Serum levels of renin, angiotensin-converting enzyme and angiotensin II in patients treated by surgical excision, propranolol and captopril for problematic proliferating infantile haemangioma.

The role of the renin-angiotensin system (RAS) in the biology of infantile haemangioma (IH) and its accelerated involution induced by ß-blockers was first proposed in 2010. This led to the first clinical trial in 2012 using low-dose captopril, an angiotensin-converting enzyme (ACE) inhibitor, demonstrating a similar response in these tumours. This study aimed to compare serial serum levels of the components of the RAS in patients before and after surgical excision, propranolol or captopril treatment for problematic proliferating IH. Patients with problematic proliferating IH underwent measurements of serum levels of plasma renin activity (PRA), ACE and angiotensin II (ATII) before, and 1-2 and 6 months following surgical excision, propranolol or captopril treatment. This study included 27 patients undergoing surgical excision (n = 8), propranolol (n = 11) and captopril (n = 8) treatment. Treatment with either surgical excision or propranolol resulted in significant decrease in the mean levels of PRA. Surgical excision or captopril treatment led to significant decline in the mean levels of ATII. All three treatment modalities had no significant effect on the mean levels of ACE. This study demonstrates the effect of surgical excision, propranolol and captopril treatment in lowering the levels of PRA and ATII, but not ACE, supporting a mechanistic role for the RAS in the biology of IH.

Genetic map construction and QTL mapping of resistance to blackleg (Leptosphaeria maculans) disease in Australian canola (Brassica napus L.) cultivars.

Genetic map construction and identification of quantitative trait loci (QTLs) for blackleg resistance were performed for four mapping populations derived from five different canola source cultivars. Three of the populations were generated from crosses between single genotypes from the blackleg-resistant cultivars Caiman, Camberra and (AV)Sapphire and the blackleg-susceptible cultivar Westar(10). The fourth population was derived from a cross between genotypes from two blackleg resistant varieties (Rainbow and (AV)Sapphire). Different types of DNA-based markers were designed and characterised from a collection of 20,000 EST sequences generated from multiple Brassica species, including a new set of 445 EST-SSR markers of high value to the international community. Multiple molecular genetic marker systems were used to construct linkage maps with locus numbers varying between 219 and 468, and coverage ranging from 1173 to 1800 cM. The proportion of polymorphic markers assigned to map locations varied from 70 to 89% across the four populations. Publicly available simple sequence repeat markers were used to assign linkage groups to reference nomenclature, and a sub-set of mapped markers were also screened on the Tapidor x Ningyou (T x N) reference population to assist this process. QTL analysis was performed based on percentage survival at low and high disease pressure sites. Multiple QTLs were identified across the four mapping populations, accounting for 13-33% of phenotypic variance (V (p)). QTL-linked marker data are suitable for implementation in breeding for disease resistance in Australian canola cultivars. However, the likelihood of shifts in pathogen race structure across different geographical locations may have implications for the long-term durability of such associations.

Functional annotation of genomic data with metabolic inference.

Metabolomics is an appealing new approach in systems biology aimed at enabling an improved understanding of the dynamic biochemical composition of living systems. Biological systems are remarkably complex. Importantly, metabolites are the end products of cellular regulatory processes, and their concentrations reflect the ultimate response of a biological system to genetic or environmental changes. In this article, we describe the components of lipid metabolomics and then use them to investigate the metabolic basis for increased abdominal adiposity in 2 strains of divergently selected chickens. Lipid metabolomics were chosen due to the availability of well-developed analytical platforms and the pervasive physiological importance of lipids in metabolism. The analysis suggests that metabolic shifts that result in increased abdominal adiposity are not universal and vary with genetic background. Metabolomics can be used to reverse engineer selection programs through superior metabolic descriptions that can then be associated with specific gene networks and transcriptional profiles.

Metabolomic mapping of atypical antipsychotic effects in schizophrenia.

Schizophrenia is associated with impairments in neurotransmitter systems and changes in neuronal membrane phospholipids. Several atypical antipsychotic drugs induce weight gain and hypertriglyceridemia. To date, there has not been a comprehensive evaluation and mapping of global lipid changes in schizophrenia, and upon treatment with antipsychotics. Such mapping could provide novel insights about disease mechanisms and metabolic side effects of therapies used for its treatment. We used a specialized metabolomics platform 'lipidomics' that quantifies over 300 polar and nonpolar lipid metabolites (across seven lipid classes) to evaluate global lipid changes in schizophrenia and upon treatment with three commonly used atypical antipsychotics. Lipid profiles were derived for 50 patients with schizophrenia before and after treatment for 2-3 weeks with olanzapine (n=20), risperidone (n=14) or aripiprazole (n=16). Patients were recruited in two cohorts (study I, n=27 and study II, n=23) to permit an internal replication analyses. The change from baseline to post-treatment was then compared among the three drugs. Olanzapine and risperidone affected a much broader range of lipid classes than aripiprazole. Approximately 50 lipids tended to be increased with both risperidone and olanzapine and concentrations of triacylglycerols increased and free fatty acids decreased with both drugs but not with aripiprazole. Phosphatidylethanolamine concentrations that were suppressed in patients with schizophrenia were raised by all three drugs. Drug specific differences were also detected. A principal component analysis (PCA) identified baseline lipid alterations, which correlated with acute treatment response. A more definitive long-term randomized study of these drugs correlating global lipid changes with clinical outcomes could yield biomarkers that define drug-response phenotypes.

Extraction of RNA from archival tissues and measurement of thrombospondin-1 mRNA in normal, dysplastic, and malignant oral tissues.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein implicated in the regulation of angiogenesis and tumour development. Our objectives were to ascertain the quantity and quality of RNA extracted from archival, formalin-fixed, paraffin embedded, oral tissues and their application in measuring the concentrations of TSP-1 mRNA in these tissues. We compared three techniques of isolation of RNA as well as related experimental variables. TSP-1 mRNA was measured in specimens of normal, dysplastic, and malignant oral tissues by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RNA suitable for analysis by real-time RT-PCR was obtained by the three techniques tested, although the yield varied depending on the protocol used (range 0.2-3.6 microg/mm(3)). The mean (S.D.) concentrations of TSP-1 mRNA relative to 18S were 21.1 (7.2) in normal oral tissues (n=9), 11.0 (8.2) in dysplastic tissue (n=8) and 7.3 (5.3) in carcinomatous tissue (n=17). The difference between normal and carcinomatous specimens was significant (p=0.01). This reduction in expression of TSP-1 mRNA from normal to dysplasia to carcinoma may favour the angiogenic drive that accompanies the development of oral tumours.

Vascular endothelial growth factor (VEGF) expression in oral tissues: possible relevance to angiogenesis, tumour progression and field cancerisation.

The aim of this study was to assess whether vascular endothelial growth factor (VEGF) expression in oral tissues is associated with angiogenesis, disease progression or field cancerisation. Vascularity and VEGF immunoreactivity were quantified in 68 archival specimens including normal oral mucosa (NOM), dysplasia (DYS) and squamous cell carcinoma (SCC). Vascularity increased significantly with disease progression; it was also higher in NOM adjacent to SCC than in NOM from healthy tissue, suggesting an association with field cancerisation. VEGF expression in epithelial cells was evaluated using two antibodies and three indices. VEGF indices and vascularity were not directly correlated. The expression of VEGF was similar in all DYS and NOM specimens, whether or not adjacent to a concurrent lesion. A comparison of SCC with NOM or DYS led to opposite results, depending on the VEGF antibody and index used. We conclude that VEGF expression in the oral mucosa may play a physiological role, but does not appear to be associated with angiogenesis, field cancerisation or transition to dysplasia. Further studies concerned with tumour development require examining specific VEGF isoforms and standardisation of the methodology.

Expression of vascular endothelial growth factor in normal and tumour oral tissues assessed with different antibodies.

Expression of vascular endothelial growth factor (VEGF) in oral tissues was assessed using different antibodies. Quantitative and topographical differences were observed between paraffin and cryostat sections. Two polyclonal antibodies (PC36, PC37) differing in their cross-reactivity with VEGF121 (not recognized by PC36), were used to stain serial cryostat sections of normal oral mucosa (n = 8) and squamous cell carcinoma (n = 7). The expression of VEGF in the epithelium was overall higher with PC37 than with PC36, the difference being significant in normal oral mucosa (p = 0.001) but not in squamous cell carcinoma samples (p = 0.094). With PC36, VEGF expression was significantly higher in squamous cell carcinoma than in normal oral mucosa specimens, whereas the opposite was true with PC37. Our results suggest that the relative levels of isoform 121 to that of 165 (and possibly others) may be different in the tissues examined, with VEGF121 preferentially expressed in normal oral mucosa. Previously published conflicting results may, therefore, be due to the presence of variable ratios of VEGF isoforms in the tissues examined, combined with differences in the cross-reactivity of the antibodies used. VEGF isoforms 121, 165 and (for the first time) 189 were detected in two frozen oral tissues by polymerase chain reaction amplification. Quantification of specific VEGF isoforms will be required in future studies concerned with the clinical value of VEGF expression.

VEGF expression in skin warts. Relevance to angiogenesis and vasodilation.

Verrucae vulgaris (skin warts) are benign proliferative lesions which are generally associated with human papillomavirus type 2 (HPV-2) infection. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen able to induce angiogenesis and vasodilation. Our previous findings indicate that these two processes take place during the formation of skin warts. The purpose of this study was to determine whether VEGF expression in these lesions was associated with HPV infection, angiogenesis or vasodilation. To this end, paraffin-embedded specimens of skin warts which were either negative for HPV-1, -2, -3 and -4 (HPV-; n = 18), or positive for HPV-2 (HPV+; n = 21) were compared with histologically normal perilesional skin (n = 13). Serial sections were stained with antibodies to von Willebrand Factor (vWF) and to VEGF. Vascularity was quantified by point counting vWF-positive blood vessels. Small and large vessels were quantified separately, using a cut-off value of 50 microm diameter. VEGF expression in the epidermis was estimated by consensus of two independent observers according to three indices: (1) percentage of cells stained, (2) intensity of the staining, and (3) product of area and intensity (final score). Results were analysed by nonparametric tests. Similar levels of VEGF were found in specimens of normal skin, HPV- and HPV+ warts, irrespective of the index used. There was no significant correlation between VEGF expression and vascularity values for either small or large vessels. These results indicate that, on its own, VEGF expression is not associated with angiogenesis, vasodilation or HPV infection in skin warts. The presence of VEGF in normal skin suggests that it may play a role in tissue homeostasis.

Prognostic value of vascularity and vascular endothelial growth factor expression in non-small cell lung cancer.

AIMS: High expression of the angiogenic factor vascular endothelial growth factor (VEGF) in tumours has been found to be associated with poor prognosis in some studies, but not in others. The aims of this study were to determine the prognostic value of VEGF in operable non-small cell lung cancer (NSCLC) and its possible association with vascularity. METHODS: Sections from 81 NSCLC archival specimens were stained with antibodies to von Willebrand factor (vWF) and VEGF. Vascularity was measured by the average density of vWF positive vessels. VEGF expression in tumour cells was assessed by consensus of two independent observers according to three indices, namely: (1) percentage of area stained, (2) intensity of staining, and (3) final score (product of area and intensity). RESULTS: VEGF immunoreactivity was present in all tumours and adjacent normal lung tissue. None of the three VEGF indices was associated with vascularity or the clinical parameters examined. Mean survival times were shorter in patients with high VEGF expression, but the difference was not significant. This applied to the full cohort of patients, or when analysed separately according to tumour type or stage. However, high VEGF expression was associated with poor survival in patients with high vascularity (p = 0.02). VEGF had no discriminant value among patients with low vascularity. Vascularity had no prognostic value, except for late stage patients (UICC stages II and IIIa combined; n = 36), where high vascularity was associated with longer survival (p = 0.01). CONCLUSIONS: VEGF on its own has no prognostic value in NSCLC, but may become a useful indicator when combined with vascularity. VEGF may play a physiological role in the normal lung.

Copper deficiency induces hepatic fatty acid synthase gene transcription in rats by increasing the nuclear content of mature sterol regulatory element binding protein 1.

Dietary copper (Cu) deficiency results in an accelerated rate of hepatic fatty acid synthase gene transcription and an enhanced rate of hepatic lipid synthesis. Because the nuclear transcription factor sterol regulatory element binding protein-1 (SREBP-1) is a strong enhancer of fatty acid synthase promoter activity, it was hypothesized that Cu deficiency induces fatty acid synthase gene transcription by increasing the nuclear localization of mature SREBP-1. Male weanling rats were pair-fed a Cu-adequate (6.0 mg/kg) or Cu-deficient (0.6 mg/kg) diet (AIN-93) for 28 d. DNase I hypersensitivity site mapping of the hepatic fatty acid synthase gene revealed the presence of four major hypersensitivity sites located at -8700 to -8600, -7300 to -6900, -600 to -400 and -100 to +50. Although Cu deficiency did not change the hypersensitivity site pattern or intensity, in vitro footprinting of the region between -100 and +50 indicated that Cu deficiency enhanced DNA protein interactions within this region. The sequence between -68 and -58 contains the DNA recognition sequence for SREBP-1 and upstream stimulatory element-1 (USF-1). Western blot analysis revealed that the dietary Cu deficiency increased the hepatic nuclear content of mature SREBP-1 by 150% (P: < 0.05), and it concomitantly decreased the membrane content of precursor SREBP-1 by 45% (P: < 0.05). Changes in the hepatic distribution of SREBP-1 associated with Cu deficiency were not accompanied by changes in SREBP-1 mRNA. The nuclear content of USF-1 was unaffected by dietary Cu status. The hepatic increase in mature SREBP-1 of Cu-deficient rats was accompanied by a 400% increase and an 80% decrease in the abundance of fatty acid synthase and cholesterol 7-alpha hydroxylase mRNA, respectively. hepatic These data indicate that a Cu deficiency stimulates hepatic lipogenic gene expression by increasing the hepatic translocation of mature SREBP-1.

High levels of apoptosis are associated with improved survival in non-small cell lung cancer.

Tumour growth is accompanied by angiogenesis and reduced apoptosis in experimental animals. The aim of this study was to examine the prognostic value of apoptosis and the association between apoptosis and vascularity in non-small cell lung cancer (NSCLC). Following in-situ end-labelling of DNA, apoptotic cells were quantified by three different indices: as a percentage, either counting total cells (AI-tc) or point-counting (AI-pc), or as cells per area (AI-area). Blood vessels were stained with vWF antibody and vascularity was quantified by three methods. Median values for AI-tc, AI-pc and AI-area were 0.38, 0.32 and 10.7, respectively. High values were associated with improved survival, reaching statistical significance for AI-area (p < 0.05). All three apoptotic indices were significantly correlated with each other, but no correlation was found between indices of apoptosis and vascularity. As previously reported, vascularity had no prognostic value. These results indicate that, in NSCLC, vascularity is not informative, but apoptotic index may be a useful prognostic factor.

Risk of breast cancer in relation to the use of injectable progestogen contraceptives and combined estrogen/progestogen contraceptives.

Recent studies have suggested that progestogen-only contraceptives and combined estrogen/progestogen oral contraceptives (COCs) may increase the risk of breast cancer among women less than 35 years of age or among recent users. The authors conducted a case-control study, in which cases of breast cancer (n = 484) [corrected] and controls (n = 1,625) hospitalized for conditions unrelated to contraceptive use were interviewed from 1994 to 1997 in hospitals in greater Cape Town, South Africa. The women were aged 20-54 years, resided in a defined area around Cape Town, and were Black or of mixed racial descent. The relative risk for exposure to injectable progestogen contraceptives (IPCs), mostly depot medroxyprogesterone acetate, was 0.9 (95% confidence interval (CI) 0.7, 1.2). There were no consistent associations within categories of age or recency or duration of use. For exposure to COCs, the overall relative risk was 1.2 (95% CI 1.0, 1.5). Among women below age 35 years, the relative risk was 1.7 (95% CI 1.0, 3.0), and it was unrelated to the duration or recency of use. The findings suggest that IPCs do not increase the risk of breast cancer, and that COCs may increase the risk among women below age 35 years, although bias cannot be excluded.

Peroxisome proliferator-activated receptors: a family of lipid-activated transcription factors.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear transcription factors that belong to the steroid receptor superfamily. This family of PPARs includes PPARalpha, PPARdelta, PPARgamma1, and PPARgamma2. These PPARs are related to the T3 and vitamin D(3) receptors and bind to a hexameric direct repeat as a heterodimeric complex with retinoid receptor Xalpha. PPARs regulate the expression of a wide array of genes that encode proteins involved in lipid metabolism, energy balance, eicosanoid signaling, cell differentiation, and tumorigenesis. A unique feature of these steroid-like receptors is that the physiologic ligands for PPARs appear to be fatty acids from the n-6 and n-3 families of fatty acids and their respective eicosanoid products. This review describes the characteristics, regulation, and gene targets for PPARs and relates their effects on gene expression to physiologic outcomes that affect lipid and glucose metabolism, thermogenesis, atherosclerosis, and cell differentiation.

Coordinate induction of peroxisomal acyl-CoA oxidase and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition.

Rats fed dietary fats rich in 20- and 22-carbon polyenoic fatty acids deposit less fat and expend more energy at rest than rats fed other types of fats. We hypothesized that this decrease in energetic efficiency was the product of: (a) enhanced peroxisomal fatty acid oxidation and/or (b) the up-regulation of genes encoding proteins that were involved with enhanced heat production, i.e. mitochondrial uncoupling proteins (UCP-2, UCP-3) and peroxisomal fatty acid oxidation proteins. Two groups of male Fisher 344 rats 3-4 week old (n=5 per group) were pair fed for 6 weeks a diet containing 40% of its energy fat derived from either fish oil or corn oil. Epididymal fat pads from rats fed the fish oil diet weighed 25% (P < 0.05) less than those found in rats fed corn oil. The decrease in fat deposition associated with fish oil ingestion was accompanied by a significant increase in the abundance of skeletal muscle UCP-3 mRNA. The level of UCP-2 mRNA skeletal muscle was unaffected by the type of dietary oil, but the abundance of UCP-2 mRNA in the liver and heart were significantly lower (P < 0.05) in rats fed fish oil than in rats fed corn oil. In addition to inducing UCP-3 expression, dietary fish oil induced peroxisomal acyl-CoA oxidase gene expression 2-3 fold in liver, skeletal muscle and heart. These data support the hypothesis that dietary fish oil reduces fat deposition by increasing the expression of mitochondrial uncoupling proteins and increasing fatty acid oxidation by the less efficient peroxisomal pathway.

Predictors of bereavement outcome for family carers of cancer patients.

AIMS: The psychological outcome of family carers after bereavement is an important issue in evaluating palliative care services. Palliative care services have the potential to provide preventive psychosocial intervention to family carers prior to bereavement, but are faced with the need to identify those who may have greatest risk of adverse outcome. This prospective study examines predictors of psychological outcome for family carers of cancer patients following bereavement based on factors identified at referral to a palliative care agency. METHODS: Cancer patients and their family carer were consecutively recruited and assessed on a range of clinical and psychological measures at referral to a palliative home care service in a metropolitan centre (Time 1). Carers were again assessed following the death of the patient, on average at 4 months post-bereavement (Time 2), using measures of bereavement symptoms and psychological morbidity. RESULTS: 178 carers were assessed on both occasions. The chief predictors of carer psychological symptoms and severity of grief at follow-up were psychological symptom scores at the time of referral (Time 1). Factors also measured at Time 1 were significant predictors of symptoms and grief scores at Time 2: greater number of adverse life events, carer's coping responses, past bereavement and separation experiences, the relationship with the patient, and greater severity of patient's illness at the time of palliative care referral. CONCLUSIONS: The findings indicate clinical risk factors for adverse short-term bereavement outcome that can be identified in family carers during palliative care treatment, that have implications for identifying the psychological needs of carers, and that form a potential basis for interventions to enhance the psychological outcome for family carers.

Role of elevated plasma transforming growth factor-beta1 levels in wound healing.

Transforming growth factor (TGF)-beta1 plays a central role in wound healing. Wounds treated with neutralizing antibody to TGF-beta1 have a lower inflammatory response, reduced early extracellular matrix deposition, and reduced later cutaneous scarring, indicating the importance of local tissue TGF-beta1. By contrast, increasing the local, tissue levels of TGF-beta1 increases the early extracellular matrix deposition but does not alter scar formation. Increased levels of plasma TGF-beta1 correlate with increased fibrogenesis in the lung, kidneys, and liver. The aim of the present study was to investigate the role of elevated systemic levels of TGF-beta1 on wound healing. We used transgenic mice that express high levels of active TGF-beta1 and have elevated plasma levels of TGF-beta1 and wild-type mice of the same strain as controls. Incisional wounds and subcutaneously implanted polyvinyl alcohol (PVA) sponges were analyzed. Surprisingly, cutaneous wounds in transgenic, TGF-beta1-overexpressing mice healed with reduced scarring accompanied by an increase in the immunostaining for TGF-beta3 and TGF-beta-receptor RII and a decrease in immunostaining for TGF-beta1 compared with wounds in control mice. By contrast, the PVA sponges showed the opposite response, with PVA sponges from transgenic mice demonstrating an enhanced rate of cellular influx and matrix deposition into the sponges accompanied by an increase in the immunostaining for all three TGF-beta isoforms and their receptors compared with PVA sponges from control mice. Together, the data demonstrate that increased circulating levels of TGF-beta1 do not always result in increased expression or activity in selected target tissues such as the skin. The two wound models, subcutaneously implanted PVA sponges and cutaneous incisional wounds, differ significantly in terms of host response patterns. Finally, the data reinforce our previous observations that the relative ratios of the three TGF-beta isoforms is critical for control of scarring.

Cytosolic and nuclear distribution of PPARgamma2 in differentiating 3T3-L1 preadipocytes.

In light of the pivotal role that PPARgamma2 plays in the expression of fat specific genes (e.g., A-FABP), we have examined the hypothesis that a rise in PPARgamma2 protein is required for the expression of A-FABP, and that the acceleration of fat cell differentiation by the thiazolidinedione agent, pioglitazone (PIOG), reflects an increase in the abundance of PPARgamma2 mRNA and protein. Western analyses surprisingly revealed that undifferentiated 3T3-L1 fibroblasts contained significant levels of PPARgamma2 protein; that the amount of total cellular PPARgamma2 only increased 2-fold during differentiation; and that the levels of PPARgamma2 protein and mRNA were not increased by PIOG even though fat cell differentiation was accelerated by PIOG as revealed by a 20-fold increase in A-FABP expression. Cell fractionation studies revealed that PPARgamma2 was evenly distributed between the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPARgamma2-specific antibody indicated that PPARgamma2 was diffusely distributed throughout the cytosol of undifferentiated 3T3-L1 cells, but as the differentiation progressed, the PPARgamma2 became focused around the developing lipid droplets. In contrast to PPARgamma2, undifferentiated 3T3-L1 cells contained no measurable quantities of RXRalpha, but once fat cell differentiation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXRalpha increased several fold. The rise in RXRalpha content paralleled the induction of A-FABP, but the expression of RXRalpha was not enhanced by PIOG. Although the amount of PPARgamma2 and RXRalpha was unaffected by PIOG, gel shift assays revealed that PIOG stimulated PPARgamma2/RXRalpha binding to the adipose response element of A-FABP by 5-fold in less than 12 h. Apparently, RXRalpha rather than PPARgamma2 is the pivotal trans-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPARgamma2 and its association with the lipid droplet of differentiating 3T3-L1 cells suggests PPARgamma2 may possess a cytosolic function in the developing fat cell.

A novel 3T3-L1 preadipocyte variant that expresses PPARgamma2 and RXRalpha but does not undergo differentiation.

This report describes a novel adipocyte-like cell line termed 3T3-L1/RB1 that was derived from preadipocyte cell line, 3T3-L1. The 3T3-L1/RB1 cells continued to divide after reaching confluence, formed foci, and constitutively expressed a low level of adipose fatty acid binding protein (A-FABP) mRNA. However, 3T3L-1/RB cells did not undergo terminal differentiation as indicated by the failure of insulin and thiazolidendiones to induce the expression of A-FABP, lipoprotein lipase, and fatty acid synthase. We hypothesized that the 3T3-L1/RB1 variant did not respond to differentiation stimuli because it did not express either peroxisomal proliferator activated receptor gamma2 (PPARgamma2) or its heterodimer partner, retinoid X receptor alpha (RXRalpha). Surprisingly, Western blots revealed that 3T3-L1/ RB1 cells contained both PPARgamma2 and RXRalpha proteins at levels equal to or greater than that of the parent cell line. However, gel retardation assays using the adipose response element from A-FABP and nuclear protein extracts from 3T3-L1/RB1 cells treated with insulin or pioglitazone revealed that nuclear protein extracts from 3T3-L1/RB1 cells had very little ability to bind the PPARgamma2 recognition sequence of the A-FABP gene. These data suggest that the 3T3-L1/RB1 variant contains a mutation that may prevent ligand activation of PPARgamma2, and the subsequent conversion of 3T3-L1/RB1 cells to mature fat cells.

Regulation of the action of steroid/thyroid hormone receptors by medium-chain fatty acids.

Triiodothyronine (T3) causes a 30-fold increase in transcription of the malic enzyme gene in chick embryo hepatocytes; medium-chain fatty acids (MCFAs) inhibit this increase. T3 action is mediated by T3 receptors (TRs) that bind to T3 response elements (T3REs) in this gene's 5'-flanking DNA. In transiently transfected hepatocytes, fragments of 5'-flanking DNA of the malic enzyme gene or artificial T3REs that conferred T3 stimulation also conferred MCFA inhibition to linked reporter genes. Thus, MCFA inhibition may be mediated through cis-acting T3REs and trans-acting TRs, distinguishing MCFA action from that of other fatty acids which act through unique sequence elements. Using binding assays and overexpression of TR, we showed that MCFAs inhibited the transactivating but not the silencing function of TR and did not alter binding of T3 to TR or of TR to T3RE. The C-terminal ligand-binding domain of TR was sufficient to confer stimulation by T3, but not inhibition by MCFA. Inhibition of transactivation by MCFA was specific: ligand-stimulated transcription from T3 or estrogen response elements was inhibited, but that from glucocorticoid or cyclic AMP response elements was not. We propose that MCFAs or metabolites thereof influence the activity of a factor(s) that interacts with the T3 and estrogen receptors to inhibit ligand-stimulated transcription.

Nutritional and hormonal regulation of the gene for malic enzyme.

In vivo, refeeding starved chickens stimulates transcription of the avian gene for malic enzyme in liver; in hepatocytes in culture, triiodothyronine (T3) and insulin stimulate transcription of this gene. In vivo, starvation, and in hepatocytes in culture, glucagon, medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFA) inhibit transcription of the malic enzyme gene. We have defined a T3-response unit in the 5'-flanking DNA of the malic enzyme gene; it contains one major T3 response element and several minor ones; maximum responsiveness is dependent on the presence of all of these elements. LCFA probably act by inhibiting binding of T3 to its nuclear receptor. MCFA appear to act by a different mechanism. Inhibitory MCFA have chain lengths of six, seven or eight carbons; a common feature of other inhibitory compounds is that they can be metabolized to MCFA. Eight-carbon fatty acids with a hydroxyl on the 2- or 3-carbon are more potent inhibitors than octanoate, whereas 2-bromo-fatty acids and 2-hydroxy hexanoate are not inhibitory. In transfection experiments with a large variety of constructs derived from the malic enzyme 5'-flanking DNA, the ability of fatty acids to inhibit promoter function localizes to regions of DNA that contain T3REs. Promoter function of artificial T3REs also is inhibited by MCFA. Inhibition of promoter function using malic enzyme DNA is relatively constant in magnitude irrespective of the size of the T3 response. We postulate that MCFA directly regulates one of the functions of the T3 receptor.