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.

Delivery of miRNAs to the adipose organ for metabolic health.

Despite the increasing prevalence of obesity and diabetes, there is no efficient treatment to combat these epidemics. The adipose organ is the main site for energy storage and plays a pivotal role in whole body lipid metabolism and energy homeostasis, including remodeling and dysfunction of adipocytes and adipose tissues in obesity and diabetes. Thus, restoring and balancing metabolic functions in the adipose organ is in demand. MiRNAs represent a novel class of drugs and drug targets, as they are heavily involved in the regulation of many cellular and metabolic processes and diseases, likewise in adipocytes. In this review, we summarize key regulatory activities of miRNAs in the adipose organ, discuss various miRNA replacement and inhibition strategies, promising delivery systems for miRNAs and reflect the future of novel miRNA-based therapeutics to target adipose tissues with the ultimate goal to combat metabolic disorders.

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).

Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue.

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.

Lipid nanocarriers for microRNA delivery.

Non-coding RNAs (ncRNAs) like microRNAs (miRNAs) or small interference RNAs (siRNAs) with their power to selectively silence any gene of interest enable the targeting of so far 'undruggable' proteins and diseases. Such RNA molecules have gained much attention from biotech and pharmaceutical companies, which led to the first Food and Drug Administration (FDA) approved ncRNA therapeutic in 2018. However, the main barrier in clinical practice of ncRNAs is the lack of an effective delivery system that can protect the RNA molecules from nuclease degradation, deliver them to specific tissues and cell types, and release them into the cytoplasm of the targeted cells, all without inducing adverse effects. For that reason, drug delivery approaches, formulations, technologies and systems for transporting pharmacological ncRNA compounds to achieve a diagnostic or therapeutic effect in the human body are in demand. Here, we review the development of therapeutic lipid-based nanoparticles for delivery of miRNAs, one class of endogenous ncRNAs with specific regulatory functions. We outline challenges and opportunities for advanced miRNA-based therapies, and discuss the complexity associated with the delivery of functional miRNAs. Novel strategies are addressed how to deal with the most critical points in miRNA delivery, such as toxicity, specific targeting of disease sites, proper cellular uptake and endosomal escape of miRNAs. Current fields of application and various preclinical settings involving miRNA therapeutics are discussed, providing an outlook to future clinical approaches. Following the current trends and technological developments in nanomedicine exciting new delivery systems for ncRNA-based therapeutics can be expected in upcoming years.

Label-free metabolic imaging by mid-infrared optoacoustic microscopy in living cells.

We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.

The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis.

Aberrant activity of the glucocorticoid (GC)/glucocorticoid receptor (GR) endocrine system has been linked to obesity-related metabolic dysfunction. Traditionally, the GC/GR axis has been believed to play a crucial role in adipose tissue formation and function in both, white (WAT) and brown adipose tissue (BAT). While recent studies have challenged this notion for WAT, the contribution of GC/GR signaling to BAT-dependent energy homeostasis remained unknown. Here, we have generated and characterized a BAT-specific GR-knockout mouse (GRBATKO ), for the first time allowing to genetically interrogate the metabolic impact of BAT-GR. The HPA axis in GRBATKO mice was intact, as was the ability of mice to adapt to cold. BAT-GR was dispensable for the adaptation to fasting-feeding cycles and the development of diet-induced obesity. In obesity, glucose and lipid metabolism, insulin sensitivity, and food intake remained unchanged, aligning with the absence of changes in thermogenic gene expression. Together, we demonstrate that the GR in UCP1-positive BAT adipocytes plays a negligible role in systemic metabolism and BAT function, thereby opposing a long-standing paradigm in the field.

A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

The glucocorticoid receptor (GR) represents the crucial molecular mediator of key endocrine, glucocorticoid hormone-dependent regulatory circuits, including control of glucose, protein, and lipid homeostasis. Consequently, aberrant glucocorticoid signaling is linked to severe metabolic disorders, including insulin resistance, obesity, and hyperglycemia, all of which also appear upon chronic glucocorticoid therapy for the treatment of inflammatory conditions. Of note, long-term glucocorticoid exposure under these therapeutic conditions typically induces glucocorticoid resistance, requiring higher doses and consequently triggering more severe metabolic phenotypes. However, the molecular basis of acquired glucocorticoid resistance remains unknown. In a screen of differential microRNA expression during glucocorticoid-dependent adipogenic differentiation of human multipotent adipose stem cells, we identified microRNA 29a (miR-29a) as one of the most down-regulated transcripts. Overexpression of miR-29a impaired adipogenesis. We found that miR-29a represses GR in human adipogenesis by directly targeting its mRNA, and downstream analyses revealed that GR mediates most of miR-29a's anti-adipogenic effects. Conversely, miR-29a expression depends on GR activation, creating a novel miR-29-driven feedback loop. miR-29a and GR expression were inversely correlated both in murine adipose tissue and in adipose tissue samples obtained from human patients. In the latter, miR-29a levels were additionally strongly negatively correlated with body mass index and adipocyte size. Importantly, inhibition of miR-29 in mice partially rescued the down-regulation of GR during dexamethasone treatment. We discovered that, in addition to modulating GR function under physiologic conditions, pharmacologic glucocorticoid application in inflammatory disease also induced miR-29a expression, correlating with reduced GR levels. This effect was abolished in mice with impaired GR function. In summary, we uncovered a novel GR-miR-29a negative feedback loop conserved between mice and humans, in health and disease. For the first time, we elucidate a microRNA-related mechanism that might contribute to GR dysregulation and resistance in peripheral tissues.-Glantschnig, C., Koenen, M., Gil-Lozano, M., Karbiener, M., Pickrahn, I., Williams-Dautovich, J., Patel, R., Cummins, C. L., Giroud, M., Hartleben, G., Vogl, E., Blüher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

Regulatory Small and Long Noncoding RNAs in Brite/Brown Adipose Tissue.

Brite/brown adipose tissue (BAT) is a thermogenic tissue able to dissipate energy via non-shivering thermogenesis. It is naturally activated by cold and has been demonstrated to increase thermogenic capacity, elevate energy expenditure, and to ultimately contribute to fat mass reduction. Thus, it emerges as novel therapeutic concept for pharmacological intervention in obesity and other metabolic disorders. Therefore, the comprehensive understanding of the regulatory network in thermogenic adipocytes is in demand.The surprising findings that (1) all human protein-coding genes make up not more than 2% of our genome, (2) organismal complexity goes well along with the percentage of nonprotein-coding sequences, and that (3) three quarters of our genome are pervasively transcribed, provide evidence that noncoding RNAs (ncRNAs) are not junk, but a significant and even predominant part of our transcriptome representing a treasure chest worth retrieving regulatory determinants in biological processes and diseases.In this chapter, the impact of regulatory small and long ncRNAs (lncRNAs) in particular microRNAs and lncRNAs on BAT formation and metabolic function and their involvement in physiological and pathological conditions has been reviewed.

Norepinephrine triggers an immediate-early regulatory network response in primary human white adipocytes.

BACKGROUND: Norepinephrine (NE) signaling has a key role in white adipose tissue (WAT) functions, including lipolysis, free fatty acid liberation and, under certain conditions, conversion of white into brite (brown-in-white) adipocytes. However, acute effects of NE stimulation have not been described at the transcriptional network level. RESULTS: We used RNA-seq to uncover a broad transcriptional response. The inference of protein-protein and protein-DNA interaction networks allowed us to identify a set of immediate-early genes (IEGs) with high betweenness, validating our approach and suggesting a hierarchical control of transcriptional regulation. In addition, we identified a transcriptional regulatory network with IEGs as master regulators, including HSF1 and NFIL3 as novel NE-induced IEG candidates. Moreover, a functional enrichment analysis and gene clustering into functional modules suggest a crosstalk between metabolic, signaling, and immune responses. CONCLUSIONS: Altogether, our network biology approach explores for the first time the immediate-early systems level response of human adipocytes to acute sympathetic activation, thereby providing a first network basis of early cell fate programs and crosstalks between metabolic and transcriptional networks required for proper WAT function.

Comparative Gene Expression Analysis in WM164 Melanoma Cells Revealed That ß-ß-Dimethylacrylshikonin Leads to ROS Generation, Loss of Mitochondrial Membrane Potential, and Autophagy Induction.

Skin cancer is currently diagnosed as one in every three cancers. Melanoma, the most aggressive form of skin cancer, is responsible for 79% of skin cancer deaths and the incidence is rising faster than in any other solid tumor type. Previously, we have demonstrated that dimethylacrylshikonin (DMAS), isolated from the roots of Onosma paniculata (Boraginaceae), exhibited the lowest IC50 values against different tumor types out of several isolated shikonin derivatives. DMAS was especially cytotoxic towards melanoma cells and led to apoptosis and cell cycle arrest. In this study, we performed a comprehensive gene expression study to investigate the mechanism of action in more detail. Gene expression signature was compared to vehicle-treated WM164 control cells after 24 h of DMAS treatment; where 1192 distinct mRNAs could be identified as expressed in all replicates and 89 were at least 2-fold differentially expressed. DMAS favored catabolic processes and led in particular to p62 increase which is involved in cell growth, survival, and autophagy. More in-depth experiments revealed that DMAS led to autophagy, ROS generation, and loss of mitochondrial membrane potential in different melanoma cells. It has been reported that the induction of an autophagic cell death represents a highly effective approach in melanoma therapy.

Comparative Secretome Analyses of Primary Murine White and Brown Adipocytes Reveal Novel Adipokines.

The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that carbohydrate-metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete a large variety of proteins. Upon NE stimulation, an increased secretion of known adipokines is favored by white adipocytes while brown adipocytes secreted higher amounts of novel adipokines. Furthermore, the secretory response between NE-stimulated and basal state was multifaceted addressing lipid and glucose metabolism, adipogenesis, and antioxidative reactions. Intriguingly, NE stimulation drastically changed the secretome in brown adipocytes. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE. Given the beneficial effects of brown adipose tissue activation on its endocrine function and systemic metabolism, this study provides an archive of novel batokine candidates and biomarkers for activated brown adipose tissue.

Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype.

Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of >80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescence-specific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.

Blocking negative effects of senescence in human skin fibroblasts with a plant extract.

There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris, which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

Age-Induced Changes in White, Brite, and Brown Adipose Depots: A Mini-Review.

Aging is a time-related process of functional decline at organelle, cellular, tissue, and organismal level that ultimately limits life. Cellular senescence is a state of permanent growth arrest in response to stress and one of the major drivers of aging and age-related disorders. Senescent cells accumulate with age, and removal of these cells delays age-related disorders in different tissues and prolongs healthy lifespan. One of the most studied aging mechanisms is the accumulation of reactive oxygen species damage in cells, organs, and organisms over time. Elevated oxidative stress is also found in metabolic diseases such as obesity, metabolic syndrome and associated disorders. Moreover, dysregulation of the energy homeostasis is also associated with aging, and many age-related genes also control energy metabolism, with the adipose organ, comprising white, brite, and brown adipocytes, as an important metabolic player in the regulation of whole-body energy homeostasis. This review summarizes transformations in the adipose organ upon aging and cellular senescence and sheds light on the reallocation of fat mass between adipose depots, on the metabolism of white and brown adipose tissue, on the regenerative potential and adipogenic differentiation capacity of preadipocytes, and on alterations in mitochondria and bioenergetics. In conclusion, the aging process is a lifelong, creeping process with gradual decline in (pre-)adipocyte function over time. Thus, slowing down the accumulation of (pre-)adipocyte damage and dysfunction, removal of senescent preadipocytes as well as blocking deleterious compounds of the senescent secretome are protective measures to maintain a lasting state of health at old age.

Long Non-Coding RNAs in Metabolic Organs and Energy Homeostasis.

Single cell organisms can surprisingly exceed the number of human protein-coding genes, which are thus not at the origin of the complexity of an organism. In contrast, the relative amount of non-protein-coding sequences increases consistently with organismal complexity. Moreover, the mammalian transcriptome predominantly comprises non-(protein)-coding RNAs (ncRNA), of which the long ncRNAs (lncRNAs) constitute the most abundant part. lncRNAs are highly species- and tissue-specific with very versatile modes of action in accordance with their binding to a large spectrum of molecules and their diverse localization. lncRNAs are transcriptional regulators adding an additional regulatory layer in biological processes and pathophysiological conditions. Here, we review lncRNAs affecting metabolic organs with a focus on the liver, pancreas, skeletal muscle, cardiac muscle, brain, and adipose organ. In addition, we will discuss the impact of lncRNAs on metabolic diseases such as obesity and diabetes. In contrast to the substantial number of lncRNA loci in the human genome, the functionally characterized lncRNAs are just the tip of the iceberg. So far, our knowledge concerning lncRNAs in energy homeostasis is still in its infancy, meaning that the rest of the iceberg is a treasure chest yet to be discovered.

Endocrine and autocrine/paracrine modulators of brown adipose tissue mass and activity as novel therapeutic strategies against obesity and type 2 diabetes.

The dramatically increasing world-wide prevalence of obesity is recognized as a risk factor for the development of various diseases. The growing research on the role of adipose tissue in controlling energy homeostasis and insulin sensitivity has revealed that the promotion of brown adipose tissue (BAT) activity and the browning of white adipose tissue (WAT) leads to multiple health benefits and prevents obesity and type 2 diabetes (T2D). Inducible thermogenic adipocytes do exist in adult humans and are linked with increased energy combustion and lower body fat mass. Thus brown adipocytes are currently placed at the center of attention for novel therapeutic strategies against metabolic diseases such as obesity and diabetes. Besides the classical, norepinephrine-mediated sympathetic recruitment and activation of thermogenic adipocytes, a number of novel circulating factors have been recently identified to have a positive or negative impact on thermogenic adipocyte formation and activity. In this review their mechanism of action and the plausible therapeutic applications will be summarized and discussed.

Small non coding RNAs in adipocyte biology and obesity.

Obesity has reached epidemic proportions world-wide and constitutes a substantial risk factor for hypertension, type 2 diabetes, cardiovascular diseases and certain cancers. So far, regulation of energy intake by dietary and pharmacological treatments has met limited success. The main interest of current research is focused on understanding the role of different pathways involved in adipose tissue function and modulation of its mass. Whole-genome sequencing studies revealed that the majority of the human genome is transcribed, with thousands of non-protein-coding RNAs (ncRNA), which comprise small and long ncRNAs. ncRNAs regulate gene expression at the transcriptional and post-transcriptional level. Numerous studies described the involvement of ncRNAs in the pathogenesis of many diseases including obesity and associated metabolic disorders. ncRNAs represent potential diagnostic biomarkers and promising therapeutic targets. In this review, we focused on small ncRNAs involved in the formation and function of adipocytes and obesity.

SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells.

Aging is accompanied by loss of subcutaneous adipose tissue. This may be due to reduced differentiation capacity or deficiency in DNA damage repair (DDR) factors. Here we investigated the role of SNEVhPrp19/hPso4, which was implicated in DDR and senescence evasion, in adipogenic differentiation of human adipose stromal cells (hASCs). We showed that SNEV is induced during adipogenesis and localized both in the nucleus and in the cytoplasm. Knockdown of SNEV perturbed adipogenic differentiation and led to accumulation of DNA damage in hASCs upon oxidative stress. In addition, we demonstrated that SNEV is required for fat deposition in Caenorhabditis elegans. Consequently, we tested other DDR factors and found that WRN is also required for adipogenesis in both models. These results demonstrate that SNEV regulates adipogenesis in hASCs and indicate that DDR capacity in general might be a pre-requisite for this process.