Normal Adipose Research Articles

Silencing of perilipin by short hairpin RNA inhibits proliferation and induces apoptosis in liposarcoma cells.

Previous studies have identified that perilipin-1 (PLIN1) is a highly specific marker for liposarcoma. However, its functions have yet to be fully elucidated. The aim of the present study was to investigate the potential role of PLIN1 in the proliferation, migration and apoptosis of liposarcoma cells. Short hairpin RNA was designed to inhibit PLIN1 levels. Cell proliferation was monitored by Cell Counting Kit‑8 assay and cell migration determined by wound healing assay. Flow cytometry was performed to assess the cell cycle distributions and apoptosis in liposarcoma cells. The results demonstrated that the expression of PLIN1 was significantly upregulated in liposarcoma tumor tissues compared with normal adipose tissues. Silencing of PLIN1 by short hairpin RNA significantly inhibited proliferation and migration and induced G1 phase cell cycle arrest and apoptosis in liposarcoma cell lines. It was identified that PLIN1 serves a crucial role in the pathogenesis and progression of liposarcoma and may be a potential therapeutic target for its clinical management.

Suppressive Role of Boron on Adipogenic Differentiation and Fat Deposition in Human Mesenchymal Stem Cells

Over the past years, adipose tissue has become an invaluable source of mesenchymal stem cells (MSCs) due to development of improved isolation methodologies. In a recent work, our group established a primary culture of human adipose-derived stem cells (hADSCs), which were characterized for their stem cell characteristics in detail and studied their myogenic differentiation potential in presence of boron. In the current study, we focused on the effects of a boron-containing compound, sodium pentaborate pentahydrate (NaB), on the adipogenic differentiation of hADSCs. Incorporation of boron in various chemical derivates has been a novel interest in drug-discovery attempts due to increasing number of reports on their anticancer, antibacterial, antiviral, and antifungal activities. In this report, a striking suppressive activity of boron on adipogenic differentiation of hADSCs is observed in a dose-dependent manner. Higher concentrations of NaB (20, 50, and 100μg/mL (68, 170 and 340μM)) resulted in a progressive decrease of lipid deposition, suppressed master regulators of adipogenesis transcriptional programming at the mRNA and protein levels, while having no evident cytotoxicity on the cells. The findings of this study are encouraging to undertake further investigations on potential beneficial effects boron in terms of its impact on normal and dysfunctional adipose biology. In that respect, these results pave the path to evaluate boron-based compounds in prevention and treatment of obesity which is a modern age pandemic that is predominant worldwide and found in strong association with comorbidities, including type 2 diabetes, hypertension, cardiovascular disease, cancers, and others."

Abstract 5835: Adipocyte-released agents induce resistance of breast cancer cells to lapatinib

Abstract Adipose tissue is often reduced to its role in fat storage and metabolic support for tumor cells. However, several studies have shown an adipocyte-mediated resistance effect to various anticancer therapies. However, the responsible agent(s), the mechanism(s) and the signaling pathway(s) involved in this resistance remain unclear. In the present study, we assessed the cytotoxic effect of lapatinib on several breast cancer cell lines in the presence or absence of adipocyte-conditioned medium. We performed different physical and chemical treatments on these conditioned media in order to separate and identify the different molecules and agents involved. In parallel, we investigated the changes that occurs in breast tumor cells following the contact with adipocyte-conditioned medium and the exposure to lapatinib both at transcriptional and protein level. In order to validate our hypothesis in vivo, we created a mouse model reproducing the contact between adipose tissue and tumors. Our results showed that tumor cells exposed to adipocyte-conditioned medium were less sensitive to lapatinib-mediated cytotoxic effects and cell cycle blockade than cells grown in standard culture medium. For example, we found that the IC50 was approximately seven-fold increased with lapatinib for tumor cells exposed to adipocyte-conditioned medium compared to the cells kept in control medium. This resistance was observed with the conditioned medium obtained both from human and murine adipocytes and confirmed on different breast tumor cell lines overexpressing HER2. Using a xenograft of normal human adipose tissue with implantation of tumor cells in contact to this adipose tissue, we also confirmed the protective effect against the antitumoral activity of lapatinib in vivo. The nature of the responsible agents of the resistance has been partly elucidated as well as the mechanisms. Indeed, it seems that the presence of soluble factors released from adipocytes such as lipolysis and metabolism products are required to limit the effect of lapatinib on tumor cells. The exposure of tumor cells to adipocyte-conditioned medium triggered changes on the lapatinib-mediated cell cycle blockade and on the expression of cell cycle regulation dependent proteins such as AKT, P27 and cyclins. In conclusion, compounds released from adipocytes reduced the lapatinib induced cytotoxicity on breast cancer cells overexpressing HER2 by interfering with the AKT signaling pathway. These results suggest possible strategies to counteract tumor cell resistance to lapatinib, by targeting adipocyte-released mediators. Citation Format: Aline Geneste, Minh Ngoc Duong, Aurore Cleret, Sabine Beaumel, Kamel Chettab, Emmanuel Delay, Philippe Valet, Lars Petter Jordheim, Charles Dumontet. Adipocyte-released agents induce resistance of breast cancer cells to lapatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5835.

Effects of transplanted adipose derived stem cells on the expressions of α-SMA and DCN in fibroblasts of hypertrophic scar tissues in rabbit ears.

To study the effects of transplanted adipose derived stem cells (ADSCs) on the expressions of α-smooth muscle actin (α-SMA) and decorin (DCN) in fibroblasts of hypertrophic scar tissues in rabbit ears. Twelve New Zealand white rabbits were selected; the normal subcutaneous adipose tissues in inguinal region were removed, ADSCs were extracted via enzyme digestion, cultured in Dulbecco's modified Eagle's medium (DMEM) and inoculated into the culture dish (3–5×104 cells/ml). After the rabbit ear hypertrophic scar model was established successfully, the fibroblasts of hypertrophic scar tissues in rabbit ears were separated and cultured using the mechanical method combined with enzyme digestion, and the ADSCs and scar fibroblasts were cultured in non-contact Transwell co-culture system for 21 days (experimental group); the corresponding scar fibroblasts were cultured in an ordinary 6-well plate without any treatment for 21 days (control group). The content of collagen I in fibroblasts was detected using the enzyme-linked immunosorbent assay (ELISA) kit, the mRNA expressions of α-SMA and DCN were detected via reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of α-SMA and DCN were detected via western blot analysis, and the expressions and distribution of α-SMA and DCN were detected via immunofluorescence assay. The results of ELISA showed that the content of collagen I in experimental group was decreased significantly (p<0.01). The results of RT-PCR and western blot analysis revealed that the mRNA and protein expression levels of α-SMA were significantly decreased (P<0.01, but those of DCN were significantly increased (p<0.01). Moreover, the results of immunofluorescence assay showed that the expression of α-SMA in experimental group was significantly decreased, while the expression of DCN was significantly increased. ADSCs can inhibit the mRNA and protein expressions of α-SMA and promote the mRNA and protein expressions of DCN in in vitro culture system, and they are expected to be used in the prevention and treatment of pathological scars.

Photoactivation of Autologous Transplantation of Subcutaneous Adipose Tissue Improves Glucose Homeostasis

Photoactivation of autologous transplantation of subcutaneous adipose tissue improves glucose homeostasis Increasing evidences indicated that normal adipose tissue transplantation improves whole-body energy metabolism and glucose homeostasis in a high-fat diet (HFD)-induced obese mice model. Adipose tissue macrophage is associated with glucose homeostasis and insulin resistance in type 2 diabetes and obese humans, offering a potential target for therapeutics. However, whether transplantation of autologous AT that changed macrophage phenotype directly contribute to systemic glucose intolerance has not been determined. Here we specifically developed our device, with more specific wavelengths of light, to activate macrophage phenotype in isolated sWAT from HFD mice. We found that the macrophage polarization, M1 marker genes expression, such as CD11c, IL-6 and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by photoactivation treatment. Furthermore, autologous transplantation of photoactivated sWAT into a HFD mice reduced blood glucose level and caused significant improvement in glucose tolerance, which was not shown in a sham-operated or non-photoactivated sWAT HFD mice. Moreover, Positron emission/computed tomography scans indicated higher glucose uptake in heart, but not liver, hindlimb muscles, and abdominal subcutaneous white adipose tissue. These data suggested that the correlation with photoactivation to shift ATMs polarization of HFD mice caused significant improvement in glucose homeostasis, and that autologous transplantation might be a promising therapeutic option for the treatment of diabetes. Disclosure J. Wu: None.

Corrigendum: Transplantation of Normal Adipose Tissue Improves Blood Flow and Reduces Inflammation in High Fat Fed Mice With Hindlimb Ischemia.

[This corrects the article on p. 197 in vol. 9, PMID: 29568274.].

NAD+-dependent deacetylase SIRT3 in adipocytes is dispensable for maintaining normal adipose tissue mitochondrial function and whole body metabolism.

Mitochondrial dysfunction in adipose tissue is involved in the pathophysiology of obesity-induced systemic metabolic complications, such as type 2 diabetes, insulin resistance, and dyslipidemia. However, the mechanisms responsible for obesity-induced adipose tissue mitochondrial dysfunction are not clear. The aim of present study was to test the hypothesis that nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin-3 (SIRT3) in adipocytes plays a critical role in adipose tissue mitochondrial biology and obesity. We first measured adipose tissue SIRT3 expression in obese and lean mice. Next, adipocyte-specific mitochondrial Sirt3 knockout (AMiSKO) mice were generated and metabolically characterized. We evaluated glucose and lipid metabolism in adult mice fed either a regular-chow diet or high-fat diet (HFD) and in aged mice. We also determined the effects of Sirt3 deletion on adipose tissue metabolism and mitochondrial biology. Supporting our hypothesis, obese mice had decreased SIRT3 gene and protein expression in adipose tissue. However, despite successful knockout of SIRT3, AMiSKO mice had normal glucose and lipid metabolism and did not change metabolic responses to HFD-feeding and aging. In addition, loss of SIRT3 had no major impact on putative SIRT3 targets, key metabolic pathways, and mitochondrial function in white and brown adipose tissue. Collectively, these findings suggest that adipocyte SIRT3 is dispensable for maintaining normal adipose tissue mitochondrial function and whole body metabolism. Contrary to our hypothesis, loss of SIRT3 function in adipocytes is unlikely to contribute to the pathophysiology of obesity-induced metabolic complications.

Pansteatitis Induced Differential Expression of Inflammatory and Apoptosis Genes in Adipose Tissue: A Glimpse into Pathophysiology of the Condition in the Nile Crocodile.

In the quest to understand the pathophysiology of pansteatitis in the Nile crocodile, a condition that was attributed to the recent crocodile die off at the Olifant river, Mpumalanga, the whole transcriptome of the Nile crocodile was sequenced and assembled using whole transcriptome shotgun sequencing data by Illumina RNAseq from RNA obtained from fat body and other adipose tissues.Total RNA, from abdominal fat body and other adipose tissue from normal farmed Nile crocodiles and samples collected from Nile crocodiles that had died of pansteatitis, were extracted and purified using Qiagen RNA mini prep (Qiagen, USA). RNA samples library were prepped using the Truseq stranded total RNA sample preparation and sequenced on the Illumina Hiseq 2500 with v4 SBS chemistry. Reads were assembled using Tophat (http://tophat.cbcb.umd.edu/) and aligned with reference transcriptome from Crocodylus porosus using Cufflinks (http://cufflinks.cbcb.umd.edu/) for downstream analysis while the differential gene expression analysis was done using Cuffdiff and RSEM (http://deweylab.github.io/RSEM/).A total of 13,329 genes were identified out which 1541 genes related to inflammatory response and apoptosis were identified. A total of 641 genes were up regulated in the pansteatitis samples as compared to normal adipose tissues using a fold change of 16.0. A total of 70 inflammatory genes were up regulated in the pansteatitis samples including, PDCD5 (programmed cell death 5), NFIL3 (nuclear factor, interleukin 3 regulated protein), HSP90B1 (heat shock protein 90kDa beta (Grp94), member 1), VCAM1 (vascular cell adhesion molecule 1), IL1R1 (interleukin 1 receptor, type I), TNIP1 (TNFAIP3 interacting protein 1), JAK1 (Janus kinase 1), TNFAIP2 (tumor necrosis factor, alpha‐induced protein 2) IL6ST (interleukin 6 signal transducer (gp130, oncostatin M receptor)) CFD (complement factor D (adipsin) and PPARG (peroxisome proliferator‐activated receptor gamma) among others. These genes are responsible for inflammation, apoptosis, regulation of Toll‐like receptors in inflammatory response, leucocyte migration and fibrosis in chronic inflammation.We present for the first time in the Nile crocodile, a comprehensive lists of inflammatory response genes that are expressed and up regulated in pansteatitis in the Nile crocodile.Support or Funding InformationPart of the study was funded by Tertiary Education Trust Fund (TETFund) AST and D, Nigeria and Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic Profiling of Serum Reveals Significant Changes in Lipid and Amino Acid Metabolism in A Lipodystrophy Mouse Model

ObjectiveAdipose tissue serves as not only an energy reservoir to store excess energy in form of triglyceride but also as an important endocrine organ producing adipokines such as leptin and adiponectin. Lipodystrophy, characterized by abnormal or degeneration of adipose tissue, leads to metabolic diseases including hypertriglyceridemia, insulin resistance, and hepatic steatosis. Lipotoxicity is known as a major driver for the metabolic dysregulation in lipodystrophy, however, which specific lipid species and/or other factors contributes to the disease progression is not well characterized. In this study, we employed untargeted metabolomics to identify small metabolites that are altered in a lipodystrophy mouse model.MethodsWe have recently generated the lipodystrophy mouse model by which adipocyte mouse double minute 2 homolog (Mdm2) is deleted by Cre‐loxP approach. Circulating metabolites were measured and compared in the serum of adipocyte‐specific Mdm2 knockout mice at 3‐week (Before lipodystrophy development) and 12‐week old (After lipodystrophy development) with age‐matched wild‐type mice, using an untargeted metabolomics profiling by LC‐MS. Data were explored using principal component analysis and statistical analysis.ResultsOur untargeted metabolomics profiling had successfully identified and quantified more than 300 circulating metabolites in the serum samples. The results promptly point towards a significant increase in metabolites groups including carnitine and amino acids in the lipodystrophy mouse mice. During the development of lipodystrophy, serum level of phosphatidylcholines species (PC) was significantly reduced whereas increased serum sphingolipid species (including sphingosine and sphinganine) were observed in Mdm2 knockout mice is positively associated with insulin resistance. The high rate of incomplete fatty acid oxidation and accumulation of acylcarnitine metabolites may indicate the damage of skeletal muscle due to an excessive fatty‐acid metabolites supply in the lipodystrophy mice.ConclusionTogether, these data show that metabolic diseases in lipodystrophy mice is associated with the alteration of metabolites supply to other tissues. Moreover, the utilization of lipodystrophy model in investigating the mechanisms of novel therapeutic targets of metabolic disease via maintaining normal adipose tissue functions was demonstrated.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biphasic calcium phosphate scaffolds with controlled pore size distribution prepared by in-situ foaming

In this study, a reproducible method of fabricating hierarchically 3D porous scaffolds with high porosity and pore interconnectivity is reported. The method is based on in-situ foaming of a dispersion of diisocyanate, polyol, water and hydroxyapatite (HA) to form a hard foamed HA/polyurethane composite which after heat treatment provided a bi-phase calcium phosphate scaffold. This technique, combining the advantages of polymer sponge and direct foaming methods, provides a better control over the macrostructure of the scaffold. A modification of the multi-scaled porous macrostructure of scaffolds produced by changing the ratio of input reactants and by sintering temperature was studied. The pore morphology, size, and distribution were characterized using a scanning electron microscope and mercury porosimetry. The pores were open and interconnected with multi-scale (from several nanometres to millimetres) sizes convenient for using in tissue engineering applications. The bioactivity was confirmed by growing an apatite layer on the surfaces after immersion in simulated body fluid. The material was biocompatible, as shown by using normal human adipose tissue-derived stem cells (ASC). When seeded onto the scaffolds, the ASC adhered and remained healthy while maintaining their typical morphology.

Transplantation of Normal Adipose Tissue Improves Blood Flow and Reduces Inflammation in High Fat Fed Mice With Hindlimb Ischemia.

Background: Fat deposition is associated with peripheral arterial disease. Adipose tissue has recently been implicated in vascular remodeling and angiogenic activity. We hypothesized that the transplantation of adipose tissues from normal mice improves blood flow perfusion and neovascularization in high-fat diet fed mice.Methods: After 14 weeks of high-fat diet (HFD)-fed mice, unilateral hind limb ischemia was performed. Subcutaneous white adipose tissue (WAT) and brown adipose tissue (BAT) fat pads were harvested from normal EGFP mice, and subcutaneously transplanted over the region of the adductor muscles of HFD mice. Blood flow was measured using Laser Doppler Scanner. Vascular density, macrophages infiltration, and macrophage polarization were examined by RT-qPCR, and immunohistochemistry.Results: We found that the transplantation of WAT derived from normal mice improved functional blood flow in HFD-fed mice compared to mice transplanted with BAT and sham-treated mice. WAT transplantation increased the recruitment of pericytes associated with nascent blood vessels, but did not affect capillary formation. Furthermore, transplantation of WAT ameliorated HFD-induced insulin resistance, M2 macrophage predominance and the release of arteriogenic factors in ischemic muscles. Mice receiving WAT also displayed a marked reduction in several proinflammatory cytokines. In contrast, mice transplanted with BAT were glucose intolerant and demonstrated increased IL-6 levels in ischemic muscles.Conclusion: These results indicate that transplantation of adipose tissue elicits improvements in blood perfusion and beneficial effects on systemic glucose homeostasis and could be a promising therapeutic option for the treatment of diabetic peripheral arterial disease.

Abstract B07: Regaining epigenetic control of the Hippo pathway to inhibit sarcomagenesis

Abstract Nearly 11,000 individuals in the United States and 200,000 individuals worldwide are diagnosed with a form of soft-tissue sarcoma (STS) every year. STS is a rare heterogeneous and complex family of disease associated with no uniform underlying genetic abnormality. However, sarcoma treatment has not changed significantly in 25 years. Patients are limited to surgery, radiotherapy, and chemotherapy. Therefore, the discovery of novel targets and mechanisms is critical. The Hippo pathway functions as a signaling hub facilitating cellular responses to multiple stimuli during tumorigenesis. The most important downstream effector of the Hippo pathway is Yes-activated protein (YAP), a transcriptional coactivator that promotes pro-proliferation and suppresses apoptosis. Inactivation of the Hippo pathway promotes nuclear localization of YAP. Whereas many studies have defined YAP1's transcriptional targets in epithelial tumors and normal tissues, its role in mesenchymal tumors is unclear. Here, we confirm that increased YAP1 mRNA expression correlates with worse overall survival in undifferentiated pleomorphic sarcoma (UPS) patients and that YAP1 protein levels are frequently elevated in human sarcomas. Furthermore, immunohistochemical (IHC) analysis of human sarcoma tissue microarray (TMA) showed that YAP levels are dramatically increased in the nuclei of high-grade UPS tumor cells, compared with normal skeletal, adipose, and arterial tissue. UPS is one of the more commonly diagnosed and aggressive subtypes of sarcomas found in adults. To define mechanisms of YAP1-mediated sarcomagenesis, we developed a novel genetic mouse model in which YAP1 is conditionally deleted from KrasG12D+; Trp53fl/fl (KP) UPS tumors (KPY). Microarray analysis of both tumors revealed that YAP1 loss inhibits expression of NF-κB signaling components and transcriptional targets. Consistent with these findings, ChIP-seq and super-enhancer analysis of human UPS tumors (n=3) revealed that many of the 900 identified UPS super enhancers regulate expression of NF-κB targets dependent on YAP1 in our system. This finding is particularly relevant as NF-κB is a key regulator of muscle cell proliferation and suppresses differentiation by inhibiting expression of the myogenic transcription factor, MYOD1. Together, these data suggest that upregulation of YAP1 activity promotes NF-κB-mediated proliferation and inhibits differentiation. Importantly, we have discovered that a combination of JQ1, a BET family inhibitor, and vorinostat (SAHA), a histone deacetylase (HDAC) inhibitor, decreases YAP1 expression, YAP1 protein stability, and sarcoma cell proliferation. Mechanistically, SAHA/JQ1 treatment significantly increases expression of the tumor suppressor angiomotin (AMOT), which binds YAP1, sequesters it in the cytoplasm, and facilitates its degradation. We have also found that AMOT expression is lost in sarcomas compared with normal muscle tissue, likely due to epigenetic suppression. SAHA/JQ1 treatment also decreased gene expression of YAP1 targets and caused reexpression of the muscle differentiation markers p57, MEF2C, and MYOD1. Based on these data we will investigate the efficacy of SAHA/JQ1 against UPS and other sarcomas in vivo using subcutaneous and autochthonous mouse models. We will probe the ability of SAHA/JQ1 treatment to inhibit YAP1 in vivo, to suppress NF-κB dependent mechanisms of cell proliferation and tumorigenesis, and to promote terminal differentiation of sarcoma cells. Ultimately, we will determine whether this approach represents a novel course of targeted treatment for sarcoma patients. Citation Format: Shuai Ye, Koreana Pak, Jennifer Shah, Susan Chor, Shaun Egolf, Gloria Marino, T.S. Karin Eisinger-Mathason. Regaining epigenetic control of the Hippo pathway to inhibit sarcomagenesis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B07.

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 μm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Normal human adipose tissue functions and differentiation in patients with biallelic LPIN1 inactivating mutations

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocyte metabolism, and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known about the roles of lipin-1 in human adipocyte physiology. Apparently, fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical, and gene expression analysis of adipose tissue biopsies from human patients harboring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of adipocyte size. Nevertheless, the adipose tissue develops without obvious histological signs of lipodystrophy and with normal qualitative composition of storage lipids. The increased expression of key adipogenic determinants such as SREBP1, PPARG, and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes with deficiency of functional lipin-1.

Adipocyte size variability in benign and malignant lipomatous tumors and morphologic mimics: a quantitative definition using digital pathology

Among well-differentiated lipomatous lesions, variability in adipocyte size has been proposed as a morphologic feature of malignancy. Specifically, normal adipose tissue and benign lipomas tend to contain adipocytes of uniform size, whereas atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) is described as containing adipocytes with a conspicuous variation in cell size. However, this proposed variance has never been objectively, quantitatively correlated with diagnosis. Using whole-slide scanning combined with semiautomated digital image analysis, we aimed to quantitatively test the hypothesis that variance in adipocyte size is a feature of malignancy in well-differentiated lipomatous tumors. Whole-slide scanning was performed on representative hematoxylin and eosin-stained slides selected from 130 cases representing benign (lipoma, spindle cell lipoma) and malignant lipomatous neoplasms (ALT/WDL) and morphologic mimics (normal adipose tissue, fat necrosis, fat atrophy). A previously validated, open source software package (Fiji-based Adiposoft) was used in semiautomated analysis of adipocyte size from a representative 1-cm2 portion of each slide. Median, range, and variance of cell sizes were compared across all diagnoses. Fat atrophy demonstrated smaller adipocyte cell size compared with other diagnoses. Among the remaining diagnostic groups, no significant differences were identified in adipocyte size or variance by objective quantitative morphologic analysis. However, the maximum range of adipocyte size was significantly higher in ALT/WDL than conventional lipoma and spindle cell lipoma. These data quantitate the morphology of ALT/WDL and its mimics and more specifically define the somewhat subjective "variability" of cell size as maximum range, rather than variance, of adipocyte size.

Adipose and Liver Function in Primate Offspring with Insulin Resistance Induced by Estrogen Deprivation in Utero

We recently demonstrated that offspring delivered to baboons deprived of estrogen during the second half of gestation exhibited insulin resistance. Therefore, because skeletal muscle accounts for >80% of insulin dependent glucose disposal, we suggested that estrogen in utero programs factors in fetal skeletal muscle important for insulin sensitivity in offspring. However, liver and adipose are also sites of insulin action and adipose insulin resistance can increase serum free fatty acid (FFA) levels and thereby reduce skeletal muscle insulin sensitivity. Therefore, in the current study we determined whether estrogen-deprived offspring exhibit normal adipose and hepatic function. The fasting serum levels of adiponectin, leptin, glucose, and analytes of liver function as well as the basal levels of serum FFA were similar in offspring of estrogen replete/suppressed baboons. Moreover, the normal glucose-induced decline in serum FFA levels measured in untreated offspring was also measured in offspring of letrozole-treated baboons. Fetal serum levels of adiponectin and leptin in late gestation also were similar and expression of nitrotyrosine negligible in fetal liver and adipose of untreated and letrozole-treated animals. These results indicate that offspring of letrozole-treated baboons have normal adipose and liver function and do not exhibit adipose insulin resistance. Therefore, we suggest that the insulin resistance observed in estrogen-deprived offspring primarily reflects a decline in insulin-stimulated glucose clearance by skeletal muscle and which supports our original suggestion that estrogen in utero programs factors in fetal skeletal muscle that promote insulin sensitivity in offspring.

Altered Adipose-Derived Stem Cell Characteristics in Macrodactyly

Macrodactyly is a congenital disease characterized by aggressive overgrowth of adipose tissue in digits or limbs frequently accompanied with hyperostosis and nerve enlargement; its pathological mechanism is poorly understood. Adipose-derived stem cells (ASCs) have been extensively studied in tissue engineering and regenerative medicine as an ideal alternative substitute for bone marrow-derived mesenchymal stem cells (BM-MSCs), but their pathological role is largely unknown. In this study, ASCs from macrodactyly adipose tissues (Mac-ASCs) were isolated and compared to ASCs derived from the normal abdominal subcutaneous adipose tissue (Sat-ASCs) for cell morphology, surface marker expression, proliferation rate, and tri-lineage differentiation potential. Despite similar cell morphology and cell surface marker expression, Mac-ASCs showed higher cell proportion in the S phase and increased proliferation compared with Sat-ASCs. Moreover, osteogenic and chondrogenic differentiation capacities were enhanced in Mac-ASCs, with reduced adipogenic potential. In addition, the expression levels of adipogenic genes were lower in undifferentiated Mac-ASCs than in Sat-ASCs. These findings unraveled enhanced proliferation activity, a regression in the differentiation stage, and greater potentiality of ASCs in macrodactyly, which could contribute to hyperostosis and nerve enlargement in addition to adipose tissue overgrowth in patients.

Injectable Mussel-Inspired Immobilization of Platelet-Rich Plasma on Microspheres Bridging Adipose Micro-Tissues to Improve Autologous Fat Transplantation by Controlling Release of PDGF and VEGF, Angiogenesis, Stem Cell Migration.

Platelets-rich plasma (PRP) can produce growth factors (GFs) to improve angiogenesis. However, direct injection of PRP does not lead to highly localized GFs. The current study employs a mussel-inspired polydopamine to immobilize PRP on gelatin microspheres (GMs) with the purpose of bridging adipose micro-tissues to help implanted fat survive (GM-pDA-PRP). Enhanced PRP adhesion leads to a prolonged and localized production of GFs, which is verified by platelet counting and by ELISA of vascular endothelial growth factors (VEGFs) and of platelet derived growth factors (PDGFs). The GM-pDA-PRP "hatches" a microenvironment for the proliferation of adipose-derived stem cells. After the adipose micro-tissue has bridged with GM-pDA-PRP after 16 weeks, triple-fluorescence staining reveals that the mature adipocytes, blood vessels, and capillaries are arranged like in normal adipose tissue. The survival fat increases significantly compared to that in control, PRP, and GM-PRP groups (84.8 ± 11.4% versus 47.8 ± 8.9%, 56.9 ± 9.7%, and 60.2 ± 10.5%, respectively). Both histological assessments and CD31 immunofluorescence indicate that the improvement of angiogenesis in GM-pDA-PRP is higher than in the fat graft group (6.4-fold in quantitative CD31 positive cells). The CD34 positive cells in the GM-pDA-PRP group are around 3.5-fold the amount in the fat graft group, which suggests that more stem cells migrate to the implant area. Cell proliferation staining shows that the number of Ki67 positive cells is around five times as high as that in the fat graft group.

PD-1 and PD-L1 expression in bone and soft tissue sarcomas

PD-1 and its ligands have been shown to play a significant role in evasion of malignant tumour cells from the immune system. Last year, the Unites States Food and Drug Administration (FDA) approved anti-PD-1 inhibitors for treatment of non-small cell lung carcinoma and recently expanded the use of immunotherapy for metastatic urothelial cell carcinoma and Hodgkin lymphoma. However, studies on expression of PD-1 and its ligand in malignant bone and soft tissue sarcoma are sparse. In this study, we evaluated PD-1 and PD-L1 expression on variants of liposarcomas and rhabdomyosarcomas, osteosarcomas and chondrosarcomas. Tissue microarrays (TMAs) for liposarcomas (well differentiated, myxoid/round cell, and pleomorphic), rhabdomyosarcomas (alveolar, embryonal, pleomorphic, and spindle cell), conventional osteosarcomas and chondrosarcomas were stained for PD-1 and PD-L1 antibodies. Adipose tissue, skeletal muscle, bone, osteochondroma and lipoma were used as control and benign counterparts. Western blot was performed to evaluate expression of PD-1 and PD-L1 in four sarcoma cell lines. Osteosarcomas, chondrosarcomas, and all variants of liposarcomas and rhabdomyosarcomas over-expressed PD-1 relative to normal tissue. Expression of PD-1 in rhabdomyosarcomas was associated with higher tumour stage. Only one case of pleomorphic liposarcoma, one case of pleomorphic rhabdomyosarcoma and two cases of alveolar rhabdomyosarcomas were positive for PD-L1. Normal adipose tissue, skeletal muscle, and bone were negative for both PD-1 and PD-L1 and lipomas and osteochondroma weakly expressed PD-1 but not PD-L1. Western blot confirmed the presence of PD-1 protein in all four sarcoma cell lines. Overall, our results showed cytoplasmic expression of PD-1 in the bone and soft tissue sarcomas, while PD-L1 was negative. Whether these data are an indication for effectiveness of immunotherapy in the management of malignant bone and soft tissue sarcomas remains to be elucidated.

Puberty is an important developmental period for the establishment of adipose tissue mass and metabolic homeostasis

ABSTRACTOver the past 2 decades, the incidence of childhood obesity has risen dramatically. This recent rise in childhood obesity is particularly concerning as adults who were obese during childhood develop type II diabetes that is intractable to current forms of treatment compared with individuals who develop obesity in adulthood. While the mechanisms responsible for the exacerbated diabetic phenotype associated with childhood obesity is not clear, it is well known that childhood is an important time period for the establishment of normal white adipose tissue in humans. This association suggests that exposure to obesogenic stimuli during adipose development may have detrimental effects on adipose function and metabolic homeostasis. In this study, we identify the period of development associated with puberty, postnatal days 18–34, as critical for the establishment of normal adipose mass in mice. Exposure of mice to high fat diet only during this time period results in metabolic dysfunction, increased leptin expression, and increased adipocyte size in adulthood in the absence of sustained increased fat mass or body weight. These findings indicate that exposure to obesogenic stimuli during critical developmental periods have prolonged effects on adipose tissue function that may contribute to the exacerbated metabolic dysfunctions associated with childhood obesity.