Lactobacillus plantarum HF02 alleviates lipid accumulation and intestinal microbiota dysbiosis in high-fat diet-induced obese mice.

BACKGROUND: Obesity is closely associated with lipid accumulation and intestinal microbiota dysbiosis. It has been proved that probiotics supplement contributes to alleviate obesity. The objective of this study was to investigate the mechanism by which Lactobacillus plantarum HF02 (LP-HF02) alleviated lipid accumulation and intestinal microbiota dysbiosis in high-fat diet-induced obese mice. RESULTS: Our results showed that LP-HF02 ameliorated body weight, dyslipidemia, liver lipid accumulation, and liver injury in obese mice. As expected, LP-HF02 inhibited pancreatic lipase activity in small intestinal contents and increased fecal triglyceride levels, thereby reducing dietary fat hydrolysis and absorption. Moreover, LP-HF02 ameliorated the intestinal microbiota composition, as evidenced by the enhanced ratio of Bacteroides to Firmicutes, the decreased abundance of pathogenic bacteria (including Bacteroides, Alistipes, Blautia, and Colidextribacter) and the increased abundance of beneficial bacteria (including Muribaculaceae, Akkermansia, Faecalibaculum, and Rikenellaceae_RC9_gut_group). LP-HF02 also increased fecal short-chain fatty acids (SCFAs) levels and colonic mucosal thickness, and subsequently decreased serum lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels in obese mice. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot results demonstrated that LP-HF02 ameliorated hepatic lipid accumulation via activating the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway. CONCLUSION: Therefore, our results indicated that LP-HF02 could be considered as a probiotic preparation for preventing obesity. © 2023 Society of Chemical Industry.

Effects of organic chemicals from diesel exhaust particles on adipocytes differentiated from human mesenchymal stem cells.

Exposure to fine particulate matter (PM2.5 ) from incomplete fossil fuel combustion (coal, oil, gas and diesel) has been linked to increased morbidity and mortality due to metabolic diseases. PM2.5 exaggerate adipose inflammation and insulin resistance in mice with diet-induced obesity. Here, we elucidate the hypothesis that such systemic effects may be triggered by adhered particle components affecting adipose tissue directly. Studying adipocytes differentiated from primary human mesenchymal stem cells, we found that lipophilic organic chemicals (OC) from diesel exhaust particles induced inflammation-associated genes and increased secretion of the chemokine CXLC8/interleukin-8 as well as matrix metalloprotease 1. The oxidative stress response gene haem oxygenase-1 and tumour necrosis factor alpha were seemingly not affected, while aryl hydrocarbon receptor-regulated genes, cytochrome P450 1A1 (CYP1A1) and CYP1B1 and plasminogen activator inhibitor-2, were clearly up-regulated. Finally, expression of ß-adrenergic receptor, known to regulate adipocyte homoeostasis, was down-regulated by exposure to these lipophilic OC. Our results indicate that low concentrations of OC from combustion particles have the potential to modify expression of genes in adipocytes that may be linked to metabolic disease. Further studies on mechanisms linking PM exposure and metabolic diseases are warranted.

Identification and characterization of hADSC-derived exosome proteins from different isolation methods.

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.

An RNAi Screening of Clinically Relevant Transcription Factors Regulating Human Adipogenesis and Adipocyte Metabolism.

CONTEXT: Healthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents. OBJECTIVE: A systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously. METHODS: TFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs. RESULTS: Analysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes. CONCLUSION: This comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes.

Immunosuppressive and Adipogenesis Inhibitory Sesterterpenoids with a Macrocyclic Ether System from Eurysolen gracilis.

Eurysoloids A (1) and B (2), two novel diastereomeric sesterterpenoids possessing a pentacyclic 5/6/5/10/5 framework with an unusual macrocyclic ether system, were isolated from Eurysolen gracilis Prain. Their structures were unambiguously determined by spectroscopic, single-crystal X-ray diffraction and DP4+ analyses. A plausible biosynthetic pathway for compounds 1 and 2 was proposed. Both compounds exhibited immunosuppressive activity via inhibiting the production of cytokine IFN-γ of T cells, and compound 2 inhibited adipogenesis in 3T3-L1 adipocytes.

Nuclear expression of MDM2 in hibernoma: a potential diagnostic pitfall.

Hibernoma is a rare benign adipocytic tumor composed of a proliferation of brown and white fat cells varying in their proportions. The tumor may also contain fat cells resembling lipoblasts, which makes it difficult to distinguish it from atypical lipomatous tumor/well differentiated liposarcoma (ALT/WDLS). Although nuclear expressions of murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) are widely used as immunohistochemical surrogate markers for ALT/WDLS, the utility of these proteins in distinguishing between hibernoma and ALT/WDLS still remains to be elucidated. We evaluated immunohistochemical expressions of MDM2 and CDK4 in 10 hibernomas expressing uncoupling protein-1 (UCP-1), a mitochondrial protein transporter consistently expressed in brown fat cells, and lacking MDM2 gene amplification, which was analyzed by fluorescence in situ hybridization (FISH). In contrast to the data previously obtained, nuclear expression of MDM2 was observed in 100% (10/10 cases) of the hibernomas irrespective of the proportion of brown fat cells, whereas no cases were positive for CDK4. The tumors also showed almost concurrent expression of p53 (in 9/10 cases) and ubiquitin-specific-processing protease 7 (USP7) (in 10/10 cases), which deubiquitinates and stabilizes MDM2, potentially resulting in its nuclear expression without MDM2 gene amplification. MDM2 expression may thus be a diagnostic pitfall for hibernoma particularly in differentiating it from ALT/WDLS.

Obesity related microRNA­424 is regulated by TNF­α in adipocytes.

In recent years, obesity has become a major public health concern. Obesity has been previously associated with low­grade inflammation and TNF­α induction in adipose tissue, which subsequently disrupts adipocyte metabolism. MicroRNAs (miRNAs/miRs) are important metabolic factors and their dysregulation has been associated with obesity­related metabolic syndromes. In fact, it has been directly suggested that miR­424 may be functionally associated with adipogenesis, although its exact role in this process remains unclear. The present study aimed to identify the function of miR­424 in adipogenesis. In the present study, miR­424 expression levels were analyzed during adipogenesis and the differential expression of this miRNA in the adipose tissue of obese and non­obese children was also assessed. Furthermore, the interaction between miR­424 and the adipocytokine TNF­α was determined. Finally, miR­424 target genes and downstream signaling pathways were predicted via bioinformatics and analyzed by performing a luciferase reporter assay to elucidate the functional mechanisms of miR­424 in adipogenesis of visceral adipocytes. The results revealed that the expression levels of miR­424 upregulated in the adipose tissue biopsies from obese children compared with the biopsies of non­obese children. However, in cultured adipocytes, the expression levels of miR­424 were discovered to be gradually downregulated during the adipogenesis process. TNF­α treatment significantly downregulated the expression levels of miR­424 via binding to its promoter region and reducing its transcriptional activity. Through bioinformatic prediction analysis, miR­424 target genes were analyzed, of which several were identified to be involved in signaling pathways that are known to regulate adipogenesis, such as the Wnt signaling pathway. In conclusion, the present study indicated that miR­424 was regulated by TNF­α and served an important role in adipogenesis.

Histological validation of adipogenic differentiation potential of ASC on collagen-based 2D scaffolds.

Determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when regeneration is supported by biomaterials or scaffolds. Scaffolds need to be able to induce tissue repair and limit undesired adipogenic differentiation. Depending on the scaffold employed, determination of cell behavior may be hindered by material interference with staining, which will limit either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however, histological analysis of cells cultured on collagen-based scaffolds is challenging. Here we describe a new histological method based on iron hematoxylin combined with Oil red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, and then stained with either ORO or iron hematoxylin and ORO combined. The collagen films avidly absorbed the ORO dye; conventional staining and quantification by dye extraction failed to discriminate between differentiated and undifferentiated cells on the films. On the contrary, the iron hematoxylin-ORO combination provided a quantitative and more reliable determination of adipocytes based on single cell count. This method is particularly recommended for determining the adipogenic differentiation potential of ASCs and other cell types grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine.

Effect of ELOVL6 on the lipid metabolism of bovine adipocytes.

This study investigated the effect of ELOVL6 (elongation of very long chain fatty acids protein 6) and its underlying mechanism on lipid metabolism in bovine adipocytes. The ELOVL6 gene was overexpressed in bovine adipocytes by adenoviruses, and RNA sequencing was performed. Overexpression of ELOVL6 showed reduced proportions of C14:0 (Myristic) and C16:0 (palmitate) fatty acids and increased proportions of C18.0 (stearate) and C20:4n6 (arachidonic) fatty acids in adipocytes. In addition, a total of 2170 differentially expressed genes (DEGs) were found, containing 1802 up-regulated and 368 down-regulated genes. KEGG pathway analysis revealed that the down-regulated genes were linked with the regulation of lipolysis and the Wnt signaling pathway. The up-regulated genes were mainly involved in the FoxO signaling pathway; the PI3K-Akt signaling pathway; and the cAMP signaling pathway. In conclusion, our results suggest that ELOVL6 could affect the fatty acid composition in bovine adipocytes. We identified numerous related DEGs and pathways, which may provide a basis for studying the function and molecular mechanism of the ELOVL6 gene in regulating lipid metabolism.

Differences in the lipid patterns during maturation of 3T3-L1 adipocytes investigated by thin-layer chromatography, gas chromatography, and mass spectrometric approaches.

Populations of industrialized countries have registered a dramatically increasing prevalence in obesity for many years. Despite continuous research, mechanisms involved in the storage and utilization of chemical energy in adipocytes are still under investigation. Adipocytes have the task to store excessive energy in the form of triacylglycerols (TG) and it is already well-known that the fatty acyl composition of TG is largely determined by the composition of the diet. In contrast to TG, the composition of adipocyte phospholipids was less comprehensively investigated. In this study, the compositions of the most abundant phospholipid classes of 3T3-L1 undifferentiated (preadipocytes) and differentiated cells (adipocytes) were determined. The lipid fractions were isolated by normal phase high-performance thin-layer chromatography and subsequently analyzed by electrospray ionization mass spectrometry. Additionally, the fatty acyl (FA) compositions were determined by gas chromatography. The positions of the FA residues were additionally confirmed by phospholipase A2 digestion. The advantages and disadvantages of the different analytical approaches will be discussed. It will be shown that undifferentiated 3T3-L1 and mature adipocytes differ extremely regarding their compositions. This goes along with an increase in odd-chain fatty acids. Graphical abstract.

Effects of Adipocyte-derived Factors on the Adrenal Cortex.

BACKGROUND AND OBJECTIVE: Obesity is highly complicated by hypertension and hyperglycemia. In particular, it has been proposed that obesity-related hypertension is caused by adipocyte-derived factors that are recognized as undetermined proteins secreted from adipocytes. Adipocyte-derived factors have been known to be related to aldosterone secretion in the adrenal gland. So far, Wnt proteins, CTRP-1, VLDL, LDL, HDL and leptin have been demonstrated to stimulate aldosterone secretion. In contrast, it has not yet been clarified whether adipocyte-derived factors also affect adrenal cortisol secretion. METHODS AND RESULTS: In the present study, we investigated the effect of adipocyte-derived factors on cortisol synthase gene CYP11B1 mRNA expression in vitro study using adrenocortical carcinoma H295R cells and mouse fibroblast 3T3-L1cells. Interestingly, adipocyte-derived factors were demonstrated to have the ability to stimulate CYP11B1 mRNA expression. CONCLUSION: Since CYP11B1 is well known as a limiting enzyme of cortisol synthesis, our study suggests that adipocyte-derived factors may stimulate cortisol secretion, as well as aldosterone secretion. Taken together, adipocyte-derived factors may be the cause of metabolic syndrome due to their stimulating effects on aldosterone/cortisol secretion. Therefore, the innovation of novel drugs against them may possibly be a new approach against metabolic syndrome.

YAP as a key regulator of adipo-osteogenic differentiation in human MSCs.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into several cell types, including cartilage, fat, and bone. As a common progenitor, MSC differentiation has to be tightly regulated to maintain the balance of their differentiation commitment. It has been reported that the decision process of MSCs into fat and bone cells is competing and reciprocal. Several factors have been suggested as critical factors that affect adipo-osteogenic decision, including melatonin and smad4. Yes-associated protein (YAP) is an important effector protein in the Hippo signaling pathway that acts as a transcriptional regulator by activating the transcription of the genes involved in cell proliferation and anti-apoptosis. The non-canonical role of YAP in regulating bone homeostasis by promoting osteogenesis and suppressing adipogenesis was recently demonstrated in a mouse model. However, it is unclear whether YAP is also crucial for modulating human MSC differentiation to fat and bone. METHODS: The expression level of YAP during MSC differentiation was modulated using pharmaceutical molecule and genetic experiments through gain- and loss-of-function approaches. RESULTS: We demonstrated for the first time that YAP has a non-canonical role in regulating the balance of adipo-osteogenic differentiation of human MSCs. The result from synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy showed unique metabolic fingerprints generated from YAP-targeted differentiated cells that were clearly distinguished from non-manipulated control. CONCLUSIONS: These results, thus, identify YAP as an important effector protein that regulates human MSC differentiation to fat and bone and suggests the use of FTIR microspectroscopy as a promising technique in stem cell research.

Plasmatic adipocyte biomarkers and foot pain associated with flatfoot in schoolchildren with obesity.

OBJECTIVE: The aim of this study was to determine the potential association of foot pain and plasmatic adipocytes as physiological biomarkers of childhood obesity with the incidence of flatfoot in a cohort of Egyptian school children aged 6 -12 years. METHODS: A total of 550 Egyptian schoolchildren (220 boys and 330 girls) aged 6-12 years were randomly invited to participate in this descriptive survey analysis. For all children, we assessed the diagnosis and severity of flatfoot as well as plasma adipocytes, as well as adiponectin, leptin, resistin, IL-6, and TNF-α, using the Dennis method and immunoassay techniques respectively. Foot pain was assessed by using a standard VAS of 100 mm and Faces Pain Scale, respectively. RESULTS: Flat foot was predicted in 30.4% of school-age children, most of them showed a higher frequency of overweight (33.3%) and obesity (62.5%). Boys showed higher ranges of flat foot than girls. Foot pain significantly correlated with flat foot and obesity among the studied populations. In overweight-obese children, plasmatic adipocyte variables, as well as adiponectin, leptin, resistin, IL-6, TNF-α showed significant correlations with foot stance, especially in boys. Also, the studied adipocyte variables along with BMI, age, gender explained about~65% of the variance of flatfoot with pain among our school-age students. CONCLUSION: Foot pain showed an association with flat foot and childhood obesity in 30.4% of school-age students (6-12 years). Foot pain was shown to correlate positively with the incidence of flat foot and changes in adiposity markers, as well as adiponectin, leptin, resistin, Il-6, TNF-α.

Dynamic microfactories co-encapsulating osteoblastic and adipose-derived stromal cells for the biofabrication of bone units.

Cells with differentiation potential into mesodermal types are the focus of emerging bone tissue engineering (TE) strategies as an alternative autologous source. When the source of cells is extremely limited or not readily accessible, such as in severe injuries, a tissue biopsy may not yield the required number of viable cells. In line, adipose-derived stromal cells (ASCs) quickly became attractive for bone TE, since they can be easily and repeatably harvested using minimally invasive techniques with low morbidity. Inspired by the multiphenotypic cellular environment of bone, we propose the co-encapsulation of ASCs and osteoblasts (OBs) in self-regulated liquefied and multilayered microcapsules. We explore the unique architecture of such hybrid units to provide a dynamic environment using a simple culture in spinner flasks. Results show that microtissues were successfully obtained inside the proposed microcapsules with an appropriate diffusion of essential molecules for cell survival and signaling. Remarkably, microcapsules cultured in the absence of supplemental osteogenic differentiation factors presented osteopontin immunofluorescence, evidencing that the combined effect of the dynamic environment, and the paracrine signaling between ASCs and OBs may prompt the development of bone-like microtissues. Furthermore, microcapsules cultured under dynamic environment presented an enhanced mineralized matrix and a more organized extracellular matrix ultrastructure compared to static cultures used as control. Altogether, data in this study unveil an effective engineered bioencapsulation strategy for the in vitro production of bone-like microtissues in a more realistic and cost-effective manner. Accordingly, we intend to use the proposed system as hybrid devices implantable by minimally invasive procedures for bone TE applications.

Plasmatic adipocyte biomarkers and foot pain associated with flatfoot in schoolchildren with obesity

SUMMARY OBJECTIVE The aim of this study was to determine the potential association of foot pain and plasmatic adipocytes as physiological biomarkers of childhood obesity with the incidence of flatfoot in a cohort of Egyptian school children aged 6 -12 years. METHODS A total of 550 Egyptian schoolchildren (220 boys and 330 girls) aged 6-12 years were randomly invited to participate in this descriptive survey analysis. For all children, we assessed the diagnosis and severity of flatfoot as well as plasma adipocytes, as well as adiponectin, leptin, resistin, IL-6, and TNF-α, using the Dennis method and immunoassay techniques respectively. Foot pain was assessed by using a standard VAS of 100 mm and Faces Pain Scale, respectively. RESULTS Flat foot was predicted in 30.4% of school-age children, most of them showed a higher frequency of overweight (33.3%) and obesity (62.5%). Boys showed higher ranges of flat foot than girls. Foot pain significantly correlated with flat foot and obesity among the studied populations. In overweight-obese children, plasmatic adipocyte variables, as well as adiponectin, leptin, resistin, IL-6, TNF-α.; showed significant correlations with foot stance, especially in boys. Also, the studied adipocyte variables along with BMI, age, gender explained about~65% of the variance of flatfoot with pain among our school-age students. CONCLUSION Foot pain showed an association with flat foot and childhood obesity in 30.4% of school-age students (6-12 years). Foot pain was shown to correlate positively with the incidence of flat foot and changes in adiposity markers, as well as adiponectin, leptin, resistin, Il-6, TNF-α

RESUMO OBJETIVO O objetivo deste estudo foi determinar a potencial associação de dor no pé e adipócitos plasmáticos como biomarcadores fisiológicos da obesidade infantil com incidência de pé plano em uma coorte de escolares egípcios de 6 a 12 anos. MÉTODOS Um total de 550 escolares egípcios (220 meninos e 330 meninas) com idades entre 6 e 12 anos foram convidados aleatoriamente para participar desta análise descritiva. Para todas as crianças, diagnóstico e gravidade do flatfoot, bem como adipócitos plasmáticos; adiponectina, leptina, resistina, IL-6 e TNF-α; foram avaliados pelo método de Dennis e técnicas de imunoensaio, respectivamente. A dor no pé foi avaliada usando uma EVA padrão de 100 mm e a Faces Pain Scale, respectivamente. RESULTADOS O pé plano foi predito em 30,4% das crianças em idade escolar; a maioria apresentou maior frequência de sobrepeso (33,3%) e obesidade (62,5%). Os meninos apresentaram maiores faixas de pé plano do que as meninas. A dor no pé correlacionou-se significativamente com pé plano e obesidade entre as populações estudadas. Em crianças obesas com sobrepeso, variáveis adipocitárias plasmáticas; adiponectina, leptina, resistina, IL-6 e TNF-α; apresentaram correlação significativa com a postura do pé, em meninos e meninas. Além disso, as variáveis estudadas dos adipócitos, juntamente com o IMC, idade e sexo, explicaram cerca de 65% da variância do pé plano com a dor entre os nossos alunos em idade escolar. CONCLUSÃO A dor no pé mostrou associação com pé plano e obesidade infantil em 30,4% dos estudantes em idade escolar (6-12 anos). A dor no pé se correlacionou positivamente com a incidência de pé plano e a mudança nos marcadores de adiposidade; adiponectina, leptina, resistina, IL-6, TNF-α.

GALNT2 as a novel modulator of adipogenesis and adipocyte insulin signaling.

BACKGROUND/OBJECTIVES: A better understanding of adipose tissue biology is crucial to tackle insulin resistance and eventually coronary heart disease and diabetes, leading causes of morbidity and mortality worldwide. GALNT2, a GalNAc-transferase, positively modulates insulin signaling in human liver cells by down-regulating ENPP1, an insulin signaling inhibitor. GALNT2 expression is increased in adipose tissue of obese as compared to that of non-obese individuals. Whether this association is secondary to a GALNT2-insulin sensitizing effect exerted also in adipocytes is unknown. We then investigated in mouse 3T3-L1 adipocytes the GALNT2 effect on adipogenesis, insulin signaling and expression levels of both Enpp1 and 72 adipogenesis-related genes. METHODS: Stable over-expressing GALNT2 and GFP preadipocytes (T0) were generated. Adipogenesis was induced with (R+) or without (R-) rosiglitazone and investigated after 15 days (T15). Lipid accumulation (by Oil Red-O staining) and intracellular triglycerides (by fluorimetric assay) were measured. Lipid droplets (LD) measures were analyzed at confocal microscope. Gene expression was assessed by RT-PCR and insulin-induced insulin receptor (IR), IRS1, JNK and AKT phosphorylation by Western blot. RESULTS: Lipid accumulation, triglycerides and LD measures progressively increased from T0 to T15R- and furthermore to T15R+. Such increases were significantly higher in GALNT2 than in GFP cells so that, as compared to T15R+GFP, T15R- GALNT2 cells showed similar (intracellular lipid and triglycerides accumulation) or even higher (LD measures, p < 0.01) values. In GALNT2 preadipocytes, insulin-induced IR, IRS1 and AKT activation was higher than that in GFP cells. GALNT2 effect was totally abolished during adipocyte maturation and completely reversed at late stage maturation. Such GALNT2 effect trajectory was paralleled by coordinated changes in the expression of Enpp1 and adipocyte-maturation key genes. CONCLUSIONS: GALNT2 is a novel modulator of adipogenesis and related cellular phenotypes, thus becoming a potential target for tackling the obesity epidemics and its devastating sequelae.

Caveolins; An Assailant or An Ally of Various Cellular Disorders.

Caveolae have impressive morphological highlights of the cytomembrane of mammalian cells which involve in wide diversity of cellular functions involving signaling pathways and cholesterol hastening. Caveolin proteins possess a 'scaffolding' domain which for caveolin-1 and caveolin-3 appear to act a dominant role in signal regulation through caveolae. Caveolin-1 is treated to be protein in the cytomembrane entrapped with caveolae in endothelial cells and vascular smooth muscle cells which diminish nitric oxide (NO) by fill up the calcium/calmodulin (Ca2+/CaM) confining point of endothelial nitric oxide synthase (eNOS), decrease NO generation produce endothelial dysfunction and atherosclerotic injury development. It is a cholesterol-binding layer protein associated with cell cholesterol transport and also shows cardioprotective action through ischemic preconditioning (IPC) in diabetic and postmenopausal rat heart. Additionally it is ensnared in the procedures of tumorigenesis, prostate disease, and inflammation. The present study in the paper is to explore the structural functionalities of caveolins and their contributory role in CVS disorders and various other diseases.

Molecular characterization of ABHD5 gene promoter in intramuscular preadipocytes of Qinchuan cattle: Roles of Evi1 and C/EBPα.

The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/ß hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5'-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of -109/+307 was identified as proximal minimum core promoter region of ABHD5 in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.

Dysplastic Lipoma: A Distinctive Atypical Lipomatous Neoplasm With Anisocytosis, Focal Nuclear Atypia, p53 Overexpression, and a Lack of MDM2 Gene Amplification by FISH; A Report of 66 Cases Demonstrating Occasional Multifocality and a Rare Association With Retinoblastoma.

In our routine and consultative pathology practices, we have repeatedly encountered an unusual subcutaneous fatty tumor with notable anisocytosis, single-cell fat necrosis, and patchy, often mild, adipocytic nuclear atypia. Because of the focal atypia, consultative cases have most often been received with concern for a diagnosis of atypical lipomatous tumor. Similar tumors have been described in small series under the designations "subcutaneous minimally atypical lipomatous tumors" and "anisometric cell lipoma." Sixty-six cases of this tumor type were collected and reviewed. Immunohistochemistry for p53, MDM2, CDK4, Retinoblastoma 1 (RB1) protein, CD34, S100, and CD163 was performed. Cases were tested for MDM2 gene amplification and RB1 gene deletion with fluorescence in situ hybridization (FISH) and for TP53 mutations by Sanger sequencing. Next-generation sequencing analysis using a panel of 271 cancer-related genes, including TP53, RB1, and MDM2, was also carried out. Our patient cohort included 57 male patients, 8 female patients, and 1 patient of unstated sex, who ranged in age from 22 to 87 years (mean: 51.2 y). All tumors were subcutaneous, with most examples occurring on the upper back, shoulders, or posterior neck (86.4%). Ten patients had multiple (2 to 5) lipomatous tumors, and the histology was confirmed to be similar in the different sites in 4 of them, including 1 patient who had a retinoblastoma diagnosed at age 1. The tumors were generally well circumscribed. At low magnification, there was notable adipocytic size variation with single-cell fat necrosis in the background associated with reactive histiocytes. Adipocytic nuclear atypia was typically patchy and characterized by chromatin coarsening, nuclear enlargement, and focal binucleation or multinucleation. Focal Lochkern change was frequent. In most instances, the degree of atypia was judged to be mild, but in 3 instances, it was more pronounced. Spindle cells were sparse or absent, and when present, cytologically bland. Thick ropy collagen bundles were absent. In all cases, p53 immunoexpression was noted (range: 2% to 20% of adipocytic nuclei), characteristically highlighting the most atypical cells. Twenty of 50 cases had MDM2 immunoreactivity, usually in <1% of the neoplastic cells, but in 4 cases, up to 10% of the cells were positive. Of 32 cases tested, 22 showed a near total loss of RB1 immunoexpression, and the remainder showed partial loss. Three of 13 cases showed RB1 gene deletion in >45% of the cells by FISH (our threshold value for reporting a positive result) with an additional 3 cases being very close to the required cutoff value. MDM2 gene amplification was absent in all 60 cases tested, including those with the greatest MDM2 immunoexpression and most pronounced atypia. All 5 tested cases showed no TP53 mutation with Sanger sequencing. Because of material quality issues, next-generation sequencing analysis could be performed in only 3 cases, and this did not reveal any recurrent mutations. All tumors were managed by simple local excision. Follow-up was available for 47 patients (range: 1 to 192 mo; mean: 27 mo) and revealed 2 local recurrences and no metastases. Dysplastic lipoma is a distinctive atypical fatty tumor variant that has p53 overexpression and RB1 gene abnormalities and lacks MDM2 gene amplification by FISH. These tumors have a strong male predominance and a notable tendency to involve the subcutaneous tissue of the shoulders, upper back and posterior neck. Multifocality is frequent (18.9% of patients with follow-up information), and there is a rare association with retinoblastoma. This tumor warrants separation from ordinary lipoma with fat necrosis, fat-rich spindle cell lipoma and the conventional form of atypical lipomatous tumor that features MDM2 gene amplification.

Adipocyte-derived Lysophosphatidylcholine Activates Adipocyte and Adipose Tissue Macrophage Nod-Like Receptor Protein 3 Inflammasomes Mediating Homocysteine-Induced Insulin Resistance.

The adipose Nod-like receptor protein 3 (NLRP3) inflammasome senses danger-associated molecular patterns (DAMPs) and initiates insulin resistance, but the mechanisms of adipose inflammasome activation remains elusive. In this study, Homocysteine (Hcy) is revealed to be a DAMP that activates adipocyte NLRP3 inflammasomes, participating in insulin resistance. Hcy-induced activation of NLRP3 inflammasomes were observed in both adipocytes and adipose tissue macrophages (ATMs) and mediated insulin resistance. Lysophosphatidylcholine (lyso-PC) acted as a second signal activator, mediating Hcy-induced adipocyte NLRP3 inflammasome activation. Hcy elevated adipocyte lyso-PC generation in a hypoxia-inducible factor 1 (HIF1)-phospholipase A2 group 16 (PLA2G16) axis-dependent manner. Lyso-PC derived from the Hcy-induced adipocyte also activated ATM NLRP3 inflammasomes in a paracrine manner. This study demonstrated that Hcy activates adipose NLRP3 inflammasomes in an adipocyte lyso-PC-dependent manner and highlights the importance of the adipocyte NLRP3 inflammasome in insulin resistance.