Mice deleted for fatty acid transport protein 5 have defective bile acid conjugation and are protected from obesity

BACKGROUND & AIMS: Fatty Acid Transport Protein 5 (FATP5) is a liver-specific member of the FATP/Slc27 family, which has been shown to exhibit both fatty acid transport and bile acid-CoA ligase activity in vitro. Here, we investigate its role in bile acid metabolism and body weight homeostasis in vivo by using a novel FATP5 knockout mouse model. METHODS: Bile acid composition was analyzed by mass spectroscopy. Body weight, food intake, energy expenditure, and fat absorption were determined in animals fed either a low- or a high-fat diet. RESULTS: Although total bile acid concentrations were unchanged in bile, liver, urine, and feces of FATP5 knockout mice, the majority of gallbladder bile acids was unconjugated, and only a small percentage was conjugated. Primary, but not secondary, bile acids were detected among the remaining conjugated forms in FATP5 deletion mice, suggesting a specific requirement for FATP5 in reconjugation of bile acids during the enterohepatic recirculation. Fat absorption in FATP5 deletion mice was largely normal, and only a small increase in fecal fat was observed on a high-fat diet. Despite normal fat absorption, FATP5 deletion mice failed to gain weight on a high-fat diet because of both decreased food intake and increased energy expenditure. CONCLUSIONS: Our findings reveal an important role for FATP5 in bile acid conjugation in vivo and an unexpected function in body weight homeostasis, which will require further analysis. FATP5 deletion mice provide a new model to study the intersection of bile acid metabolism, lipid metabolism, and body weight regulation.     

Evidence suggesting 11 beta-hydroxylase inhibition during aminoglutethimide administration

Steroid hormone excretion during amino-glutethimide administration was studied in a patient with Cushing's syndrome due to bilateral adrenocortical hyperplasia. Plasma and urinary 17-OHCS showed a persistent decrease, as did urinary total 17-KS. Chromatographic fractionation of urinary 17-KS demonstrated a dramatic reduction of 11-oxy-17-KS, while 11-deoxy-17-KS, after a transient fall, tended to recover. Urinary THS showed an absolute increase, while pregnanetriolone diminished. Δ⁵-pregnenetriol and pregnanetriol, after initial decreases, showed a gradual trend upward. The sum of these data, and particularly the increase in THS excretion, suggests that, in this case, aminoglutethimide exerted an inhibitory effect not only on the early steps of adrenocortical steroidogenesis but also on 11 β-hydroxylation.     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic receptor (CR) in rat epididymal 105,000 $\text{g}$ supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-³H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration 01 hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.     

Effect of aminoglutethimide on extraglandular metabolism of exogenous testosterone

Aminoglutethimide (AG), an inhibitor of steroid biosynthesis, seems to have an extraglandular site of action on steroid catabolism. To study this effect, five males with peripheral hypogonadism were first given testosterone propionate and then the same dose was repeated combined with AG and urinary testosterone, and its metabolites were measured. AG was shown to have a very evident effect on the peripheral degradation of exogenous testosterone. This may be responsible for a few signs of virilization and fetal masculinization in women taking AG.     

干扰素调节因子5在系统性红斑狼疮的表达及临床相关性研究

目的 比较干扰素调节因子5(IRF5)mRNA在系统性红斑狼疮(SLE)患者和健康对照组的表达水平,分析IRF5表达与SLE疾病活动性及自身抗体和临床症状的相关性.方法 Ficoll密度梯度离心法分离SLE患者及健康对照外周血单个核细胞(PBMC),Trizol法分离提取总RNA,反转录mRNA为eDNA;实时定量聚合酶链反应(PCR)法测定SLE患者和正常对照组IRF5表达量;并分析SLE患者IRF5表达量与疾病活动性及临床症状的相关性.结果 SLE组IRF5表达量(2.1+2.2)高于正常对照组(1.5±1.2),但差异无统计学意义(P=0.161);SLE患者IRF5表达量与其疾病活动指数(SLEDAI)显著相关(r=0.616,P<0.01);SLE患者中抗dsDNA抗体阳性组IRF5表达量(3.2±2.8)明显高于抗体阴性组(1.3±1.6).差异有统计学意义(P=0.018);SLE患者有发热、神经精神症状者IRF5表达量明显高于无此类临床症状者.结论 IRF5在SLE患者中表达偏高,且与SLEDAI显著相关,其可能通过调节下游基因的转录表达诱一导免疫失调,并由此参与SLE的发病过程.     

High-affinity binding of the antiestrogen [3H]tamoxifen to the 8S estradiol receptor

The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.     

Some properties of androgen-binding activity in rat testis.

High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.     

Evolution of steroid-5α-reductases and comparison of their function with 5β-reductase

Steroid-5α-reductases (SRD5α) and steroid-5β-reductase (SRD5β) represent a convergence in evolution: they share similar biological functions, but do not have a common ancestor. In vertebrates, SRD5α and SRD5β are involved in C-19 and C-21 steroid biosynthesis, bile acid biosynthesis and erythropoiesis. We compare and contrast the history, evolution, tissue distribution, enzyme characteristics and biological functions of SRD5α and SRD5β and suggest possible future directions for research efforts. Both, the unique and overlapping roles that SRD5α and SRD5β play in steroid hormone metabolism, are indicated. We also present the phylogeny of the SRD5α. The main SRD5α subfamilies obtained include, not only the well-known SRD5α type 1, type 2 and type 3, but also the synaptic glycoprotein (GPSN2)/trans-2,3-enoly-CoA reductase group. Phylogenetic analysis indicated that a eukaryotic ancestor likely underwent duplication events to generate these three subfamilies (type 1/2, type 3 and GPSN2 ancestors); both SRD5α type 1/2 and GPSN2 subfamilies may have evolved by ancient duplication events at the early stage of vertebrate and chordate evolution.     

Is it safe to use TNF-α blockers for systemic inflammatory disease in patients with heart failure? Importance of dosage and receptor specificity

Tumor necrosis factor-alpha (TNF-α) blockers are widely used in the treatment of chronic inflammatory diseases, especially chronic arthritis. Current guidelines advise against the use of such agents in patients who have a concomitant heart failure. Consequently, a group of patients with a devastating inflammatory disease cannot benefit from an excellent treatment option. After a critical review of the current literature, we conclude that there is not sufficient evidence to warn against such a regimen if recommended standard doses are used. A negative effect on the heart function seems to occur if unconventional high doses of TNF-α blockers are given. The theoretical background for this is discussed.     

Meta-analysis of immunohistochemical expression of hypoxia inducible factor-1α as a prognostic role in gastric cancer

AIM:To conduct a meta-analysis to evaluate the prognostic role of hypoxia inducible factor-1α(HIF-1α)expression in gastric cancer.METHODS:The PubMed,EMBASE,and Web of Science databases were searched systematically for all articles published in English before August,2013.Pooled effect was calculated from the available data to evaluate the association between HIF-1αexpression and 5-year overall survival and tumor clinicopathological features in gastric cancer patients.Pooled odds ratios(ORs)with95%CIs were calculated using either a fixed-effects or a random-effects model.RESULTS:Nine studies matched the selection criteria,which reported on 1103 subjects,548 of whom had HIF-1αpositive expression(50%).This meta-analysis indicated that HIF-1αpositive expression in gastric cancer correlated with lower 5-year overall survival(OR=0.36;95%CI:0.21-0.64),worse tumor differentiation(OR=0.38;95%CI:0.23-0.64),deeper invasion(OR=0.42;95%CI:0.32-0.57),higher rates of lymph node metastasis(OR=2.23;95%CI:1.46-3.40),lymphatic invasion(OR=2.50;95%CI:1.46-4.28),and vascular invasion(OR=1.80;95%CI:1.29-2.51),and higher TNM stage(Ⅲ+Ⅳ)(OR=0.31;95%CI:0.15-0.60).CONCLUSION:HIF-1αpositive expression indicates a poor prognosis for patients with gastric cancer.Further studies are required to confirm these results.     

Regulation of cell proliferation and malignant potential by irisin in endometrial,colon, thyroid and esophageal cancer cell lines

Objective

Irisin is a novel hormone that has been proposed to mediate the beneficial effects of exercise on metabolism, including body weight regulation and insulin resistance. No previous studies have evaluated whether irisin may regulate cell proliferation and malignant potential of obesity-related cancer cell lines.

Materials/Methods

Cell proliferation and malignant potential i.e. cell adhesion and colony formation were studied in vitro using human and mouse obesity-related cancer cell lines i.e. endometrial (KLE and RL95-2), colon (HT29 and MCA38), thyroid (SW579 and BHP7) and esophageal (OE13 and OE33).

Results

We observed that, in contrast to metformin, cell proliferation is not regulated by irisin in a dose-dependent manner in human and mouse obesity-related cancer cell lines. Specifically, physiological (5 to 10 nmol/L) and high physiological/pharmacological (50 to 100 nmol/L) concentrations of irisin had no effect on cell proliferation when compared to control in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines. Also, we observed that, in contrast to metformin, neither physiological nor high physiological/pharmacological concentrations of irisin regulate cell adhesion and/or colony formation in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines.

Conclusions

Our data suggest that irisin, in physiological and high physiological/pharmacological concentrations, has no in vitro effect on cell proliferation and malignant potential of obesity-related cancer cell lines. Future work is needed to determine the regulation of irisin levels and any physiological effects it may have on obesity-related cancers in vivo in animals and humans.     

Interaction of glucocorticoid hormones with rat skeletal muscle: catabolic effects and hormone binding.

The mechanism of action of glucocorticoid hormones on rat skeletal muscle was studied by following their effect on muscle weight, free amino acid content, activity of amino acid-metabolizing enzymes, and binding to cytoplasmic receptor proteins. A significant reduction of gastrocnemius muscle and body weight occurred following administration of cortisol, triamcinolone diacetate, and triamcinolone acetonide to adrenalectomized rats. Treatment with triamcinolone diacetate also reduced the level of several free amino acids and enhanced the activity of a myofibrillar protease in skeletal muscle. The hormone had, however, no effect on the activity of various enzymes involved in amino acid catabolism in muscle. In nephrosis, another condition of muscle wasting, the level of several muscle amino acids were also reduced to a lesser extent. Cortisol and triamcinolone acetonide, both of which induce muscle wasting, were found to bind to two distinct cytoplasmic proteins in muscle. Binding of the labeled hormones was followed at 0 C and could be observed in presence of a 1000-fold excess of the catabolically inactive steroid epicortisol. Binding of 3H-triamcinolone acetonide. In vitro competition experiments further suggest a correlation between steroid binding to the 3H-dexamethasone or 3H-triamcinolone acetonide site and their potency to induce muscle catabolism. It is concluded that skeletal muscle is a direct target organ for glucocorticoids, and that muscle responsiveness involves binding of the active hormones to cytoplasmic receptor sites.     

Prognostic impact of microsatellite instability and DNA ploidy in human colon carcinoma patients

BACKGROUND & AIMS: Genomic instability in colon cancers is a consequence of chromosomal instability characterized by aneuploidy or defective DNA mismatch repair (MMR) indicated by microsatellite instability (MSI). Given that high-frequency MSI (MSI-H) and diploidy are correlated, we determined whether they are independent prognostic variables. METHODS: Astler-Coller stage B2 and C colon cancers (N = 528) from patients treated in 5-fluorouracil-based adjuvant therapy trials were analyzed for MSI using 11 microsatellite markers. Immunostaining for hMLH1, hMSH2, and p53 proteins was performed. DNA ploidy was analyzed by flow cytometry. Associations with disease-free and overall survival were determined. RESULTS: MSI-H was detected in 95 tumors (18%), and 70 (74%) of these were diploid. Tumors showing MSI-H (hazard ratio, 0.65; 95% confidence interval, 0.44-0.96; P = .023) or loss of MMR proteins (P = .024) were associated with better overall survival. Improved disease-free and overall survival were found for diploid versus aneuploid/tetraploid tumors (overall survival: hazard ratio, 0.59; 95% confidence interval, 0.43-0.79; P = .0003). In the subgroups of MSI-H and microsatellite stable (MSS)/low-frequency MSI (MSI-L) tumors, diploidy was associated with better survival. The prognostic impact of ploidy was similar in stage B2 and C tumors. Ploidy did not predict the benefit of 5-fluorouracil-based treatment. When ploidy, MSI, and MMR proteins were analyzed in the same multivariate model, only ploidy remained significant. CONCLUSIONS: DNA ploidy and MSI-H status were independent prognostic variables, yet ploidy was the strongest marker. Diploidy was associated with better survival in MSI-H and in MSS/MSI-L patient subgroups.     

Accumulation of carnitine in rat epididymis after injection of [3H]butyrobetaine in vivo: quantitative aspects, and the effects of androgens and antiandrogens

After i.m. injection of [3H]butyrobetaine into intact and castrated rats, the specific activity of plasma carnitine remained nearly constant over 24--96 h and epididymal uptake of carnitine was constant per unit time up to 72 h. The uptake ratio of intact to castrated rats was high at 48, 72 and 96 h after injection. Administration of estradiol valerate over 20 days reduced carnitine uptake in epididymis. This reduction was dose-dependent when estrogen was administered i.m. at 0.33--10 microgram/day levels. A maximum reduction of 90% was obtained with the 10 microgram dose. A dose increase from 33 to 100 microgram/day caused no further reduction. Norspiroxenone (2--10 mg/day) and SK 7670 (1.5 and 7.5 mg/day) were less effective than estradiol valerate (10 microgram/day) in suppressing carnitine uptake in epididymis. Epididymal carnitine uptake in estradiol valerate treated rats (33 microgram/day for 20 days) increased in a time- and dose-dependent manner under testosterone propionate treatment (50, 250, 1250 microgram/day). Carnitine uptake increased to 80% of the nonsuppressed levels when testosterone propionate was adminsitered over a 6-day period at 1250 microgram/day. Dihydrotestosterone increased epididymal carnitine uptake to the same extent as testosterone propionate. delta4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol (50 microgram/day) were less effective, stimulating uptake to only 15% and 40% respectively of the testosterone propionate (250 microgram/day) stimulated levels. Changes in epididymal carnitine uptake evoked by various experimental procedures were closely paralleled by weight changes in the ventral prostate. This response resemblance indicates a similarity between the androgen sensitivity of the prostate gland and that of the carnitine uptake system in epididymis. The dose-dependent effect of estrogen on the accumulation of epididymal carnitine, together with the marked responses induced in this system by manipulation of its androgen status, support a possible use for the system as an assay for androgen or antiandrogen potency in vivo.     

Small doses of 5α-dihydrotestosterone mimic the effects of 5α-androstane-3β,17β-diol on acid phosphatase activity in the adult rat prostate gland

These studies were designed to further investigate whether 5α-androstane-3β,17β-diol was exerting unique effects on rat prostate acid phosphatase activity or could possibly be exerting its actions by a small peripheral conversion to 5α-dihydrotestosterone. Intraperitoneal administration of 5α-dihydrotestosterone in doses of 1 mg, 100 μg or 50 μg per day starting 7 days after castration led to the restoration of normal characteristics of acid phosphatase activity. However, when 5α-dihydrotestosterone was given in a dose of only 25 μg per day starting 7 days after castration, the changes in acid phosphatase activity were indistinguishable from those found when 5α-androstane-3β,17β-diol was administered in a dose of 2 mg per day. This suggests that the effects of 5α-androstane-3β,17β-diol can be explained by its conversion to small amounts of 5α-dihydrotestosterone.     

Cortisol metabolism in lymphocytes from cancer-bearing patients

A study was conducted to determine the pattern of cortisol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1,2-³H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-bearing patients: 20α-hydroxycortisol, 20β-hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.     

Complete response by a combination of 5-fluorouracil and interferon-alpha chemotherapy for lung metastasis of hepatocellular carcinoma after hepatic resection with portal and hepatic vein tumor thrombectomy.

A 75-year-old female was admitted to our hospital and diagnosed with hepatocellular carcinoma (HCC). Computed tomography (CT) revealed a liver tumor with tumor thrombi in the portal trunk and main hepatic vein, as well as small lung metastases. The patient had good liver function with no sign of hepatitis B or C infection. She underwent right trisectionectomy of the liver with tumor thrombectomy. Intrahepatic recurrence and progression of lung metastases were observed 4 months later. Intrahepatic recurrent tumors were treated with transcatheter arterial chemoembolization (TACE), and lung metastases were treated with systemic combination chemotherapy of 5-fluorouracil (5FU) and interferon-alpha (IFN-alpha). Computed tomography showed no viable lesions in the liver and lung 6 months after these treatments. The patient has been disease free for 18 months. Prognosis is poor for patients with hepatocellular carcinoma with portal vein tumor thrombus (PVVT) or extrahepatic metastasis. This systemic combination chemotherapy with 5-fluorouracil and interferon-alpha might be effective for patients with good liver function when intrahepatic lesions are well controlled by multidisciplinary treatments, including hepatic resection with tumor thrombectomy.     

Prolonged survival of a patient with advanced colonic cancer

A 39-year-old man who survived 14 years following the diagnosis of advanced colonic carcinoma is reported. The presence of metastatic lesions had been well documented at the time of abdominal surgery on three different occasions and at autopsy. The possible mechanism for his unusually prolonged survival time is discussed.     

Malignant melanoma of the anorectum

Melanoma is a virulent tumor that can be widely disseminated and is genrally incurable even when the primary growth is small. Review of the cases of 34 patients with primary melanoma of the anorectum, with follow-up ranging from two to five years, suggests that improvement in survival depends on acute surgical awareness and aggressive surgical intervention. Read at the meeting of the American Society of Colon and Rectal Surgeons, Atlanta, Georgia, June 10 to 14, 1979.