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Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.
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Linfócitos B/imunologia , Complexo II de Transporte de Elétrons/genética , Inflamação/metabolismo , Linfocitose/imunologia , Mitocôndrias/metabolismo , Mutação/genética , Anti-Inflamatórios/farmacologia , Respiração Celular , Células Cultivadas , Fumaratos/metabolismo , Glicólise , Humanos , Inflamação/genética , Interleucina-6/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Consumo de Oxigênio , Estudos Prospectivos , Transdução de Sinais , Sequenciamento do ExomaRESUMO
While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.
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Transtorno do Espectro Autista , Transtornos do Neurodesenvolvimento , Síndrome de Prader-Willi , Adolescente , Humanos , Transtorno do Espectro Autista/genética , Hiperfagia/genética , Hiperfagia/complicações , Transtornos do Neurodesenvolvimento/genética , Obesidade/complicações , Síndrome de Prader-Willi/complicações , Síndrome de Prader-Willi/genética , ProteínasRESUMO
Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, thermogenic adipocytes are linked to cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this plasticity could help improving metabolism. Here, we show that the lncRNA Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Comparing transcriptional profiles of human adipose tissues and cultured adipocytes we discovered that AATBC was enriched in thermogenic conditions. Using primary and immortalized human adipocytes we found that AATBC enhanced the thermogenic phenotype, which was linked to increased respiration and a more fragmented mitochondrial network. Expression of AATBC in adipose tissue of mice led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, BMI, and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.
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Inflammatory bowel disease (IBD) occurring following allogeneic stem cell transplantation (aSCT) is a very rare condition. The underlying pathogenesis needs to be better defined. There is currently no systematic effort to exclude loss- or gain-of-function mutations in immune-related genes in stem cell donors. This is despite the fact that more than 100 inborn errors of immunity may cause or contribute to IBD. We have molecularly characterized a patient who developed fulminant inflammatory bowel disease following aSCT with stable 100% donor-derived hematopoiesis. A pathogenic c.A291G; p.I97M HAVCR2 mutation encoding the immune checkpoint protein TIM-3 was identified in the patient's blood-derived DNA, while being absent in DNA derived from the skin. TIM-3 expression was much decreased in the patient's serum, and in vitro-activated patient-derived T cells expressed reduced TIM-3 levels. In contrast, T cell-intrinsic CD25 expression and production of inflammatory cytokines were preserved. TIM-3 expression was barely detectable in the immune cells of the patient's intestinal mucosa, while being detected unambiguously in the inflamed and non-inflamed colon from unrelated individuals. In conclusion, we report the first case of acquired, "transplanted" insufficiency of the regulatory TIM-3 checkpoint linked to post-aSCT IBD.
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Receptor Celular 2 do Vírus da Hepatite A , Doenças Inflamatórias Intestinais , Transplante de Células-Tronco , Humanos , Citocinas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/genética , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/etiologia , Mucosa Intestinal , Transplante de Células-Tronco/efeitos adversosRESUMO
Martorell hypertensive ischaemic leg ulcer (Martorell HYTILU) is a rare but significant cause of distal leg ulcers. Although hypertension and diabetes are known factors in its development, the precise pathogenesis of Martorell HYTILU remains elusive. To reach a better understanding of Martorell HYTILU, transcriptomic analysis was conducted through RNA sequencing and immunohistochemical comparison of Martorell HYTILU (n = 17) with chronic venous ulcers (n = 4) and healthy skin (n = 4). Gene expression analysis showed a marked activation of immune-related pathways in both Martorell HYTILU and chronic venous ulcers compared with healthy skin. Notably, neutrophil activity was substantially higher in Martorell HYTILU. While pathway analysis revealed a mild downregulation of several immune pathways in Martorell HYTILU compared with chronic venous ulcers, keratinization, cornification, and epidermis development were significantly upregulated in Martorell HYTILU. Additionally, STAC2, a gene encoding for a protein promoting the expression of the calcium channel Cav1.1, was significantly upregulated in Martorell HYTILU and was detected perivascularly in situ (Martorell HYTILU n = 24; chronic venous ulcers n = 9, healthy skin n = 11). The high expression of STAC2 in Martorell HYTILU suggests that increased calcium influx plays an important role in the pathogenesis of the disease. Consequently, calcium channel antagonists could be a promising treatment avenue for Martorell HYTILU.
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Hipertensão , Úlcera Varicosa , Humanos , Masculino , Feminino , Úlcera Varicosa/imunologia , Idoso , Doença Crônica , Hipertensão/complicações , Hipertensão/genética , Pessoa de Meia-Idade , Pele/patologia , Pele/imunologia , Isquemia/genética , Isquemia/imunologia , Perfilação da Expressão Gênica , Transcriptoma , Estudos de Casos e Controles , Úlcera da Perna/etiologia , Úlcera da Perna/imunologia , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.
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DNA Ligases , Síndromes de Imunodeficiência , Humanos , DNA Ligases/genética , Autoimunidade/genética , Haploinsuficiência , DNA Ligase Dependente de ATP/genética , Síndromes de Imunodeficiência/genética , Mutação , DNARESUMO
Mutations in CD46 predispose to atypical hemolytic uremic syndrome (aHUS) with low penetrance. Factors driving immune-dysregulatory disease in individual mutation carriers have remained ill-understood. In addition to its role as a negative regulator of the complement system, CD46 modifies T cell-intrinsic metabolic adaptation and cytokine production. Comparative immunologic analysis of diseased vs. healthy CD46 mutation carriers has not been performed in detail yet. In this study, we comprehensively analyzed clinical, molecular, immune-phenotypic, cytokine secretion, immune-metabolic, and genetic profiles in healthy vs. diseased individuals carrying a rare, heterozygous CD46 mutation identified within a large single family. Five out of six studied individuals carried a CD46 gene splice-site mutation causing an in-frame deletion of 21 base pairs. One child suffered from aHUS and his paternal uncle manifested with adult-onset systemic lupus erythematosus (SLE). Three mutation carriers had no clinical evidence of CD46-related disease to date. CD4+ T cell-intrinsic CD46 expression was uniformly 50%-reduced but was comparable in diseased vs. healthy mutation carriers. Reconstitution experiments defined the 21-base pair-deleted CD46 variant as intracellularly-but not surface-expressed and haploinsufficient. Both healthy and diseased mutation carriers displayed reduced CD46-dependent T cell mitochondrial adaptation. Diseased mutation carriers had lower peripheral regulatory T cell (Treg) frequencies and carried potentially epistatic, private rare variants in other inborn errors of immunity (IEI)-associated proinflammatory genes, not found in healthy mutation carriers. In conclusion, low Treg and rare non-CD46 immune-gene variants may contribute to clinically manifest CD46 haploinsufficiency-associated immune-dysregulation.
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Família , Haploinsuficiência , Adulto , Criança , Humanos , Nível de Saúde , Heterozigoto , Citocinas , Proteína Cofatora de Membrana/genéticaRESUMO
White adipose tissue (WAT) fibrosis, characterized by an excess of extracellular (ECM) matrix components, is strongly associated with WAT inflammation and dysfunction due to obesity. Interleukin (IL)-13 and IL-4 were recently identified as critical mediators in the pathogenesis of fibrotic diseases. However, their role in WAT fibrosis is still ill-defined. We therefore established an ex vivo WAT organotypic culture system and demonstrated an upregulation of fibrosis-related genes and an increase of α-smooth muscle actin (αSMA) and fibronectin abundance upon dose-dependent stimulation with IL-13/IL-4. These fibrotic effects were lost in WAT lacking il4ra, which encodes for the underlying receptor controlling this process. Adipose tissue macrophages were found to play a key role in mediating IL-13/IL-4 effects in WAT fibrosis as their depletion through clodronate dramatically decreased the fibrotic phenotype. IL-4-induced WAT fibrosis was partly confirmed in mice injected intraperitoneally with IL-4. Furthermore, gene correlation analyses of human WAT samples revealed a strong positive correlation of fibrosis markers with IL-13/IL-4 receptors, whereas IL13 and IL4 correlations failed to confirm this association. In conclusion, IL-13 and IL-4 can induce WAT fibrosis ex vivo and partly in vivo, but their role in human WAT remains to be further elucidated.
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Interleucina-13 , Interleucina-4 , Humanos , Camundongos , Animais , Interleucina-13/genética , Interleucina-4/genética , Tecido Adiposo/patologia , Tecido Adiposo Branco/patologia , FibroseRESUMO
Proteoglycans are central components of the extracellular matrix (ECM) and binding partners for inflammatory chemokines. Morphological differences in the ECM and increased inflammation are prominent features of the white adipose tissues in patients with obesity. The impact of obesity and weight loss on the expression of specific proteoglycans in adipose tissue is not well known. This study aimed to investigate the relationship between adiposity and proteoglycan expression. We analyzed transcriptomic data from two human bariatric surgery cohorts. In addition, RT-qPCR was performed on adipose tissues from female and male mice fed a high-fat diet. Both visceral and subcutaneous adipose tissue depots were analyzed. Adipose mRNA expression of specific proteoglycans, proteoglycan biosynthetic enzymes, proteoglycan partner molecules, and other ECM-related proteins were altered in both human cohorts. We consistently observed more profound alterations in gene expression of ECM targets in the visceral adipose tissues after surgery (among others VCAN (p = 0.000309), OGN (p = 0.000976), GPC4 (p = 0.00525), COL1A1 (p = 0.00221)). Further, gene analyses in mice revealed sex differences in these two tissue compartments in obese mice. We suggest that adipose tissue repair is still in progress long after surgery, which may reflect challenges in remodeling increased adipose tissues. This study can provide the basis for more mechanistic studies on the role of proteoglycans in adipose tissues in obesity.
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Tecido Adiposo , Proteoglicanas , Feminino , Humanos , Masculino , Animais , Camundongos , Proteoglicanas/genética , Proteoglicanas/metabolismo , Tecido Adiposo/metabolismo , Obesidade/genética , Obesidade/metabolismo , Gordura Subcutânea/metabolismo , Adiposidade , Proteínas da Matriz Extracelular/metabolismo , Dieta Hiperlipídica/efeitos adversosRESUMO
BACKGROUND: Patch testing of contact allergens to diagnose allergic contact dermatitis (ACD) is a traditional, useful tool. The most important decision is the distinction between allergic and irritant reactions, as this has direct implications on diagnosis and management. Our objective was to evaluate a new method of non-contact infrared reading of patch tests. Secondary objectives included a possible correlation between the intensity of the patch test reaction and temperature change. METHODS: 420 positive reactions from patients were included in our study. An independent patch test reader assessed the positive reactions and classified them as allergic (of intensity + to +++) or irritant (IR). At the same time, a forward-looking infrared (FLIR) camera attachment for an iPhone was used to acquire infrared thermal images of the patch tests, and images were analyzed using the FLIR ONE app. RESULTS: Allergic patch test reactions were characterized by temperature increases of 0.72 ± 0.67 °C compared to surrounding skin. Irritant reactions only resulted in 0.17 ± 0.31 °C temperature increase. The mean temperature difference between the two groups was highly significant (p < 0.0001) and therefore was used to predict the type of contact dermatitis. CONCLUSIONS: Thermography is a reliable and effective way to distinguish between allergic and irritant contact dermatitis.
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Dermatite Alérgica de Contato/diagnóstico , Dermatite Irritante/diagnóstico , Testes do Emplastro , Termografia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alérgenos/administração & dosagem , Feminino , Humanos , Irritantes/administração & dosagem , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Temperatura Cutânea , Adulto JovemAssuntos
Calcinose/etiologia , Doenças do Tecido Conjuntivo/etiologia , Síndrome de Down/complicações , Síndrome de Down/genética , Feminino , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Interferon Tipo I/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pessoa de Meia-Idade , Transdução de SinaisRESUMO
Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal in vitro and in vivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation.
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BACKGROUND: Studies on DNA methylation following bariatric surgery have primarily focused on blood cells, while it is unclear to which extend it may reflect DNA methylation profiles in specific metabolically relevant organs such as adipose tissue. Here, we investigated whether adipose tissue depots specific methylation changes after bariatric surgery are mirrored in blood. METHODS: Using Illumina 850K EPIC technology, we analysed genome-wide DNA methylation in paired blood, subcutaneous and omental visceral AT (SAT/OVAT) samples from nine individuals (N = 6 female) with severe obesity pre- and post-surgery. FINDINGS: The numbers and effect sizes of differentially methylated regions (DMRs) post-bariatric surgery were more pronounced in AT (SAT: 12,865 DMRs from -11.5 to 10.8%; OVAT: 14,632 DMRs from -13.7 to 12.8%) than in blood (9267 DMRs from -8.8 to 7.7%). Cross-tissue DMRs implicated immune-related genes. Among them, 49 regions could be validated with similar methylation changes in blood from independent individuals. Fourteen DMRs correlated with differentially expressed genes in AT post bariatric surgery, including downregulation of PIK3AP1 in both SAT and OVAT. DNA methylation age acceleration was significantly higher in AT compared to blood, but remained unaffected after surgery. INTERPRETATION: Concurrent methylation pattern changes in blood and AT, particularly in immune-related genes, suggest blood DNA methylation mirrors AT's inflammatory state post-bariatric surgery. FUNDING: The funding sources are listed in the Acknowledgments section.
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Tecido Adiposo , Cirurgia Bariátrica , Metilação de DNA , Epigênese Genética , Humanos , Cirurgia Bariátrica/métodos , Feminino , Masculino , Tecido Adiposo/metabolismo , Adulto , Pessoa de Meia-Idade , Epigenômica/métodosRESUMO
OBJECTIVE: Picalm (phosphatidylinositol-binding clathrin assembly protein), a ubiquitously expressed clathrin-adapter protein, is a well-known susceptibility gene for Alzheimer's disease, but its role in white adipose tissue (WAT) function has not yet been studied. Transcriptome analysis revealed differential expression of Picalm in WAT of diabetes-prone and diabetes-resistant mice, hence we aimed to investigate the potential link between Picalm expression and glucose homeostasis, obesity-related metabolic phenotypes, and its specific role in insulin-regulated GLUT4 trafficking in adipocytes. METHODS: Picalm expression and epigenetic regulation by microRNAs (miRNAs) and DNA methylation were analyzed in WAT of diabetes-resistant (DR) and diabetes-prone (DP) female New Zealand Obese (NZO) mice and in male NZO after time-restricted feeding (TRF) and alternate-day fasting (ADF). PICALM expression in human WAT was evaluated in a cross-sectional cohort and assessed before and after weight loss induced by bariatric surgery. siRNA-mediated knockdown of Picalm in 3T3-L1-adipocytes was performed to elucidate functional outcomes on GLUT4-translocation as well as insulin signaling and adipogenesis. RESULTS: Picalm expression in WAT was significantly lower in DR compared to DP NZO female mice, as well as in insulin-sensitive vs. resistant NZO males, and was also reduced in NZO males following TRF and ADF. Four miRNAs (let-7c, miR-30c, miR-335, miR-344) were identified as potential mediators of diabetes susceptibility-related differences in Picalm expression, while 11 miRNAs (including miR-23a, miR-29b, and miR-101a) were implicated in TRF and ADF effects. Human PICALM expression in adipose tissue was lower in individuals without obesity vs. with obesity and associated with weight-loss outcomes post-bariatric surgery. siRNA-mediated knockdown of Picalm in mature 3T3-L1-adipocytes resulted in amplified insulin-stimulated translocation of the endogenous glucose transporter GLUT4 to the plasma membrane and increased phosphorylation of Akt and Tbc1d4. Moreover, depleting Picalm before and during 3T3-L1 differentiation significantly suppressed adipogenesis, suggesting that Picalm may have distinct roles in the biology of pre- and mature adipocytes. CONCLUSIONS: Picalm is a novel regulator of GLUT4-translocation in WAT, with its expression modulated by both genetic predisposition to diabetes and dietary interventions. These findings suggest a potential role for Picalm in improving glucose homeostasis and highlight its relevance as a therapeutic target for metabolic disorders.
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Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.
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In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans.
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Resistência à Insulina , Jejum Intermitente , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Resistência à Insulina/genética , Obesidade/genética , Obesidade/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Redução de PesoRESUMO
Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed "committed pre-adipocytes" from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling, and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism.
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Adipose tissue can recruit catabolic adipocytes that utilize chemical energy to dissipate heat. This process occurs either by uncoupled respiration through uncoupling protein 1 (UCP1) or by utilizing ATP-dependent futile cycles (FCs). However, it remains unclear how these pathways coexist since both processes rely on the mitochondrial membrane potential. Utilizing single-nucleus RNA sequencing to deconvolute the heterogeneity of subcutaneous adipose tissue in mice and humans, we identify at least 2 distinct subpopulations of beige adipocytes: FC-adipocytes and UCP1-beige adipocytes. Importantly, we demonstrate that the FC-adipocyte subpopulation is highly metabolically active and utilizes FCs to dissipate energy, thus contributing to thermogenesis independent of Ucp1. Furthermore, FC-adipocytes are important drivers of systemic energy homeostasis and linked to glucose metabolism and obesity resistance in humans. Taken together, our findings identify a noncanonical thermogenic adipocyte subpopulation, which could be an important regulator of energy homeostasis in mammals.
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Transcription factor-based cellular reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.