CCAAT/enhancer-binding protein beta (C/EBPß) knockdown reduces inflammation, ER stress, and apoptosis, and promotes autophagy in oxLDL-treated RAW264.7 macrophage cells.

Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPß) is a transcription factor, and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPß regulates inflammation, endoplasmic reticulum (ER) stress, and apoptosis, in macrophage foam cells and its underlying molecular mechanism remains unknown. Here, we investigated the effect of C/EBPß knockdown on proteins and genes implicated in inflammation, ER stress, apoptosis, and autophagy in macrophage foam cells. RAW264.7 macrophage cells were transfected with control and C/EBPß-siRNA and then treated with nLDL and oxLDL. Key proteins and genes involved in inflammation, ER stress, apoptosis, and autophagy were analyzed by western blot and qPCR. We found that short interfering RNA (siRNA)-mediated knockdown of C/EBPß attenuated atherogenic lipid-mediated induction of proteins and genes implicated in inflammation (P-NFkB-p65, NFkB-p65, and TNFα), ER stress (ATF4 and ATF6), and apoptosis (CHOP, caspase 1, 3, and 12). Interestingly, C/EBPß knockdown upregulated the expression of autophagy proteins (LC3A/B-II, ATG5) and genes (LC3B, ATG5) but decreased the mammalian target of rapamycin (mTOR) protein phosphorylation and mTORC1 gene expression in oxLDL-loaded RAW264.7 macrophage cells. More importantly, treatment with rapamycin (inhibitor of mTOR) increased expression of proteins implicated in autophagy and cholesterol efflux in oxLDL-loaded RAW 264.7 macrophage cells. The present results suggest that C/EBPß inactivation regulates macrophage foam cell formation in atherogenesis by reducing inflammation, ER stress, and apoptosis and by promoting autophagy and inactivating mTOR.

Lipolytic Effects of 3-Iodothyronamine (T1AM) and a Novel Thyronamine-Like Analog SG-2 through the AMPK Pathway.

3-Iodothyronamine (T1AM) and its synthetic analog SG-2 are rapidly emerging as promising drivers of cellular metabolic reprogramming. Our recent research indicates that in obese mice a sub-chronic low dose T1AM treatment increased lipolysis, associated with significant weight loss independent of food consumption. The specific cellular mechanism of T1AM's lipolytic effect and its site of action remains unknown. First, to study the mechanism used by T1AM to gain entry into cells, we synthesized a fluoro-labeled version of T1AM (FL-T1AM) by conjugating it to rhodamine (TRITC) and analyzed its cellular uptake and localization in 3T3-L1 mouse adipocytes. Cell imaging using confocal microscopy revealed a rapid intercellular uptake of FL-T1AM into mitochondria without localization to the lipid droplet or nucleus of mature adipocytes. Treatment of 3T3-L1 adipocytes with T1AM and SG-2 resulted in decreased lipid accumulation, the latter showing a significantly higher potency than T1AM (10 µM vs. 20 µM, respectively). We further examined the effects of T1AM and SG-2 on liver HepG2 cells. A significant decrease in lipid accumulation was observed in HepG2 cells treated with T1AM or SG-2, due to increased lipolytic activity. This was confirmed by accumulation of glycerol in the culture media and through activation of the AMPK/ACC signaling pathways.

Carbohydrate consumption and variable-intensity exercise responses in boys and men.

PURPOSE: The effect of carbohydrate (CHO) supplementation on physiological and perceptual responses to steady-state exercise has been studied in children. However, little is known about these responses to variable-intensity exercise (VIE) and how these responses might differ from adults. This study examined the physiological and perceptual effects of CHO on VIE in boys and men. METHODS: Eight boys (11.1 ± 0.9 years) and 11 men (23.8 ± 2.1 years) consumed CHO or a placebo (PL) beverage before and throughout VIE (three 12-min cycling bouts with intensity varying every 20-30 s between 25, 50, 75, and 125% peak work rate). Pulmonary gas exchange was assessed during the second 12-min bout. RPE was assessed twice per bout. RESULTS: In CHO, blood glucose increased and then decreased more from pre-exercise to 12 min and was higher in this trial at the end of exercise in men versus boys. In boys, blood glucose in CHO was higher at 24 and 36 min of exercise than in PL. RER during the CHO trial was higher in both groups; the other physiological responses were unaffected by CHO. All RPE measures (whole body, legs and chest) increased over time, but were not different between groups or trials. CONCLUSION: Blood glucose patterns during VIE were differentially affected by CHO in boys and men, but most physiological and perceptual responses to VIE were unaffected by CHO in either group. Knowledge of the underlying mechanisms of glucose regulation and effects on physical performance during this type of exercise in children is warranted.

Uptake of 3-iodothyronamine hormone analogs inhibits the growth and viability of cancer cells.

3-Iodothyronamine (T1AM) is a structural analog of thyroid hormone that has been demonstrated to have potent affects on numerous physiological systems. Most studies on T1AM have explored its effects in healthy functional systems; while its potential therapeutic uses and safety, and efficacy in pathological conditions are largely unknown. We sought to evaluate the effects of T1AM and its structural analog SG-2 on cancer cell growth and viability. We analyzed the cytotoxicity of these analogs on MCF7 breast cancer cells, HepG2 hepatocellular cancer cells as well as normal control cells using primary human foreskin fibroblasts and mouse preadipocytes control cells. The cytotoxicity of T1AM and SG-2 was determined by cell growth curves, and validated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assays. Cellular uptake analysis was conducted using confocal microscopy. Real-time (RT)-PCR was conducted to identify gene pathways affected by SG-2 in cancer cells. The IC 50 of T1AM was approximately double the concentration of its analog SG-2 in cancer cells. Cytotoxicity studies on normal cells revealed that IC 50 concentrations of SG-2 in cancer cells had no significant impact on cell viability in these cell types. Cell-imaging experiments demonstrated rapid uptake and localization to the mitochondrial membrane. T1AM and SG-2 are able to reduce cancer cell growth and viability. These findings support the potential for use of these compounds and related analogs for their antiproliferation properties in cancer cells.

Desmin loss and mitochondrial damage precede left ventricular systolic failure in volume overload heart failure.

Heart failure due to chronic volume overload (VO) in rats and humans is characterized by disorganization of the cardiomyocyte desmin/mitochondrial network. Here, we tested the hypothesis that desmin breakdown is an early and continuous process throughout VO. Male Sprague-Dawley rats had aortocaval fistula (ACF) or sham surgery and were examined 24 h and 4 and 12 wk later. Desmin/mitochondrial ultrastructure was examined by transmission electron microscopy (TEM) and immunohistochemistry (IHC). Protein and kinome analysis were performed in isolated cardiomyocytes, and desmin cleavage was assessed by mass spectrometry in left ventricular (LV) tissue. Echocardiography demonstrated a 40% decrease in the LV mass-to-volume ratio with spherical remodeling at 4 wk with ACF and LV systolic dysfunction at 12 wk. Starting at 24 h and continuing to 4 and 12 wk, with ACF there is TEM evidence of extensive mitochondrial clustering, IHC evidence of disorganization associated with desmin breakdown, and desmin protein cleavage verified by Western blot analysis and mass spectrometry. IHC results revealed that ACF cardiomyocytes at 4 and 12 wk had perinuclear translocation of αB-crystallin from the Z disk with increased α, ß-unsaturated aldehyde 4-hydroxynonelal. Use of protein markers with verification by TUNEL staining and kinome analysis revealed an absence of cardiomyocyte apoptosis at 4 and 12 wk of ACF. Significant increases in protein indicators of mitophagy were countered by a sixfold increase in p62/sequestosome-1, which is indicative of an inability to complete autophagy. An early and continuous disruption of the desmin/mitochondrial architecture, accompanied by oxidative stress and inhibition of apoptosis and mitophagy, suggests its causal role in LV dilatation and systolic dysfunction in VO.NEW & NOTEWORTHY This study provides new evidence of early onset (24 h) and continuous (4-12 wk) desmin misarrangement and disruption of the normal sarcomeric and mitochondrial architecture throughout the progression of volume overload heart failure, suggesting a causal link between desmin cleavage and mitochondrial disorganization and damage.

Metabolic profiling reveals reprogramming of lipid metabolic pathways in treatment of polycystic ovary syndrome with 3-iodothyronamine.

Complex diseases such as polycystic ovary syndrome (PCOS) are associated with intricate pathophysiological, hormonal, and metabolic feedbacks that make their early diagnosis challenging, thus increasing the prevalence risks for obesity, cardiovascular, and fatty liver diseases. To explore the crosstalk between endocrine and lipid metabolic pathways, we administered 3-iodothyronamine (T1AM), a natural analog of thyroid hormone, in a mouse model of PCOS and analyzed plasma and tissue extracts using multidisciplinary omics and biochemical approaches. T1AM administration induces a profound tissue-specific antilipogenic effect in liver and muscle by lowering gene expression of key regulators of lipid metabolism, PTP1B and PLIN2, significantly increasing metabolites (glucogenic, amino acids, carnitine, and citrate) levels, while enhancing protection against oxidative stress. In contrast, T1AM has an opposing effect on the regulation of estrogenic pathways in the ovary by upregulating STAR, CYP11A1, and CYP17A1. Biochemical measurements provide further evidence of significant reduction in liver cholesterol and triglycerides in post-T1AM treatment. Our results shed light onto tissue-specific metabolic vs. hormonal pathway interactions, thus illuminating the intricacies within the pathophysiology of PCOS This study opens up new avenues to design drugs for targeted therapeutics to improve quality of life in complex metabolic diseases.

SCD1 activity in muscle increases triglyceride PUFA content, exercise capacity, and PPARδ expression in mice.

Stearoyl-CoA desaturase (SCD)1 converts saturated fatty acids into monounsaturated fatty acids. Using muscle overexpression, we sought to determine the role of SCD1 expression in glucose and lipid metabolism and its effects on exercise capacity in mice. Wild-type C57Bl/6 (WT) and SCD1 muscle transgenic (SCD1-Tg) mice were generated, and expression of the SCD1 transgene was restricted to skeletal muscle. SCD1 overexpression was associated with increased triglyceride (TG) content. The fatty acid composition of the muscle revealed a significant increase in polyunsaturated fatty acid (PUFA) content of TG, including linoleate (18:2n6). Untrained SCD1-Tg mice also displayed significantly increased treadmill exercise capacity (WT = 6.6 ± 3 min, Tg = 71.9 ± 9.5 min; P = 0.0009). SCD1-Tg mice had decreased fasting plasma glucose, glucose transporter (GLUT)1 mRNA, fatty acid oxidation, mitochondrial content, and increased peroxisome proliferator-activated receptor (PPAR)δ and Pgc-1 protein expression in skeletal muscle. In vitro studies in C2C12 myocytes revealed that linoleate (18:2n6) and not oleate (18:1n9) caused a 3-fold increase in PPARδ and a 9-fold increase in CPT-1b with a subsequent increase in fat oxidation. The present model suggests that increasing delta-9 desaturase activity of muscle increases metabolic function, exercise capacity, and lipid oxidation likely through increased PUFA content, which increases PPARδ expression and activity. However, the mechanism of action that results in increased PUFA content of SCD1-Tg mice remains to be elucidated.

Lipocalin-2 increases fat oxidation in vitro and is correlated with energy expenditure in normal weight but not obese women.

OBJECTIVE: The role of lipocalin-2 (Lcn2) was determined in regulating metabolism in cell, animal, and human models. DESIGN AND METHODS: Adipocytes were treated with recombinant lipocalin-2 (rLcn2) to determine the effect on lipid metabolism. rLcn2 was injected into mice to determine the effect on metabolism in vivo. To assess the relationship between Lcn2 and fat oxidation (FatOx) in humans, normal weight (NW) and obese (OB) women were given three separate high fat (HF) meals followed by indirect calorimetry. The relationship between postprandial Lcn2 with macronutrient metabolism and total energy expenditure (TEE) using Pearson correlations was determined. RESULTS: Lcn2 increased expression of genes involved in ß-oxidation including peroxisome proliferator-activated receptor-δ in adipocytes, as well as (3) H labeled oleate ß-oxidation. Lcn2 injected into chow-fed mice directly increased TEE by 18% after the first dark cycle (232 ± 1.4 cal vs. 341 ± 1.4 cal; PBS vs. Lcn2) and remained significantly elevated by 10% after the second dark cycle (296 ± 1.4 cal vs. 326 ± 1.4 cal; PBS vs. Lcn2). Lcn2 was correlated with TEE in all three HF meal challenges in NW but not OB females. CONCLUSIONS: Lipocalin-2 is a novel adipokine that promotes FatOx and TEE and its function may be impaired in obesity.

The influence of maturation on the oxygen uptake efficiency slope.

This study examined the influence of maturation on the oxygen uptake efficiency slope (OUES) in healthy male subjects. Seventy-six healthy male subjects (8-27 yr) were divided into groups based on maturation status: prepubertal (PP), midpubertal (MP), late-pubertal (LP), and young-adult (YA) males. Puberty status was determined by physical examination. Subjects performed a graded exercise test on a cycle ergometer to determine OUES. Group differences were assessed using a one-way ANOVA. OUES values (VO(2)L·min(-1)/log(10)V(E)L·min(-1)) were lower in PP and MP compared with LP and YA (p < .05). When OUES was expressed relative to body mass (VO(2)mL·kg-1·min(-1)/log(10)V(E)mL·kg(-1)·min(-1)) differences between groups reversed whereby PP and MP had higher mass relative OUES values compared with LP and YA (p < .05). Adjusting OUES by measures of body mass failed to eliminate differences across maturational groups. This suggests that qualitative factors, perhaps related to oxidative metabolism, account for the responses observed in this study.