Early Adipogenesis and Upregulation of UCP1 in Mesenchymal Stromal Cells Stimulated by Devitalized Microfragmented Fat (MiFAT).

Adipose tissue is mainly composed by adipocytes. Moreover, mesenchymal stromal/stem cells (MSCs), macrophages, endothelial cells, and extracellular matrix components are present. The variety of molecules as cytokines and growth factors of its structure very rich in blood vessel makes it also similar to a true endocrine organ that however needs still to be fully investigated. In our study, we used human lipoaspirate to obtain mechanically microfragmented fat (MiFAT) which was washed and then devitalized by freezing-thawing cycles. In our experiments, thawed MiFAT was used to stimulate cultures of MSCs from two different sources (adipose tissue and gingiva papilla) in comparison with a traditional stimulation in vitro obtained by culturing MSCs with adipogenic medium. MSCs stimulated with MiFAT showed a very early production of lipid droplets, after only 3 days, that correlated with an increased expression of adipokines. Furthermore, a significant upregulation of PPAR gamma 1 alpha coactivator (PPARGC1A) was observed with an overexpression of uncoupling protein 1 (UCP1) that suggest a pattern of differentiation compatible with the beige-brown fat.

Effects of Exhaustive Exercise on Adiponectin and High-Molecular-Weight Oligomer Levels in Male Amateur Athletes.

Physical activity promotes metabolic health and prevents lifestyle-related diseases. Adiponectin is specifically produced by adipose tissue and comes in three forms, differing in terms of weight: trimers (LMW), hexamers (MMW), and high-molecular-weight (HMW) oligomers. The oligomers are associated with the beneficial effects of adiponectin. In this study, we aimed to investigate the impact of a single bout of exhaustive exercise on adiponectin expression in 25 male amateur athletes, divided into two groups, one comprising young adults (YAs) (n = 15), and the other comprising middle-aged adults (MAs) (n = 10). Body fat was estimated through skinfold thickness. Adiponectin levels were assessed at baseline and at 15 min and 24 h post-exercise, while HMW oligomer levels were evaluated at baseline and at 24 h post-exercise. We observed a significant increase in total adiponectin at both 15 min and 24 h after exercise, with there being a more evident effect among the YA subjects. HMW oligomers also increased significantly after exercise both in the total sample and among the YA subjects, but this was not the case in the MA subjects. The increase in adiponectin levels was significantly associated with Powerpeak. Furthermore, a significant inverse correlation was found between basal adiponectin and VO2peak and Powerpeak. In conclusion, a single bout of exhaustive exercise can rapidly and significantly enhance the basal circulating adiponectin concentration, which seems to be negatively associated with maximal aerobic capacity.

The time course of irisin release after an acute exercise: relevant implications for health and future experimental designs.

This study aimed to analyze the acute impact of exercise on serum irisin levels in 22 young (YA, 24.6 ± 3.5 yrs) and in 12 middle-aged male adults (MA, 54.6 ± 5.7 yrs) 15 min and 24 h after an incremental cycling exercise test to exhaustion. ELISA assay was used for serum irisin detection. Circulating irisin increased significantly from baseline (9.0 ± 2.0 ng/ml) to 15 min post-exercise (10.2 ± 2.0 ng/ml, P < 0.001), but the greatest increment was detected after 24 h (13.5 ± 2.5 ng/ml, P < 0.001) reaching more than 50% of the basal release. Levels were significantly higher in YA (9.7 ± 1.7 to 11.1 ± 1.8 to 14.5 ± 2.2 ng/ml) than MA (7.6 ± 1.6 to 8.7 ± 1.5 to 11.8± 2.2 ng/ml) for all measured time-points (P < 0.05). Nevertheless, MA showed a comparable increase in serum irisin levels when compared to YA. These findings highlight the importance of acute physical exercise as a countermeasure against age-related deterioration of skeletal muscle mass and function in both YA and MA.

Effects of Triheptanoin on Mitochondrial Respiration and Glycolysis in Cultured Fibroblasts from Neutral Lipid Storage Disease Type M (NLSD-M) Patients.

Neutral lipid storage disease type M (NLSD-M) is an ultra-rare, autosomal recessive disorder that causes severe skeletal and cardiac muscle damage and lipid accumulation in all body tissues. In this hereditary pathology, the defective action of the adipose triglyceride lipase (ATGL) enzyme induces the enlargement of cytoplasmic lipid droplets and reduction in the detachment of mono- (MG) and diglycerides (DG). Although the pathogenesis of muscle fiber necrosis is unknown, some studies have shown alterations in cellular energy production, probably because MG and DG, the substrates of Krebs cycle, are less available. No tests have been tried with medium-chain fatty acid molecules to evaluate the anaplerotic effect in NLSD cells. In this study, we evaluated the in vitro effect of triheptanoin (Dojolvi®), a highly purified chemical triglyceride with seven carbon atoms, in fibroblasts obtained from five NLSD-M patients. Glycolytic and mitochondrial functions were determined by Seahorse XF Agylent Technology, and cellular viability and triglyceride content were measured through colorimetric assays. After the addition of triheptanoin, we observed an increase in glycolysis and mitochondrial respiration in all patients compared with healthy controls. These preliminary results show that triheptanoin is able to induce an anaplerotic effect in NLSD-M fibroblasts, paving the way towards new therapeutic strategies.

Salivary and serum irisin in healthy adults before and after exercise.

Irisin is an exercise-induced cytokine mainly secreted by myocytes. Circulating level of irisin can increase in response to acute exercise, promoting pleiotropic effects on health. Generally, irisin is evaluated in blood, however, its collection is invasive. Saliva sample would not have any risk associated with blood collection and would represent a less invasive method for irisin detection. Until now, there are only a few studies that have analyzed irisin levels in saliva. In the present research, five healthy male adults performed an incremental exercise until exhaustion on cycle ergometer. Serum and saliva samples were collected before exercise and 15min, 24h and 48h after reaching the exhaustion. Irisin was detected by ELISA assay. Serum and salivary irisin levels increased from baseline to 24h post exercise and reverted to basal levels after 48h of rest. A significant rise of both serum and salivary irisin level at 24h (p≤0.05) compared to baseline levels was found. Furthermore, a significant correlation between irisin percentage change in serum and saliva from baseline to 24h post exercise was detected (r=0.92, p<0.05). Despite the relatively limited sample, this research suggests that collecting saliva samples might represent a valid and less invasive method to detect irisin level changes induced by exercise.

Corrigendum: CDKN2A determines mesothelioma cell fate to EZH2 inhibition.

[This corrects the article DOI: 10.3389/fonc.2021.678447.].

Neutral lipid storage disease with myopathy: A 10-year follow-up case report.

Mutations in PNPLA2 gene encoding for adipose triglyceride lipase (ATGL), involved in triglyceride degradation, lead to an inborn error of neutral lipid metabolism. The disorder that results in abnormal storage of neutral lipid is known as neutral lipid storage disease with myopathy (NLSDM). We report the follow-up of a 30-year-old woman with NLSDM, asymptomatic until age 23. At the age of 18, a high level of CPK and neutral lipid abnormal accumulation in muscle and skin cells suggested NLSDM diagnosis, afterwards confirmed by PNPLA2 analysis. After 5 years, she developed weakness in the upper and lower extremities. She was put on a low-fat diet with medium-chain triglycerides (MCT) oil supplementation but, although her CPK level decreased, myopathy continued to progress. At present, she presents severe skeletal myopathy without cardiac involvement. In this patient, no beneficial effects on progressive skeletal muscle weakness were detected after the MCT diet, probably due to complete loss of PNPLA2 expression.

Influence of 5-HTTLPR polymorphism on postpartum depressive and posttraumatic symptoms.

INTRODUCTION: Postpartum depression (PPD) is a multifactor disorder caused by psychological, social, and also biological factors. 5-HTTLPR polymorphism in the promoter region of serotonin transporter gene seems to influence PPD onset. In this study, we examined the effect of 5-HTTLPR polymorphism on prenatal and postnatal symptoms of depression and posttraumatic stress in women. METHODS: A longitudinal design with three points - time 1 (32-40 weeks gestation); time 2 (2 or 3 weeks after birth), and time 3 (3 months after birth) - was made. A total of 141 women were recruited during childbirth preparation courses. At time 1, women completed the Beck Depression Inventory (BDI) and the Los Angeles Symptoms Checklist (LASC). At time 2, they fulfilled BDI and Edinburgh Postnatal Depression Scale (EDPS), LASC and the Perinatal Posttraumatic stress disorder (PTSD) Questionnaire (PPQ); midwives and nurses collected biological test tubes by blood sampling for the genetic analysis. At time 3, the women were reassessed for BDI, LASC, EDPS, and PPQs. Analysis of variance and moderation analysis were used to correlate genotype and psychological investigations. RESULTS: Results showed that, compared with LL/LS genotypes, SS genotype moderated cognitive depressive symptoms onset at T2 and T3. Moreover, this genotype correlated, directly or indirectly, with PTSD postpartum aspects (re-experience, avoidance, and hyperarousal). DISCUSSION: Findings revealed that a lower expression of serotonin transporter gene, associated with SS genotype, seems to render women more vulnerable to depressive and PTSD symptoms after childbirth.

CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition.

Malignant pleural mesothelioma is an aggressive cancer, heterogeneous in its presentation and behaviour. Despite an increasing knowledge about molecular markers and their diagnostic and prognostic value, they are not used as much as they might be for treatment allocation. It has been recently reported that mesothelioma cells that lack BAP1 (BRCA1 Associated Protein) are sensitive to inhibition of the EZH2 (Enhancer of Zeste Homolog 2) histone methyltransferase. Since we observed strong H3K27me3 (histone H3 lysine 27 trimetylation) immunoreactivity in BAP1 wild-type mesothelioma biopsies, we decided to characterize in vitro the response/resistance of BAP1 wild-type mesothelioma cells to the EZH2 selective inhibitor, EPZ-6438. Here we demonstrate that BAP1 wild-type mesothelioma cells were rendered sensitive to EPZ-6438 upon SIRT1 (Sirtuin 1) silencing/inhibition or when cultured as multicellular spheroids, in which SIRT1 expression was lower compared to cells grown in monolayers. Notably, treatment of spheroids with EPZ-6438 abolished H3K27me3 and induced the expression of CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A), causing cell growth arrest. EPZ-6438 treatment also resulted in a rapid and sustained induction of the genes encoding HIF2α (Hypoxia Inducible Factor 2α), TG2 (Transglutaminase 2) and IL-6 (Interleukin 6). Loss of CDKN2 is a common event in mesothelioma. CDKN2A silencing in combination with EPZ-6438 treatment induced apoptotic death in mesothelioma spheroids. In a CDKN2A wild-type setting apoptosis was induced by combining EPZ-6438 with 1-155, a TG2 selective and irreversible inhibitor. In conclusion, our data suggests that the expression of CDKN2A predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis.

Recurrent N209* ABHD5 mutation in two unreported families with Chanarin Dorfman Syndrome.

ABHD5 protein is widely involved in lipid and energy homeostasis. Mutations in the ABHD5 gene are associated with the onset of Neutral Lipid Storage Disease with Ichthyosis (NLSDI), historically known as Chanarin Dorfman Syndrome (CDS). CDS is a rare autosomal recessive lipid storage disease, characterized by non-bullous congenital ichthyosiform eritrhoderma (NCIE), hepatomegaly and liver steatosis. Myopathy, neurosensory hearing loss, cataracts, nystagmus, strabismus, and mental impairment are considered additional findings. To date, 151 CDS patients have been reported all over the world. Here we described two additional families with patients affected by CDS from Turkey. Our patients were a 42 and 22-years old men, admitted to the Hospital for congenital ichthyosis. Hepatic steatosis and myopathy were also detected in both patients. ABHD5 molecular analysis revealed the presence of N209* mutation. Our data enlarge the cohort of CDS patients and provide a revision of muscle clinical findings for this rare inborn error of neutral lipid metabolism.

ETF dehydrogenase advances in molecular genetics and impact on treatment.

Electron transfer flavoprotein dehydrogenase, also called ETF-ubiquinone oxidoreductase (ETF-QO), is a protein localized in the inner membrane of mitochondria, playing a central role in the electron-transfer system. Indeed, ETF-QO mediates electron transport from flavoprotein dehydrogenases to the ubiquinone pool. ETF-QO mutations are often associated with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD, OMIM#231680), a multisystem genetic disease characterized by various clinical manifestations with different degrees of severity. In this review, we outline the clinical features correlated with ETF-QO deficiency and the benefits obtained from different treatments, such as riboflavin, L-carnitine and/or coenzyme Q10 supplementation, and a diet poor in fat and protein. Moreover, we provide a detailed summary of molecular and bioinformatic investigations, describing the mutations identified in ETFDH gene and highlighting their predicted impact on enzymatic structure and activity. In addition, we report biochemical and functional analysis, performed in HEK293 cells and patient fibroblasts and muscle cells, to show the relationship between the nature of ETFDH mutations, the variable impairment of enzyme function, and the different degrees of RR-MADD severity. Finally, we describe in detail 5 RR-MADD patients carrying different ETFDH mutations and presenting variable degrees of clinical symptom severity.

Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations.

Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.

A novel PNPLA2 mutation causing total loss of RNA and protein expression in two NLSDM siblings with early onset but slowly progressive severe myopathy.

Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, due to an enzymatic error of lipid metabolism. Patients present always with skeletal muscle myopathy and variable cardiac and hepatic involvement. NLSDM is caused by mutations in the PNPLA2 gene, which encodes the adipose triglyceride lipase (ATGL). Here we report the molecular characterization and clinical findings of two NLSDM siblings carrying the novel c.187+1G > C homozygous PNPLA2 mutation, localized in the splice site of intron 2. Molecular analyses revealed that neither aberrant PNPLA2 mRNA isoforms, nor ATGL mutated protein were detectable in patient's cells. Clinically, both patients presented early onset muscle weakness, in particular of proximal upper limb muscles. In almost 15 years, muscle damage affected also distal upper limbs. This is a NLSDM family, displaying a severe PNPLA2 mutation in two siblings with clinical presentation characterized by an early onset, but a slowly evolution of severe myopathy.

FOXC2 Disease Mutations Identified in Lymphedema Distichiasis Patients Impair Transcriptional Activity and Cell Proliferation.

FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants (p.L80F, p.H199Pfs*264, p.I213Tfs*18, p.Y109*) compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death.

Correlation between ETFDH mutations and dysregulation of serum myomiRs in MADD patients.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare fatty acids oxidation disorder which is often associated with deficiency of electron transfer flavoprotein dehydrogenase (ETFDH). In this study we reported clinical features and evaluation of expression profile of circulating muscle-specific miRNAs (myomiRs) in two MADD patients carrying different ETFDH gene mutations. Patient 1 was a compound heterozygote for two missense mutations. She showed a late onset MADD clinical phenotype and a significant increase of serum myomiRs. Patient 2, carrying a missense and a frameshift mutation, displayed early onset symptoms and a slight increase of some serum myomiRs.

MiRNAs as biomarkers of phenotype in neutral lipid storage disease with myopathy.

BACKGROUND: Neutral lipid storage disease with myopathy (NLSDM) is a rare lipid metabolism disorder. In this study, we evaluated some circulating miRNAs levels in serum samples and the MRI of three affected siblings. METHODS: Three members of one NLSDM family were identified: two brothers and one sister. Muscles of lower and right upper extremities were studied by MRI. Expression profile of miRNAs, obtained from serum samples, was detected using qRT-PCR. RESULTS: Two brothers presented with progressive skeletal myopathy, while the sister had severe hepatosteatosis and diabetes. NLSDM patients showed a significant increase of muscle-specific miRNAs expression compared with healthy subjects. We found a correlation between hepatic damage and elevation of miRNAs expression profile of liver origin. CONCLUSIONS: The dysregulation of miRNAs might represent an indicator of skeletal and hepatic damage and it might be useful to monitor the progression of NLSDM.

A novel mutation of ABHD5 gene in a Chanarin Dorfman patient with unusual dermatological findings.

BACKGROUND: Chanarin Dorfman Syndrome (CDS) is a rare autosomal recessive disorder characterized by the multisytemic accumulation of neutral lipids inside the cytoplasmic lipid droplets. This condition is caused by mutations in the abhydrolase domain containing 5 gene (ABHD5). In CDS the skin involvement is the prevalent and always observed clinical feature, consisting of a non-bullous congenital ichthyosiform erythroderma (NCIE). Moreover, a variable involvement of the liver and neuromuscular system can be also observed. In this report, we aimed to perform the clinical and genetic characterization of a patient affected by CDS with atypical dermatological findings, considering this rare inborn error of neutral lipid metabolism. METHODS: Genomic DNA samples obtained from patient and his parents were used to perform the sequencing of the ABHD5 exons and their intron/exon boundaries. Bioinformatic analyses were performed to investigate the possible effect of the identified mutation on protein structure. RESULTS: Here we present the case of a 29-year-old male patient with CDS, who, for long time, has been misdiagnosed as pityriasis rubra pilaris (PRP). He has a history of increasing hyperlipidemia; hepatomegaly associated with hepatosteatosis was also detected. ABHD5 molecular analysis revealed a novel missense mutation, the c.811G > A (p.G271R). Bioinformatic investigations showed that the variant has a deleterious effect on ABHD5 function, probably causing an incorrect folding of the mutant protein. CONCLUSIONS: These results highlihts the importance of genetic testing for ABHD5 in unresolved cases of patients presenting unusual skin lesions, that resemble PRP, associated with a history of hyperlipidemia and nonalcoholic fatty liver.

Metabolic lipid muscle disorders: biomarkers and treatment.

Lipid storage myopathies (LSMs) are metabolic disorders of the utilization of fat in muscles due to several different defects. In this review, a molecular update of LSMs is presented and recent attempts of finding treatment options are discussed. The main topics discussed are: primary carnitine deficiency, riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency, neutral lipid storage disorders and carnitine palmitoyl transferase deficiency. The most frequent presentations and genetic abnormalities are summarized. We present their diagnosis utilizing biomedical and morphological biomarkers and possible therapeutic interventions. The treatment of these metabolic disorders is a subject of active translational research but appears, in some cases, still elusive.