Viable Adipocytes Research Articles

Heterogeneity of extracellular vesicles in porcine myoblasts regulates adipocyte differentiation

sThe interactions between myogenic cells and adipocytes play an important role in improving carcass traits and the efficiency of energy utilization. However, there are few reports about the interaction between them mediated by small extracellular vesicles (sEV). In this study, sEV derived from porcine primary skeletal muscle stem cells (MuSCs) was found to be involved in the inhibition of porcine primary adipocyte viability, triglyceride content, Oil Red O enrichment and the expression of adipogenic genes. When the MuSCs were treated with insulin (INS) and oleic acid (OA), the effects of their secreted sEVs on adipose precursor cells were reversed, suggesting that the signaling effects of sEV are related to their own heterogeneity. Further by component heterogeneity analysis, miR-146a-5p was found to be enriched in sEVs of MuSCs and to regulate and suppress adipogenesis through its heterogeneity. This study provides an important mechanism and molecular target for small extracellular vesicles to regulate the interaction between muscle and adipose tissue and improve carcass traits at the intercellular level.

Nervonic acid improves fat transplantation by promoting adipogenesis and angiogenesis.

Adipose tissue engraftment has become a promising strategy in the field of regenerative surgery; however, there are notable challenges associated with it, such as resorption of 50‑90% of the transplanted fat or cyst formation due to fat necrosis after fat transplantation. Therefore, identifying novel materials or methods to improve the engraftment efficiency is crucial. The present study investigated the effects of nervonic acid (NA), a monounsaturated very long‑chain fatty acid, on adipogenesis and fat transplantation, as well as its underlying mechanisms. To assess this, NA was used to treat cells during adipogenesis in vitro, and the expression levels of markers, including PPARγ and CEBPα, and signaling molecules were detected through reverse transcription‑quantitative PCR and western blotting. In addition, NA was mixed with fat grafts in in vivo fat transplantation, followed by analysis through Oil Red O staining, hematoxylin & eosin staining and immunohistochemistry. It was demonstrated that NA treatment accelerated adipogenesis through activation of the Akt/mTOR pathway and inhibition of Wnt signaling. NA treatment enriched the expression of Akt/mTOR signaling‑related genes, and increased the expression of genes involved in angiogenesis and fat differentiation in human mesenchymal stem cells (MSCs). Additionally, NA effectively improved the outcome of adipose tissue engraftment in mice. Treatment of grafts with NA at transplantation reduced the resorption of transplanted fat and increased the proportion of perilipin‑1+ adipocytes with a lower portion of vacuoles in mice. Moreover, the NA‑treated group exhibited a reduced pro‑inflammatory response and had more CD31+ vessel structures, which were relatively evenly distributed among viable adipocytes, facilitating successful engraftment. In conclusion, the present study demonstrated that NA may not only stimulate adipogenesis by regulating signaling pathways in human MSCs, but could improve the outcome of fat transplantation by reducing inflammation and stimulating angiogenesis. It was thus hypothesized that NA could serve as an adjuvant strategy to enhance fat engraftment in regenerative surgery.

Oral Administration of Lutein Improves Fat Graft Survival by Alleviating Oxidative Stress in Mice.

Oxidative stress induced by ischemia and hypoxia in fat transplantation is a major obstacle to graft retention. Previous studies have shown that lutein has excellent adipose tissue affinity and anti-oxidative stress ability, however, the effects of oral lutein on fat transplantation have not been studied yet. We aimed to investigate whether oral lutein can improve fat transplantation retention by regulating oxidative stress, apoptosis, and inflammatory cytokine levels in graft tissues. Nude mice were assigned to the control group (normal saline), low-dose lutein group (10 mg/kg/day) and high-dose lutein group (20 mg/kg/day) randomly. All mice were given by gavage 1 week before fat grafting and continued for 2 weeks. The grafts were collected 1, 2, and 12 weeks after treatment. By conducting histological analyses, western blotting, quantitative polymerase chain reaction and cell metabolic function detections, the regulatory effects of lutein on apoptosis and oxidative stress in grafts were demonstrated. Besides, RNA sequencing was conducted to further clarify the efficacy of lutein on fat grafting. Lutein induced superior graft retention, histological structures and more viable adipocytes than control group. It relieved tissue oxidative stress and lipid oxidative damage by decreasing reactive oxygen species, and significantly reduced inflammation and apoptosis of grafts. RNA sequencing analysis confirmed that lutein could down-regulate the genes expression of oxidative stress and related inflammation and apoptosis. Our study suggested that oral administration of lutein can improve fat graft survival by reducing the levels of oxidative stress, inflammation and apoptosis in grafted fat.

The Extract of Humulus japonicus Inhibits Lipogenesis and Promotes Lipolysis via PKA/p38 Signaling

Introduction: Previous research has shown that an aqueous extract of Humulus japonicus (EH) can ameliorate hypertension, nonalcoholic fatty liver disease, and oxidative stress in adipocytes by activating the thermogenic pathway. However, the effects of an ethanol (30%) extract of EH on obesity are unknown. Methods: Various protein expression levels in fully differentiated 3T3-L1 adipocytes were assessed by Western blotting. Lipid deposition in 3T3-L1 adipocytes was examined by oil red O staining. The MTT assay was used to evaluate adipocyte viability. Caspase 3 activity and glycerol release were determined using commercial assay kits. Results: In this study, we discovered that EH treatment inhibited lipogenesis and promoted lipolysis in both differentiated 3T3-L1 adipocytes and adipose tissue of mice fed a high-fat diet. EH treatment also increased phosphorylated protein kinase A (PKA) levels while reducing p38 phosphorylation. When H89, a PKA inhibitor, was used, the effects of EH on lipogenic lipid accumulation and lipolysis in 3T3-L1 adipocytes were eliminated. Treatment with luteolin 7-O-β-d-glucoside (LU), the major active compound in EH, also suppressed lipid deposition and p38 phosphorylation but enhanced lipolysis in 3T3-L1 adipocytes. These changes were abrogated by H89. Conclusion: These findings indicate that EH containing LU reduces lipogenesis and stimulates lipolysis via the PKA/p38 signaling pathway, leading to an improvement in obesity in mice. Therefore, our study suggested that EH could be a promising therapeutic agent for treating obesity.

Attenuated angiogenesis and macrophage infiltration during adipose tissue regeneration in a megavolume human fat grafting nude mouse model.

Survival and regeneration mechanisms of large (>250 mL) fat grafts remain incompletely understood. In fat grafts from volunteers with megavolume fat transfer breast augmentation, neovascularization and inflammatory cell infiltration decreased within 7 days according to histological analysis. We further investigated this phenomenon using a nude mouse model. To simulate clinical contexts, chambers containing 1 mL human fat were implanted into nude mice. Chambers allowed selective transfer of tissue fluid from recipient nude mice into chambers, but not capillaries or macrophages. Seven days later, fat was removed from the chamber and reimplanted into a new nude mouse in the open-chambered fat group (OCFG, n=45). Adipose samples from volunteers and explanted grafts from OCFG were subjected to histological analyses. Graft weight, vascularization, and immune response were also compared between the OCFG and conventional direct fat grafting (control group (CG)). Percent tissue integrity, percent fibrosis, adipocyte viability, and neovascularization did not significantly differ between volunteer samples and OCFG grafts at day 7. On day 90, OCFG retention rate was decreased relative to CG and the fibrosis area was larger in the OCFG than in the CG. However, the macrophage and capillary counts were lower in the OCFG group relative to CG at days 7 and 14 after transplantation. The present study provides histological analyses of megavolume fat grafts sampled from clinical breast augmentation tissues and a xenograft nude mouse model. However, these preliminary results in a small clinical cohort should be further assessed in large allogeneic animal models.

Optimization and validation of a fat-on-a-chip model for non-invasive therapeutic drug discovery.

Obesity is a significant public health concern that is closely associated with various comorbidities such as heart disease, stroke, type II diabetes (T2D), and certain cancers. Due to the central role of adipose tissue in many disease etiologies and the pervasive nature in the body, engineered adipose tissue models are essential for drug discovery and studying disease progression. This study validates a fat-on-a-chip (FOAC) model derived from primary mature adipocytes. Our FOAC model uses a Micronit perfusion device and introduces a novel approach for collecting continuous data by using two non-invasive readout techniques, resazurin and glucose uptake. The Micronit platform proved to be a reproducible model that can effectively maintain adipocyte viability, metabolic activity, and basic functionality, and is capable of mimicking physiologically relevant responses such as adipocyte hypertrophy and insulin-mediated glucose uptake. Importantly, we demonstrate that adipocyte size is highly dependent on extracellular matrix properties, as adipocytes derived from different patients with variable starting lipid areas equilibrate to the same size in the hyaluronic acid hydrogel. This model can be used to study T2D and monitor adipocyte responses to insulin for longitudinally tracking therapeutic efficacy of novel drugs or drug combinations.

Advancing Standardization in Fat Graft Survival Studies: Assessing the Superiority of Paraepididymal Fat Graft as a Donor Site.

There is currently no standardization in the field of research on fat grafts in rats, which is one of the most popular topics in plastic surgery. The aim of our study was to demonstrate the effects of selecting paraepididymal fat grafts as the donor area on enhancing the reliability of fat graft studies. In this study, 12 male Sprague-Dawley rats were used to obtain adipose grafts from both inguinal and paraepididymal regions. After measuring the graft weights, they were subjected to histological examination using hematoxylin-eosin staining, as well as immunohistochemical staining with antiperilipin antibody. Purity of the samples, viability of adipose cells, and the presence of lymph nodes within the grafts were analyzed. The purity of adipose cells in graft samples obtained from the paraepididymal region was found to be 98.1% compared with the total sample. In contrast, the purity of adipose cells in graft samples obtained from the inguinal region was 58.37%. Hematoxylin-eosin staining revealed significantly higher adipocyte viability and vascularity in the paraepididymal region compared with the inguinal region (P = 0.0134). Conversely, lymphatic tissue content in samples obtained from the inguinal region was significantly higher compared with paraepididymal adipose tissue samples (P < 0.0001). Immunohistochemical staining with antiperilipin antibody showed a denser and more uniform staining pattern in paraepididymal adipose grafts (P < 0.0001). Using paraepididymal fat, devoid of lymphatic tissue, naturally eliminates 2 critical biases (estrogen and lymphatic tissue), enhancing the standardization and reliability of fat graft survival studies.

Assessing Long-Term Volume Retention in Breast Fat Grafting: A Comparative Study of Lipoaspirate Processing Techniques.

Autologous fat grafting is a method of improving aesthetic outcomes after both breast reconstruction and aesthetic surgery through volume enhancement and tissue contouring. Long-lasting effects are linked to greater patient satisfaction and more optimal augmentation results. Harvesting, processing, and injection techniques may all affect the longevity of deformity filling. Our objective is to evaluate the effect of lipoaspirate processing modality on longitudinal volume retention after surgery. A prospective, single-institution, randomized control trial placed consented postmastectomy fat grafting patients into 1 of 3 treatment arms (active filtration, low-pressure decantation, and standard decantation) in a 1:1:1 ratio. A preoperative 3-dimensional scan of the upper torso was taken as baseline. At the 3-month postoperative visit, another 3D scan was taken. Audodesk Meshmixer was used to evaluate the volume change. The volume of fat injected during the initial procedure did not differ significantly between the treatment arms (P > 0.05). Both active filtration and low-pressure decantation resulted in higher percentage volume retention than traditional decantation (P < 0.05). Active filtration and low-pressure decantation exhibited comparable degrees of fat maintenance at 3 months (P > 0.05). Compared with using traditional decantation as the lipoaspirate purification technique, active filtration and low-pressure decantation may have led to higher levels of cell viability by way of reduced cellular debris and other inflammatory components that may contribute to tissue resorption and necrosis. Further immunohistochemistry studies are needed to examine whether active filtration and low-pressure decantation lead to lipoaspirates with more concentrated viable adipocytes, progenitor cells, and factors for angiogenesis.

Enhancing Fat Graft Survival via Upregulating Autophagy of Adipocytes.

Autophagy is a cellular self-protection mechanism. The upregulation of adipose-derived stem cells' (ADSCs) autophagy can promote fat graft survival. However, the effect of interfering with adipocyte autophagy on graft survival is still unknown. In addition, autophagy is involved in adipocyte dedifferentiation. We investigated the effect of autophagy on adipocyte dedifferentiation and fat graft survival. The classic autophagy regulatory drugs rapamycin (100nM) and 3-methyladenine (3-MA; 10mM) were used to treat adipocytes, adipocyte dedifferentiation was observed, and their effects on ADSCs were detected. In our experiments, 100nM rapamycin, 10mM 3-MA and saline were mixed with human adipose tissue and transplanted into nude mice. At 2, 4, 8 and 12 weeks postoperatively, the grafts were harvested for histological and immunohistochemical analysis. Rapamycin and 3-MA can promote and inhibit adipocyte dedifferentiation by regulating autophagy. Both drugs can inhibit ADSC proliferation, and 10mM 3-MA can inhibit ADSC adipogenesis. At weeks 8 and 12, the volume retention rate of the rapamycin group (8 weeks, 64.77% ± 6.36%; 12 weeks, 56.13% ± 4.73%) was higher than the control group (8 weeks, 52.62% ± 4.04%; P < 0.05; 12 weeks, 43.17% ± 6.02%; P < 0.05) and the rapamycin group had more viable adipocytes and better vascularization. Compared with the control group, the volume retention rate, viable adipocytes and vascularization of the 3-MA group decreased. Rapamycin can promote adipocyte dedifferentiation by upregulating autophagy to promote fat graft survival. 3-MA can inhibit graft survival, but its mechanism includes the inhibition of adipocyte dedifferentiation and ADSC proliferation and adipogenesis. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

In Vitro Characterization of Fat Grafts Processed Using the REVOLVE ENVI System versus Decantation

Background: This preclinical study evaluated benchtop/in vitro properties and fat viability and activity of grafts processed using the REVOLVE ENVI 600 system compared with decantation and evaluated properties of REVOLVE ENVI waste. Methods: Lipoaspirate from six donors was processed using REVOLVE ENVI or decantation. The composition of each graft, hematocrit/red blood cell content, fat particle size/macrostructure, viable adipocyte count, and adipocyte activity were analyzed. Stromal vascular fraction was analyzed for viable progenitor cell count and colony-forming units. Results: REVOLVE ENVI grafts had a higher mean (±SD) fat content at 85.6% ± 6.1% than decanted grafts at 72.1% ± 4.0% (P &lt; 0.001), with negligible free oil (0.4% ± 1.1%) and cellular debris (&lt;0.1%), whereas REVOLVE ENVI waste contained primarily aqueous fluid (91.0% ± 2.2%) with negligible viable fat. REVOLVE ENVI grafts had significantly lower hematocrit levels (P &lt; 0.001) and contained significantly more large fat globules (P &lt; 0.001) than decanted grafts or REVOLVE ENVI waste. The percentage of tissue particles of more than 1000 µm was highest for REVOLVE ENVI grafts at 61.6% ± 9.2% (decantation: 52.5% ± 13.4%; REVOLVE ENVI waste: 0.49% ± 1.50%), and the percentage of particles less than 200 µm was lowest for REVOLVE ENVI grafts at 15.7% ± 2.6% (decantation: 32.2% ± 8.9%; REVOLVE ENVI waste: 97.9% ± 4.5%). REVOLVE ENVI grafts contained 145.2% ± 36.0% more viable adipocytes, 145.7% ± 46.2% greater activity, 195.5% ± 104.2% more progenitors in SVF, and 363.5% ± 161.2% more SVF colony-forming units than decanted grafts. Conclusion: Fat grafts processed using REVOLVE ENVI demonstrated greater viability and activity than decanted grafts in vitro.

The Synergetic Roles of Stromal Vascular Fraction (SVF) and Extracellular Matrix (ECM) on Fat Graft Retention in Nude Mice

Background. Both stromal vascular fraction (SVF) and extracellular matrix (ECM) are of great concern to adipogenesis and angiogenesis. SVF and ECM are rich in adipose tissue and may provide structural and biochemical support and form a microenvironment for free granular fat. The present study was to investigate whether SVF‐gel, a mixture of SVF and ECM harvested by mechanical emulsification, could improve the long‐term volume retention of fat grafts. Methods. Human SVF‐gel of different percentages was mixed with microfat. According to the percentages of SVF‐gel into microfat, 4 groups were included in the study; they are the microfat group, 10% gel group, 30% gel group, and gel group. The fat grafts were transplanted in the subcutaneous layer on each flank in nude mice. The sample volume was measured to evaluate the fat retention rate 90 days post‐transplantation. Tissue integrity, collagen content, numbers of viable adipocytes, and density of blood vessels were examined by further detection. Results. The retention volume rates in the 30% gel group and gel group were significantly higher than the microfat group and the 10% gel group (p &lt; 0.05). Equivalent fat integrity was observed in the four groups. Higher collagen volume, enhanced mRNA expression of VEGF, TNF‐α and adiponectin, more CD31‐positive blood vessels, and more regenerative adipocytes were observed in the 30% gel group and the gel group. The fat tissue in the 30% gel group showed similar structures as the normal fat tissue, while almost of the tissue in the gel group exhibited as fibrous tissue. Conclusion. SVF‐gel could improve fat graft retention while it came to a certain ratio into microfat via proangiogenic effect and fat regeneration which may be provided by SVF and ECM synergistically. SVF‐gel‐assisted fat grafting is a promising strategy to be used in clinical operations.

Effect of SARS-CoV-2 spike protein exposure on ACE2 and interleukin 6 productions in human adipocytes: An in-vitro study.

Since adipocytes play a crucial role in pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to their interaction with angiotensin-converting enzyme 2 (ACE2) and interleukin 6 (IL-6), obesity is associated with an increased risk of coronavirus disease 2019 (COVID-19) mortality. Discovery of ACE2 as a SARS-CoV-2 receptor raises a controversy about whether to use ACE inhibitors (ACEIs) could be an optional therapy to prevent cytokine storms. Studies assessing the expressions of ACE2 and IL-6 upon exposure to SARS-CoV-2 is therefore important as a basis for therapeutical trials in the future. The aim of this study was to determine the effect of SARS-CoV-2 spike protein exposure on the production of ACE2 and IL-6 in adipocyte cells. Adipocytes were collected from abdominal adipose tissues of healthy and obese 45-year-old male donor having neither a history of SARS-CoV-2 infection nor COVID-19 vaccination. After being stained using the oil red O protocol, the viable adipocytes were then exposed to S1 subunit of SARS-CoV-2 spike protein. The levels of ACE2 and IL-6 were then examined using the enzyme-linked immunosorbent assay (ELISA). The results showed significant increase of ACE2 (90.22 µg/mL) and IL-6 level (60.01 µg/mL) in human adipocytes upon exposure compared to unexposed control cells (ACE2 13.33 µg/mL; IL-6 21.33 µg/mL), both comparisons had p<0.001). This study provides insight into the basic mechanism of severe COVID-19 symptoms in obese patients and provides a basic information of the potential of ACE inhibitors as an optional therapy for COVID-19 patients with obesity.

Engineering 3D-Printed Bioresorbable Scaffold to Improve Non-Vascularized Fat Grafting: A Proof-of-Concept Study.

Autologous fat grafting is the gold standard for treatment in patients with soft-tissue defects. However, the technique has a major limitation of unpredictable fat resorption due to insufficient blood supply in the initial phase after transplantation. To overcome this problem, we investigated the capability of a medical-grade poly L-lactide-co-poly ε-caprolactone (PLCL) scaffold to support adipose tissue and vascular regeneration. Deploying FDM 3D-printing, we produced a bioresorbable porous scaffold with interconnected pore networks to facilitate nutrient and oxygen diffusion. The compressive modulus of printed scaffold mimicked the mechanical properties of native adipose tissue. In vitro assays demonstrated that PLCL scaffolds or their degradation products supported differentiation of preadipocytes into viable mature adipocytes under appropriate induction. Interestingly, the chorioallantoic membrane assay revealed vascular invasion inside the porous scaffold, which represented a guiding structure for ingrowing blood vessels. Then, lipoaspirate-seeded scaffolds were transplanted subcutaneously into the dorsal region of immunocompetent rats (n = 16) for 1 or 2 months. The volume of adipose tissue was maintained inside the scaffold over time. Histomorphometric evaluation discovered small- and normal-sized perilipin+ adipocytes (no hypertrophy) classically organized into lobular structures inside the scaffold. Adipose tissue was surrounded by discrete layers of fibrous connective tissue associated with CD68+ macrophage patches around the scaffold filaments. Adipocyte viability, assessed via TUNEL staining, was sustained by the presence of a high number of CD31-positive vessels inside the scaffold, confirming the CAM results. Overall, our study provides proof that 3D-printed PLCL scaffolds can be used to improve fat graft volume preservation and vascularization, paving the way for new therapeutic options for soft-tissue defects.

Enhanced Fat Graft Viability and Remodeling Using a Helium-based Radiofrequency Device to Prepare the Recipient Site

BackgroundImprovements to autologous fat grafting for soft tissue augmentation are needed to overcome the unpredictable volume retention. Approaches such as fat harvesting and processing, injection technique, preparation of the recipient site, and supplemental biologics are topics of ongoing research. Here, an energy-based device was investigated as a stimulatory tool for recipient site preparation for improving fat graft retention.ObjectiveThe objective was to measure the stimulatory responses in fat grafts after 4 weeks when using a helium-based radiofrequency device to pretreat the recipient tissue.MethodsUsing an autologous fat grafting mouse model, the inguinal fat pad was grafted in a small cranial pocket after either a saline injection alone (control) or a saline injection followed by pretreatment (treated). The fat pad was resected after 4 weeks, sectioned and stained with immunofluorescence markers to investigate tissue remodeling.ResultsPretreatment resulted in higher viability of adipocytes, a higher concentration of viable ASCs in areas of adipose tissue regeneration, and localized macrophages in the areas of regeneration when compared to the control. There was no observable difference in vascularity or angiogenesis. The staining for ASCs was higher in the pretreated group in comparison with the control group (5.0% vs. 3.3%, p=0.36) when using a pixel classifier in QuPath in the viable adipose tissue regions.ConclusionsThe use of a helium-based radiofrequency device as a pretreatment tool appears to increase the viability of the adipose tissue likely due to higher concentration of ASCs. The apparent increase in viable ASCs may be due to enhanced proliferation or paracrine recruitment of these cells in response to the helium-based radiofrequency treatment.No Level AssignedThis journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.Bullet List of Important Points:Pretreatment of the fat graft recipient site increases the viability of the adipose tissue after 4 weeks in comparison with the control grafts.The increased viability is likely due to the observed increase in adipose-derived stem cells in the pretreated group.Pretreatment enhanced the adipose tissue remodeling as colocalization of adipose-derived stem cells and macrophages showed an active remodeling, whereas the control group exhibited more necrotic and fibrotic tissue.

Hesperetin attenuates the expression of markers of adipose tissue fibrosis in pre-adipocytes

BackgroundExcessive extracellular matrix (ECM) deposition in adipose tissue is a hallmark of fibrosis, leading to disrupted adipose tissue homeostasis and metabolic dysfunction. Hesperetin, a flavonoid compound, has shown promising anti-inflammatory, anti-obesity and anti-diabetic properties. Therefore, we investigated the anti-fibrotic effects of hesperetin, through targeting ECM components and matrix metalloproteinase enzymes.Methods3T3-L1 cells were cultured in DMEM, containing 10% FBS and 1% penicillin/streptomycin. Cells were treated with a range of hesperetin concentrations, and the cell viability was determined using MTT assay. Subsequently, the expression of genes encoding collagen VI, osteopontin, matrix metalloproteinase-2 (Mmp-2) and Mmp-9 was analyzed using specific primers and real-time PCR technique. To evaluate protein levels of collagen VI and osteopontin, Western blotting was performed.ResultsHesperetin affected the viability of 3T3-L1 adipocytes with IC50 of 447.4 µM, 339.2 µM and 258.8 µM (24 h, 48 and 72 h, respectively). Hesperetin significantly reduced the gene and protein expression of both collagen VI and osteopontin in 3T3-L1 pre-adipocytes, in a time- and dose-dependent manner. Hesperetin was also able to cause a remarkable decline in gene expression of Mmp2 and Mmp9.ConclusionHesperetin could potently reduce the production of markers of adipose tissue fibrosis and might be considered a potential anti-fibrotic compound in obesity. Thus, hesperetin has the potency to be used for the treatment of obesity-associated fibrosis.

Identifying G protein-coupled receptors involved in adipose tissue function using the innovative RNA-seq database FATTLAS

G protein-coupled receptors (GPCRs) modulate the function of adipose tissue (AT) in general and of adipocytes, specifically. Although it is well-established that GPCRs are widely expressed in AT, their repertoire as well as their regulation and function in (patho)physiological conditions (e.g., obesity) is not fully resolved. Here, we established FATTLAS, an interactive public database, for improved access and analysis of RNA-seq data of mouse and human AT. After extracting the GPCRome of non-obese and obese individuals, highly expressed and differentially regulated GPCRs were identified. Exemplarily, we describe four receptors (GPR146, MRGPRF, FZD5, PTGER2) and analyzed their functions in a (pre)adipocyte cell model. Besides all receptors being involved in adipogenesis, MRGPRF is essential for adipocyte viability and regulates cAMP levels, while GPR146 modulates adipocyte lipolysis via constitutive activation of Gi proteins. Taken together, by implementing and using FATTLAS we describe four hitherto unrecognized GPCRs associated with AT function and adipogenesis.

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

ObjectiveEnhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. MethodNAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). ResultNAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. ConclusionNAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

Pyrroloquinoline Quinone (PQQ) Improves Long-term Survival of Fat Grafts by Alleviating Oxidative Stress and Promoting Angiogenesis During the Early Phase After Transplantation.

Reducing absorption after autologous fat grafting is a current challenge. Pyrroloquinoline quinone (PQQ) is the strongest known catalyst of redox reactions, which can scavenge reactive oxygen species (ROS) and alleviate oxidative stress. The aim of this study was to establish an in vivo model of PQQ-assisted lipotransfer and clarify the role of PQQ in reducing oxidative stress, alleviating apoptosis, and promoting angiogenesis during the acute hypoxic phase after grafting. In addition the study was performed to assess whether this intervention would have a positive effect on the improvement of long-term volume retention. Different concentrations of PQQ (low: 10 μM, medium: 100 μM, and high: 1000 μM) were mixed with human adipose tissue and transplanted subcutaneously into nude mice. Meanwhile, a control group of phosphate-buffered saline in an equal volume to PQQ was set up. On the third day after grafting, whole mount fluorescence staining was applied to detect ROS, mitochondrial membrane potential (MMP), apoptosis, adipocyte activity, and angiogenesis. Graft volume retention rate and electron microscopic morphology were evaluated at the third month. Immunohistochemistry and polymerase chain reaction (PCR) were further employed to elucidate the mechanism of action of PQQ. PQQ-assisted fat grafting improved the long-term volume retention, promoted the quality and viability of the adipose tissue, and reduced the level of fibrosis. The underlying mechanism of PQQ assisted in scavenging the accumulated ROS, restoring MMP, enhancing adipocyte viability, alleviating tissue apoptosis, and promoting timely angiogenesis during the hypoxia stress phase. The most effective concentration of PQQ was 100 μM. Immunohistochemistry and PCR experiments confirmed that PQQ reduced the expression of Bax and cytochrome c in the mitochondrial apoptotic pathway and increased the level of the antiapoptotic molecule Bcl-2. PQQ could improve the long-term survival of adipocytes by alleviating hypoxic stress and promoting timely angiogenesis in the early phase following lipotransfer.

Assessing Adipocyte Viability and Surgeons' Work Efficiency by Comparing Different Liposuction Methods.

Autologous fat grafting is widely used in plastic and reconstructive surgery. Liposuction methods play a key role in surgeons' work efficiency, adipocyte viability, graft survival, and outcomes. We investigated the effect of four liposuction methods on adipocyte viability, debris, and surgeons' work efficiency by measuring the active energy expenditure and changes in heart rate. Human lipoaspirate was harvested from patients' removed abdominal flaps using four different liposuction methods, and we counted calories per aspirated volume and surgeons' heart rate. Adipocytes were separated from the lipoaspirate immediately by digestion with 0.1% type I collagenase. After digestion, parts of the cells and debris were measured. Adipocytes were plated in an adipocyte maintenance medium containing Alamar blue reagent. The adipocyte metabolic activity was measured using a spectrophotometer. After evaluating the active energy expenditure and changes in surgeons' heart rate, the ultrasonic-assisted liposuction (UAL) method was determined to be the most ergonomic liposuction device for surgeons. In addition, adipocyte viability was higher in the UAL group than in the other groups, and debris was the lowest in the power-assisted liposuction 1 group (PAL1). Adipocyte viability is crucial for improving fat grafting outcomes. This study revealed that the viability of adipocytes is best preserved using the UAL and PAL1 liposuction methods. The UAL and PAL1 methods caused the least damage to the cells. The UAL method yielded the best results for surgeons' work efficiency.

Injectable pulverized electrospun poly(lactic-co-glycolic acid) fibers improve human adipose tissue engraftment and volume retention.

Autologous adipose tissue is commonly used for tissue engraftment for the purposes of soft tissue reconstruction due to its relative abundance in the human body and ease of acquisition using liposuction methods. This has led to the adoption of autologous adipose engraftment procedures that allow for the injection of adipose tissues to be used as a "filler" for correcting cosmetic defects and deformities in soft tissues. However, the clinical use of such methods has several limitations, including high resorption rates and poor cell survivability, which lead to low graft volume retention and inconsistent outcomes. Here, we describe a novel application of milled electrospun poly(lactic-co-glycolic acid) (PLGA) fibers, which can be co-injected with adipose tissue to improve engraftment outcomes. These PLGA fibers had no significant negative impact on the viability of adipocytes in vitro and did not elicit long-term proinflammatory responses in vivo. Furthermore, co-delivery of human adipose tissue with pulverized electrospun PLGA fibers led to significant improvements in reperfusion, vascularity, and retention of graft volume compared to injections of adipose tissue alone. Taken together, the use of milled electrospun fibers to enhance autologous adipose engraftment techniques represents a novel approach for improving upon the shortcomings of such methods.