Platelet lysate suppresses the expression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) have been shown to display a considerable therapeutic potential in cellular therapies. However, harmful adipogenic maldifferentiation of transplanted MSCs may seriously threaten the success of this therapeutic approach. We have previously demonstrated that using platelet lysate (PL) instead of widely used fetal calf serum (FCS) diminished lipid accumulation in adipogenically stimulated human MSCs and identified, among others, lipocalin-type prostaglandin D2 synthase (L-PGDS) as a gene suppressed in PL-supplemented MSCs. Here, we investigated the role of PL and putatively pro-adipogenic L-PGDS in human MSC adipogenesis. Next to strongly reduced levels of L-PGDS we show that PL-supplemented MSCs display markedly decreased expression of adipogenic master regulators and differentiation markers, both before and after induction of adipocyte differentiation. The low adipogenic differentiation capability of PL-supplemented MSCs could be partially restored by exogenous addition of L-PGDS protein. Conversely, siRNA-mediated downregulation of L-PGDS in FCS-supplemented MSCs profoundly reduced adipocyte differentiation. In contrast, inhibiting endogenous prostaglandin synthesis by aspirin did not reduce differentiation, suggesting that a mechanism such as lipid shuttling but not the prostaglandin D2 synthase activity of L-PGDS is critical for adipogenesis. Our data demonstrate that L-PGDS is a novel pro-adipogenic factor in human MSCs which might be of relevance in adipocyte metabolism and disease. L-PGDS gene expression is a potential quality marker for human MSCs, as it might predict unwanted adipogenic differentiation after MSC transplantation.     

Laminin Production and Basement Membrane Deposition by Mesenchymal Stem Cells upon Adipogenic Differentiation

The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-β1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α₆β₁. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α₆β₁ were seen using IF imaging at day 14. Laminin-411 and Int α₆β₁ were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element.     

Effects of arachidonic acid on the concentration of hydroxyeicosatetraenoic acids in culture media of mesenchymal stromal cells differentiating into adipocytes or osteoblasts

Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction.     

Biochemical changes induced by strontium ranelate in differentiating adipocytes

Low bone formation in osteoporosis is associated with a shift from osteoblastic to adipogenic differentiation of mesenchymal stem cells (MSC) inducing a concomitant lipotoxic milieu within the bone marrow. Strontium ranelate (SrRN), a treatment for osteoporosis, has both anti-resorptive and anabolic effects on bone. The anabolic effect of SrRN has been associated with its effect on both osteoblastogenesis and adipogenesis. However, the effect of SrRN on the potentially lipotoxic factors produced by differentiating marrow adipocytes remains poorly understood. To expand the knowledge on the effect of SrRN treatment on the bone microenvironment, we assessed changes in adipogenic factors and adipokine expression in adipocytic differentiation of MSC in vitro. Primary human MSC were induced to differentiate in adipogenic conditions in the presence or absence of SrRN (1–2 mM). We tested the dose-dependent effects of SrRN on adipocyte differentiation including changes in the expression of adipogenic markers and adipokines. We report that adipogenesis was negatively affected in the presence of SrRN with a concomitant dose-dependent decrease in the expression of adipogenic markers and changes in adipokine profile. Taken together, our data suggests that SrRN induces biochemical changes in differentiating adipocytes that could generate a favorable osteogenic effect within the bone marrow milieu.     

Anandamide-derived Prostamide F2α Negatively Regulates Adipogenesis

Lipid mediators variedly affect adipocyte differentiation. Anandamide stimulates adipogenesis via CB₁ receptors and peroxisome proliferator-activated receptor γ. Anandamide may be converted by PTGS2 (COX2) and prostaglandin F synthases, such as prostamide/prostaglandin F synthase, to prostaglandin F_2α ethanolamide (PGF_2αEA), of which bimatoprost is a potent synthetic analog. PGF_2αEA/bimatoprost act via prostaglandin F_2αFP receptor/FP alt4 splicing variant heterodimers. We investigated whether prostamide signaling occurs in preadipocytes and controls adipogenesis. Exposure of mouse 3T3-L1 or human preadipocytes to PGF_2αEA/bimatoprost during early differentiation inhibits adipogenesis. PGF_2αEA is produced from anandamide in preadipocytes and much less so in differentiating adipocytes, which express much less PTGS2, FP, and its alt4 splicing variant. Selective antagonism of PGF_2αEA receptors counteracts prostamide effects on adipogenesis, as does inhibition of ERK1/2 phosphorylation. Selective inhibition of PGF_2αEA versus prostaglandin F_2α biosynthesis accelerates adipogenesis. PGF_2αEA levels are reduced in the white adipose tissue of high fat diet-fed mice where there is a high requirement for new adipocytes. Prostamides also inhibit zebrafish larval adipogenesis in vivo. We propose that prostamide signaling in preadipocytes is a novel anandamide-derived antiadipogenic mechanism.     

Strontium ranelate rebalances bone marrow adipogenesis and osteoblastogenesis in senescent osteopenic mice through NFATc/Maf and Wnt signaling

With aging, bone marrow mesenchymal stromal cell (MSC) osteoblast differentiation decreases whereas MSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. Here, we investigated whether activation of cell signaling by strontium ranelate (SrRan) can reverse the excessive adipogenic differentiation associated with aging. In murine MSC cultures, SrRan increased Runx2 expression and matrix mineralization and decreased PPARγ2 expression and adipogenesis. This effect was associated with increased expression of the Wnt noncanonical representative Wnt5a and adipogenic modulator Maf and was abrogated by Wnt- and nuclear factor of activated T-cells (NFAT)c antagonists, implying a role for Wnt and NFATc/Maf signaling in the switch in osteoblastogenesis to adipogenesis induced by SrRan. To confirm this finding, we investigated the effect of SrRan in SAMP6 senescent mice, which exhibit decreased osteoblastogenesis, increased adipogenesis, and osteopenia. SrRan administration at a clinically relevant dose level increased bone mineral density, bone volume, trabecular thickness and number, as shown by densitometric, microscanning, and histomorphometric analyses in long bones and vertebrae. This attenuation of bone loss was related to increased osteoblast surface and bone formation rate and decreased bone marrow adipocyte volume and size. The restoration of osteoblast and adipocyte balance induced by SrRan was linked to increased Wnt5a and Maf expression in the bone marrow. The results indicate that SrRan acts on lineage allocation of MSCs by antagonizing the age-related switch in osteoblast to adipocyte differentiation via mechanisms involving NFATc/Maf and Wnt signaling, resulting in increased bone formation and attenuation of bone loss in senescent osteopenic mice.     

Zfp423 Promotes Adipogenic Differentiation of Bovine Stromal Vascular Cells

Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423) as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates) were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR) α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05) in high adipogenic cells, while transforming growth factor (TGF)-β was higher (156.1±48.7%, P<0.05) in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05) in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular adipogenesis and marbling in beef cattle.     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1–7). Co-expression of AngII receptors (AT₁ and AT₂) and Ang(1–7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1–7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1–7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1–7)-Mas antagonizes the antiadipogenic effect of AngII-AT₁ by inhibiting the AngII-AT₁-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT₁-ACE2-Ang(1–7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 Promote the Differentiation of Human Subcutaneous Preadipocytes

1,25(OH)₂D₃ inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D₃ to 1,25(OH)₂D₃, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)₂D₃, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)₂D₃ enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)₂D₃ was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0–3), suggesting that 1,25(OH)₂D₃ promoted maturation. 25(OH)D₃ also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)₂D₃. Consistent with this possibility, incubation of preadipocytes with 25(OH)D₃ led to 1,25(OH)₂D₃ accumulation in the media. 1,25(OH)₂D₃ also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.     

Oral intake of Lactobacillus plantarum L‐14 extract alleviates TLR2‐ and AMPK‐mediated obesity‐associated disorders in high‐fat‐diet‐induced obese C57BL/6J mice

ObjectivesWhether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity‐associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L‐14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high‐fat‐diet (HFD) mouse model.Materials and methodsMouse pre‐adipocyte 3T3‐L1 cells and human bone marrow mesenchymal stem cells (hBM‐MSC) were treated with L‐14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti‐adipogenic effects was analysed at cellular and molecular levels. L‐14 extract was orally administrated to HFD‐feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti‐adipogenic mechanism of exopolysaccharide (EPS) isolated from L‐14 extract was also analysed using Toll‐like receptor 2 (TLR2) inhibitor C29.ResultsL‐14 extract inhibited 3T3‐L1 and hBM‐MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L‐14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L‐14 extract also significantly decreased the serum triacylglycerol/high‐density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis.ConclusionsEPS from L‐14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L‐14 extract improves obesity and obesity‐associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.