Differentiation Of Adipose Precursor Cells Research Articles

Molecular and Cellular Mechanisms of Intramuscular Fat Development and Growth in Cattle.

Intramuscular fat, also referred to as marbling fat, is the white fat deposited within skeletal muscle tissue. The content of intramuscular fat in the skeletal muscle, particularly the longissimus dorsi muscle, of cattle is a critical determinant of beef quality and value. In this review, we summarize the process of intramuscular fat development and growth, the factors that affect this process, and the molecular and epigenetic mechanisms that mediate this process in cattle. Compared to other species, cattle have a remarkable ability to accumulate intramuscular fat, partly attributed to the abundance of sources of fatty acids for synthesizing triglycerides. Compared to other adipose depots such as subcutaneous fat, intramuscular fat develops later and grows more slowly. The commitment and differentiation of adipose precursor cells into adipocytes as well as the maturation of adipocytes are crucial steps in intramuscular fat development and growth in cattle. Each of these steps is controlled by various factors, underscoring the complexity of the regulatory network governing adipogenesis in the skeletal muscle. These factors include genetics, epigenetics, nutrition (including maternal nutrition), rumen microbiome, vitamins, hormones, weaning age, slaughter age, slaughter weight, and stress. Many of these factors seem to affect intramuscular fat deposition through the transcriptional or epigenetic regulation of genes directly involved in the development and growth of intramuscular fat. A better understanding of the molecular and cellular mechanisms by which intramuscular fat develops and grows in cattle will help us develop more effective strategies to optimize intramuscular fat deposition in cattle, thereby maximizing the quality and value of beef meat.

Accumulation of 4-Hydroxynonenal Characterizes Diabetic Fat and Modulates Adipogenic Differentiation of Adipose Precursor Cells

Accumulation of 4-Hydroxynonenal Characterizes Diabetic Fat and Modulates Adipogenic Differentiation of Adipose Precursor Cells

Adipose Tissue Dysfunction as Determinant of Obesity-Associated Metabolic Complications.

Obesity is a critical risk factor for the development of type 2 diabetes (T2D), and its prevalence is rising worldwide. White adipose tissue (WAT) has a crucial role in regulating systemic energy homeostasis. Adipose tissue expands by a combination of an increase in adipocyte size (hypertrophy) and number (hyperplasia). The recruitment and differentiation of adipose precursor cells in the subcutaneous adipose tissue (SAT), rather than merely inflating the cells, would be protective from the obesity-associated metabolic complications. In metabolically unhealthy obesity, the storage capacity of SAT, the largest WAT depot, is limited, and further caloric overload leads to the fat accumulation in ectopic tissues (e.g., liver, skeletal muscle, and heart) and in the visceral adipose depots, an event commonly defined as “lipotoxicity.” Excessive ectopic lipid accumulation leads to local inflammation and insulin resistance (IR). Indeed, overnutrition triggers uncontrolled inflammatory responses in WAT, leading to chronic low-grade inflammation, therefore fostering the progression of IR. This review summarizes the current knowledge on WAT dysfunction in obesity and its associated metabolic abnormalities, such as IR. A better understanding of the mechanisms regulating adipose tissue expansion in obesity is required for the development of future therapeutic approaches in obesity-associated metabolic complications.

Defective differentiation of adipose precursor cells from lipodystrophic mice lacking perilipin 1.

Perilipin 1 (Plin1) localizes at the surface of lipid droplets to regulate triglyceride storage and hydrolysis in adipocytes. Plin1 defect leads to low adiposity in mice and partial lipodystrophy in human. This study investigated the roles of Plin1 in adipocyte differentiation. Plin1 null (-/-) mice showed plenty of multilocular adipocytes and small unilocular adipocytes in adipose tissue, along with lack of a subpopulation of adipose progenitor cells capable of in vivo adipogenesis and along with downregulation of adipogenic pathway. Before initiation of differentiation, adipose stromal-vascular cells (SVCs) from Plin1-/- mice already accumulated numerous tiny lipid droplets, which increased in number and size during the first 12-h induction but thereafter became disappeared at day 1 of differentiation. The adipogenic signaling was dysregulated despite protein level of PPARγ was near normal in Plin1-/- SVCs like in Plin1-/- adipose tissue. Heterozygous Plin1+/- SVCs were able to develop lipid droplets, with both the number and size more than in Plin1-/- SVCs but less than in Plin1+/+ SVCs, indicating that Plin1 haploinsufficiency accounts for attenuated adipogenesis. Aberrant lipid droplet growth and differentiation of Plin1-/- SVCs were rescued by adenoviral Plin1 expression and were ameliorated by enhanced or prolonged adipogenic stimulation. Our finding suggests that Plin1 plays an important role in adipocyte differentiation and provides an insight into the pathology of partial lipodystrophy in patients with Plin1 mutation.

New role of oxysterols as regulators of adipogenic differentation in adipose-derived mesenchymal stem cells

Growing evidence indicates that adipose tissue (AT) represents a potential source of pluripotent mesenchymal stem cells. However, the mechanisms underlying the lineage-specific commitment of human adipose-derived stem cells (ASC) remain still not fully elucidated. Oxysterols are cholesterol oxide products resulting from non-enzymatic (ie, 7-Ketocolesterol) or enzymatic (ie, 5,6-Secosterol) oxidation of cholesterol, which are now emerging as reliable markers of adipose “oxidative stress” in vivo. Recent data suggest that, by regulating the adipogenic differentiation of ASC, lipid peroxidation products may play an important role in linking the adipose dysfunction with impairment of glucose homeostasis. In this study we combined a lipidomic approach with the subcutaneous (sc) microdialysis technique to characterize the adipose-derived profile of fatty acids (FA) and oxysterols in vivo. ASC were isolated from abdominal sc, mesemteric (MES) and omental (OM) fat specimens obtained from obese nondiabetic (OB) and type 2 diabetic (OBT2D) patients. Flow cytometry (FC) was used for the evaluation of cell viability, mitochondrial status and cell immunophenotyping. In AT interstitial fluid, abundant concentrations of oxysterols (7κC and 5,6-S) and fatty acids (lipokines) were found. Experimental challenging with 7κC and 5,6-S showed a different time-dose-response effect. Indeed, the MTT assay, we found that in ASC isolated from the sc depot 5,6-S (50 and 10μM) reduced cell viability after 24, 48 and 72 h, respectively. In contrast, in the same cell-type population, the effect of 7κC at 10μM was observed only after 72 h. FC analysis indicated a similar effect of both oxysterols even after short-time exposure either in ASC from the MES or the OM fat depot. Notably, cell challenge with 7κC and 5,6-S at 10, 5 and 1 μM, respectively, was accompanied by an impairment of mitochondrial status only in OM, but not in MES. Furthermore, both the oxysterols (10μM) downregulated the expression of stemness surface markers suggesting a different “susceptibility” of the ASC to lipid peroxidation cell damage. Accordingly, 7κC at 10 and 1μM impaired the adipogenic differentiation of sc and OM ASC isolated from either OB or T2DOB, and demodulated the mitochondrial activity of the differentiated adipocytes. Altogether, our results suggest human AT as a critical compartment for storage and secretion of lipokines and oxysterols, which, when in excess, appear to detrimentally modulate the mitochondrial activity and the adipogenic differentiation of adipose precursor cells. This work was partly funded from Fondazione Cassa di Risparmio di Perugia, project 2010.020.0098, “Ricerca Scientifica e Tecnologica”

Prostacyclin IP receptor up-regulates the early expression of C/EBPβ and C/EBPδ in preadipose cells

Prostacyclin (PGI 2) and its stable analogue carbacyclin (cPGI 2) are known to trigger the protein kinase A pathway after binding to the cell surface IP receptor and to promote or enhance terminal differentiation of adipose precursor cells to adipose cells. The early expression of C/EBPβ and C/EBPδ is known to be critical for adipocyte differentiation in vitro as well as in vivo. We report herein that in Ob1771 and 3T3-F442A preadipose cells, activation of the IP receptor by specific agonists (PGI 2, cPGI 2 and BMY 45778) is sufficient to up-regulate rapidly the expression of C/EBPβ and C/EBPδ. Cyclic AMP-elevating agents are able to substitute for IP receptor agonists, in agreement with the coupling of IP receptor to adenylate cyclase. Consistent with the fact that PGI 2 is released from preadipose cells and behaves as a paracrine/autocrine effector of adipose cell differentiation, the present results favor a key role of prostacyclin by means of the IP receptor and its intracellular signaling pathway in eliciting the critical early expression of both transcription factors.

Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent.

The mitogenic-adipogenic activity of carbaprostacyclin (cPGI2), a stable analogue of prostacyclin (PGI2), has been proposed to be related to its ability to elicit cAMP production and to activate the protein kinase A cascade (Négrel, R., Gaillard, D., and Ailhaud, G. (1989) Biochem. J. 257, 399-405). In the present study, cPGI2 has been compared with other activators of the cAMP pathway, namely isoproterenol, forskolin and 8-bromo-cAMP, with respect to adipose cell differentiation. Carbaprostacyclin behaved as a much more potent and efficient effector of mouse Ob1771 preadipocyte differentiation than the latter agents. Moreover, cPGI2 also exerted a specific amplifying mitogenic-adipogenic role, as compared with isoproterenol in rat as well as human adipose precursor cells in primary culture, suggesting that the prostanoid was able to generate an additional second messenger. The fact that ionomycin was able to potentiate and amplify the differentiation induced by 8-bromo-cAMP led us to give evidence, using preadipocytes preloaded with the fluorescent calcium chelator Indo-1, that cPGI2, besides its ability to activate adenyl cyclase, was also able to induce a transient increase in intracellular free calcium. This phenomenon was independent of cAMP production or inositol phospholipid breakdown and appeared to be mediated after binding to a single class of PGI2 receptor. The potential to generate simultaneously two synergistic intracellular signals allows us to ascribe to PGI2 a key and specific role in the differentiation of adipose precursor cells in vitro that would likely lead in vivo to the recruitment of "dormant" preadipocytes to become adipocytes.

Control of terminal differentiation of adipose precursor cells by glucocorticoids

The role of glucocorticoids on adipose conversion has been studied using confluent Ob1771 mouse preadipose cells maintained in a serum-free culture medium able to support the emergence of early but not that of late markers of differentiation. Under these culture conditions, glucocorticoids play, at physiological concentrations, a permissive role for terminal differentiation, characterized by glycerol-3-phosphate dehydrogenase expression and triacylglycerol accumulation within 12 days, whereas progesterone, testosterone, and estradiol are inactive. Glucocorticoids behave as mitogenic-adipogenic stimuli able to trigger growth-arrested, early marker-expressing cells to enter the terminal phase of the differentiation program and thus appear to mimic the mitogenic-adipogenic activity already described for arachidonic acid and cyclic AMP-elevating agents, especially prostacyclin. When compared to corticosterone alone, exposure of Ob1771 cells to both corticosterone and arachidonic acid leads to an additional increase in the glycerol-3-phosphate dehydrogenase activity and number of differentiated cells; this potentiation is further enhanced when the culture medium is supplemented with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. This suggests indirectly the involvement of prostacyclin as a metabolite of arachidonic acid able to induce cyclic AMP accumulation. In agreement with this hypothesis, it is found that a promoting effect is exerted by corticosterone on the metabolism of arachidonic acid, leading in turn to an increase in the production of prostacyclin. These findings allow a better understanding of the role of glucocorticoids on adipose cell differentiation and explain a posteriori the effectiveness of the combination of dexamethasone-isobutyl-methylxanthine used in innumerable studies.

Extracellular factors, signalling pathways and differentiation of adipose precursor cells

Growth of adipose depots varies according to sex, age and diet. The irreversible acquisition of new fat cells is a continuous process throughout life, even into senescence. At the cellular level, this phenomenon raises the question of the origin and biological status of adipose precursor cells behaving as dormant cells. This leads in turn to the question of the nature of the factors which regulate formation of new fat cells from dormant cells. The process of adipose cell ditferentiation has been primarily investigated in adipose precursor cells from established clonal lines, such as 3T3-Ll, 3T3-F442A, Ob17, 1246,3T3-T, and ST13, which are all aneuploid. Some investigators have turned to the study of adipose precursor cells derived from the stromal-vascular fraction of adipose tissues from various species, including the human (Ailhaud et al In Obesity: Towards a Moc these cells are diploid but have a limited life span. When transplanted into animals, cells from both sources develop into mature fat cells. In M-o they differentiate, in the presence of an appropriate hormonal milieu, to yield cells that have most, if not all, the morphological and biochemical characteristics of adipocytes. Adipose precursor cells resemble fibroblasts while growing at low density but at growth arrest induced by xxious means (serumor calciumdeprivation, thymidine block, or merely by cotiuence), the differentiation process takes place and culminates in a large trlacylglycerol accumulation. It is worth pointing out that ‘differentiation’ as applied to adipose precursor cells can have distinct meanings, and that this can lead to confusion.

The Adipocyte: Relationships between Proliferation and Adipose Cell Differentiation

The differentiation of adipose precursor cells can be divided into early and late events. Growth arrest at the G1/S boundary triggers the activation of early genes, i.e., pOb24 and lipoprotein lipase; the expression of both genes is primarily regulated at a transcriptional level. The expression of late markers, which lead to terminal differentiation and accumulation of neutral lipids, takes place after a limited number of mitoses of early-marker-expressing cells. Only terminal differentiation requires the presence of growth hormone and triiodothyronine as obligatory hormones and insulin as a modulating hormone, and results in the formation of triacylglycerol-filled, non-dividing cells. It appears that terminal differentiation involves the cyclic AMP pathway, the diacylglycerol pathway, and a third pathway triggered by insulinlike growth factor-I and insulin. It is thus proposed that a combination of mitogenic-adipogenic signals is required to trigger terminal differentiation of preadipose cells.

Regulation of gene expression during adipocyte differentiation: a review.

The differentiation of adipose precursor cells is accompanied by the acquisition of adipocyte-specific messenger (m) RNAs allowing characteristic changes in protein composition. The development of methods for cloning and characterizing individual genes has provided the opportunity to study selective gene expression by adipocytes at the molecular level. In this review, the information obtained to date regarding transcriptional and post-transcriptional regulatory mechanisms utilized by adipocytes is summarized. Included are descriptions of conserved DNA sequences found in noncoding regions of adipose genes and of how protein-DNA interactions at these regions are thought to regulate the initiation of transcription. Among the transcription factors implemented in regulation of adipocyte-specific gene expression are the protein product of the proto-oncogene c-fos, the triiodothyronine receptor and steroid hormone receptors. Data also are discussed that indicate that RNA editing and determinants of mRNA stability contribute to the adipocyte phenotype. It is hoped that a review of the regulatory aspects of gene expression during adipocyte differentiation will identify areas deserving further study by researchers interested in understanding adipose development.

Prostacyclin as a potent effector of adipose-cell differentiation

The terminal differentiation of Ob1771 pre-adipose cells induced by arachidonic acid in serum-free hormone-supplemented medium containing insulin, transferrin, growth hormone, tri-iodothyronine and fetuin (5F medium) was strongly diminished in the presence of inhibitors of prostaglandin synthesis, namely aspirin or indomethacin. Carbaprostacyclin, a stable analogue of prostacyclin (prostaglandin I2) known to be synthesized by pre-adipocytes and adipocytes, behaved as an efficient activator of cyclic AMP production and was able, when added to 5F medium, to mimic the adipogenic effect of arachidonic acid. Prostaglandins E2, F2 alpha and D2, unable to affect the cyclic AMP production, failed to substitute for carbaprostacyclin. However, prostaglandin F2 alpha, which is another metabolite of arachidonic acid in pre-adipose and adipose cells, able to promote inositol phospholipid breakdown and protein kinase C activation, potentiated the adipogenic effect of carbaprostacyclin. In addition, carbaprostacyclin enhanced both a limited proliferation and terminal differentiation of adipose precursor cells isolated from rodent and human adipose tissues maintained in primary culture. These results demonstrate the critical role of prostacyclin and prostaglandin F2 alpha on adipose conversion in vitro and suggest a paracrine/autocrine role of both prostanoids in the development of adipose tissue in vivo.

Effects of sex steroid hormones on differentiation of adipose precursor cells in primary culture

The effects of estradiol-17β and progesterone on multiplication, differentiation and lipid filling of adipose precursor cells were examined in primary cell cultures of cells prepared from adipose tissue of both male and ovariectomized female rats. Progesterone down to a concentration of 10 −7 mol/liter, alone or in the presence of estradiol-17β stimulated the development of glycerophosphate dehydrogenase and lipoprotein lipase activity. Estradiol-17β alone had no effects. These effects were essentially parallel to increases in the rate of lipid filling of the cells. Furthermore, the formation of cells with a lipid vacuole >20 μm was markedly stimulated, suggesting that new fat cells were formed by the stimulation of differentiation of the adipose precursor cells. No effects of the sex steroid hormones were seen on the rate of multiplication. These results suggest a role of sex steroid hormones in the regulation of triglyceride storage capacity in adipose tissue by facilitating the differentiation of precursor cells to form new adipocytes.

Characterization of ouabain-resistant mutants of the preadipocyte Ob17 clonal line Adipose conversion in vitro and in vivo

After exposure to a mutagenic drug (ethylmethanesulfonate), mutant clones of Ob17 preadipose cells resistant to the cytotoxic action of ouabain have been isolated. Their ability to grow in serum-supplemented medium containing ⩾3 mM ouabain is consistent with the decreased ouabain sensitivity of K + transport observed in one of these clones (Ob17-OR11). Ouabain-resistant mutants retain their ability to convert into adipose-like cells in vitro with a frequency similar to that of original Ob17 cells. Long-term maintenance of Ob17-OR11 cells in ouabain-enriched medium leads to a higher frequency of adipose conversion, which is linked to a rather specific and limited growth-promoting effect of the drug. Undifferentiated Ob17-OR11 cells, when injected subcutaneously into athymic mouse, give rise in vivo to a fat pad containing ouabain-resistant mature adipocytes. Therefore, Ob17-OR11 clone should be a useful tool to distinguish in vivo between intrinsic and extrinsic factors involved in the differentiation of adipose precursor cells.

Lipoprotéine lipase et différenciation adipocytaire

Some hormonal factors, possibly involved in the proliferation and differentiation of adipose precursor cells in vivo, have been characterized in vitro using different preadipocyte cell lines established from rodent adipose tissue. The process of adipose conversion has also been studied using these cell lines; in this process, stem cells (adipoblasts) were committed at any cell division during the growth phase. At confluence, committed cells (preadipocytes) underwent a limited number of mitoses and differentiated into adipose cells, whereas the uncommitted cells remained as stem cells in the cell population. This stochastic model could be extended to the development of rat adipose tissue in vivo. The study of adipose conversion showed the early emergence of lipoprotein lipase (LPL) and monoglyceride lipase (MGL). LPL activity appeared in the cells before any triglyceride accumulation. In contrast, this accumulation seemed dependent upon the emergence of glycerol-3-phosphate dehydrogenase. In vitro experiments clearly established that LPL-containing (differentiating) cells underwent postconfluent mitoses. This limited proliferation was in agreement with previous data obtained in vivo and indicates that only triglyceride-containing (mature) cells could not divide.