A comparative assessment of adipose‐derived stem cells from subcutaneous and visceral fat as a potential cell source for knee osteoarthritis treatment

The intra‐articular injection of adipose‐derived stem cells (ASCs) is a novel potential therapy for patients with osteoarthritis (OA). However, the efficacy of ASCs from different regions of the body remains unknown. This study investigated whether ASCs from subcutaneous or visceral adipose tissue provide the same improvement of OA. Mouse and human subcutaneous and visceral adipose tissue were excised for ASC isolation. Morphology, proliferation, surface markers and adipocyte differentiation of subcutaneous ASCs (S‐ASCs) and visceral ASCs (V‐ASCs) were analysed. A surgically induced rat model of OA was established, and 4 weeks after the operation, S‐ASCs, V‐ASCs or phosphate‐buffered saline (PBS, control) were injected into the articular cavity. Histology, immunohistochemistry and gene expression analyses were performed 6 weeks after ASC injection. The ability of ASCs to differentiate into chondrocytes was assessed by in vitro chondrogenesis, and the immunosuppressive activity of ASCs was evaluated by co‐culturing with macrophages. The proliferation of V‐ASCs was significantly greater than that of S‐ASCs, but S‐ASCs had the greater adipogenic capacity than V‐ASCs. In addition, the infracted cartilage treated with S‐ASCs showed significantly greater improvement than cartilage treated with PBS or V‐ASCs. Moreover, S‐ASCs showed better chondrogenic potential and immunosuppression in vitro. Subcutaneous adipose tissue is an effective cell source for cell therapy of OA as it promotes stem cell differentiation into chondrocytes and inhibits immunological reactions.     

Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity,and Inflammation in Fat

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H₂O₂, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.     

Direct effect of ghrelin on leptin production by cultured rat white adipocytes

Objective: Because ghrelin is known to stimulate adipogenesis, we tested whether ghrelin could contribute to the maintenance of homeostasis, directly affecting rat white adipocyte leptin production. Research Methods and Procedures: Isolated retroperitoneal adipocytes were cultured for 0.5 to 48 hours without (baseline) or with (0.001 to 1 nM) ghrelin alone or in combination with insulin (0.01 to 10 nM) or dexamethasone (1 to 100 nM). Adipocytes were also incubated with ghrelin and inhibitors either of RNA (actinomycin D) or protein synthesis (cycloheximide) or with several concentrations (10 to 1000 nM) of a specific ghrelin antagonist. When cultures were terminated, we evaluated adipocyte leptin secretion and ob mRNA expression. Results: Our data indicate that ghrelin directly enhanced adipocyte leptin release and ob mRNA expression, that the leptin‐releasing activity of ghrelin was additive to the action of both insulin and dexamethasone and was abrogated by protein synthesis inhibitors, and that effects of ghrelin on adipocyte ob mRNA expression and release were blocked by coincubation with the specific growth hormone secretagogue receptor 1a antagonist. Discussion: Our study supports the ability of ghrelin to enhance white adipose tissue leptin production by a direct receptor‐mediated effect. This activity of ghrelin could play a potentially significant role in rapid restoration of homeostasis after food intake.     

The Effect of the Interleukin‐1 Cytokine Family Members IL‐1F6 and IL‐1F8 on Adipocyte Differentiation

Obesity is characterized by chronic low‐grade inflammation originating from expanding adipose tissue. In the present study, we examined the adipogenic expression levels of IL‐1F6 and IL‐1F8, both members of the IL‐1 family of cytokines, and their effects on adipose tissue gene expression. Although IL‐1F6 is primarily present in adipose tissue resident macrophages and induced by inflammation, IL‐1F8 is absent. IL‐1F6, but not IL‐1F8, reduces adipocyte differentiation, as shown by a significant decrease in PPARγ gene expression. Finally, both IL‐1F6 and IL‐1F8 are able to induce inflammatory gene expression in mature adipocytes. In conclusion, we demonstrate for the first time that IL‐1F6 is present in adipose tissue and that IL‐1F6 and IL‐1F8 are involved in the regulation of adipose tissue gene expression. Importantly, IL‐1F6 inhibits PPARγ expression which may lead to reduced adipocyte differentiation suggesting metabolic effects of this cytokine.     

β2‐Adrenoceptor Agonists Induce the Mammalian Clock Gene,hPer1, mRNA in Cultured Human Bronchial Epithelium Cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β₂‐adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β₂‐adrenoceptor agonists in BEAS‐2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS‐2B cells as verified by immunoblot analysis. β₂‐adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP‐CREB in BEAS‐2B cells.     

Circadian clock rhythms in different adipose tissue model systems

ABSTRACT

Circadian clock-controlled 24-h oscillations in adipose tissues play an important role in the regulation of energy homeostasis, thus representing a potential drug target for prevention and therapy of metabolic diseases. For pharmacological screens, scalable adipose model systems are needed that largely recapitulate clock properties observed in vivo. In this study, we compared molecular circadian clock regulation in different ex vivo and in vitro models derived from murine adipose tissues. Explant cultures from three different adipose depots of PER2::LUC circadian reporter mice revealed stable and comparable rhythms of luminescence ex vivo. Likewise, primary pre- and mature adipocytes from these mice displayed stable luminescence rhythms, but with strong damping in mature adipocytes. Stable circadian periods were also observed using Bmal1-luc and Per2-luc reporters after lentiviral transduction of wild-type pre-adipocytes. SV40 immortalized adipocytes of murine brown, subcutaneous and epididymal adipose tissue origin showed rhythmic mRNA expression of the core clock genes Bmal1, Per2, Dbp and REV-erbα in pre- and mature adipocytes, with a maturation-associated increase in overall mRNA levels and amplitudes. A comparison of clock gene mRNA rhythm phases revealed specific changes between in vivo and ex vivo conditions. In summary, our data indicate that adipose culture systems to a large extent mimic in vivo tissue clock regulation. Thus, both explant and cell systems may be useful tools for large-scale screens for adipose clock regulating factors.     

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X‐linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose‐derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X‐linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co‐cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)‐positive ASCs and DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

High Local Concentrations and Effects on Differentiation Implicate Interleukin‐6 as a Paracrine Regulator

Objective: To examine the possibility that interleukin‐6 (IL‐6) can act as a paracrine regulator in adipose tissue by examining effects on adipogenic genes and measuring interstitial IL‐6 concentrations in situ. Research Methods and Procedures: Circulating and interstitial IL‐6 concentrations in abdominal and femoral adipose tissue were measured using the calibrated microdialysis technique in 20 healthy male subjects. The effects of adipose cell enlargement on gene expression and IL‐6 secretion were examined, as well as the effect of IL‐6 in vitro on gene expression of adiponectin and other markers of adipocyte differentiation. Results: The IL‐6 concentration in the interstitial fluid was ～100‐fold higher than that in plasma, suggesting that IL‐6 may be a paracrine regulator of adipose tissue. This was further supported by the finding that adding IL‐6 in vitro at similar concentrations down‐regulated the expression of adiponectin, aP2, and PPARγ‐2 in cultured human adipose tissue. In addition, gene expression and release of IL‐6, both in vivo and in vitro, correlated with adipose cell size. Discussion: These data suggest that IL‐6 may be a paracrine regulator of adipose tissue. Furthermore, increased adipose tissue production of IL‐6 after hypertrophic enlargement of the adipose cells may detrimentally affect systemic insulin action by inducing adipose tissue dysfunction with impaired differentiation of the pre‐adipocytes and/or adipocytes and lower adiponectin.     

Overexpressing STAMP2 attenuates adipose tissue angiogenesis and insulin resistance in diabetic ApoE−/−/LDLR−/− mouse via a PPARγ/CD36 pathway

The aim of this study was to investigate whether overexpression of STAMP2 improves insulin resistance by regulating angiogenesis in adipose tissues. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Histological and morphological analysis demonstrated that STAMP2 gene overexpression reduced adipocyte size, angiogenesis in epididymal and brown adipose tissues. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited after STAMP2 gene overexpression. The cellular effect of STAMP2 on angiogenesis was explored in human umbilical vein endothelial cells (HUVECs) model. Correlation of STAMP2 and angiogenesis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. In vitro, overexpression of STAMP2 significantly inhibited endothelial cell migration, tube formation. The effects of Ad‐STAMP2 transfection on HUVECs were abolished by treatment with PPARγ antagonist GW9662 (2.5 μM), and the roles of STAMP2 siRNA on HUVECs were also reversed by treatment with PPARγ agonist rosiglitazone (RSG) (0.1 mM). RT‐PCR indicated that STAMP2 could regulate levels of adhesion molecules, vascular endothelial growth factor A and CD36. The expression of PPARγ and CD36 was decreased when STAMP2 was inhibited by siRNA, while PPARγ and CD36 were highly expressed after overexpression of STAMP2. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via attenuating angiogenesis in epididymal and brown adipose tissues through the PPARγ/CD36 signalling pathway.