Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

Prostaglandin F2 stimulates the generation of lipoxygenase products in guinea pig isolated trachea

The generation of lipoxygenase products on the contraction elicited by prostaglandin (PG) F2 alpha was investigated in the guinea-pig isolated trachea. Indomethacin (5 x 10(-6) M) inhibited the response at low concentrations of PGF2 alpha while enhanced the response at higher concentrations of PGF2 alpha. Phenidone (10(-4) M) and nordihydroguaiaretic acid (NDGA, 3 x 10(-5) M) appeared to inhibit the PGF2 alpha response. The PGF2 alpha response augmented by indomethacin was dose-dependently inhibited by NDGA and a leukotriene (LT) antagonist, FPL55712. NDGA had no effect on the contraction elicited by histamine but markedly inhibited the contraction elicited by LTD4. The inhibition by NDGA of the LTD4-induced contraction was abolished in the presence of indomethacin (5 x 10(-6) M). FPL55712 inhibited the LTD4-induced contraction but the extent of the antagonism was not changed by indomethacin. In the presence of indomethacin PGF2 alpha (10(-8) M) did not affect the LTD4 (3 x 10(-9) M) response but significantly enhanced the arachidonic acid (AA, 6.6 x 10(-5) M)-induced contraction. FPL55712 (3 x 10(-6) M) completely inhibited the AA response augmented by PGF2 alpha. These results suggest that lipoxygenase-mediated LT-like substances are released in the response at higher concentrations of PGF2 alpha on the guinea-pig isolated trachea, and the mode of action of PGF2 alpha is different from those of histamine and LTD4.     

Lactoferrin gene knockdown leads to similar effects to iron chelation in human adipocytes

In human and mice adipose tissue, lactoferrin (LTF) has been found to be associated with increased adipogenesis and decreased inflammatory markers. Here, we aimed to investigate the effects of LTF knockdown (KD) in human adipocyte differentiation. In addition, the effects of exogenous LTF administration and iron chelation [using deferoxamine (DFO, 10 μM)] were tested. In both subcutaneous and visceral pre‐adipocytes, LTF KD led to decrease significantly adipogenic, lipogenic and insulin signalling‐related gene expression and a significant increase in the gene expression of inflammatory mediators. Human lactoferrin (hLf, 1 μM) administration led to recover adipocyte differentiation in LTF KD pre‐adipocytes. Interestingly, iron chelation triggered similar effects to LTF KD, decreasing significantly adipogenic gene expressions. Of note, DFO (10 μM) and hLf (1 and 10 μM) co‐administration led to a dose‐dependent recovery of adipocyte differentiation. These new data reveal that endogenous LTF biosynthesis during human adipocyte differentiation is essential to achieve this process, possibly, modulating adipocyte iron homoeostasis. hLf administration might be a useful therapeutic target in obesity‐associated adipose tissue dysfunction.     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

Phospholipase A2 is a differentiation-dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.     

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5, 6-cis-double bond as well. Bovine luteal function was affected following treatment of heifers with 25 mg PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained in vitro on PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Caffeine reduces TNFα up-regulation in human adipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity.     

Effect of alterations in the cyclopentane ring on bone resorptive activity of prostaglandin

Previous studies have shown that the natural prostanoids, PGE2, PGE1 and PGF2 alpha are potent stimulators of bone resorption. In this study, we have examined the effects of alterations in the cyclopentane ring of these prostanoids for their effect on the resorptive response of cultured long bones from 19-day fetal rats as measured by the release of previously incorporated 45Ca. Indomethacin (10(-6)M) was added to minimize endogenous prostaglandin production. In this system PGE2 and PGE1, the 9 keto, 11 alpha hydroxy compounds, were approximately equally effective at concentrations of 10(-8) to 10(-6) M. The 9 alpha hydroxy, 11 alpha hydroxy compound, PGF2 alpha, was active at 10(-7) to 10(-5) M. In contrast, the 9 alpha hydroxy, 11-keto compound, PGD2, showed only a minimal stimulation of bone resorption at 10(-5) M. While these data suggested that the 11 alpha hydroxy group was important for bone resorbing activity, 11 beta PGE2 and 11-deoxy PGE1 were only slightly less potent than their physiologic counterparts. Both 9 beta, 11 alpha PGF2 and 9 alpha, 11 beta PGF2 were less potent than PGF2 alpha but did cause substantial stimulation of bone resorption and were equally effective at 10(-6) to 10(-5) M. 9 alpha, 11 beta PGF2 alpha is of particular interest since it is major metabolite of PGD2. These results suggest that the binding of prostanoids to the receptor which mediates bone resorption is affected by changes at the 9 and 11 positions of the pentane ring but do not support the hypothesis that the 11 alpha OH function is essential for this biological activity.     

Decrease in transforming growth factor beta 1 binding during differentiation of rat adipocyte precursors in primary culture

Transforming growth factor beta 1 (TGF-beta 1) binding and action were investigated during differentiation of adipocyte precursors freshly isolated from rat inguinal fat-pad cultivated in defined medium. The data presented in this paper indicate that TGF-beta 1 inhibits differentiation of adipocyte precursors with a 50% effective dose of 9 pM. Time course experiments demonstrate that TGF-beta 1 is active only when it is added to the cells while they are still undifferentiated. If added after the cells have started to differentiate, TGF-beta 1 is less active or becomes inactive. 125I-TGF-beta 1 binding studies on adipocyte precursors before and after differentiation indicate a 10-fold decrease in the number of TGF-beta 1 binding sites after the cells have differentiated. Blocking of the differentiation process by treating the cells with fetal bovine serum or with prostaglandin F2 alpha prevented the decrease in the number of TGF-beta 1 receptors, thereby demonstrating that this change in binding was specifically linked to the differentiation process. Experiments cross-linking 125I-TGF-beta 1 to adipocyte precursors showed that 125I-TGF-beta 1 is specifically cross-linked to two bands with molecular weights of 92,000 and 70,000. After differentiation, a decrease in the intensity of the cross-linked bands was observed. These results demonstrate that loss of cell surface TGF-beta 1 binding sites follows differentiation of adipocyte precursors.     

The dietary fatty acid 10E12Z-CLA induces epiregulin expression through COX-2 dependent PGF(2α) synthesis in adipocytes

Conjugated linoleic acids (CLAs) are a group of dietary fatty acids that are widely marketed as weight loss supplements. The isomer responsible for this effect is the trans-10, cis-12 CLA (10E12Z-CLA) isomer. 10E12Z-CLA treatment during differentiation of 3T3-L1 adipocytes induces expression of prostaglandin-endoperoxide synthase-2 (Cyclooxygenase-2; COX-2). This work demonstrates that COX-2 is also induced in fully differentiated 3T3-L1 adipocytes after a single treatment of 10E12Z-CLA at both the mRNA (20-40 fold) and protein level (7 fold). Furthermore, prostaglandin (PG)F(2α), but not PGE(2), is significantly increased 10 fold. In female BALB/c mice fed 0.5% 10E12Z-CLA for 10 days, COX-2 was induced in uterine adipose (2 fold). In vitro, pharmacological COX-2 inhibition did not block the effect of 10E12Z-CLA on adipocyte-specific gene expression although PGF(2α) was dose-dependently decreased. These studies demonstrate that PGF(2α) was not by itself responsible for the reduction in adipocyte character due to 10E12Z-CLA treatment. However, PGF(2α), either exogenously or endogenously in response to 10E12Z-CLA, increased the expression of the potent mitogen and epidermal growth factor (EGF) receptor (EGFR) ligand epiregulin in 3T3-L1 adipocytes. Blocking PGF(2α) signaling with the PGF(2α) receptor (FP) antagonist AL-8810 returned epiregulin mRNA levels back to baseline. Although this pathway is not directly responsible for adipocyte dependent gene expression, these results suggest that this signaling pathway may still have broad effect on the adipocyte and surrounding cells.     

Mitochondrial reactive oxygen species control the transcription factor CHOP-10/GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect

Recent reports emphasize the importance of mitochondria in white adipose tissue biology. In addition to their crucial role in energy homeostasis, mitochondria are the main site of reactive oxygen species generation. When moderately produced, they function as physiological signaling molecules. Thus, mitochondrial reactive oxygen species trigger hypoxia-dependent gene expression. Therefore the present study tested the implication of mitochondrial reactive oxygen species in adipocyte differentiation and their putative role in the hypoxia-dependent effect on this differentiation. Pharmacological manipulations of mitochondrial reactive oxygen species generation demonstrate a very strong and negative correlation between changes in mitochondrial reactive oxygen species and adipocyte differentiation of 3T3-F442A preadipocytes. Moreover, mitochondrial reactive oxygen species positively and specifically control expression of the adipogenic repressor CHOP-10/GADD153. Hypoxia (1% O2) strongly increased reactive oxygen species generation, hypoxia-inducible factor-1 and CHOP-10/GADD153 expression, and inhibited adipocyte differentiation. All of these hypoxia-dependent effects were partly prevented by antioxidants. By using hypoxia-inducible factor-1alpha (HIF-1alpha)-deficient mouse embryonic fibroblasts, HIF-1alpha was shown not to be required for hypoxia-mediated CHOP-10/GADD153 induction. Moreover, the comparison of hypoxia and CoCl2 effects on adipocyte differentiation of wild type or HIF-1alpha deficient mouse embryonic fibroblasts suggests the existence of at least two pathways dependent or not on the presence of HIF-1alpha. Together, these data demonstrate that mitochondrial reactive oxygen species control CHOP-10/GADD153 expression, are antiadipogenic signaling molecules, and trigger hypoxia-dependent inhibition of adipocyte differentiation.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to 3 x 10(-8) M--3 x 10(-4) M of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), prostacyclin (PGI2), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE2, PGF2 alpha, PGI2, and AA. Although preincubation of dispersed adrenal cells with indomethacin (3 x 10(-5) M) markedly inhibited PGE2 synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.     

Interaction between phenylephrine and prostaglandins E1 and F1 alpha on the contractile responses of vascular muscle.

Prostaglandins (PGs) E1 or F1 alpha (1.4--8.4 x 10(-8) M) contracted strips of rabbit aorta and increased the contractions produced by 1--6 x 10(-7) M phenylephrine (PE). The addition of the PGs simultaneously with PE or after a low concentration of PE (2 x 10(-7) M) significantly increased the PE-induced contractions. However, when the PGs were added after a higher concentration of PE (6 x 10(-7) M) an additional increase in the PE-induced contraction was produced with PGF1 alpha but not with PGE1. Isobolic plots of the data obtained from the simultaneous addition of PE and the PGs indicate that both PGs interact with PE in a synergistic or potentiative manner, suggesting that their effects are mediated through different receptor mechanisms. Addition of the PGs after a high dose of PE indicates that there may also be either qualitative or quantitative differences between PGE1 and PGF1 alpha.     

Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.     

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.     

The primary culture of mouse adipocyte precursor cells in defined medium.

1. A defined medium supporting the proliferation and differentiation of adipocyte precursors isolated from inguinal fat pads of 8-12-day-old mice was developed. 2. It consists of a 1:1 mixture of DME and WAJC404A media supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), fibroblast growth factor (10 ng/ml) and high density lipoproteins (HDL) (90 micrograms protein/ml). 3. DME-F12 medium (1:1 mixture) used as a nutrient mixture in the defined medium of rat and human adipocyte precursors was inadequate for cultivating mouse adipocyte precursors. 4. HDL had a definite beneficial effect on both preadipocyte growth and differentiation. 5. Differentiation was enhanced by addition of dexamethasone (10(-9) M) but could be almost completely inhibited by transforming growth factor beta 1 (TGF-beta 1). 6. TGF-beta 1 was shown to be effective only when present in the early stages of differentiation.