Lipid synthesis in macrophages during inflammation in vivo: Effect of agonists of peroxisome proliferator activated receptors α and γ and of retinoid X receptors

The effects of peroxisome proliferator activated receptors α and γ (PPAR-α and PPAR-γ) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57B1 macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [¹⁴C]oleate incorporation into cholesteryl esters and triglycerides and [¹⁴C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18–24 h after injection and was decreased 5–7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of ¹²⁵I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of ¹²⁵I-labeled acetylated LDL (AcLDL) was 2–3-fold decreased. Addition of NLDL (50 μg/ml) or AcLDL (25 μg/ml) into the incubation medium of activated macrophages induced 9–14-and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-α, or PPAR-γ agonists—9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively—30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 μM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-α, or PPAR-γ agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 364–374.     

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E₂ 9-ketoreductase, which catalyzes the conversion of PGE₂ to PGF₂, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE₂, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min^–1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA₂, PGE₁, and 13,14-dihydro-15-keto PGF₂, but not that of PGD₂, 11-PGE₂, PGH₂, PGJ₂, or ¹²-PGJ₂. The reaction product formed from PGE₂ was identified as PGF₂, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE₂ was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE₂ 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF₂ in human brain.Special issue dedicated to Dr. Sidney Udenfriend.     

Midtrimester abortions induced by intraamniotic prostaglandin F2α treatment

A group of 10 patients, 16.2±0.5 weeks pregnant, received intraamniotically 10mg followed at 3 hours intervals by 5mg PG F2α. The total dose of 31.5±3.2mg PG F2α successfully induced abortion in 15.1±1.8 hours. Seven patients aborted completely and 3 incompletely. The rapid rise in RP was followed by a gradual increase in IUP and a continuing decrease in estradiol-17β and progesterone after a delay of about 6 hours. The systemic side effects were minimal and the vital signs and laboratory tests revealed no significant changes. The case reports of 4 additional patients are presented, and the mechanism of the abortifacient action of PG F2α is discussed. When further improved, intraamniotic PG F2α therapy may favorably compete with methods currently used for midtrimester legal abortions.     

Different skeletal effects of the peroxisome proliferator activated receptor (PPAR)α agonist fenofibrate and the PPARγ agonist pioglitazone

Background

All the peroxisome proliferator activated receptors (PPARs) are found to be expressed in bone cells. The PPARγ agonist rosiglitazone has been shown to decrease bone mass in mice and thiazolidinediones (TZDs) have recently been found to increase bone loss and fracture risk in humans treated for type 2 diabetes mellitus. The aim of the study was to examine the effect of the PPARα agonist fenofibrate (FENO) and the PPARγ agonist pioglitazone (PIO) on bone in intact female rats.

Methods

Rats were given methylcellulose (vehicle), fenofibrate or pioglitazone (35 mg/kg body weight/day) by gavage for 4 months. BMC, BMD, and body composition were measured by DXA. Histomorphometry and biomechanical testing of excised femurs were performed. Effects of the compounds on bone cells were studied.

Results

The FENO group had higher femoral BMD and smaller medullary area at the distal femur; while trabecular bone volume was similar to controls. Whole body BMD, BMC, and trabecular bone volume were lower, while medullary area was increased in PIO rats compared to controls. Ultimate bending moment and energy absorption of the femoral shafts were reduced in the PIO group, while similar to controls in the FENO group. Plasma osteocalcin was higher in the FENO group than in the other groups. FENO stimulated proliferation and differentiation of, and OPG release from, the preosteoblast cell line MC3T3-E1.

Conclusion

We show opposite skeletal effects of PPARα and γ agonists in intact female rats. FENO resulted in significantly higher femoral BMD and lower medullary area, while PIO induced bone loss and impairment of the mechanical strength. This represents a novel effect of PPARα activation.     

The peroxisome proliferator activated receptor δ is required for the differentiation of THP-1 monocytic cells by phorbol ester

BACKGROUND: PPARdelta (NR1C2) promotes lipid accumulation in human macrophages in vitro and has been implicated in the response of macrophages to vLDL. We have investigated the role of PPARdelta in PMA-stimulated macrophage differentiation.The THP-1 monocytic cell line which displays macrophage like differentiation in response to phorbol esters was used as a model system. We manipulated the response to PMA using a potent synthetic agonist of PPARdelta, compound F. THP-1 sub-lines that either over-expressed PPARdelta protein, or expressed PPARdelta anti-sense RNA were generated. We then explored the effects of these genetic modulations on the differentiation process. RESULTS: The PPARdelta agonist, compound F, stimulated differentiation in the presence of sub-nanomolar concentrations of phorbol ester. Several markers of differentiation were induced by compound F in a synergistic fashion with phorbol ester, including CD68 and IL8. Over-expression of PPARdelta also sensitised THP-1 cells to phorbol ester and correspondingly, inhibition of PPARdelta by anti-sense RNA completely abolished this response. CONCLUSIONS: These data collectively demonstrate that PPARdelta plays a fundamental role in mediating a subset of cellular effects of phorbol ester and supports observations from mouse knockout models that PPARdelta is involved in macrophage-mediated inflammatory responses.     

Doses of prostaglandin F2α effective for induction of parturition in goats

Twelve mixed breed does were injected with different doses of prostaglandin F₂α (PGF₂α) or saline on day 144 of gestation. Four each received single intramuscular injections of 5.0 or 2.5 mg PGF_2α, or 1.0 ml saline (controls). Systemic progesterone (P₄) concentrations were determined daily from day 144 until the day of kidding. Does receiving 5.0 mg PGF₂α, 2.5 mg PGF₂α, or saline kidded within mean (± SD) hours and range (hours) of 35 ± 8.6 and 28–48, 43 ± 11.8 and 29–57, and 111 ± 79.1 and 41–200, respectively. Mean (± SD) concentrations of P₄ (ng/ml) on the day of injection and on day 1 postinjection were 5.2 ± 2.6 and 0.7 ± 0.9, 5.3 ± 2.2 and 1.1 ± 1.0, and 6.4 ± 3.9 and 4.1 ± 2.6 for does receiving 5.0 mg PGF₂α, 2.5 mg PGF₂α, or saline, respectively. It was concluded that 5.0 mg and 2.5 mg PGF₂α effectively shortened the interval from injection to parturition, but that this interval was not as predictable as that previously reported with 20 mg PGF₂α.     

Pregnancy termination by prostaglandin F2α stimulates maternal behavior in the rat

Treatment with PGF_2α resulted in the termination of pregnancy in 16- and 19-day pregnant rats, but not in 10- or 13-day pregnant rats. Rats that aborted displayed a rapid onset of maternal behavior when tested with foster pups. Aborted rats also displayed sexual receptivity and ovulation: these phenomena resemble the sequence of events following hysterectomy on the same days of pregnancy. Both can be related to the events surrounding normal parturition in the rat. The results are interpreted as due to a pregnancy-induced deactivation of the factor in the uterus that prevents estrogen from stimulating maternal behavior in nonpregnant females. In the absence of this factor, the PGF_2α-induced rise in estrogen secretion facilitates maternal behavior and sexual behavior and induces ovulation.     

Effect of prostaglandin F2α on anterior pituitary function in man

Five healthy adult men received iv PGF_2α at dosages of 0.05, 0.20 and 2.0 μg/kg/min for 30 min. There were no significant changes in serum FSH, LH or TSH levels. Serum GH and cortisol levels were slightly increased at the highest dosage. These responses were associated with, and presumably a result of, stressful side effects. Thus, PGF_2α cannot be used as a provocative test of pituitary hormone reserve.Prostaglandins (PG's) have recently been implicated in the release of a number of hormones from the anterior pituitary gland. The stimulation of GH release by PG's of the E series from incubated rat pituitary slices has been demonstrated. $\text{In vivo}$ stimulation by PGE₁ of ACTH in rats and of GH release in man has also been shown.The present study was undertaken in order to examine the efficacy of iv administration of PGF_2α as a provocative test of anterior pituitary hormone reserve in man. The responses in circulating levels of gonadotropins, TSH, GH, and cortisol (as an index of ACTH) were measured.     

Antiluteogenic effects of serial prostaglandin F2α administration in cycling mares

A single dose of PGF2α does not consistently induce luteolysis in the equine CL until at least 5 days after ovulation, leading to the erroneous assumption that the early CL is refractory to the luteolytic effects of PGF2α. We hypothesized that serial administration of PGF2α in early diestrus would induce a return to estrus similar to mares treated with a single injection in mid-diestrus, and fertility of the induced estrus would not differ. The objectives of the study were to evaluate the effects of the 2 approaches as reflected by: (1) concentrations of plasma progesterone; (2) interovulatory and treatment-to-ovulation intervals; (3) the proportion of mares pregnant after artificial insemination. The study consisted of a balanced crossover design in which 10 reproductively normal Quarter Horse Mares were exposed to 2 treatments on 2 consecutive reproductive cycles. At detected ovulation (Day 0), mares were randomly allotted to 1 of 2 treatment groups: I, mid-diestrus treatment, administration of a single 10-mg dose of dinoprost tromethamine (PGF2α) im on Day 10; II, early diestrus treatment, administration of 10-mg PGF2α im twice daily on Days 0, 1, and 2 and once daily on Days 3 and 4. Mares in estrus and with a follicle 35 mm or greater in diameter were artificially inseminated with at least 2 billion motile sperm from a fertile stallion. Pregnancy was defined as detection of a growing embryonic vesicle on 2 consecutive examinations approximately 14 days after ovulation. Serial plasma samples were collected throughout the study period, and concentration of plasma progesterone was determined by RIA. A mixed-model ANOVA for repeated measures was used to analyze hormonal data. Interovulatory and treatment-to-ovulation intervals were compared by a paired t test and fertility by a McNemar chi-square analysis. All mares in group I underwent luteolysis after PGF2α administration denoted by mean (±SD) concentration of plasma progesterone of 0.25 ± 0.21 ng/mL detected 2 days after treatment. In group II, mean concentration of plasma progesterone remained below 1.0 ng/mL during treatment and until the onset of the next estrus. The mean interovulatory interval in group I was 18.5 ± 2.0 days compared with 13.1 ± 3.7 days in group II (P < 0.01). Treatment-to-ovulation intervals were 8.5 ± 2.0 days and 13.1 ± 3.7 days for groups I and II, respectively (P < 0.05). In both groups, 9 of 10 mares were pregnant (P = 1.0). Serial PGF2α administration beginning at ovulation consistently prevented luteal function in 10 of 10 mares in the present study without adversely affecting pregnancy rate of post-treatment cycles.     

Luteal progesterone and prostaglandin F2α production invitro during fluprostenol-induced luteolysis in the mare

Prostaglandin F₂α (PGF₂α) release $\text{in}$$\text{vitro}$ by luteal tissue from mares was quantified to determine if exogenous prostaglandin analog increased endogenous luteal PGF₂α production during induced luteolysis. On day 8 after ovulation, luteal tissue was collected by flank laparotomy and endometrium was collected by uterine biopsy. Mares were assigned to one of four treatments: (1) no intramuscular injection at 0-hr (n = 5), (2) 250 μg Fluprostenol (ICI 81008 PGF₂α analog) at 4-hr (n = 4), (3) 250 μg Fluprostenol at 12-hr (n = 5), or (4) 250 μg Fluprostenol at 28-hr (n = 5) prior to tissue collection at laparotomy. Blood was collected from a jugular vein at laparotomy. Luteal and endometrial tissues (100-mg minces) were incubated in duplicate in 5 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) in an ice bath in an air atmosphere or at 37°C in an atmosphere of 95% O₂:5% CO₂. The incubation treatments consisted of: no treatment, indomethacin 1.3 × 10^?4M, 1 μg/ml of arachidonic acid, 10 μg/ml of Fluprostenol, and 100 μM dbc-AMP (Fluprostenol was not added to endometrial tissue incubations). The injection of Fluprostenol induced luteolysis in these mares as indicated by decreased plasma progesterone and luteal tissue progesterone production (P<0.01). Luteal PGF₂α production was only detectable in tissue from mares that had been injected with Fluprostenol; production reached a maximum by 12 hr post-injection and had returned to pre-treatment levels by 28 hr (P<0.01). Endometrial tissue produced PGF₂α, but this activity was not significantly affected by injection of mares with Fluprostenol. Increased production of PGF₂α by luteal tissue of mares during PGF₂α analog induced luteolysis was similar to that observed in the pig and ewe.     

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina

Cyclooxygenases are responsible for the production of prostaglandin H₂ (PGH₂) from arachidonic acid. PGH₂ can be converted into some bioactive prostaglandins, including prostaglandin F_2α (PGF_2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF_2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF_2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF_2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF_2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.     

Desensitization of prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Desensitization of prostaglandin (PG) F₂ receptor-mediated phosphoinositide (PI) hydrolysis was investigated in cultured rat astrocytes. Prolonged exposure of astrocytes differentiated by dibutyryl cyclic AMP-treatment to PGF₂ caused the desensitization of subsequent PGF₂-induced PI hydrolysis. The desensitization was time- and PGF₂ dose-dependent; maximal decrease in the PI hydrolysis was observed after exposure to 10 M PGF₂ for 4 h and the degree of the desensitization was 31.7±2.7% of control. Pretreatment with either PGD₂ or PGE₂ also induced the desensitization of subsequent PGF₂-stimulated PI hydrolysis and conversely pretreatment of PGF₂ decreased the PI responses to PGD₂ and PGE₂. The desensitization prevented by phloretin and was reversible upon removal of the agonist. Protein synthesis inhibitors blocked the recovery of the desensitization. Treatment of the cells with phorbol 12-myristate 13-acetate had no effect on the desensitization. These results suggest that prolonged exposure of the astrocytes to PGF₂ caused the desensitization of the receptors.     

Utero-ovarian venous concentrations of prostaglandin E2 (PGE2 and prostaglandin F2α (PGF2α) following PGE2 intrauterine infusions

The uterine horns and utero-ovarian veins of nine crossbred mature gilts were bilaterally cannulated on day 9 of the estrous cycle (day 0 - first day of estrus). Each uterine horn in treated gilts (N=5) was infused with 150 μg PGE₂ in 3 ml of saline at 0900 h on day 12, 15 and 18 of the estrous cycle. Control gilts (N=4) received 3 ml saline intrauterine infusions on the corresponding day. Blood samples were collected from the utero-ovarian veins 15 min before each infusion and for the following 6 h with 15, 30 and 60 min intervals through the first, second and third two-hour periods, respectively. Venous concentrations of PGE₂ and PGF_2α were determined by radioimmunoassay procedures. Infusion of PGE₂ resulted in an immediate elevation in PGE₂ concentration in utero-ovarian venous drainage. Coincident elevations of PGF_2α utero-ovarian venous concentrations were observed after PGE₂ infusion. Plasma PGF_2α concentrations in the utero-ovarian veins were elevated (P<.01) in PGE₂ treated gilts for one hour post-treatment. The duration of PGE₂ and PGE₂α elevations as well as the peak values were influenced by day of the cycle.     

Expression of the peroxisome proliferator activated receptor γ gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes

Background  

Adipose tissues serve not only as a store for energy in the form of lipid, but also as endocrine tissues that regulates metabolic activities of the organism by secreting various kinds of hormones. Peroxisome proliferator activated receptor γ (PPARγ) is a key regulator of adipocyte differentiation that induces the expression of adipocyte-specific genes in preadipocytes and mediates their differentiation into adipocytes. Furthermore, PPARγ has an important role to maintain the physiological function of mature adipocyte by controlling expressions of various genes properly. Therefore, any reduction in amount and activity of PPARγ is linked to the pathogenesis of metabolic syndrome.     

Effects of inhibition of prostaglandin F2α biosynthesis during preluteolysis and luteolysis in heifers

Flunixin meglumine (FM; 2.5 mg/kg) was given to heifers at three 8-h intervals, 16 d after ovulation (first treatment = Hour 0) to inhibit the synthesis of prostaglandin F_2α (PGF), based on plasma concentrations of a PGF metabolite (PGFM). Blood samples were collected at 8-h intervals from 15 to 18 d in a vehicle (control) and FM group (n = 16/group). Hourly samples were collected from Hours −2 to 28 in 10 heifers in each group. Heifers that were in preluteolysis or luteolysis at Hour 0 based on plasma progesterone (P4) concentrations at 8-h intervals were partitioned into subgroups. Concentration of PGFM was reduced (P < 0.05) by FM treatment in each subgroup. For the preluteolytic subgroup, the first decrease (P < 0.05) in P4 concentration after Hour 0 occurred at Hours 24 and 40 in the vehicle and FM groups, respectively. Plasma P4 concentrations 32 and 40 h after the beginning of luteolysis in the luteolytic subgroup were greater (P < 0.05) in the FM group. Concentration at the peak of a PGFM pulse in the FM group was greater (P < 0.05) in the luteolytic than in the preluteolytic subgroup. The peak of a PGFM pulse occurred more frequently (P < 0.001) at the same hour as the peak of an LH fluctuation than at the ending nadir of an LH fluctuation. In conclusion, a reduction in prominence of PGFM pulses during luteolysis delayed completion of luteolysis, and treatment with FM inhibited PGFM production more during preluteolysis than during luteolysis.     

The effect of oxytocin,estrogen, calcium ionophore A23187 and hydrocortisone on prostaglandin F2α and 6-oxo-prostaglandin F1α production by cultured human endometrial and myometrial explants

Normal human endometrium (classified by histology and date after last menstrual period) was cultured for 72h, and the output of prostaglandin F₂α and 6-oxo-prostaglandin Fla detected by radioimmunoassay. Hormones/stimuli were added to the culture during the second day of culture for 5h and 19h periods.

• 1.1) The output of prostaglandin F₂α from cultured endometrium was significantly higher (p<0.05) at the beginning (d4–8) and end (d25–30) of the menstrual cycle, compared to mid-cycle (d13–24) endometrium. Significantly more prostaglandin F₂α was released from proliferative than from secretory phase endometrium (p<0.02).
• 2.2) Prostaglandin F₂α release was rapidly stimulated by sodium arachidonate (20–300 μg/ml), and by calcium ionophore A23187 (5 μg/ml) at an extracellular calcium ion concentration of 1.8mM.The ionophore stimulation was greater in mid-cycle endometrium than in endometrium from the beginning or the end of the menstrual cycle.
• 3.3) Estradiol-17β (10 ng/ml) gradually increased the output of prostaglandin F₂α from secretory phase endometrium, and this stimulation was observed in the post-incubation period after hormone had been removed from the incubation medium.
• 4.4) Oxytocin (1 × 10⁻⁵U/ml caused a more rapid stimulation of prostaglandin F₂α output from secretory phase tissue (p<0.05 during the first 5h incubation period with hormone).
• 5.5) Oxytocin (1 × 10⁻⁵ U/ml) and estradiol (long/ml) together significantly stimulated prostaglandin F2a production by proliferative as well as secretory phase endometria.
• 6.6) A high dose of hydrocortisone (loo μg/ml) inhibited the output of prostaglandin F₂α from proliferative and secretory phase endometrium and also from ionophore-stimulated endometrium. However, this dose of hydrocortisone did not inhibit the synthesis of prostaglandin F2a from exogenous arachidonic acid, or the estradiol-induced increase in prostaglandin F₂α production.
• 7.7) Co-culture of endometrium with myometrium did not modify the output of prostaglandin F₂α or of 6-oxo-prostaglandin Fla from cultured tissues.
• 8.8) These experiments suggest that arachidonic acid supply to the cyclooxygenase enzyme may vary during the menstrual cycle: and indicate a gradual increase in prostaglandin synthesising capacity in response to estrogen, more rapid control via oxytocin, and an interaction between estrogen and oxytocin to modulate prostaglandin F2a synthesis in human endometrium.