The obesity-linked human lncRNA AATBC stimulates mitochondrial function in adipocytes.

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.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

AIMS/HYPOTHESIS: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. METHODS: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests. RESULTS: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR-HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. CONCLUSIONS/INTERPRETATION: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. DATA AVAILABILITY: Array data have been submitted to the GEO database at NCBI (GSE148699).

Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death initiated by oxidative stress and lipid peroxidation. Recent evidence has linked ferroptosis to the action of the transcription factor Nuclear factor erythroid-2 derived,-like-1 (NFE2L1). NFE2L1 regulates proteasome abundance in an adaptive fashion, maintaining protein quality control to secure cellular homeostasis, but the regulation of NFE2L1 during ferroptosis and the role of the ubiquitin-proteasome system (UPS) herein are still unclear. In the present study, using an unbiased proteomic approach charting the specific ubiquitylation sites, we show that induction of ferroptosis leads to recalibration of the UPS. RSL3-induced ferroptosis inhibits proteasome activity and leads to global hyperubiquitylation, which is linked to NFE2L1 activation. As NFE2L1 resides in the endoplasmic reticulum tethered to the membrane, it undergoes complex posttranslational modification steps to become active and induce the expression of proteasome subunit genes. We show that proteolytic cleavage of NFE2L1 by the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is a critical step for the ferroptosis-induced feed-back loop of proteasome function. Cells lacking DDI2 cannot activate NFE2L1 in response to RSL3 and show global hyperubiquitylation. Genetic or chemical induction of ferroptosis in cells with a disrupted DDI2-NFE2L1 pathway diminishes proteasomal activity and promotes cell death. Also, treating cells with the clinical drug nelfinavir, which inhibits DDI2, sensitized cells to ferroptosis. In conclusion, our results provide new insight into the importance of the UPS in ferroptosis and highlight the role of the DDI2-NFE2L1 as a potential therapeutic target. Manipulating DDI2-NFE2L1 activity through chemical inhibition might help sensitizing cells to ferroptosis, thus enhancing existing cancer therapies.

Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

Immune-mediated denervation of the pineal gland underlies sleep disturbance in cardiac disease.

Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons. Depletion of macrophages in the SCG prevented disease-associated denervation of the pineal gland and restored physiological melatonin secretion. Our data identify the mechanism by which diurnal rhythmicity in cardiac disease is disturbed and suggest a target for therapeutic intervention.

Apremilast prevents blistering in human epidermis and stabilizes keratinocyte adhesion in pemphigus.

Pemphigus vulgaris is a life-threatening blistering skin disease caused by autoantibodies destabilizing desmosomal adhesion. Current therapies focus on suppression of autoantibody formation and thus treatments directly stabilizing keratinocyte adhesion would fulfill an unmet medical need. We here demonstrate that apremilast, a phosphodiesterase 4 inhibitor used in psoriasis, prevents skin blistering in pemphigus vulgaris. Apremilast abrogates pemphigus autoantibody-induced loss of keratinocyte cohesion in ex-vivo human epidermis, cultured keratinocytes in vitro and in vivo in mice. In parallel, apremilast inhibits keratin retraction as well as desmosome splitting, induces phosphorylation of plakoglobin at serine 665 and desmoplakin assembly into desmosomal plaques. We established a plakoglobin phospho-deficient mouse model that reveals fragile epidermis with altered organization of keratin filaments and desmosomal cadherins. In keratinocytes derived from these mice, intercellular adhesion is impaired and not rescued by apremilast. These data identify an unreported mechanism of desmosome regulation and propose that apremilast stabilizes keratinocyte adhesion and is protective in pemphigus.

NFE2L1-mediated proteasome function protects from ferroptosis.

OBJECTIVE: Ferroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood. METHODS AND RESULTS: Here we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis. CONCLUSION: Our data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.

Role of Ubiquilins for Brown Adipocyte Proteostasis and Thermogenesis.

The acclimatization of brown adipose tissue (BAT) to sustained cold exposure requires an adaptive increase in proteasomal protein quality control. Ubiquilins represent a recently identified family of shuttle proteins with versatile functions in protein degradation, such as facilitating substrate targeting and proteasomal degradation. However, whether ubiquilins participate in brown adipocyte function has not been investigated so far. Here, we determine the role of ubiquilins for proteostasis and non-shivering thermogenesis in brown adipocytes. We found that Ubqln1, 2 and 4 are highly expressed in BAT and their expression was induced by cold and proteasomal inhibition. Surprisingly, silencing of ubiquilin gene expression (one or multiple in combinations) did not lead to aggravated ER stress or inflammation. Moreover, ubiquitin level and proteasomal activity under basal conditions were not impacted by loss of ubiquilins. Also, non-shivering thermogenesis measured by norepinephrine-induced respiration remained intact after loss of ubiquilins. In conclusion, ubiquilin proteins are highly abundant in BAT and regulated by cold, but they are dispensable for brown adipocyte proteostasis and thermogenesis.