Adipose Cell Development Research Articles

The role of long non-coding RNAs in the development of adipose cells.

In recent times, the rising prevalence of obesity and its associated comorbidities have had a severe impact on human health and social progress. Therefore, scientists are delving deeper into the pathogenesis of obesity, exploring the role of non-coding RNAs. Long non-coding RNAs (lncRNAs), once regarded as mere "noise" during genome transcription, have now been confirmed through numerous studies to regulate gene expression and contribute to the occurrence and progression of several human diseases. LncRNAs can interact with protein, DNA, and RNA, respectively, and participate in regulating gene expression by modulating the levels of visible modification, transcription, post-transcription, and biological environment. Increasingly, researchers have established the involvement of lncRNAs in regulating adipogenesis, development, and energy metabolism of adipose tissue (white and brown fat). In this article, we present a literature review of the role of lncRNAs in the development of adipose cells.

Influence of folate on lipid accumulation in human primary subcutaneous adipocytes (827.8)

Epidemiological studies report inverse associations between serum folate concentrations and body mass index, however the potential influence of changes in folate status on adipose cell development and metabolism has only been examined in animal models or a murine cell line. The objective of this study was to determine the influence of alterations in folate availability on differentiation of human primary preadipocytes. Human primary preadipocytes, derived from subcutaneous adipose tissue of normal weight female donors (ZenBio Inc), were exposed to 0.06, 6 or 60 uM folic acid (FA; Sigma), 5‐methyltetrahydrofolate (MTHF; Merck) or tetrahydrofolate (THF; Merck) during the differentiation period. The impact of treatment on lipid accumulation was determined using AdipoRedTM Adipogenesis assay reagent (Lonza). Results indicate that FA, MTHF and THF decreased lipid accumulation in mature adipocytes, after adjusting for cell number. Interestingly, MTHF and THF, reduced forms of folate, exhibited a greater inhibitory effect on lipid accumulation. These results indicate that biologically active reduced forms of folate may have a greater effect on adipocyte differentiation in human adipocytes. This study also supports preliminary findings in murine cell lines demonstrating an effect of changes in folate availability on adipose cell development.Grant Funding Source: Funded in part by HATCH #GEO00707. MTHF and THF were a kind gift from Merck & Cie, Switzerland.

Banting Lecture 2012

The Banting Medal for Scientific Achievement Award is the American Diabetes Association's highest scientific award and honors an individual who has made significant, long-term contributions to the understanding of diabetes, its treatment, and/or prevention.The award is named after Nobel Prize winner Sir Frederick Banting, who codiscovered insulin treatment for diabetes.Bruce M. Spiegelman, PhD, of Harvard Medical School and the Dana-Farber Cancer Institute in Boston, received the American Diabetes Association's Banting Medal for Scientific Achievement at the Association's 72nd Scientific Sessions, 8–12 June 2012, Philadelphia, Pennsylvania. He presented the Banting Lecture, “Transcriptional Control of Adipogenesis—Toward a New Generation of Therapeutics for Metabolic Disease,” on Sunday, 10 June 2012. In his lecture, Dr. Spiegelman described the discovery of several transcriptional components that control adipose cell development: PPAR-γ, PGC1-α, and PRDM16. He also described the cloning and characterization of beige fat cells, the thermogenic “brown-like” cells that can develop in white fat depots. Lastly, Dr. Spiegelman discussed irisin, a newly discovered regulatory hormone that converts white fat into the more thermogenic beige fat. Dr. Spiegelman’s research has found that irisin, which is induced by exercise, appears to activate some of the same health benefits as exercise, including improvement of glycemic control. Understanding the regulation of adipose tissue, white, brown, and beige, can potentially lead to the development of a new generation of therapeutics for diabetes prevention and treatment.

P196 - Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation

Introduction Mitochondria have been shown to be impaired in insulin resistance-related diseases, but have not been extensively studied during the first steps of adipose cell development. This study aimed to describe the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. Materiels et methodes 3T3-L1 preadipocytes were differentiated into adipocytes. At days 0, 3, 6, 9 and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Resultats Compared to preadipocytes (day 0), new adipocytes (day 6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9 days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1 (and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, CPT1) was increased. ROS production displayed a nadir at day 6 with a concomitant increase in antioxidant enzyme gene expression. Conclusion This 3T3-L1 based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.

Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation

Mitochondria have been shown to be impaired in insulin resistance-related diseases but have not been extensively studied during the first steps of adipose cell development. This study was designed to determine the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. 3T3-L1 preadipocytes were differentiated into adipocytes without using glitazone compounds. At days0, 3, 6, 9, and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Compared to preadipocytes (day0), new adipocytes (days6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1α and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, and CPT1) was increased. Reactive oxygen species (ROS) production displayed a nadir at day6 with a concomitant increase in antioxidant enzyme gene expression. This 3T3-L1-based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.

EID1-induces brown-like adipocyte traits in white 3T3-L1 pre-adipocytes

PPARγ and pRB play an important role in the development of adipose cells, and functional modification of these proteins may lead to beneficial changes in adipose cell physiology. In the present work, we show that over-expression of EID1 (E1A-like inhibitor of differentiation), an inhibitor of muscle cell differentiation, reduces PPARγ ligand-dependent transactivation and decreases triglyceride stores in pre-adipocytes (3T3-L1 cells). Additionally, we found that EID1 binds to pRB at the onset of adipocyte differentiation and may act to reduce pRB levels. Over-expression of EID1 in 3T3-L1 cells leads to increased expression of UCP1 and PGC-1α, both of which are involved in caloric dissipation and thermogenesis, in brown adipose tissue. These results indicate that EID1 is able to reduce fat accumulation in adipose cells and induce expression of brown fat genes in pre-adipocytes (3T3-L1 cells) normally destined to become white fat cells. The functional reduction of PPARγ and pRB mediated by EID1 in adipose cells may play an important role in insulin resistance and the metabolic syndrome.

Decreased expression of the IGF-II gene during porcine adipose cell differentiation

IGF-I and IGF-II are known to regulate cell development and recent data suggest a possible role of IGF-II on adipose tissue development. This study was undertaken to examine the IGF system gene expression in porcine differentiating adipocytes. Both adipocytes and stromal-vascular (s/v) cells were isolated from subcutaneous adipose tissue collected from 7-day-old piglets. s/v cells were cultured in chemically defined medium. Compared with isolated adipocytes, IGF-II and IGFBP-5 mRNA levels were very high in freshly isolated s/v cells, whereas IGF-I mRNA levels were lower in s/v cells than in adipocytes. Between day 0 and day 6 of culture, IGF-II and IGFBP-5 gene expression decreased whereas expression levels of late markers of adipocyte differentiation were up-regulated. Cell differentiation was also associated with an increase in the expression of IGF-I, insulin and IGF receptor genes. The current findings suggest that IGF-I and IGF-II have different effects on porcine adipose cell development.

Fat and Beyond: The Diverse Biology of PPARγ

The nuclear receptor PPARgamma is a ligand-activated transcription factor that plays an important role in the control of gene expression linked to a variety of physiological processes. PPARgamma was initially characterized as the master regulator for the development of adipose cells. Ligands for PPARgamma include naturally occurring fatty acids and the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPARgamma improves insulin sensitivity in rodents and humans through a combination of metabolic actions, including partitioning of lipid stores and the regulation of metabolic and inflammatory mediators termed adipokines. PPARgamma signaling has also been implicated in the control of cell proliferation, atherosclerosis, macrophage function, and immunity. Here, we review recent advances in our understanding of the diverse biological actions of PPARgamma with an eye toward the expanding therapeutic potential of PPARgamma agonist drugs.

Effect of dietary fat or starch supply during gestation and/or lactation on the performance of sows, piglets’ survival and on the performance of progeny after weaning

Two trials were carried out to compare the effects of fat or starch inclusion in sow’s diet on sow and litter performance. In each trial, sows were assigned to one of two treatments. In trial 1, the sows were fed diets containing either soybean oil (5%, treatment GL5) or cornstarch (11.3%, GL0) from day 35 of gestation to weaning. Daily net energy and nutrient allowance were equalised during gestation. In trial 2, the same treatments were applied only after farrowing (treatments L5 and L0, respectively). Within each trial, a batch of piglets was studied until slaughter. In trial 1, adipose cell development and total lipid content were determined on some pigs at weaning (n = 6/treatment) and at slaughter in dorsal subcutaneous adipose tissue (n = 13/group at least) and in muscle (n = 46/group at least). Piglets’ birth weight was not affected by treatment in trial 1. Survival rates at birth and after 24 h of life were higher in treatment GL5 (4.0% v. 7.5% stillborn piglets in GL0 treatment, P < 0.05; 8.7% v. 12.6% of piglets alive at 24 h of age died in treatment GL0, P = 0.06). Subsequently, overall survival rate until weaning was higher in treatment GL5 (81.4% v. 75.7% of total born piglets, P = 0.03), but litter size at weaning was not significantly affected (11.3). Litter growth rate before weaning was increased when a fat-enriched diet was provided during gestation and lactation (+140 g/day per litter; P < 0.01) and to a lower extent when provided only after farrowing (+90 g/day; P < 0.05). Energy supply through fat did not decrease the mobilisation of the sow’s body reserve and backfat thickness loss was even higher with treatment GL5 (P < 0.05). After weaning, pigs’ average daily gain, feed : gain ratio and carcass lean content were not affected by the energy source supplied before and/or after farrowing. At weaning, the number of adipose cells in the dorsal subcutaneous adipose tissue and in the Longissimus dorsi muscle was higher in the GL5 pigs. Muscle lipid content at weaning did not differ between treatments, but it was higher at slaughter, around 110 kg, in the GL5 pigs (3.46% v. 2.58%, P < 0.001).

Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into Adipocytes

The authors describe protocols for culture conditions in which mouse ES cells can be maintained in an undifferentiated state or committed to undergo adipocyte differentiation at a high rate and in a highly reproducible fashion. There is also a protocol for maintaining and differentiating human adult stem cells, isolated form adipose tissue and from bone marrow, into adipocytes. These culture systems provide a powerful means for studying the first step of adipose cell development and a means to investigate effects of drugs on the biology of adipocytes. There are also protocols for detection of adipocytes and analysis of their gene expression.

Lipid metabolism and cellular features of skeletal muscle and subcutaneous adipose tissue in pigs differing in IGF-II genotype

In pigs, a paternally (pat) imprinted mutation in the IGF-II gene is associated with increased muscle mass and decreased backfat thickness. The aim of this study was to determine whether this mutation influenced cellular, biochemical and metabolic features of skeletal muscle and adipose tissue. Muscle ( trapezius) and subcutaneous adipose tissue (SCAT) were collected from pigs (106 kg) carrying (Qpat, n = 6) or not carrying (qpat, n = 7) the mutation. Adipocytes were isolated from those tissues by collagenase treatment. Lipid content and activity of lipogenic enzymes were determined using standard assays. Gene expression levels were determined by real-time PCR. Levels of IGF-II mRNA were higher ( P < 0.01) in muscle of Qpat than in that of qpat pigs, but they did not differ significantly between the two groups in SCAT. Whereas levels of IGF-I mRNA in muscle were similar in both groups, they were higher ( P < 0.05) in SCAT of Qpat pigs than in that of qpat pigs. Muscle lipid content and intramuscular adipocyte diameters were not influenced significantly by the IGF-II genotype. In SCAT, the reduction of backfat thickness in Qpat pigs compared with qpat pigs was associated with lower ( P < 0.05) lipid content and smaller ( P < 0.05) adipocytes, with no significant genotype-effects on expressions and/or activities of lipogenic enzymes. In summary, our results suggest that the IGF-II mutation altered body composition in pigs by favoring myofiber hypertrophy and repressing adipose cell development in SCAT.

Development of Adipocytes from Differentiated ES Cells

This chapter describes an in vitro system that allows access to the development of adipocytes from pluripotent mouse ES cells. Because there are no specific markers that can be used to identify adipoblasts and preadipocytes in vivo , the white adipose tissue can be identified only when it contains adipocytes (i.e., terminally differentiated cells) and cannot be detected macroscopically during embryogenesis. Therefore, very little is known about the ontogeny of adipose tissue. Mainly, the in vitro system used to study adipogenesis is immortal preadipocyte cell lines, which represent already determined cells. The use of in vitro differentiation of ES cells to analyze the effects of mutations on adipogenesis has begun recently and master genes that commit progression from pluripotent stem cells to the adipogenic lineage have not yet been identified. The chapter also describes a protocol for culture conditions in which ES cells undergo adipocyte differentiation at a high rate and in a highly reproducible fashion. This culture system should provide a powerful means for studying the first step of adipose cell development and a route to identify regulatory genes involved in adipogenesis.

Differentiation of embryonic stem cells as a model to study gene function during the development of adipose cells.

Differentiation of embryonic stem cells as a model to study gene function during the development of adipose cells.

PPARγ-dependent and PPARγ-independent effects on the development of adipose cells from embryonic stem cells

Peroxisome proliferator-activated receptor (PPAR) γ was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARγ in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARγ is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARγ in developing EBs over-expressing PPARγ did not commit stem cells towards the adipose lineage. In differentiated PPARγ −/− EBs, only markers characteristic of preadipocytes were found to be expressed. PPARδ is present in EBs but did not compensate for the lack of PPARγ in terminal differentiation. Taken together, these results favor a critical PPARγ-independent phase culminating in preadipocyte formation that precedes a PPARγ-dependent phase in the development of adipose cells from pluripotent stem cells.

The expression of cyclin D1 during adipogenesis in pig primary stromal-vascular cultures.

Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal-vascular (S-V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Western blot analysis was performed on cultured S-V cells using 8% sodium dodecyl sulfate-polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti-mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti-CCAAT/enhancer binding protein (C/EBP alpha) and anti-cyclin D1 together. Cyclin D1 expression was evaluated in 105-day fetal dorsal subcutaneous tissues using paraffin sections. Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S-V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBP alpha beginning at day 3 and remained colocalized with C/EBP alpha through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. The elevated level of cyclin D1 expression colocalized with C/EBP alpha expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid-filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.

Cultures of adipose precursor cells and cells of clonal lines from animal white adipose tissue.

AbstractPreadipocyte cellular models have proven to be useful tools for the study of adipose cell development in vitro (1–5). Among these models are preadipocyte clonal lines from various origins and primary cultures of fibroblast-like adipose precursor cells present in the stromal-vascular fraction of adipose tissue, which can be isolated from several depots and various species, including the human (see Chapter 20). The availability of these cellular models has allowed crucial advances with respect to the characterization of the main steps of the differentiation process from the preadipocyte to the adipocyte in (1) delineating the kinetics of expression of adipose specific markers, (2) cloning some of these markers and studying their hormonal control, and (3) giving evidence of new secreted compounds from adipose cells (6–13). In addition, the availability of multipotent or totipotent stem cells (1,14,15) gives the opportunity of approaching the events that govern the process of determination of progenitors to the adipose lineage. These stem cell systems are thus expected to allow characterization of still missing key genes, as well as marker(s) of the unipotent adipoblast before its commitment to a preadipocyte. KeywordsEmbryonic Stem CellRetinoic AcidLeukemia Inhibitory FactorClonal LinePluripotent Embryonic Stem CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Role of pathways for signal transducers and activators of transcription, and mitogen-activated protein kinase in adipocyte differentiation.

Members of the signal transducer and activator of transcription (STAT) family and the mitogen-activated protein kinase (MAPK) cascade play a major role in the regulation of cell growth and differentiation. This review concentrates on the role played by these pathways in the development of adipose cells. STATs are activated by both positive and negative modulators of adipocyte differentiation leading to the hypothesis that the STAT pathway may function in adipogenesis. The role of the p42/p44 MAPK pathway in adipocyte differentiation has recently been the subject of contradictory reports. Several molecular mechanisms are proposed to explain the opposing effects of MAPK activation in the programme of adipose cell differentiation.

Leukemia Inhibitory Factor and Its Receptor Promote Adipocyte Differentiation via the Mitogen-activated Protein Kinase Cascade

Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr(-/-) ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor gamma2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPbeta and C/EBPdelta is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPbeta and C/EBPdelta genes via the mitogen-activated protein kinase cascade.

Embryonic Stem Cell-Derived Adipogenesis

Key events leading to terminal differentiation of preadipocytes into adipocytes have been characterized in the recent years. However, master genes that commit progression from multipotent mesenchymal stem cell to the adipoblast stage of development have not yet been identified. The use of embryonic stem (ES) cells as a route to study early events in adipogenesis and to characterize factors involved in the decision of stem cells to follow the adipogenic pathway is described in this paper. The capacity of lif^–/^– and lifr^–/^– ES cells to undergo adipocyte differentiation is reported as an application of mutant ES cells to study gene function during the development of adipose cells.

Regulation of adipose cell development in utero.

The condition of obesity is impacted by increases in fat cell number, fat cell size, or a combination of the two. It is generally believed that fat cell number is dependent on the age of onset and the degree of obesity. This review provides an update on intrauterine growth of fetal adipose tissue, the earliest period of proliferation onset, and the factors that interact to enhance or suppress development. Fetal adipose tissue development is regulated by the complex interaction of maternal, endocrine, and paracrine influences that initiate specific changes in angiogenesis, adipogenesis, and metabolism. Developmental stages and metabolic processes influenced by specific hormones and paracrine factors have been identified through examination of the offspring of obese and diabetic pregnancies, hormonal manipulation during late pregnancy in animal models, and the use of cell culture. Collectively, the results of the studies cited herein delineate the basis for imprinting or conditioning of fetal preadipocytes at the paracrine/autocrine level and a role of thyroxine, glucocorticoids, and other hormones in fetal adipose tissue development and metabolism.