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How do you think it was possible for the women who suffered such discrimination to persevere? Did looking beyond themselves to the next generation play a part in their ability to be so strong?
If you could have lived the life of any character in the book who would it be? Conversely, which character would you least like to have been and why?
Why is it some people are able to overcome such incredible odds, rise above, and pave the way for others? What prevents someone from doing this?
Do you see a correlation between a nation not speaking up against slavery and a world that did not speak up about Nazi Germany in their treatment of the Jews? If so, how?
Why was it women were the ones more often than men who took it upon themselves to "civilize" towns? Is it true today that women offer society refinement and polish? How so?
Do you think it is important for children to have role models from their own race? Does color matter in this regard? Why or why not?
Do you think the women in the book ever thought their trailblazing efforts would one day be recognized? Do you think they saw themselves as trailblazers? Which woman blazed a path for others the most?
Prejudice can be described as a distrust/dislike for those unlike ourselves. What are some motivating factors behind prejudice? What is it about skin color that so divides the human race?
Which story/character moved you the most and why? Which character in the book can you identify with the most and why?
Are women better equipped to cope with loss in their lives than men? If so, how? Could you find the same strength if you needed to?
What do you think is the underlying factor that has caused African American women to be overlooked for so long?
How does an abused woman find the strength to go on? What is it that beats one woman down, but empowers another?
How did the women's spirituality and faith factor in their ability to succeed?
How did the women in the stories find the strength to keep silent when they or their children were mistreated?
Which minor character in the book did you feel the most contempt for and why?
When can fear be a motivating factor? Which character in the book did not let fear run her life?
What incident/person vexed you most in the book?
Whose life did you feel was the most difficult and why?
Why do all societies stratify themselves with one group claiming superiority over the other?


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RESEARCH DESIGN AND METHODS
Metabolism| September 01 2008
Pyruvate Dehydrogenase Kinase 4: Regulation by Thiazolidinediones and Implication in Glyceroneogenesis in Adipose Tissue
Thomas Cadoudal;
Thomas Cadoudal
From the Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche-S 747, Université Paris Descartes, Centre Universitaire des Saints-Pères, Paris, France
Emilie Distel;
Emilie Distel
Sylvie Durant;
Sylvie Durant
Françoise Fouque;
Françoise Fouque
Jean-Marc Blouin;
Jean-Marc Blouin
Martine Collinet;
Martine Collinet
Sylvie Bortoli;
Sylvie Bortoli
Claude Forest;
Claude Forest
Chantal Benelli
Corresponding author: Dr. Claude Forest, claude.forest@univ-paris5.fr
Diabetes 2008;57(9):2272–2279
https://doi.org/10.2337/db08-0477
Thomas Cadoudal, Emilie Distel, Sylvie Durant, Françoise Fouque, Jean-Marc Blouin, Martine Collinet, Sylvie Bortoli, Claude Forest, Chantal Benelli; Pyruvate Dehydrogenase Kinase 4: Regulation by Thiazolidinediones and Implication in Glyceroneogenesis in Adipose Tissue. Diabetes 1 September 2008; 57 (9): 2272–2279. https://doi.org/10.2337/db08-0477
OBJECTIVE—Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled by PDK.
RESEARCH DESIGN AND METHODS—Rosiglitazone was administered to Zucker fa/fa rats, and then PDK4 and PDK2 mRNAs were examined in subcutaneous, periepididymal, and retroperitoneal WAT, liver, and muscle by real-time RT-PCR. Cultured WAT explants from humans and rats and 3T3-F442A adipocytes were rosiglitazone-treated before analyses of PDK2 and PDK4 mRNA and protein. Small interfering RNA (siRNA) was transfected by electroporation. Glyceroneogenesis was determined using [1-14C]pyruvate incorporation into lipids.
RESULTS—Rosiglitazone increased PDK4 mRNA in all WAT depots but not in liver and muscle. PDK2 transcript was not affected. This isoform selectivity was also found in ex vivo–treated explants. In 3T3-F442A adipocytes, Pdk4 expression was strongly and selectively induced by rosiglitazone in a direct and transcriptional manner, with a concentration required for half-maximal effect at 1 nmol/l. The use of dichloroacetic acid or leelamine, two PDK inhibitors, or a specific PDK4 siRNA demonstrated that PDK4 participated in glyceroneogenesis, therefore altering nonesterified fatty acid release in both basal and rosiglitazone-activated conditions.
CONCLUSIONS—These data show that PDK4 upregulation in adipocytes participates in the hypolipidemic effect of thiazolidinediones through modulation of glyceroneogenesis.
Insulin resistance is associated with alterations in the balance between glucose and fatty acid oxidative pathways. This leads to chronic hyperglycemia because of an excessive hepatic glucose production (gluconeogenesis) (1) associated with a decrease in insulin-induced glucose disposal within peripheral tissues, such as skeletal muscle (2). Furthermore, insulin resistance is associated with an excessive plasma concentration of nonesterified fatty acids (NEFAs), which is partly due to a reduction of the antilipolytic action of insulin on white adipose tissue (WAT) in postprandial situation and a decrease in fatty acid reesterification during lipolysis at fast (3,4). Numerous lines of evidence support the notion that this increase in plasma NEFA plays a pivotal role in the early onset of insulin resistance (5–7). The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate to acetyl-CoA and CO2. This complex regulates the balance between oxidation of glucose and lipids, depending on nutritional status, and thus plays the role of metabolic switch for fuel selection (8). PDC activity is tightly controlled in the short term by a continuous phosphorylation-dephosphorylation cycle (9,10). Phosphorylation of the E1 subunit of PDC is catalyzed by the PDC kinases (PDKs), which inactivate PDC, while PDC phosphatases (PDPs) activate PDC through dephosphorylation. Thus, the relative activities of PDK and PDP regulate the proportion of PDC in the active dephosphorylated form in mitochondria. Four isoforms of PDK (PDK1–4) and two isoforms of PDP (PDP1 and -2) have been described in mammals and are expressed in varying amounts in a tissue-specific manner (11,12). To prevent hyperglycemia in insulin-resistant states, inhibitors of PDK have been developed to activate PDC, thereby decreasing gluconeogenesis in liver and increasing glucose oxidative capacities in skeletal muscle (13,14). However, the role of PDC and its regulation by PDK-to-PDP ratio in other insulin-sensitive tissues, like adipose tissue, has not been extensively studied.
In WAT, lipolytic and reesterification pathways are active and both participate in the control of NEFA release (15). Reesterification into triglycerides of an important part of NEFA arising from lipolysis requires the synthesis of glycerol-3-phosphate (G3P), which mainly arises from noncarbohydrate substrates like lactate or pyruvate through a pathway named glyceroneogenesis (16,17). The key enzyme of this metabolic pathway is the cytosolic isoform of PEPCK-C (18). Pyruvate can be either carboxylated to oxaloacetate by pyruvate carboxylase and used for glyceroneogenesis or decarboxylated to acetyl-CoA by PDC for the tricarboxylic acid cycle. Hence, we hypothesized that pyruvate flux through glyceroneogenesis was negatively linked to PDC activity. As a consequence, the PDK-to-PDP ratio would participate in the fatty acid reesterification pathway in adipocytes.
We have previously shown that adipocyte PEPCK-C and the whole glyceroneogenic pathway are acute targets for peroxisome proliferator–activated receptor (PPAR)γ agonists, such as thiazolidinediones, in rodents and humans (19–21). Pck1, which encodes PEPCK-C, is the earliest and strongest rosiglitazone-activated gene in adipocytes, suggesting an essential role of PEPCK-C in the response of WAT to thiazolidinediones. Such an increase in PEPCK-C precedes activation of glyceroneogenesis, which participates in the sequestration of lipids in adipose tissue observed with these compounds and thus explains, at least in part, the hypolipidemic properties of this class of drugs (22). Because the inactivation of PDC by phosphorylation could favor glyceroneogenesis, we have studied the regulation by thiazolidinediones of the expression of PDK2 and PDK4, the two isoforms of PDK expressed in adipose tissue (11).
Here, we make the original demonstration of a rapid tissue- and isoform-selective upregulation by rosiglitazone of the expression of Pdk4 in adipocytes. We also demonstrate the implication of PDK4 in the control of glyceroneogenesis, suggesting that this upregulation participates in the thiazolidinedione-induced decrease in NEFA release from WAT.
Dulbecco modified Eagle's medium (DMEM) was from Life Technologies (Cercy-Pontoise, France). Rosiglitazone was from Alexis Biochemicals (Coger, Paris). Leelamine was from Cayman Chemicals (Interchim, Montluçon, France). Small interfering RNA (siRNA) was from Invitrogen (Carlsbad, CA). Fetal bovine serum, essentially fatty acid–free BSA, 5,6-dichloro-1B-d-ribofuranosyl benzimidazole (DRB), dichloroacetate (DCA), and all other products were purchased from Sigma (L'isle d'Abeau Chesnes, France).
Male Zucker fa/fa rats were purchased from Charles River Laboratories (L'arbresle, France), and male Sprague-Dawley rats were purchased from Janvier Laboratories (Bagneux, France) at 6 weeks of age. They were allowed to acclimate 2 weeks before initiation of treatment and were kept on a 12-h light/dark cycle at constant room temperature. Conventional laboratory diet and tap water were provided ad libitum. In vivo experiments with Zucker rats were carried out as previously described (21). Briefly, rats were given a dose of 5 mg · kg−1 · day−1 rosiglitazone (maleate) or vehicle (0.5% methylcellulose) via oral gavage in the morning. Four rats were used per treatment group. After 4 days of treatment, animals were fasted during 4 h before killing. Plasma samples were obtained via the jugular vein, and aliquots were stored at −20°C for further glucose (Accu-Check; Roche, Meylan, France), insulin (Ultrasensitive rat insulin ELISA; EuroBio, Courtaboeuf, France), triglycerides (Vitros chemistry system; Johnson & Johnson), and NEFA (Fatty Acids half micro test; Roche) determinations. Samples of subcutaneous, periepididymal, and retroperitoneal WAT, liver, and soleus muscle were dissected and rapidly frozen in liquid nitrogen and stored at −80°C for further RNA analysis. The protocol for the animal studies was conducted according to the French Guidelines for the Care and Use of Experimental Animals.
Culture of human adipose tissue explants.
Explants of subcutaneous WAT were obtained from eight women undergoing elective surgery. They were aged 45.7 ± 4.4 (mean ± SE) and had BMI of 30.29 ± 1.42 kg/m2. None of the subjects suffered from known metabolic or malignant diseases or were taking medications known to alter adipocyte metabolism. The study was performed according to the Declaration of Helsinki. All the patients gave informed written consent.
WAT specimen from the subcutaneous region was obtained within 15 min after the onset of surgery. WAT (300 mg) was cut in small fragments of ∼20 mg and cultured in DMEM containing 12.5 mmol/l glucose, 200 IU/ml penicillin, and 50 mg/l streptomycin at 37°C in 10% CO2 atmosphere. After 1 h of preincubation, rosiglitazone was added for 5 h. Explants were then frozen in liquid nitrogen before RNA extraction.
Isolation of adipocytes.
Adipocytes from Sprague-Dawley rat periepididymal fat pads were isolated in DMEM containing 5 mmol/l glucose, 0.5 mmol/l pyruvate, and 2% BSA, as described by Weisberg et al. (23). The pellet containing stromavascular fraction was discarded, whereas the floating cells were collected as adipocyte-enriched fraction.
Cell culture and treatment.
3T3-F442A adipocytes were cultured at 37°C in a humidified atmosphere of 10% CO2 in DMEM containing 25 mmol/l glucose, 10% newborn calf serum, 200 IU/ml penicillin, 50 mg/l streptomycin, 8 mg/l biotin, and 4 mg/l pantothenate. At confluence, the medium was changed to DMEM supplemented with 10% FCS and 20 nmol/l insulin to promote adipogenesis. This medium was changed every 2–3 days for 6 days. Twenty-four hours before RNA extraction, cells were placed in serum-free and hormone-free medium.
RNA interference.
RNA interference with siRNA was performed by electroporation according to the manufacturer (Cell line Nucleofector kit L; Amaxa). Briefly, 3T3-F442A adipocytes on day 8 of differentiation were detached from culture dishes with trypsin-EDTA (Invitrogen). Tubes containing about 2 million cells were centrifuged for 10 min at 127g. Pelleted cells were then resuspended in 100 μl Nucleofector solution. Control (5′-GGAGGUGGUUGACUUUCAUTT; Invitrogen) or murine PDK4-specific (5′-GGAUUUGGUGGAGUUCCAUTT, Invitrogen) siRNAs were delivered into adipocytes (250 nmol/l) by electroporation. Transfection was stopped by adding DMEM containing 10% FCS. Adipocytes were then reseeded into six-well plates. Twenty-four hours after electroporation, medium was changed to FCS-free DMEM containing rosiglitazone or not. Nineteen hours later, cells were used for RNA analysis or metabolic studies.
RNA analysis.
Total RNA was extracted from the cell lines and from rat WAT by the method of Chomczynski and Sacchi (24), whereas RNeasy total RNA kit from Qiagen (Courtaboeuf, France) was used for human WAT explants. Sample quality was controlled by 260:280 nm absorption ratio determination (between 1.8 and 2.0).
For real-time RT-PCR analyses, 1.25 μg total RNA was first reverse transcribed using the High Capacity cDNA Archive kit (Applied Biosystems, Courtaboeuf, France). Reverse transcribed RNA were amplified on a thermal cycler (ABI prism 7900 HT; Applied Biosystems) using the SYBR green fluorescence method and specific oligonucleotides. Results were analyzed with the SDS 2.1 real-time detection system software. Quantification of RNA was carried out by comparison of the number of cycles required to reach reference and target threshold values (δ-δ Ct method).
Protein analysis.
Mitochondrial fraction was prepared from 3T3-F442A adipocytes, and mitochondrial pellet was resuspended in PBS 1× buffer, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS to extract mitochondrial proteins. Protein concentrations were determined using the Bradford method using BSA as the standard. Fractions (50 μg) were subjected to SDS-PAGE using a 10% resolving gel with a 6% stacking gel. Resolved proteins were transferred electrophoretically to nitrocellulose membranes, blocked for 1 h at 22°C with Tris-buffered saline (TBS) supplemented with 5% (w/v) nonfat powdered milk and incubated overnight at 4°C with polyclonal antisera raised against mouse PDK4, PDK2 from Santa Cruz Biotechnology (Tebu-bio, France), or PDC-E2 (provided by Dr. J.G. Lindsay, University of Glasgow, U.K.). Nitrocellulose membranes were then washed with 1% Tween in TBS (three times for 5 min) and incubated with horseradish peroxidase–linked secondary antibody anti-rabbit IgG (1:5,000 dilution, in 1% [w/v] BSA in TBS with 0.05% Tween) for 1 h at 22°C. Bound antibody was visualized using ECL according to the manufacturer's instructions. The blots were exposed to Hyperfilm, and the signals were quantified by scanning densitometry.
Metabolic studies.
Isolated adipocytes were submitted to incorporation assay immediately after their collection. [1-14C]pyruvate incorporation was performed with 400 μl adipocyte suspension in 2 ml Krebs buffer containing 2% fatty acid–free BSA, 0.5 mmol/l pyruvate, and [1-14C]pyruvate at isotopic dilution of 25. When indicated, PDK inhibitors were added in the incorporation medium. Untransfected or siRNA-transfected 3T3-F442A adipocytes were treated or not for 18 h with rosiglitazone. Three hours before [1-14C]pyruvate incorporation, medium was replaced with glucose-free, serum-deprived DMEM containing 0.3% free fatty acid BSA. Pyruvate incorporation was carried out with 20 μmol/l [1-14C]pyruvate in Krebs buffer containing 0.3% BSA and leelamine, when indicated. After a 2-h incubation, cells were rinsed in PBS and scraped in 10 mmol/l Tris-Cl, pH 7.4, containing 0.25 mol/l sucrose, 0.1 mmol/l EDTA, 0.1 mmol/l dithiothreitol, and 0.1% Triton and frozen in liquid nitrogen before lipid extraction. Lipids were extracted according to the simplified method of Bligh and Dyer (25), and the subsequent [1-14C]pyruvate incorporation was estimated by counting the radioactivity associated with the chloroformic fraction. NEFA concentration was monitored in the incubation medium. Under each conditions, cells were submitted to a test of toxicity, using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method, which did not show any drug-induced toxicity.
In vitro functional PDK assay.
To test the efficiency of leelamine, a functional PDK assay was performed using a commercially available PDC purified from pig heart and containing intrinsic PDK activity, as extensively described by Jackson et al. (26), with 800 μg/ml purified PDC.
Image quantification and data analysis.
Quantitative results of Western blotting were obtained by densitometry in ImageJ software. The nonparametric Mann-Whitney U test for pairwise comparisons was applied due to the small number of experiments. Analyses were performed using the StatView 4.01 (Abacus Concepts, Berkeley, CA) statistical package. A value of P < 0.05 was considered statistically significant.
Rosiglitazone induces Pdk4 expression in adipose tissue and in adipocytes.
We showed previously that treating male Zucker fa/fa rats with rosiglitazone for 4 days significantly lowered both plasma insulin and serum lipid levels (21). This normalization in lipidemia was linked to a large induction in WAT glyceroneogenesis, thus decreasing NEFA release. Under these conditions, the expression of one of the key genes involved in glyceroneogenesis, PEPCK-C, was stimulated two- to threefold in subcutaneous, periepididymal, and retroperitoneal WAT. A similar rosiglitazone-induced normalization in lipidemia was obtained in the present study (Table 1). We used real-time RT-PCR to analyze the effect that rosiglitazone would have on the expression of Pdk2, Pdk4, Pdp1, and Pdp2 in the same three WAT depots. PDP1 and PDP2 mRNAs remained unaffected by rosiglitazone treatment (data not shown). In contrast, rosiglitazone induced a large 3- to 4.5-fold increase in PDK4 mRNA in all depots but neither in liver nor in soleus muscle, demonstrating a tissue-specific response of this gene under this acute treatment (Fig. 1A). In contrast, Pdk2 expression remained insensitive to rosiglitazone whatever tissue, demonstrating a selective action of the drug on PDK isoforms (Fig. 1B).
To determine whether rosiglitazone acts on PDK4 gene expression through a direct effect on WAT, we treated ex vivo rats and humans WAT explants for 5 h with rosiglitazone and monitored PDK4 mRNA. Both species responded to rosiglitazone by a large three- to fourfold increase in Pdk4 expression, whereas mRNA concentrations of PDK2 and pyruvate carboxylase (PC) were unaffected (Fig. 2A and B). WAT is a heterogeneous tissue containing not only adipocytes but also many other cell types, including preadipocytes, macrophages, fibroblasts, and endothelial cells. To study the mechanism of rosiglitazone action on Pdk4 gene expression in a homogeneous source of adipocytes, we used the 3T3-F442A adipocyte cell line. These cells were previously used to study Pdk1 regulation and glyceroneogenesis by PPARγ activators (27). A 2-h treatment of differentiated 3T3-F442A adipocytes with rosiglitazone induced PDK4 mRNA 2.5-fold whereas the concentrations of PDK2 and PC mRNAs were unchanged (Fig. 2C). We used Western blot experiments with specific antibodies for either PDK2 or PDK4 to monitor the relative expression of both protein isoforms in 3T3-F442A adipocytes at day 6 after confluence. Taking PDC-E2 as a control, results strongly suggest that PDK4 protein amount is about threefold that of PDK2 in adipocytes (Fig. 2D).
Rosiglitazone directly increases Pdk4 transcription and protein abundance in 3T3-F442A adipocytes.
Rosiglitazone induction in Pdk4 expression was acute, with a maximum attained at 2 h in 3T3-F442A adipocytes (Fig. 3A). We chose this 2-h treatment to determine the rosiglitazone concentration that produced the half-maximal effect (EC50). Incubation of 3T3-F442A adipocytes with rosiglitazone caused a dose-dependent increase in the abundance of PDK4 mRNA (Fig. 3B). EC50 was at about 1 nmol/l, in agreement with the affinity of rosiglitazone for PPARγ, strongly suggesting that this receptor was involved. The rosiglitazone-induced increase in PDK4 mRNA could be the result of stimulation of Pdk4 transcription and/or of mRNA stability. To address these issues, we used DRB, a transcription inhibitor, and puromycin, a protein synthesis inhibitor. 3T3-F442A adipocytes were first treated with 1 μmol/l rosiglitazone with or without 80 μmol/l DRB for 0.5–2 h before analyzing PDK4 mRNA abundance by real-time RT-PCR. Whatever the time of treatment, DRB abolished the induction of PDK4 mRNA by rosiglitazone (Fig. 3A). Under the same conditions of rosiglitazone treatment, puromycin had no effect, demonstrating that de novo protein synthesis was not required (data not shown). These results are strongly in favor of rosiglitazone directly increasing transcription rate of Pdk4. Second, we examined the possibility of a rosiglitazone-induced PDK4 mRNA stabilization by incubating 3T3-F442A adipocytes for 2 h with rosiglitazone and then for various times with DRB before monitoring PDK4 mRNA abundance by real-time RT-PCR. In the absence of rosiglitazone, PDK4 mRNA half-life was estimated at ∼1.5 h as previously obtained in other cell types (Fig. 3C). After a 2-h treatment with 1 μmol/l rosiglitazone, PDK4 mRNA half-life rose to ∼2 h, showing a slight rosiglitazone-induced stabilization of PDK4 messenger in adipocytes. mRNA induction was followed by a significant twofold increase in PDK4 protein at 18 h of rosiglitazone treatment (Fig. 3D).
Contribution of PDK4 to fatty acid reesterification in basal and rosiglitazone-treated adipocytes.
To test the potential involvement of PDC and PDK in the control of pyruvate flux toward glyceroneogenesis, pyruvate incorporation into neutral lipids was monitored in isolated adipocytes in the presence or not of PDK inhibitors. We tested both DCA, one of the few known highly specific synthetic PDK inhibitor, and leelamine, a more recent PDK inhibitor (28,29). Results of MTT test demonstrated that neither of these compounds was cytotoxic at the concentrations used (data not shown). Figure 4A shows that both inhibitors significantly reduced glyceroneogenic flux in isolated adipocytes from rat WAT. However, the concentrations used to reach a statistical significant reduction differ for the two inhibitors. At 50 μmol/l, leelamine inhibited pyruvate incorporation by 40% (P < 0.001), whereas a 10-fold higher DCA concentration was required to produce a significant 60% inhibition (P < 0.01) (Fig. 4A). Hence, leelamine is more efficient to reduce pyruvate incorporation into triglycerides than DCA. To ensure that leelamine effect was due to PDK inhibition, an in vitro assay was performed, monitoring residual PDC activity after kinase action. At the concentration of 50 μmol/l, leelamine induced an inhibition of 41.00 ± 2.14% in PDK activity (n = 8, P < 0.001).
To further study the functional involvement of PDK in both basal and rosiglitazone-activated conditions, we treated or not 3T3-F442A adipocytes with 1 μmol/l rosiglitazone for 18 h and then monitored the incorporation of [1-14C]pyruvate into lipids in the presence or not of leelamine for 2 h. The addition of 50 and 100 μmol/l leelamine reduced basal glyceroneogenic flux 25 and 56%, respectively, with a concomitant increase in NEFA release (Fig. 4B and C). As expected, rosiglitazone induced glyceroneogenesis and reduced NEFA release. The addition of 50 and 100 μmol/l leelamine on rosiglitazone-pretreated cells reduced glyceroneogenic flux 59 and 115%, respectively, with a concomitant increase in NEFA release (Fig. 4B and C). Under these conditions, glycerol release was not modified (data not shown). Hence, PDK inhibitors did not affect lipolysis. To ascertain the specific implication of PDK4 in glyceroneogenesis, a selective PDK4 siRNA was transfected in 3T3-F442A adipocytes. Forty-eight hours after transfection, a specific 60% (P < 0.001) decrease in PDK4 mRNA was observed whereas concentrations in PDK2, PDP1, PDP2, PC, and PEPCK-C mRNAs remained unchanged (Fig. 5A). Furthermore, PDK4 siRNA did not induce interferon (IFN)-β mRNA, showing the lack of IFN response (Fig. 5A). Incorporation of [1-14C]pyruvate into lipids was reduced 40% after transfection of adipocytes with PDK4 siRNA (P < 0.05) (Fig. 5B). Under these experimental conditions, rosiglitazone induced [1-14C]pyruvate incorporation 3.1-fold in control cells (P < 0.001) (Fig. 5B). A 35% decrease in rosiglitazone-induced [1-14C]pyruvate incorporation was observed in the presence of PDK4 siRNA (P < 0.01).
For a better understanding of the early mechanisms involved in PPARγ effects in WAT, we investigated the regulation of potent primary targets for thiazolidinediones. One important observation of our present report is that the expression of Pdk4 is acutely induced by the specific PPARγ agonist rosiglitazone in WAT explants from rats and humans. Induction of PDK4 mRNA occurs in the same time frame as that of PEPCK-C transcript. Furthermore, as we observed previously for PEPCK-C, a 4-day administration of rosiglitazone to Zucker fa/fa rats, a model of insulin resistance and dyslipidemia, induced an increase in Pdk4 expression specifically in WAT from several depots. A longer term in vivo effect of COOH, a PPARγ agonist, on PDK4 mRNA was observed by Laplante et al. (30). Our results are in accordance with these previous results in which, however, the issue of tissue-specific action of the drug was not addressed. In the course of our experiments, we showed that rosiglitazone induction of Pdk4 expression was tissue-specific because liver and muscle did not respond to such a treatment. Furthermore, in our study, Pdk2 expression remained unaffected by rosiglitazone treatment, whereas Laplante et al. (30) observed a simultaneous induction of both Pdk2 and Pdk4 expression in visceral WAT, probably because of the longer duration of treatment used (3 weeks). In contrast to our results, Way et al. (31) observed that short-term (1-day) treatment with GW1929, a different PPARγ agonist, reduced the concentration of PDK4 transcript in muscles from ZDF Zucker diabetic rats (31). One explanation for this discrepancy could be that ZDF rats are insulin-resistant and diabetic at earlier times than the Zucker fa/fa rats we used here. Hence when ZDF rats are 9 weeks old, their insulin-resistant state is correlated with low PDK4 activity in muscle, which could be raised by rosiglitazone. Such an effect is probably indirect as the result of thiazolidinedione-induced rapid decrease in blood NEFA. In 8-week-old fa/fa rats, blood NEFA concentrations are near to normal and therefore unable to stimulate PDK4 in muscle (21). Whatever happens in muscle, our data clearly demonstrate that PDK4 is an early-responsive positive target for PPARγ in WAT.
To further study the mechanisms by which rosiglitazone stimulates Pdk4 expression, we used the 3T3-F442A cell line. Rosiglitazone rapidly stimulated Pdk4 expression in fully differentiated 3T3-F442A adipocytes as previously found in 7,800 C1 hepatoma cells treated with a PPARα ligand (32). Using transcription and translation inhibitors, we showed that PDK4 was a transcriptional and direct target for rosiglitazone. Our results are in slight contrast with those from Sears et al. (33) who showed that in 3T3-L1 adipocytes, rosiglitazone induction of PDK4 mRNA is delayed, suggesting that in these cells, PDK4 is an indirect PPARγ target. Furthermore, we found that half-life of the PDK4 transcript was short, i.e., ∼2 h, as previously observed for hepatoma cells (32). Interestingly, PC gene expression was not modulated. PC was previously shown as a PPARγ-induced gene during adipocyte differentiation of the 3T3-L1 preadipocytes (34). Our observation that rosiglitazone does not modulate pyruvate carboxylase gene expression in differentiated 3T3-F442A adipocytes is in agreement with a delayed action of PPARγ agonist on this gene during adipocyte differentiation.
We show unambiguously here that PDK4 participates in adipocyte glyceroneogenesis because leelamine, a PDK inhibitor, and a specific PDK4 siRNA strongly reduces basal and rosiglitazone-induced pyruvate incorporation into neutral lipids, with a coordinated stimulation of NEFA release. These results are illustrated in Fig. 6 in which pyruvate crossroad is shown as the central switch for the modulation of G3P production. We postulate that the circulating substrate for the whole process is lactate, which is abundant in the blood under the physiological situation during which lipolysis is activated, i.e., fasting. Rosiglitazone induction of both PDK4 and PEPCK-C results, respectively, in reduction of pyruvate flux toward acetyl-CoA because PDC activity is strongly inhibited by PDK4 and in increased G3P synthesis via PEPCK-C. Such a combined action of thiazolidinediones allows increased reesterification during lipolysis leading to decreased NEFA release from adipocytes. The reduced PDC activity under rosiglitazone treatment raises the intriguing possibility that thiazolidinediones may reduce glucose oxidation to CO2 in adipocytes, in contrast with what occurs in muscle and at the whole-body level. Although this issue requires further development, we can emphasize here that glucose oxidation is not a predominant pathway during fasting and in the absence of insulin.
Elevated concentration of plasma NEFA is now recognized as a key factor in the onset of insulin resistance and type 2 diabetes (35). Our previous results indicate that WAT glyceroneogenesis is important to lipid homeostasis and that a disregulation in this pathway has profound pathophysiological effects (19–21,36). This would result in dramatic changes in net NEFA output, hence in the concentration of plasma NEFA, which in turn would mediate metabolic alterations, for instance by affecting insulin sensitivity of peripheral tissues. Here, we provide clear evidence that PDK4 is a new player in the glyceroneogenic process and is a thiazolidinedione target selectively in WAT. Therefore, by means of PEPCK-C and of PDK4, glyceroneogenesis is a target for thiazolidinediones, the antidiabetic action of which seems clearly to be linked to their hypolipidemic effect (22,37).
Rosiglitazone induces PDK4 gene expression in fa/fa rat adipose tissue in vivo. Male Zucker fa/fa rats were treated for 4 days with rosiglitazone (5 mg · kg−1 · day −1). Rats were killed, and tissues were removed. Total RNA was extracted from subcutaneous, periepididymal, and retroperitoneal WAT, liver, and soleus muscle. PDK4 (A) and PDK2 (B) mRNA were analyzed by real-time RT-PCR. Values were normalized to 18S rRNA and expressed as percentage of untreated rats. Results are mean ± SE from four rats in each group. *P < 0.05; **P < 0.01; ***P < 0.001.
Rosiglitazone induces Pdk4 expression in rat and human adipose tissue ex vivo and in 3T3–F442A adipocytes. Rat periepididymal (A) and human subcutaneous abdominal WAT (B) were cultured in DMEM containing 12.5 mmol/l glucose and treated or not with 1 μmol/l rosiglitazone for 5 h. C: 3T3–F442A adipocytes at day 6 of differentiation were cultured in DMEM containing 25 mmol/l glucose and treated or not for 2 h with 1 μmol/l rosiglitazone. Total RNA was extracted, and PDK4, PDK2, and PC mRNA were analyzed by real-time RT-PCR with normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. D: Protein extracts were obtained from differentiated 3T3–F442A adipocytes and subjected to Western blotting using antibodies against PDK4, PDK2, and the E2 subunit of PDC complex. Data are means ± SE from three to five independent experiments, each performed in triplicate, and are expressed as percent of control. **P < 0.01.
Mechanism of rosiglitazone stimulation of Pdk4 expression in 3T3–F442A adipocytes. Quantitative changes in the abundance of PDK4 mRNA in 3T3–F442A adipocytes either cultured for the indicated times with 1 μmol/l rosiglitazone in the absence or in the presence of 80 μmol/l DRB (A) or treated for 2 h with the indicated concentrations of rosiglitazone (B). In C, 3T3–F442A adipocytes were treated or not with 1 μmol/l rosiglitazone for 2 h before DRB addition, and then mRNA for PDK4 was estimated over time. Values were normalized to GAPDH and are expressed as percentage of control. In D, 40 μg mitochondrial proteins from adipocytes treated or not with 1 μmol/l rosiglitazone for 18 h were subjected to Western blotting. Densitometry values were normalized to PDC-E2 and expressed as percentage of control. Results are means ± SE of three to five independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.
PDK4 inhibition alters basal and rosiglitazone-activated glyceroneogenesis. A: Using [1-14C]pyruvate incorporation into neutral lipids, glyceroneogenic flux was monitored in isolated adipocytes from rat periepididymal fat pads for 2 h in the presence or not of PDK inhibitors, leelamine, or DCA at the indicated concentrations. 3T3–F442A adipocytes were treated or not with 1 μmol/l rosiglitazone for 18 h, and then glyceroneogenic flux (B) and medium NEFA concentration (C) were monitored in the presence or not of leelamine for 2 h. Results are means ± SE of three independent experiments, each performed in triplicate. **P < 0.01; ***P < 0.001 vs. control; §§§ P < 0.001 vs. rosiglitazone treatment.
Influence of PDK4 siRNA on gene expression and glyceroneogenesis in 3T3–F442A adipocytes. 3T3–F442A adipocytes were transfected with a control or a PDK4-specific siRNA. Forty-eight hours after transfection, cells were used for mRNA studies (A) or [1-14C]pyruvate incorporation experiments (B). A: Total RNA was extracted, and concentration of PDK4, PDK2, PDP1, PDP2, pyruvate carboxylase, PEPCK-C, and IFN-β mRNA was analyzed by real-time RT-PCR with normalization to 18S rRNA. Control or PDK4 siRNA-transfected adipocytes were treated or not with 0.1 μmol/l rosiglitazone for 18 h, and glyceroneogenic flux was monitored. Results are means ± SE of three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01; ***P < 0.001 vs. control; §§P < 0.01 vs. rosiglitazone treatment.
Pyruvate routing for fatty acid reesterification: influence of thiazolidinediones. Pyruvate is either decarboxylated by PDC into acetyl-CoA or carboxylated to oxaloacetate (OA) by PC in mitochondria. Oxaloacetate is the precursor of G3P, and PEPCK-C is involved in this process, allowing for reesterification into triglyceride (TG) of NEFA arising from lipolysis. This could lead to decreased NEFA release in the blood flow, without affecting glycerol release. PDC is mainly regulated by a phosphorylation/dephosphorylation cycle. PDK4 inactivates PDC, thereby regulating pyruvate availability for glyceroneogenesis in adipocytes. Rosiglitazone induces both PDK4 and PEPCK-C, thereby reducing pyruvate flux toward acetyl-CoA and favoring G3P production.
Effect of rosiglitazone treatment on metabolic parameters in male Zucker fa/fa rats
Control .
Rosiglitazone .
Body wt (g) 323 ± 14 324 ± 10
Glucose (mg/dl) 120 ± 21 153 ± 35
Insulin (ng/dl) 8.8 ± 0.6 2.7 ± 0.4*
NEFA (μmol/l) 180 ± 70 60 ± 30†
Triglycerides (mg/dl) 145 ± 21.9 87.9 ± 14.5†
Data are means ± SE (n = 4 for each group). Eight-week-old male Zucker fa/fa rats were or were not treated with rosiglitazone (5 mg · kg−1 · day−1) for 4 days. Blood samples were collected for analyses.
P < 0.01,
P < 0.05.
Published ahead of print at http://diabetes.diabetesjournals.org on 2 June 2008.
T.C. and E.D. contributed equally to this work.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
T.C. has received a fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. E.D. has received a fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. J.-M.B. has received a fellowship from the Ligue Nationale contre le Cancer. C.F. has received support from the Institut National de la Recherche Médicale and the University Paris Descartes and a grant from the Association de Langue Française pour l'Etude du Diabète et des Maladies métaboliques ALFEDIAM-GlaxoSmithKline.
We thank P. Juraver (Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche-S 747) for rat handling, Dr. V. Nivet (University Paris Descartes) for triglyceride determination, and Dr. L. Benelli (Plastic Surgery Unit, Hartman Clinic, Paris) for his helpful involvement with human studies.
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redpajama_set_name: RedPajamaCommonCrawl


text:
Why Do Mexico's Football Fans Keep Going Unpunished For Shouting Homophobic Slurs At Opposing Players
BY Justin Lessner | June 19, 2019 AT 9:56 am
By Justin Lessner June 19, 2019 at 9:56 am
angelo_marino / Instagram
If you've ever been to a sports match, it's pretty likely you'll encounter some serious macho behavior from fans and players alike.
Things are typically no different at soccer games – even world-class events like the World Cup or the Copa Americana – where homophobic slurs are often used to taunt opposing teams.
Unfortunately, that was again the case at this weekend's Copa Ora game between Mexico and Cuba.
Credit: @FCYahoo / Twitter
It's an all too familiar scene at soccer matches, but espeically at those where Mexican futbol fans go. It starts with the building chant of "Eeeehhhhhh" and ends with the homophobic punctuation of "puto."
On Saturday both the Rose Bowl and Concacaf allowed the anti-gay and homophobic "puto" chant to descend on the crowd as many as 50 times, according to video and people on Twitter.
The response by both Concacaf and the stadium to try to stop the chant were designed to fail and simply pay lip service to people trying to stop the discriminatory behavior.
The chant during Mexico's 7-0 routing of Cuba was widespread, very loud and involved a huge number of people in the stands.
For many fans it was alarming.
I've seen reports that the anti-gay "puto" chant was heard 30-50 times during the #GoldCup2019 match at the Rose Bowl tonight. Who can confirm?
— Cyd Zeigler (@CydZeigler) June 16, 2019
Here, on international television and in front of some 65,000 people, was an anti-gay slur, the Spanish equivalent of "faggot," being hurled in unison by hundreds of fans at an opposing goalkeeper.
Yet in many ways, it was routine and perhaps even easy to ignore.
After all, authorities did just that for years, as the taunt spread from Mexican club games to the national team last decade. It soon became a community staple, a rite of passage for match-going fans, a so-called tradition that follows the Mexican team almost everywhere it goes.
Just last year at the World Cup in Russia, global soccer's governing body opened disciplinary proceedings after the chant accompanied German goal kicks during Mexico's World Cup opener. FIFA not only fined the Mexican federation but threatened more sanctions. It also promised to revoke Fan IDs – essentially tickets – and eject anybody caught yelling the slur.
The "puto" chant is anti-gay and is designed to belittle opposing male players by likening them to prostitutes and women.
The chant is, at its core, indisputably anti-gay and FIFA itself has labeled the chant discriminatory.
Yet soccer organizations allow the chant to persist, negatively affecting LGBTQ fans, players and coaches.
Some on Twitter didn't mince words in their reaction to Mexico's fans.
Credit: @elizabettaReyes / Twitter
A lot of fans took to Twitter to express their anger and embarrassment at their own team for continuing to act in such a hurtful and childish way.
While a very small minority to continue to claim that the word has zero homophobic meaning.
While some pointed out the inconsistency between different leagues.
Credit: @windsof_madness / Twitter
Because apparently any homophobic slurs are completely banned at Los Angeles Football Club matches but the Rose Bowl Stadium doesn't have a similar ban.
One Twitter user even tweeted a potential solution – at least for those watching the game on TV.
Credit: @pedroheizer / Twitter
I mean muting the very moment when the chant is about to happen is one way to shield viewers from the blatant homophobia…
Or, I mean, fans could just stop.
Homophobia Mexico soccer futbol
Florida Republicans Just Introduced Four Anti-LGBTQ Bills On The Last Day Possible And People Are Asking Why Now?
By Emerald Pellot January 16, 2020 at 1:56 pm
BY Emerald Pellot | January 16, 2020 AT 1:56 pm
Ben Wassenhatch / Flickr
Seven Republican lawmakers in Florida filed four anti-LGBTQ bills hours before the deadline for the upcoming legislative session this week. The bills undo many of the protections that exist for LGBTQ Florida residents. The four bills would repeal county and municipal ordinances for LGBTQ workers, legalize gay conversion therapy, and ban transgender healthcare for children, according to NBC News.
The homophobic and transphobic legislation was introduced by representatives Anthony Sabatini, Bob Rommel, Michael Grant, and Byron Donalds, along with Senators Joe Gruters and Keith Perry. Florida Rep. Shevrin Jones, who is a member of the LGBTQ community, and other advocates are now fighting against the bills' passing.
Advocates respond to the bill calling it "discrimination and hate."
Legislators who file anti-LGBT bills – especially ones that greenlight discrimination – don't get to act shocked when guys like Mike Hill laugh at constituents who want to kill gay people.https://t.co/FYit1Sa92F
— Scott Maxwell (@Scott_Maxwell) January 16, 2020
"Clearly they've decided that discrimination and hate are central to their election-year platform despite our state's incredible diversity," Jones said in a statement. "Just as I've done since I was elected in 2012, I will continue to fight any legislation that marginalizes or threatens any Floridian's shot at a secure, safe, and bright quality of life."
Jones also accused Florida Republicans of, "wasting tax dollars attacking Florida's most vulnerable communities rather than prioritizing the issues that impact everyday people's lives."
Equality Florida released a statement highlighting many of the consequences such a bill would have.
"This is the most overtly anti-LGBTQ agenda from the Florida Legislature in recent memory," Jon Harris Maurer, the group's public policy director, said. "It runs the gamut from openly hostile legislation that would arrest and imprison doctors for providing medically necessary care, to legislation that would carelessly erase critical local LGBTQ protections."
Senator Gruters defends the bill, claiming it "includes protections."
I'm dissapointed to see some of my colleagues proposing this regressive and discriminatory anti-LGBT agenda.
What benefit to the public does this legislation actually serve, apart from tossing red meat to a political base in an election season?… https://t.co/0K26noGnul
— Senator Lori Berman (@loriberman) January 16, 2020
"The bill certainly does not authorize an employer to discriminate against employees who are members of protected classes, whether protected by federal or state law or local ordinance," Gruters told NBC News via email. "While I do not believe the bill has any impact on local anti-discrimination ordinances, in an abundance of caution, I included language in the bill's preamble to make clear that the preemption would not affect local anti-discrimination laws, and any court would interpret the preemption consistent with that preamble."
While Gruters claims it would include protections, Joe Saunders, senior political director at Equality Florida, claims that these so-called protections are merely a part of the bill's preamble and would carry no weight should the bills become law.
"We appreciate that Sen. Gruters put that in," Saunders said. "It's not policy; it's not considered part of the bill."
Democratic senator Lori Berman suggested the bill was nothing more than a political stunt to garner votes from homophobic and transphobic constituents.
"I'm disappointed to see some of my colleagues proposing this regressive and discriminatory anti-LGBT agenda," Berman wrote on Twitter. "What benefit to the public does this legislation actually serve, apart from tossing red meat to a political base in an election season?"
Trans children are particularly vulnerable to the policies.
The Proteus Fund, the Ford Foundation, the Arcus Foundation and others have earmarked at least $10 million to redefine religious freedom as discriminatory and harmful when it conflicts with LGBT and pro-abortion rights priorities. #religiousFreedomDayhttps://t.co/lXnlox314x
— Kevin J. Jones (@kevinjjones) January 16, 2020
"Transgender youth are some of the most at risk in our community," Gina Duncan, Equality Florida's director of transgender equality, told NBC News. "It is outrageous that conservative legislators would threaten their health and safety. Medical professionals, not politicians, should decide what medical care is in the best interest of a patient. Forcing a doctor to deny best practice medical care and deny support to transgender youth can be life-threatening."
Making it illegal for doctors to provide necessary care to trans children certainly goes against the Hippocratic oath, but if you think something so dystopian can't happen just look at how Republicans have slowly chipped away at abortion rights – another form of necessary care that can be life-saving.
Just yesterday South Dakora Republican lawmakers introduced a bill that would also ban doctors from performing gender-affirming surgeries or treatments on children. Similar bills have been filed in Texas, Georgia, and Kentucky.
According to the Associated Press, "The Endocrine Society, which is the leading professional organization for doctors who specialize in hormones, does not recommend gender-transition medical treatment before puberty for children who do not identify with their biological gender. For youths experiencing puberty and older adolescents, the Endocrine Society recommends that a team composed of expert medical professionals and mental health professionals manages treatment."
Opponents of these anti-trans bills believe the laws interfere with the doctor-patient relationship and contradict the widely accepted practices of the medical community.
"Sadly, the medical care of transgender youth has been sensationalized and politicized," Jack Turban, a researcher at Harvard Medical School, told NBC News. "Gender-affirming medical care for transgender adolescents is endorsed by major medical organizations, including the Endocrine Society, the American Academy of Pediatrics, and the American Academy of Child & Adolescent Psychiatry. It should go without saying, but providing standard medical care should not be a felony."
HomophobiapoliticsDiscriminationFlorida
Brazil's President Told A Reporter He Had A "Homosexual's Face" At A Truly Bizarre Press Conference
By César Albarrán Torres December 30, 2019 at 9:35 am
BY César Albarrán Torres | December 30, 2019 AT 9:35 am
Palacio de Planalto / Flickr
Brazil's LGBTQ+ community has faced significant challenges in the past few years. The primarily Catholic country tends to experience high levels of homophobia based on religious dogma. Trans women in Brazil are not only discriminated against, but also harmed and murdered at dramatic rates, some of the highest in the world. Added to this, the far-right is back in power and Jair Bolsonaro, a businessman who many compare to Donald J Trump for his populist discourse and extreme conservative views, is the president.
Like Trump, Bolsonaro often blames the media for everything, Bolsonaro is particularly wary of the conglomerate Globo (like Trump is, say, of CNN). He has constantly accused the media of bias and also made derogatory comments against women and minorities. His mishandling of the Amazon fires and the imperialist approach he has to indigenous communities are also infamous.
Bolsonaro is outspoken and sometimes seems to just wing it in speeches, saying outrageous things that are both often half-truths and testament to his intransigent worldview (does it sound eerily familiar?). A recent episode evidences just how deeply flawed Bolsonaro's views on diversity are and how threatening the current sociopolitical climate is for LGBTQ+ Brazilians is.
So during a press conference Bolsonaro was asked about his son's alleged acts of corruption.
Credit: EuroNews / Giphy
Brazil, after all, is part of Latin America, a region where politicians and presidents are infamous for being either corrupt or having shady family or friends. So one would assume that the Brazilian president would know how to respond to questions about corruption among his family members. It literally is the bread and butter of Latin American politics.
Well, Bolsonaro was confronted by a reporter, who asked him about his son, Senator Flavio Bolsonaro, and claims that point to institutional corruption that leads to him.
As The Telegraph UK reports, during a heated exchange "Mr Bolsonaro accused the press of bias against him and his son, Senator Flavio Bolsonaro, who is being investigated by prosecutors in Rio de Janeiro over allegations that he hired staff with no duties while he was a state legislator."
The Senator has also been accused of hiring "ghost workers" in the past. The younger Bolsonaro has denied these allegations and conservative groups in Brazil point towards a left-wing, Lula-supporting conspiracy against the Bolsonaro administration.
So did the president keep his cool? Nah! He lashed out against the reporter saying he had "a homosexual's face". Yes, he went there! Like, WTF dude?
Think about the worst that a politician can say and you will not even be close to what Bolsonaro told the reporter. Ready? Here it goes: "Your face looks an awful lot like a homosexual's, but that's no reason to accuse you of being a homosexual."
Let's unpack this a little bit: he basically argued that there is a "homosexual" type of face (which is the first sign of discrimination, which starts with generalizations) and he implied that homosexuality is wrong, something that people can be "accused" of. His supporters and staff laughed as he said this, while the opposition took on social media to post head shots of themselves with the caption "homosexual face".
Bolsonaro is famous for his incendiary comments, of course, as The Telegraph reminds us: "Mr Bolsonaro has a history of making derogatory remarks about women, gay people and racial minorities, including on last year's campaign trail, though such comments have been less frequent since he took office at the beginning of this year." It seems like his PR team is trying to reign him in, but he sometimes goes off script and chaos follows.
But what on Earth is a "homosexual's face"? Perhaps this?
Mr Bolsonaro, Ricky Martin is definitely much hotter than you, you gotta give him that. If this is a "homosexual face" then, damn, that reporter is lucky.
What about this gorgeous queer face?
Miss Spain Patricia Yurena has come out as gay. Is this what you meant by a "homosexual's face", Mr Bolsonaro?
Or perhaps the mug of Puerto Rican boxer Orlando Cruz?
We wonder if Bolsonaro, or anyone in his circle for that matter, would dare to mock Cruz based on him being gay. He packs a mean punch! Orlando came out a few years ago and rocked a sport which is based on a very set idea of what masculinity is. The boxing community was surprisingly supportive and homophobic comments were highly criticized.
Are these faces in Sao Paolo what you would describe as "homosexual", Mr Bolsonaro?
Credit: VamosGay.com
We got a question for you, Jair. Are you as proud about yourself as these brave Brazilians? Nah, we didn't think so.
HomophobiaJair BolsonaroBrazil
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text:
May 1, 2014 at 3:19 pm EDT | by Michael K. Lavers
Rea Carey arrested during immigration protest
Rea Carey of the National Gay and Lesbian Task Force said she wants action on comprehensive immigration reform (Photo by Kathy Plate)
National Gay and Lesbian Task Force Executive Director Rea Carey is among the 27 people who were arrested outside the U.S. Capitol on Wednesday during a protest in support of a comprehensive immigration reform bill.
She and others blocked an intersection outside the U.S. House of Representatives as part of a "Stop Separating Families Action" the Fair Immigration Reform Movement organized. Carey and her fellow activists specifically urged House Speaker John Boehner (R-Ohio) to allow a vote on the measure the U.S. Senate approved last year.
"If you're not part of the solution, you're part of the problem," said Carey before her arrest. "In this instance, Speaker Boehner is the problem. His intransigence on fixing our broken immigration system is inflicting cruel and unusual punishment on millions of vulnerable undocumented immigrants and their families."
The Fair Immigration Reform Movement protest is among the events in support of the issue that have taken place in D.C. this week.
Participants who took part in a march from the National Mall to Lafayette Park that CASA de Maryland hosted called upon President Obama to stop deporting undocumented immigrants from the country. The National Council for La Raza and Causa, an Oregon immigrant advocacy group that backs marriage rights for same-sex couples and other LGBT-specific issues, are among the groups that also took part.
Immigration Equality on Tuesday hosted a press conference in front of the White House to draw attention to the issue.
Fernanda Vallejos, an undocumented transgender woman from the Honduran city of San Pedro Sula who currently lives in New York City, is among those who spoke.
Vallejos — who worked for an HIV/AIDS service organization in the Central American country before traveling to Texas where her family lives — told the Washington Blade after the Tuesday press conference that she came to the U.S. in hopes of finding "a new chance of life." She noted in Spanish that LGBT Hondurans are frequently tortured and receive death threats.
"If I go back to my country, I will return to torture or death," said Vallejos.
Fernanda Vallejos speaks outside the White House on April 29, 2014. (Washington Blade photo by Michael Key)
Vallejos last year spent two months in Immigration and Customs Enforcement detention facilities north of Houston. She told the Blade that officers made jokes about her gender identity and expression and placed her with male inmates — even though she identifies as a woman.
Vallejos urged Obama to help her and other undocumented immigrants.
"We are people in need," she said. "We cannot return to our country."
Barack ObamabisexualCASA de MarylandCausaFair Immigration Reform MovementFernanda VallejosgayHondurasImmigration EqualityJohn BoehnerlesbianNational Council for La RazaNational Gay and Lesbian Task ForceRea Careytransgender
Michael K. Lavers
Michael K. Lavers is the international news editor of the Washington Blade. Follow Michael
Baja Rat
May 2, 2014 at 3:26 pm EDT at 3:26 pm
Illegal aliens are criminals and parasites, one and all. Build a wall and deport 'em all.
Michael Petrelis
May 2, 2014 at 11:59 am EDT at 11:59 am
The task force this week posted the IRS 990 for their 501c3 foundation on their site and it showed that in 2013 Carey was compensated $260,000 for her services. She now is a member of an exclusive club of Gay Inc executives compensated more than a quarter million dollars for their services.
Today I requested that the task force also post the IRS 990s for the past three years of their 501c4 action fund arm and I hope they will provide those to the public on their web site sometime today or early next week.
As Deep Throat Mark Felt said during the Watergate scandal, follow the money. Important advice for our times.


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Home Health & Medicine
England's youth are drinking less and less — and some have never had a drink
I didn't know you were allowed to do this in the UK.
by Alexandru Micu
in Health & Medicine, News, Nutrition, Science
Image credits Kimery Davis / Flickr.A new study published by researchers from the University College London shows that the younger generations in England drink less, and in fewer numbers, than those before them. This trend, the authors note, is largely powered by people who never start drinking.
Cracking fewer cold ones with the boys
"These trends are to be welcomed from a public-health standpoint," says the study's corresponding author, Dr. Linda Ng Fat. "Factors influencing the shift away from drinking should be capitalised on going forward to ensure that healthier drinking behaviours in young people continue to be encouraged."
The team drew on data pertaining to alcohol consumption recorded as part of the annual Health Survey for England, which looks at changes in the health and lifestyles of people all over the UK. The survey was first carried out in 1991 and involve around 8,000 adults and 2,000 children each year. Information is collected through an interview and, if the participants agree, a visit from a specially trained nurse.
The team used data from 9,699 people aged 16-24 years collected between 2005-2015. They looked at the proportion of non-drinkers among social demographic and health sub-groups, along with alcohol units consumed by those that did drink and levels of binge drinking.
The data revealed that 29% of 16-24-year-olds in the UK don't drink alcohol — a significant increase from 18% back in 2005. The lion's share of this increase is represented by those who have never consumed alcohol: their ranks swelled from 9% of their age cohort in 2005 to 17% in 2015. In other happy news from the team, fewer young people are drinking above recommended limits — from 43% in 2005 to 28% — and they're less enthusiastic about binge drinking — 18% reported to binge drinking in 2015, down from 25% in 2005. Furthermore, more young people were also engaging in weekly abstinence compared to previous generations (from 35% to 50%).
"Increases in non-drinking among young people were found across a broad range of groups, including those living in northern or southern regions of England, among the white population, those in full-time education, in employment and across all social classes and healthier groups," Dr. Ng Fat explains.
"That the increase in non-drinking was found across many different groups suggests that non-drinking may becoming more mainstream among young people which could be caused by cultural factors."
Beer pressure
Young people tend to take more risks and live less healthily than older generations, but the team's results seem to hint at a cultural shift. Risky behaviors such as binge drinking "may be becoming less normalized," the authors explain, while not-drinking "maybe becoming more acceptable". This rise in non-drinking wasn't mirrored among ethnic minorities, those with poor mental health, or smokers, however — with the last point suggesting that the risky behaviors of smoking and alcohol tend to cluster together.
Still, the authors caution that the cross-sectional, observational nature of this study does not allow for conclusions about cause and effect to be drawn at this time.
The paper " Investigating the growing trend of non-drinking among young people; analysis of repeated cross-sectional surveys in England 2005–2015″ has been published in the journal BMC Public Health.
Tags: alcoholdrinkingenglanduk


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It's Been Our Honor
Wendy's Celebrates the Former High School Heisman Program
Over the past 25 years, Wendy's has had the incredible honor of getting to know many of the most esteemed high school senior athletes from all across our country.
Wendy's founder Dave Thomas created Wendy's High School Heisman scholarship in 1994. The scholarship has provided hundreds of thousands of dollars in college funding to students and more than a million dollars to high school academic and athletic programs across the country. It began as a way to identify and honor the nation's most remarkable high school seniors; students who go above and beyond on the field, in the classroom, and in their communities.
But in the end, it was the students who honored us by sharing their personal stories of triumph, opening up about the adversity they've faced, and getting personal about exactly what it is that inspires and drives them to never settle, keep digging deeper, and always reach higher.
From the very first Heisman class of winners in 1994 to the final class of 2018, the student applicants, high school administrators, teachers, coaches, parents, caregivers and more who have been a part of the program's success have afforded Wendy's the privilege to witness firsthand what a tireless commitment to excellence really looks like. We've met countless students who live and breathe the same values that fuel Wendy's success every day: a never-ending aspiration to be better along with a passionate heart for serving others.
We've celebrated the lasting impact these students make in their communities and the world, awarding hundreds of thousands of dollars in college scholarships and more than a million dollars to improve high school academic and athletic programs across the country.
Along the way, we've watched the program's participants build a legacy of inspiration that will live on as shining example for future generations of students committed to going the extra mile, putting in the additional effort, and leaping any and every hurdle in order to realize their full potential and personal bests.
The Wendy's family thanks everyone who fully supported this program and allowed us the privilege of witnessing and sharing your stories of greatness. To every honoree, and to all those who aspire to continue their legacy, Wendy's salutes you.
You've honored us.
And we are grateful to have had this opportunity to honor you.
If you have questions or comments, please call or text us at 1-888-624-8140.
Past Stories


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Unclassified Topic
The 2014 Awards Report: See the World's Most Creative Agencies, Campaigns and Clients
Who's the Winningest of Them All? Find Out in Our Yearly Ranking of Most Creatively Celebrated Marketers, Agencies and Campaigns
Published on December 30, 2014.
How restaurant chains are duking it out to be the best in their categories
Presenting the top winners of our 2014 Awards Report, which features the most creatively celebrated companies and personalities of the year. Our survey tallies the results from the nine most-respected industry awards shows: Art Directors Club, Association of Independent Commercial Producers, the Andy Awards, the Cannes International Festival of Creativity, the Clios, D&AD, The One Show, the Webby Awards and the Festival of Media.
Each award won is assigned a point value according to the weight and preciousness of the honor. Those point totals then determine the most-awarded companies, campaigns and people, across a number of categories.
See the full lists and winning work here, including the 25 most-celebrated advertisers, as well as the top ten lists of the most-awarded agencies, campaigns, creative directors, networks, directors, production companies and more.
Here's a recap of the big winners:
Agency Network
Agency Company
Art Director / Copywriter
Most Awarded Advertiser
Fueled by numerous wins across the industry shows for its "Live Test Series" campaign, Volvo knocked last year's chart-topper, Unilever, out of the #1 slot as this year's most-awarded advertiser. Unilever took a respectable second place, however, thanks to a range of wins by several brands, including Axe/Lynx, Dove and more.
Forsman & Bodenfors
Most Awarded Agency
Long a creative powerhouse, the Swedish shop rose to the top of the agency list thanks to the stellar performance of the Volvo "Live Test Series " B-to-B campaign for the brand's trucks, featuring the massive viral hit "Epic Split." But Jean-Claude Van Damme wasn't pulling all the weight. The agency also earned nods for work for Helly Hansen, UNICEF and the Volvo XC60.
Volvo "Live Test Series"
Most Awarded Campaign
While Van Damme became the face of this campaign, his "Epic Split" was just one in a series of many outrageous product demo films showcasing the features of Volvo trucks. Together, they made for the year's most awarded campaign, earning multiple Best of Show wins across the industry festivals. Beyond plaudits, the campaign set a new standard in creativity -- and entertainment -- in the business-to-business sector.
Most Awarded Agency Network
Ogilvy & Mather pulled a repeat this year, and once again nabbed the top seat of the agency network charts. Big winners for the net included Dove's "Real Beauty" campaign, work for IBM and Coca-Cola, as well as British Airways' impressive outdoor digital campaign,"Magic of Flying."
Omnicom CEO John Wren
Most Awarded Agency Company
Omnicom reclaimed the top spot on the agency company chart in 2014, after falling #2 to WPP last year. DDB's celebrated "Sorry, I Spent It on Myself" campaign, another of the year's most awarded efforts, TBWA's work for Apple including "Misunderstood" (via MAL) and "Intention" (via MAL), the GayTM campaign for ANZ Bank (via Whybin), adidas' "D Rose Jump Store" (via London) were some of the big winners that helped Omnicom nab the top spot.
David Droga
Most Awarded Chief Creative Officer
Droga5 frontman David Droga claimed the top seat of the CCO list, thanks to his agency's diverse output of work, which included Newcastle's "If We Made It" piss take on Super Bowl advertising, tech innovations Thunderclap and Birdseye, and clever integrated work for the New Museum, Prudential and more. CAA Marketing co-CCO Jesse Coulter followed in second, thanks to continued success with Chipotle on "The Scarecrow," while Droga5 New York CCO Ted Royer ranked third.
Susan Hoffman, Wieden & Kennedy
Most Awarded Creative Director
Wieden & Kennedy global ECD Susan Hoffman, the most awarded creative director in 2012, was back on top of the CD chart this year, propelled by more excellent work for Nike, Old Spice , P&G and Dodge. Fellow ECD Joe Staples took number #2, followed by CAA Marketing's Angie Sun, Todd Hunter and Tony Fur in the #3 slot.
Anders Eklind, Sophia Lindholm, Bjorn Engstrom and Martin Ringqvist
Most Awarded Art Directors, Most Awarded Copywriters
Art directors Anders Eklind and Sophia Lindholm and copywriters Bjorn Engstrom and Martin Ringqvist took the top slots on their respective charts as the creatives behind Volvo's "Live Test Series" campaign. For two years, the team worked hand-in-hand with the client, along with Volvo's engineers, to come up with the over-the-top demo films touting the merits of the automaker's trucks. Unlike a lot of branded films we see these days, what made the films special was that, as Mr. Engstrom told Ad Age, "They're not stunts, we're demonstrating the products in a relevant way."
Andreas Nilsson
Most Awarded Director
Volvo helped make a chart-topper in the director category too, sending Andreas Nilsson to #1 for his work on "Epic Split." He was followed by Limbert Fabian and Brandon Oldenburg, the team from Moonbot Studios who directed Chipotle's "The Scarecrow."
Folke Film
Most Awarded Production Company
Folke Film, the Swedish production company that repped director Andreas Nilsson during production of Volvo's "Epic Split," surpassed big guns MJZ and Smuggler to claim the top spot this year. Last year's #1 company, MJZ, dropped to second, with wins for 10+ different clients, including Old Spice, Guinness, Nike, DirecTV, Carlsberg, Heineken, Oreo and others, while Smuggler took third thanks to its work on Newcastle's "If We Made It," Volvo's "Live Test Series" and more.
The 2014 Awards Report may not work on your platform. Please view it on AdAge.com
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Cannes Lions Jury President: Branded Content is Permeating Advertising
If I knew then what I know now ... Confessions of a self-diagnosed perfectionist
Marketer A-List 2018: Coca-Cola
Nike is Ad Age's Marketer of the Year for 2018
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This 1899 patent features a pear-shape counterweight which holds the Christmas tree candle holder and candle in place. From our collection of Christmas Tree Candle Holder Patents. Credit: www.christmasgiftsfromgermany.com.