Farnesol prevents aging-related muscle weakness in mice through enhanced farnesylation of Parkin-interacting substrate.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis. Reduced PGC-1α abundance is linked to skeletal muscle weakness in aging or pathological conditions, such as neurodegenerative diseases and diabetes; thus, elevating PGC-1α abundance might be a promising strategy to treat muscle aging. Here, we performed high-throughput screening and identified a natural compound, farnesol, as a potent inducer of PGC-1α. Farnesol administration enhanced oxidative muscle capacity and muscle strength, leading to metabolic rejuvenation in aged mice. Moreover, farnesol treatment accelerated the recovery of muscle injury associated with enhanced muscle stem cell function. The protein expression of Parkin-interacting substrate (PARIS/Zfp746), a transcriptional repressor of PGC-1α, was elevated in aged muscles, likely contributing to PGC-1α reduction. The beneficial effect of farnesol on aged muscle was mediated through enhanced PARIS farnesylation, thereby relieving PARIS-mediated PGC-1α suppression. Furthermore, short-term exercise increased PARIS farnesylation in the muscles of young and aged mice, whereas long-term exercise decreased PARIS expression in the muscles of aged mice, leading to the elevation of PGC-1α. Collectively, the current study demonstrated that the PARIS-PGC-1α pathway is linked to muscle aging and that farnesol treatment can restore muscle functionality in aged mice through increased farnesylation of PARIS.

Non-invasive assessments of the advanced glycation end products in human skin using reflectance NIR spectroscopy.

Advanced glycation end products (AGE) are produced by non-enzymatic reaction between glucose and biomolecules including proteins. AGE accumulation is known to cause alternations of structure and function in proteins and to be related with an increased risk of diabetic complications, cardiovascular diseases, and aging processes. Conventionally, AGE accumulation has been estimated by measuring auto fluorescence level using ultraviolet (UV) light excitation. In this study, we investigated an alternative approach to estimate auto fluorescence level and thus AGE accumulation in in vivo human skin using NIR (Near-Infrared) spectroscopy. To examine spectral features attributed to glycation in proteins, we first analyzed in vitro NIR spectra from native and glycated protein. Then, we further examined NIR spectra of in vivo skin from human subjects, and estimated their auto fluorescence level using several multivariate regression approaches. Our analysis in in vitro spectra from native and glycated albumin revealed that glycation may affect -CH and -NH stretching. Furthermore, we elucidated that those bands for -CH and -NH may be responsible for the variation in auto fluorescence level in human skin NIR spectra. Finally, auto fluorescence level was estimated from those NIR spectra using several multivariate regression methods: principal component regression (PCR), partial least square regression (PLS-R) and support vector regression (SVR). Among the three methods, SVR showed the best performance. We demonstrated in this study that NIR spectroscopy can be used as an alternative non-invasive method to estimate AGE accumulation in in vivo human skin tissue without UV radiation on skin tissue.

Non-invasive method to monitor molecular changes in human stratum corneum during acute barrier disruption using reflectance NIR spectroscopy.

Stratum corneum is the outer most part of skin for barrier function. Disorder in stratum corneum is related with many skin diseases including acne, atopic dermatitis and psoriasis. In developed countries, about 20% of the population has disorder in the barrier function of stratum corneum. Adhesive tape stripping is a method to disrupt skin barrier function in studying disorder in stratum corneum. In this study, we obtained NIR (Near-Infrared) spectrum of human skin after tape stripping. Changes in skin spectra after barrier disruption were investigated through principal component analysis (PCA) of spectrum. PCA analysis revealed that peaks for -NH stretching and -CH vibration mainly contributed to the spectral variation caused by barrier disruption. Furthermore, second derivative of spectrum revealed that acute barrier disruption contributes to spectral changes in the region related with secondary structure of protein, lipid and water associated with lipid in stratum corneum. We demonstrated that acute barrier disruption affected features in NIR spectrum. These spectral changes revealed that acute barrier disruption affected keratin protein and ceramide in human stratum corneum. These results suggest that NIR spectroscopy can be used to monitor changes in filamentous network and lamellar structure in stratum corneum. NIR spectroscopy can provide non-invasive method to investigate skin disease related with barrier disruption by monitoring disturbance in protein and lipid structure in stratum corneum.

High glucose alters retinal astrocytes phenotype through increased production of inflammatory cytokines and oxidative stress.

Astrocytes are macroglial cells that have a crucial role in development of the retinal vasculature and maintenance of the blood-retina-barrier (BRB). Diabetes affects the physiology and function of retinal vascular cells including astrocytes (AC) leading to breakdown of BRB. However, the detailed cellular mechanisms leading to retinal AC dysfunction under high glucose conditions remain unclear. Here we show that high glucose conditions did not induce the apoptosis of retinal AC, but instead increased their rate of DNA synthesis and adhesion to extracellular matrix proteins. These alterations were associated with changes in intracellular signaling pathways involved in cell survival, migration and proliferation. High glucose conditions also affected the expression of inflammatory cytokines in retinal AC, activated NF-κB, and prevented their network formation on Matrigel. In addition, we showed that the attenuation of retinal AC migration under high glucose conditions, and capillary morphogenesis of retinal endothelial cells on Matrigel, was mediated through increased oxidative stress. Antioxidant proteins including heme oxygenase-1 and peroxiredoxin-2 levels were also increased in retinal AC under high glucose conditions through nuclear localization of transcription factor nuclear factor-erythroid 2-related factor-2. Together our results demonstrated that high glucose conditions alter the function of retinal AC by increased production of inflammatory cytokines and oxidative stress with significant impact on their proliferation, adhesion, and migration.

PEDF expression regulates the proangiogenic and proinflammatory phenotype of the lung endothelium.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with important roles in regulation of inflammation and angiogenesis. It is produced by various cell types, including endothelial cells (EC). However, the cell autonomous impact of PEDF on EC function needs further investigation. Lung EC prepared from PEDF-deficient (PEDF-/-) mice were more migratory and failed to undergo capillary morphogenesis in Matrigel compared with wild type (PEDF+/+) EC. Although no significant differences were observed in the rates of apoptosis in PEDF-/- EC compared with PEDF+/+ cells under basal or stress conditions, PEDF-/- EC proliferated at a slower rate. PEDF-/- EC also expressed increased levels of proinflammatory markers, including vascular endothelial growth factor, inducible nitric oxide synthase, vascular cell adhesion molecule-1, as well as altered cellular junctional organization, and nuclear localization of ß-catenin. The PEDF-/- EC were also more adhesive, expressed decreased levels of thrombospondin-2, tenascin-C, and osteopontin, and increased fibronectin. Furthermore, we showed lungs from PEDF-/- mice exhibited increased expression of macrophage marker F4/80, along with increased thickness of the vascular walls, consistent with a proinflammatory phenotype. Together, our data suggest that the PEDF expression makes significant contribution to modulation of the inflammatory and angiogenic phenotype of the lung endothelium.

Diabetes and retinal vascular dysfunction.

Diabetes predominantly affects the microvascular circulation of the retina resulting in a range of structural changes unique to this tissue. These changes ultimately lead to altered permeability, hyperproliferation of endothelial cells and edema, and abnormal vascularization of the retina with resulting loss of vision. Enhanced production of inflammatory mediators and oxidative stress are primary insults with significant contribution to the pathogenesis of diabetic retinopathy (DR). We have determined the identity of the retinal vascular cells affected by hyperglycemia, and have delineated the cell autonomous impact of high glucose on function of these cells. We discuss some of the high glucose specific changes in retinal vascular cells and their contribution to retinal vascular dysfunction. This knowledge provides novel insight into the molecular and cellular defects contributing to the development and progression of diabetic retinopathy, and will aid in the development of innovative, as well as target specific therapeutic approaches for prevention and treatment of DR.

Caspase-14: a novel caspase in the retina with a potential role in diabetic retinopathy.

PURPOSE: The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions. METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3. RESULTS: Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14. CONCLUSIONS: Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms.

Chitooligosaccharide induces mitochondrial biogenesis and increases exercise endurance through the activation of Sirt1 and AMPK in rats.

By catabolizing glucose and lipids, mitochondria produce ATPs to meet energy demands. When the number and activity of mitochondria are not sufficient, the human body becomes easily fatigued due to the lack of ATP, thus the control of the quantity and function of mitochondria is important to optimize energy balance. By increasing mitochondrial capacity? it may be possible to enhance energy metabolism and improve exercise endurance. Here, through the screening of various functional food ingredients, we found that chitooligosaccharide (COS) is an effective inducer of mitochondrial biogenesis. In rodents, COS increased the mitochondrial content in skeletal muscle and enhanced exercise endurance. In cultured myocytes, the expression of major regulators of mitochondrial biogenesis and key components of mitochondrial electron transfer chain was increased upon COS treatment. COS-mediated induction of mitochondrial biogenesis was achieved in part by the activation of silent information regulator two ortholog 1 (Sirt1) and AMP-activated protein kinase (AMPK). Taken together, our data suggest that COS could act as an exercise mimetic by inducing mitochondrial biogenesis and enhancing exercise endurance through the activation of Sirt1 and AMPK.

Effects of chitooligosaccharide lactate salt on sleep deprivation-induced fatigue in mice.

Chitooligosaccharides (COS), oligosaccharides composed of two to seven glucosamine residues, are known to exhibit various biological activities. In this study, we investigated the effects of COS in an in vivo mouse sleep deprivation-induced fatigue model in an effort to develop a functional food with anti-fatigue efficacy. Male Balb/c mice were orally administered 500 mg (kg d)(-1) of COS lactate or COS HCl for 2 weeks, and severe fatigue was induced by sleep deprivation. To evaluate the extent of fatigue, the swimming time, representing the immobility time, was measured in a forced swim test. As a result, oral intake of COS lactate-manifested anti-fatigue effects could be observed by the attenuation of fatigue-induced body weight loss and shorter immobility period. In addition, COS lactate was shown to alleviate the fatigue-induced increase in cortisol and lipid peroxidation and a decrease in superoxide dismutase (SOD) activity. Of particular note, the oral administration of COS lactate increased the mitochondrial membrane potential and the mitochondrial number significantly, indicating that COS lactate may enhance mitochondrial function. In support of this, COS lactate increased the expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and cytochrome c (Cyt C) mRNA, indicating that it may increase mitochondrial biogenesis. These results suggest that COS lactate can be an effective anti-fatigue functional food, and this anti-fatigue effect may result from, at least in part, the enhancement of mitochondrial biogenesis and the inhibition of free radical generation.

Proteomic analysis of mitochondrial proteins of basal and lipolytically (isoproterenol and TNF-alpha)-stimulated adipocytes.

The regulation of adipocyte lipolysis is increasingly believed to influence insulin resistance, in a process that may be associated with mitochondrial dysfunction. However, the molecular basis of the relationship between mitochondrial protein expression, lipolytic responsiveness, and insulin resistance remains unknown. A set of proteins that shows altered abundances in the mitochondria of untreated and treated 3T3-L1 adipocytes with TNF-alpha or isoproterenol was identified. These include the proteins associated with energy production, including fatty acid oxidation, TCA cycle, and oxidative phosphorylation. Proteins associated with oxidative stress dissipation were down-regulated in lipolytically stimulated adipocytes. Lipolytic stimulation with isoproterenol and TNF-alpha, which is also a potent proinflammatory cytokine, showed some noticeable differences in mitochondrial protein expression. For example, isoproterenol markedly enhanced the expression of prohibitin which is involved in the integrity of mitochondria but TNF-alpha did not. These results provide valuable information on mitochondrial dysfunction associated with oxidative stress induced by lipolytic stimulation.

The effects of BADGE and caffeine on the time-course response of adiponectin and lipid oxidative enzymes in high fat diet-fed C57BL/6J mice: correlation with reduced adiposity and steatosis.

Adiponectin, which is expressed exclusively in adipose tissue, has been shown to increase fatty acid oxidation via activation of AMP-activated kinase (AMPK) and phosphorylation of acetyl CoA carboxylase (ACC). ACC phosphorylation and carnitine palmitoyl-transferase-1 (CPT1) activity have been shown to be rate controlling factors in fatty acid oxidation. In high fat diet (HFD)-induced obese mice, we analyzed the time-course of changes in the expression of adiponectin and lipid oxidative enzymes induced by treatment with bisphenol A diglycidyl ether (BADGE) or caffeine for 8 weeks, and investigated whether the changes of adiponectin and lipid oxidative enzymes expression correlated with reduced adiposity or steatosis after 8 weeks of treatment. After 8 weeks of treatment, BADGE and caffeine had reduced body weight and epididymal adipose tissue weight in mice fed HFD, and markedly reduced the number of fatty droplets in the liver. Interestingly, the expression of adiponectin and lipid oxidative enzymes significantly increased after 2 weeks of treatment. These results indicate that the expression of adiponectin and lipid oxidative enzymes in the early stages of BADGE or caffeine treatment correlated well with the long-term anti-obesity effects.

Catechin gallates are NADP+-competitive inhibitors of glucose-6-phosphate dehydrogenase and other enzymes that employ NADP+ as a coenzyme.

Recent studies have shown that glucose-6-phosphate dehydrogenase (G6PD) is an effectual therapeutic target for metabolic disorders, including obesity and diabetes. In this study, we used in silico and conventional screening approaches to identify putative inhibitors of G6PD and found that gallated catechins (EGCG, GCG, ECG, CG), but not ungallated catechins (ECG, GC, EC, C), were NADP(+)-competitive inhibitors of G6PD and other enzymes that employ NADP(+) as a coenzyme, such as IDH and 6PGD.

A combination of caffeine, arginine, soy isoflavones, and L-carnitine enhances both lipolysis and fatty acid oxidation in 3T3-L1 and HepG2 cells in vitro and in KK mice in vivo.

To develop an anti-obesity agent containing dietary components, we focused on the mechanisms that enhance both lipolysis and fatty acid oxidation. Caffeine and arginine (CA), a nonselective adenosine-receptor antagonist and an inducer of lipolytic hormone, respectively, were used to stimulate lipolysis. Soy isoflavones and L-carnitine (SL), stimulators of carnitine palmitoyl transferase 1A and a cofactor for beta-oxidation of fatty acids, respectively, were used to enhance fatty acid oxidation. Effects of a combination of CA and SL (CASL) on lipid metabolism were studied in vitro and in vivo. During 3T3-L1 differentiation, lipid accumulation was significantly lower in cells treated with CASL (50 micromol/L, 1 mmol/L, 1 micromol/L, and 1 mmol/L, respectively) compared with each alone. Lipolysis was also significantly greater in 3T3-L1 adipocytes treated with CASL (50 micromol/L, 1 mmol/L, 10 micromol/L and 0.5 mmol/L, respectively) compared with each alone. In addition, treatment with higher concentrations of CASL (2 mmol/L, 1 mmol/L, 10 micromol/L, and 1 mmol/L, respectively) significantly enhanced beta-oxidation in HepG2 cells. The effects of CASL-containing diets (250 mg, 6 g, 200 mg, and 1.5 g/kg diet, respectively) were studied in vivo. When KK mice were food deprived for 48 h and subsequently refed a fat-free diet for 72 h, hepatic triglyceride (TG) accumulation was significantly lower in mice fed CASL compared with the control mice. In addition, after obese KK mice were fed a low-fat diet for 2 wk, adipose tissue weights were significantly lower in those fed CASL, but not CA or SL alone, compared with the control mice. Plasma and liver TG levels were also lower in mice fed CASL than in the control mice. These results suggest that CASL is effective for controlling obesity.

Genistein downregulates SREBP-1 regulated gene expression by inhibiting site-1 protease expression in HepG2 cells.

Genistein is one of the most abundant isoflavones in soy. The effects of genistein on cholesterol synthesis and fatty acid oxidation have been well documented, but the effect of genistein on fatty acid synthesis remains unclear. Thus, we investigated the effect of genistein on fatty acid synthase (FAS) expressions in HepG2 cells. In HepG2 cells treated with 10 micromol/L genistein, mRNA and protein expressions of FAS, as well as FAS activity, were significantly decreased. The promoter region of FAS contains binding sites for the transcription factor called sterol regulated element binding protein 1 (SREBP-1); SREBP-1 must be processed by site-1 (S1P) and site-2 proteases to be activated. We also investigated the effects of genistein on S1P, SREBP-1 expression, and subsequent SREBP-1 processing by S1P in HepG2 cells. Genistein reduced the expression of S1P and the processing of SREBP-1 but did not change the expression of SREBP-1 mRNA. SREBP-1 is also a transcription factor for lipogenic genes, such as stearoyl coenzyme-A desaturase1 (SCD1), glycerol-3-phosphate acyltransferase (GPAT), and acetyl-CoA carboxylase (ACC)1, and ACC2. Genistein also significantly inhibited the expression of these lipogenic genes. Thus, genistein treatment of HepG2 cells decreased the expression of lipogenic genes such as FAS, SCD1, GPAT, and ACC, which is, at least in part, mediated through the downregulation of S1P expression and subsequent SREBP-1 proteolytic cleavage.

Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets.

Prp19p is an integral component of the heteromeric protein complex (the NineTeen complex) in the nucleus, and it is essential for the structural integrity of NineTeen complex and its subsequent activation of the spliceosome. We identified Prp19p, which has never been reported in relation to any function outside of the nucleus, as a member of proteins associated with lipid droplets. Down-regulation of Prp19p expression with RNA interference in 3T3-L1 cells repressed lipid droplet formation with the reduction in the level of expression of perilipin and S3-12. The levels of expression of SCD1 (stearoyl-CoA desaturase-1), DGAT-1 (acyl-CoA diacylglycerol acyltransferase-1), and glycerol-3-phosphate acyltransferase were also reduced in Prp19p down-regulated cells, and a significant decrease in triglycerides was observed. Unlike perilipin, which is one of the most extensively studied lipid droplet-associated proteins, Prp19p is not essential for cAMP- and hormone-sensitive lipase-dependent lipolysis pathways, even though Prp19p is a component of the lipid droplet phospholipid monolayer, and down-regulation of Prp19p represses fat accretion significantly. These results suggest that Prp19p or Prp19-interacting proteins during lipid droplet biogenesis in adipocytes may be considered as another class of potential targets for attacking obesity and obesity-related problems.

(-)-Catechin suppresses expression of Kruppel-like factor 7 and increases expression and secretion of adiponectin protein in 3T3-L1 cells.

Adiponectin is an adipocyte-specific secretory hormone that can increase insulin sensitivity and promote adipocyte differentiation. Administration of adiponectin to obese or diabetic mice reduces plasma glucose and free fatty acid levels. Green tea polyphenols possess many pharmacological activities such as antioxidant, anti-inflammatory, antiobesity, and antidiabetic activities. To investigate whether green tea polyphenols have an effect on the regulation of adiponectin, we measured expression and secretion levels of adiponectin protein after treatment of each green tea polyphenols in 3T3-L1 adipocytes. We found that (-)-catechin enhanced the expression and secretion of adiponectin protein in a dose- and time-dependent manner. Furthermore, treatment of (-)-catechin increased insulin-dependent glucose uptake in differentiated adipocytes and augmented the expression of adipogenic marker genes, including PPARgamma, CEBPalpha, FAS, and SCD-1, when (-)-catechin was treated during adipocyte differentiation. In search of the molecular mechanism responsible for inducible effect of (-)-catechin on adiponectin expression, we found that (-)-catechin markedly suppresses the expression of Kruppel-like factor 7 (KLF7) protein, which has recently been reported to inhibit the expression of adiponectin and other adipogenesis related genes, including leptin, PPARgamma, C/EBPalpha, and aP2 in adipocytes. KLF7 is a transcription factor in adipocyte and plays an important role in the pathogenesis of type 2 diabetes. Taken together, these data suggest that the upregulation of adiponectin protein by (-)-catechin may involve, at least in part, suppression of KLF7 in 3T3-L1 cells.

Ginsenoside F1 protects human HaCaT keratinocytes from ultraviolet-B-induced apoptosis by maintaining constant levels of Bcl-2.

Ginsenosides, the major active ingredients of ginseng, show a variety of biomedical efficacies such as antiaging and antioxidation. Here, we investigate the protective activity of the ginsenoside F1, an enzymatically modified derivative of ginsenoside Rg1, against ultraviolet-B-induced damage in human HaCaT keratinocytes. Ginsenoside F1 significantly reduced ultraviolet-B-induced cell death and protected HaCaT cells from apoptosis caused by ultraviolet B irradiation. Furthermore, ginsenoside F1 prevented ultraviolet-B-induced cleavage of poly(ADP-ribose) polymerase in HaCaT cells. In search of the molecular mechanism responsible for the antiapoptotic effect of ginsenoside F1, we find that protection from ultraviolet-B-induced apoptosis is tightly correlated with ginsenoside-F1-mediated inhibition of ultraviolet-B-induced downregulation of Bcl-2 and Brn-3a expression.