expression of IL-1Ra. The plasma IL-1Ra levels were positively correlated with the plasma GIP levels. Accordingly, although circulating GIP levels are increased in both DIO and Lepob/ob mice, the underlying mechanisms differ, and the anti-obesity actions of α-GIs and leptin sensitizers may be mediated partly by the suppression of GIP secretion.

GPER and ERα mediate estradiol enhancement of mitochondrial function in inflamed adipocytes through a PKA dependent mechanism.

Obesity is associated with inflammation, dysregulated adipokine secretion, and disrupted adipose tissue mitochondrial function. Estradiol (E2) has been previously reported to increase mitochondrial function and biogenesis in several cell lines, but neither the type of oestrogen receptor (ERα, ERß and GPER) involved nor the mechanism whereby such effects are exerted have been fully described. Considering the anti-inflammatory activity of E2 as well as its effects in enhancing mitochondrial biogenesis, the aim of this study was to investigate the contribution of ERα, ERß, and GPER signaling to the E2-mediated enhancement of adipocyte mitochondrial function in a pro-inflammatory situation. 3T3-L1 cells were treated for 24 h with ER agonists (PPT, DPN, and G1) and antagonists (MPP, PHTPP, and G15) in the presence or absence of interleukin 6 (IL6), as a pro-inflammatory stimulus. Inflammation, mitochondrial function and biogenesis markers were analyzed. To confirm the involvement of the PKA pathway, cells were treated with a GPER agonist, a PKA inhibitor, and IL6. Mitochondrial function markers were analyzed. Our results showed that activation of ERα and GPER, but not ERß, was able to counteract the proinflammatory effects of IL6 treatment, as well as mitochondrial biogenesis and function indicators. Inhibition of PKA prevented the E2- and G1-associated increase in mitochondrial function markers. In conclusion E2 prevents IL6 induced inflammation in adipocytes and promotes mitochondrial function through the combined activation of both GPER and ERα. These findings expand our understanding of ER interactions under inflammatory conditions in female rodent white adipose tissue.

P465L-PPARγ mutation confers partial resistance to the hypolipidaemic action of fibrates.

AIMS: Familial partial lipodystrophic syndrome 3 (FPLD3) is associated with mutations in the transcription factor PPARγ. One of these mutations, the P467L, confers a dominant negative effect. We and others have previously investigated the pathophysiology associated with this mutation using a humanized mouse model that recapitulates most of the clinical symptoms observed in patients who have been phenotyped under different experimental conditions. One of the key clinical manifestations observed, both in humans and mouse models, is the ectopic accumulation of fat in the liver. With this study we aim to dissect the molecular mechanisms that contribute to the excessive accumulation of lipids in the liver and characterize the negative effect of this PPARγ mutation on the activity of PPARα in vivo when activated by fibrates. MATERIAL AND METHODS: P465L-PPAR mutant and wild-type mice were divided into 8 experimental groups, 4 different conditions per genotype. Briefly, mice were fed a chow diet or a high-fat diet (HFD 45% Kcal from fat) for a period of 28 days and treated with WY14643 or vehicle for five days before culling. At the end of the experiment, tissues and plasma were collected. We performed extensive gene expression, fatty acid composition and histological analysis in the livers. The serum collected was used to measure several metabolites and to perform basic lipoprotein profile. RESULTS: P465L mice showed increased levels of insulin and free fatty acids (FFA) as well as increased liver steatosis. They also exhibit decreased levels of very low density lipoproteins (VLDL) when fed an HFD. We also provide evidence of impaired expression of a number of well-established PPARα target genes in the P465L mutant livers. CONCLUSION: Our data demonstrate that P465L confers partial resistance to the hypolipidemic action of fibrates. These results show that the fatty liver phenotype observed in P465L mutant mice is not only the consequence of dysfunctional adipose tissue, but also involves defective liver metabolism. All in all, the deleterious effects of P465L-PPARγ mutation may be magnified by their collateral negative effect on PPARα function.

Corrigendum: Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness.

This corrects the article DOI: 10.1038/nm.4115.

Phenyl-γ-valerolactones, flavan-3-ol colonic metabolites, protect brown adipocytes from oxidative stress without affecting their differentiation or function.

SCOPE: Consumption of products rich in flavan-3-ols, such as tea and cocoa, has been associated with decreased obesity, partially dependent on their capacity to enhance energy expenditure. Despite these phenolics having been reported to increase the thermogenic program in brown and white adipose tissue, flavan-3-ols are vastly metabolised in vivo to phenyl-γ-valerolactones. Therefore, we hypothesize that phenyl-γ-valerolactones may directly stimulate the differentiation and the activation of brown adipocytes. METHODS AND RESULTS: Immortalized brown pre-adipocytes were differentiated in presence of (R)-5-(3',4'-dihydroxyphenyl)-γ-valerolactone (VL1), (R)-5-(3´-hydroxyphenyl)-γ-valerolactone-4'-O-sulphate (VL2), (R)-5-phenyl-γ-valerolactone-3´,4´-di-O-sulphate (VL3), at concentrations of 2 or 10µM, whereas fully differentiated brown adipocyte were treated acutely (6-24h). None of the treatments regulated the expression levels of the uncouple protein 1, nor of the main transcription factors involved in brown adipogenesis. Similarly, mitochondrial content was unchanged after treatments. Moreover these compounds did not display peroxisome proliferator-activated receptor γ-agonist activity, as evaluated by luciferase assay, and did not enhance norepinephrine-stimulated lipolysis in mature adipocytes. However, both VL1 and VL2 prevented oxidative stress caused by H2 O2 . CONCLUSION: Phenyl-γ-valerolactones and their sulphated forms do not influence brown adipocyte development or function at physiological or supraphysiological doses in vitro, but they are active protecting brown adipocytes from increased reactive oxygen species production.

Stearoyl-CoA Desaturase 1 Is a Key Determinant of Membrane Lipid Composition in 3T3-L1 Adipocytes.

Stearoyl-CoA desaturase 1 (SCD1) is a lipogenic enzyme important for the regulation of membrane lipid homeostasis; dysregulation likely contributes to obesity associated metabolic disturbances. SCD1 catalyses the Δ9 desaturation of 12-19 carbon saturated fatty acids to monounsaturated fatty acids. To understand its influence in cellular lipid composition we investigated the effect of genetic ablation of SCD1 in 3T3-L1 adipocytes on membrane microdomain lipid composition at the species-specific level. Using liquid chromatography/electrospray ionisation-tandem mass spectrometry, we quantified 70 species of ceramide, mono-, di- and trihexosylceramide, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, bis(monoacylglycero)phosphate, phosphatidylinositol and cholesterol in 3T3-L1 adipocytes in which a 90% reduction in scd1 mRNA expression was achieved with siRNA. Cholesterol content was unchanged although decreases in other lipids resulted in cholesterol accounting for a higher proportion of lipid in the membranes. This was associated with decreased membrane lateral diffusion. An increased ratio of 24:0 to 24:1 in ceramide, mono- and dihexosylceramide, and sphingomyelin likely also contributed to this decrease in lateral diffusion. Of particular interest, we observed a decrease in phospholipids containing arachidonic acid. Given the high degree of structural flexibility of this acyl chain this will influence membrane lateral diffusion, and is likely responsible for the transcriptional activation of Lands' cycle enzymes lpcat3 and mboat7. Of relevance these profound changes in the lipidome were not accompanied by dramatic changes in gene expression in mature differentiated adipocytes, suggesting that adaptive homeostatic mechanisms to ensure partial maintenance of the biophysical properties of membranes likely occur at a post-transcriptional level.

Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness.

The discovery of genetic mechanisms for resistance to obesity and diabetes may illuminate new therapeutic strategies for the treatment of this global health challenge. We used the polygenic 'lean' mouse model, which has been selected for low adiposity over 60 generations, to identify mitochondrial thiosulfate sulfurtransferase (Tst; also known as rhodanese) as a candidate obesity-resistance gene with selectively increased expression in adipocytes. Elevated adipose Tst expression correlated with indices of metabolic health across diverse mouse strains. Transgenic overexpression of Tst in adipocytes protected mice from diet-induced obesity and insulin-resistant diabetes. Tst-deficient mice showed markedly exacerbated diabetes, whereas pharmacological activation of TST ameliorated diabetes in mice. Mechanistically, TST selectively augmented mitochondrial function combined with degradation of reactive oxygen species and sulfide. In humans, TST mRNA expression in adipose tissue correlated positively with insulin sensitivity in adipose tissue and negatively with fat mass. Thus, the genetic identification of Tst as a beneficial regulator of adipocyte mitochondrial function may have therapeutic significance for individuals with type 2 diabetes.

Mitochondria are required for pro-ageing features of the senescent phenotype.

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1ß-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1ß deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

Human Adipocytes Induce Inflammation and Atrophy in Muscle Cells During Obesity.

Inflammation and lipid accumulation are hallmarks of muscular pathologies resulting from metabolic diseases such as obesity and type 2 diabetes. During obesity, the hypertrophy of visceral adipose tissue (VAT) contributes to muscle dysfunction, particularly through the dysregulated production of adipokines. We have investigated the cross talk between human adipocytes and skeletal muscle cells to identify mechanisms linking adiposity and muscular dysfunctions. First, we demonstrated that the secretome of obese adipocytes decreased the expression of contractile proteins in myotubes, consequently inducing atrophy. Using a three-dimensional coculture of human myotubes and VAT adipocytes, we showed the decreased expression of genes corresponding to skeletal muscle contractility complex and myogenesis. We demonstrated an increased secretion by cocultured cells of cytokines and chemokines with interleukin (IL)-6 and IL-1ß as key contributors. Moreover, we gathered evidence showing that obese subcutaneous adipocytes were less potent than VAT adipocytes in inducing these myotube dysfunctions. Interestingly, the atrophy induced by visceral adipocytes was corrected by IGF-II/insulin growth factor binding protein-5. Finally, we observed that the skeletal muscle of obese mice displayed decreased expression of muscular markers in correlation with VAT hypertrophy and abnormal distribution of the muscle fiber size. In summary, we show the negative impact of obese adipocytes on muscle phenotype, which could contribute to muscle wasting associated with metabolic disorders.

Transducin-like enhancer of split 3 (TLE3) in adipose tissue is increased in situations characterized by decreased PPARγ gene expression.

UNLABELLED: Transgenic overexpression of adipose tissue (AT) transducin-like enhancer of split 3 (TLE3) mimicked peroxisome proliferator-activated receptor gamma (PPARγ) agonists, improving insulin resistance in mice. This study aimed to investigate TLE3 gene expression (qRT-PCR) and protein (Western blot) in subjects with a wide spectrum of obesity and insulin sensitivity and in an independent cohort of obese subjects following surgery-induced weight loss. TLE3 was analyzed in human adipocytes and after treatment with rosiglitazone. Given the findings in humans, TLE3 was also investigated in mice after a high-fat diet (HFD) and in PPARγ knockout mice. Subcutaneous (SC) AT TLE3 was increased in subjects with type 2 diabetes (T2D). In fact, SC TLE3 was associated with increased fasting glucose (r = 0.25, p = 0.015) and S6K1 activity (r = 0.671, p = 0.003), and with decreased Glut4 (r = -0.426, p = 0.006) and IRS-1 expression (-31 %, p = 0.007) and activation (P-IRS-1/IRS-1, -17 %, p = 0.024). TLE3 was preferentially expressed in mature adipocytes and increased during in vitro differentiation in parallel to PPARγ. Weight loss led to improved insulin sensitivity, increased AT PPARγ and decreased TLE3 (-24 %, p = 0.0002), while rosiglitazone administration downregulated TLE3 gene expression in fully differentiated adipocytes (-45 %, p < 0.0001). The concept that TLE3 may act as a homeostatic linchpin in AT was also supported by its increased expression in HFD-fed mice (39 %, p = 0.013) and PPARγ knockout (74 %, p = 0.001). In summary, increased AT TLE3 in subjects with T2D and in AT from HFD-fed and PPARγ knockout mice suggest that TLE3 may play an adaptive regulatory role that improves AT function under decreased PPARγ expression. KEY MESSAGE: TLE3 is expressed in mature adipocytes concomitantly with PPARγ. Subcutaneous adipose TLE3 is increased in T2D patients. Adipose TLE3 is upregulated in genetically ablated PPARγ and HFD-fed mice. TLE3 may be a homeostatic linchpin in insulin resistance and defective PPARγ.

Increased dihydroceramide/ceramide ratio mediated by defective expression of degs1 impairs adipocyte differentiation and function.

Adipose tissue dysfunction is an important determinant of obesity-associated, lipid-induced metabolic complications. Ceramides are well-known mediators of lipid-induced insulin resistance in peripheral organs such as muscle. DEGS1 is the desaturase catalyzing the last step in the main ceramide biosynthetic pathway. Functional suppression of DEGS1 activity results in substantial changes in ceramide species likely to affect fundamental biological functions such as oxidative stress, cell survival, and proliferation. Here, we show that degs1 expression is specifically decreased in the adipose tissue of obese patients and murine models of genetic and nutritional obesity. Moreover, loss-of-function experiments using pharmacological or genetic ablation of DEGS1 in preadipocytes prevented adipogenesis and decreased lipid accumulation. This was associated with elevated oxidative stress, cellular death, and blockage of the cell cycle. These effects were coupled with increased dihydroceramide content. Finally, we validated in vivo that pharmacological inhibition of DEGS1 impairs adipocyte differentiation. These data identify DEGS1 as a new potential target to restore adipose tissue function and prevent obesity-associated metabolic disturbances.

Identification of novel type 2 diabetes candidate genes involved in the crosstalk between the mitochondrial and the insulin signaling systems.

Type 2 Diabetes (T2D) is a highly prevalent chronic metabolic disease with strong co-morbidity with obesity and cardiovascular diseases. There is growing evidence supporting the notion that a crosstalk between mitochondria and the insulin signaling cascade could be involved in the etiology of T2D and insulin resistance. In this study we investigated the molecular basis of this crosstalk by using systems biology approaches. We combined, filtered, and interrogated different types of functional interaction data, such as direct protein-protein interactions, co-expression analyses, and metabolic and signaling dependencies. As a result, we constructed the mitochondria-insulin (MITIN) network, which highlights 286 genes as candidate functional linkers between these two systems. The results of internal gene expression analysis of three independent experimental models of mitochondria and insulin signaling perturbations further support the connecting roles of these genes. In addition, we further assessed whether these genes are involved in the etiology of T2D using the genome-wide association study meta-analysis from the DIAGRAM consortium, involving 8,130 T2D cases and 38,987 controls. We found modest enrichment of genes associated with T2D amongst our linker genes (p = 0.0549), including three already validated T2D SNPs and 15 additional SNPs, which, when combined, were collectively associated to increased fasting glucose levels according to MAGIC genome wide meta-analysis (p = 8.12×10(-5)). This study highlights the potential of combining systems biology, experimental, and genome-wide association data mining for identifying novel genes and related variants that increase vulnerability to complex diseases.