extfieldsofstudy: Medicine Biology

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Diabetic Nephropathy Induced by Increased Ace Gene Dosage Is Associated with High Renal Levels of Angiotensin (1–7) and Bradykinin

Population studies have shown an association between diabetic nephropathy (DN) and insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene (ACE in humans, Ace in mice). The aim was to evaluate the modulation of Ace copies number and diabetes mellitus (DM) on renal RAS and correlate it with indicators of kidney function. Increased number of copies of the Ace gene, associated with DM, induces renal dysfunction. The susceptibility to the development of DN in 3 copies of animals is associated with an imbalance in activity of RAS enzymes leading to increased synthesis of Ang II and Ang-(1–7). Increased concentration of renal Ang-(1–7) appears to potentiate the deleterious effects triggered by Ang II on kidney structure and function. Results also show increased bradykinin concentration in 3 copies diabetic group. Taken together, results indicate that the deleterious effects described in 3 copies diabetic group are, at least in part, due to a combination of factors not usually described in the literature. Thus, the data presented here show up innovative and contribute to understanding the complex mechanisms involved in the development of DN, in order to optimize the treatment of patients with this complication.

Introduction
According to recent studies, 382 million people have diabetes and by 2035 this will rise to 592 million, posing the disease as one of the most challenging health problems of the 21st century [1,2]. Diabetic nephropathy (DN) is a serious complication of diabetes and is the leading cause of end-stage renal disease (ESRD), accounting for approximately 40% of all cases [1,3]. The renin angiotensin system (RAS) plays a key role in both renal physiology and the pathogenesis of chronic kidney disease and several studies to date have provided compelling support for the existence of an independent renal RAS whose activation appears to be critical in the development of DN [4,5]. Angiotensin II (Ang II) is generated at higher levels in the kidney than in the systemic circulation and angiotensin converting enzyme 2 (ACE2), mainly related to angiotensin (1)(2)(3)(4)(5)(6)(7) [Ang-(1-7)] generation, is highly expressed in the kidney. It has been extensively described that activation of the RAS is associated with glomerular and tubular cell injury in chronic kidney diseases, including DN [6]. Within kidney cells Ang II stimulates a variety of signaling pathways linked to increased cell growth and production of extracellular matrix proteins, via binding to its AT1 receptor. In mesangial cells, binding of Ang II to AT1 receptors stimulates protein synthesis, cell hypertrophy, and the production of the profibrotic cytokine transforming growth factor, which is thought to be a critical mediator of progressive glomerulosclerosis [7].
On the other hand, it has been suggested that Ang-(1-7) is an important counterbalancing mechanism within 2 Journal of Diabetes Research the RAS having vasodepressor and antihypertensive actions in hypertensive animals or humans [8,9]. Studies support a protective role for Ang-(1-7); this peptide prevents Ang II induced MAPK activation in proximal tubular cells [10] and its infusion has been reported to attenuate experimental glomerulonephritis [11]. Indeed, Giani et al. [8] showed that Ang-(1-7) reduced proteinuria and renal fibrosis in hypertensive rats. Similarly, Zhang et al. [11] found that Ang-(1-7) infusion attenuated glomerulosclerosis in rats and more recently attenuated kidney injury in mice with type 2 diabetes mellitus [12]. However, other studies suggest that some of the cellular effects of Ang-(1-7) may be deleterious; for example, stimulation of growth factor expression and cell proliferation by angiotensin in MC have been reported [13]. In addition, deletion of the gene for the Mas receptor in mice is associated with decreased inflammation in kidneys subjected to unilateral ureteral obstruction [14]. In rats with streptozotocin (STZ) induced diabetes, Benter et al. [15] have reported that chronic Ang-(1-7) treatment attenuated proteinuria and restored vascular reactivity. In contrast, Shao et al. [16] recently showed that chronic administration of Ang-(1-7) caused an increase in kidney TGF-1 mRNA and protein, associated with increased proteinuria and renal injury in STZ-diabetic rats.
Although hyperglycemia is an important factor, it does not account fully for the risk of DN and alone it is not causative. A significant proportion of patients with diabetes do not develop DN despite long-standing severe hyperglycemia indicating that other factors, apart from chronic hyperglycemia per se, may equally contribute susceptibility for the development of DN. The familial aggregation of the DN demonstrates the involvement of genetic factors in the pathogenesis of this complication, and available data provide convincing evidence that the angiotensin converting enzyme I/D (ACE I/D) polymorphism is significantly associated with overt nephropathy being the insertion/insertion (II) genotype and low plasma ACE levels protective against the disease both in type I and type II diabetics [17][18][19]. These associations suggest that a high constitutive level of ACE is deleterious to the kidney in the setting of chronic hyperglycemia. However, a causal link between higher levels of ACE, angiotensin peptides, and kidney malfunction in association with diabetes has not been completely established.
Determining the contribution of a single gene to a complex phenotype such as DN remains a challenge since this is a context dependent response. The generation of genetically engineered mice with discrete lower and above normal ACE levels [20] overcomes some of these limitations, enabling, under controlled genetic and environmental conditions, testing the role Ace gene plays in DN. Huang et al. [21] showed that after 12 weeks of STZ induced diabetes, the 3 copies diabetic mice developed higher blood pressure and proteinuria, proving that a modest genetic increase in ACE levels is sufficient to initiate the chain of events leading to DN. The current study has extended these observations by characterizing the role of bradykinin and angiotensin peptides in face of increased and decreased dosage of ACE in the pathogenesis of diabetic lesions. Our results suggest that Ace locus associated with hyperglycemia disrupt RAS balance, generating a complex novel set of peptides synthesis. This includes not only a markedly increase on renal Ang II generation but also high tissue levels of bradykinin and Ang-(1-7), which in turn may have an important contribution on the development of diabetic lesions associated with increased Ace gene dosage.

Generation of Mice with 1 and 3 Copies of the Ace
Gene. Mice (8-10 weeks old) were kindly provided from the Laboratory of Experimental Hypertension and Molecular Cardiology of University of São Paulo. Genetically engineered mice carrying either an inactivation (1 copy) or duplication (3 copies) of the Ace gene at its endogenous locus [20] were used for the study. Heterozygous mice of each strain were crossed for eight generations with wild-type C57BL6 mice and among themselves to generate the experimental groups. Identification of genetically modified offspring was made at 21 days of age by PCR amplification of DNA isolated from ear biopsies as previously described [22]. Animals were fed with standard laboratory chow and given water ad libitum while housed (4-5 per cage) in a temperature-controlled room (22 ∘ C) with a dark-light cycle of 12-12 h. All experimental procedures followed institutional guidelines for care, use, and euthanasia of laboratory animals, and the protocols were approved by the Institutional Review Board of Federal University of São Paulo, Brazil (1861/11). Efforts were exerted to reduce the number of animals used.

Experimental
Procedure. Male mice of each genotype fasted 6 hours and then received a single intraperitoneal injection of STZ (150 mg/kg body wt. Sigma Chemical Company, St. Louis, MO, USA). The age-matched control mice were injected with an equivalent volume of saline. One week after STZ injection, blood samples were collected through the tail vein, and plasma glucose level was determined (Accu-Check Advantage Blood Glucose Monitor, Roche Diagnostic Corporation, Indianapolis, IN). Mice with plasma glucose ≥250 mg/dL and symptoms of polyuria, polyphagia, and polydipsia were considered to be diabetic and used in the present study. After the onset of diabetes, mice were monitored every other week for body weight and blood glucose, and every 4 weeks mice were placed in metabolic cages (Tecniplast, USA) for the determination of water and food intake, albumin, creatinine, and urea excretion in 24-hour urine output over a period of twelve weeks, without insulin treatment. At 12 weeks, 3 days after the last urine collection, mice were euthanized by decapitation and trunk blood was collected in ice-chilled heparinized tubes. Blood was centrifuged at 10,000 g for 10 minutes at 4 ∘ C. Plasma was separated, aliquoted, and stored at −80 ∘ C. Kidneys were rapidly excised, weighted, and processed for several purposes. Left kidney was frozen in liquid nitrogen for biochemical analysis (kept at −80 ∘ C until its use) and right kidney was fixed by immersion in formalin solution 10%, neutrally buffered, and processed for paraffin embedding according to standard procedures (for histological studies).

Metabolic Analysis.
Mice were placed in metabolic cages for 24 h for the determination of urine excretion, water, and food intake. Albuminuria, urinary urea, and creatinine were quantified using respective enzyme-linked immunosorbent assay (ELISA, Mouse Albumin ELISA Quantification Set, Bethyl Laboratories, USA) and colorimetric commercially available kits (Labtest Diagnostic, Brazil). Metabolic analyses were performed at monthly intervals, during the 3-month protocol period. Incomplete data of metabolic parameters due to death of animals was deleted, and finally the total metabolic data of 6-11 mice in each group was recorded.

Tissue Preparation.
At 12 weeks, 3 days after the last metabolic cage, animals were decapitated and trunk blood was collected. Kidneys were then removed, weighted, and processed for several purposes. Left kidney was rapidly excised, frozen in liquid nitrogen for biochemical analysis, and kept at −80 ∘ C until its use. The tissue was homogenized as described by Oliveira et al. [23] with some modifications related to tissue weight and buffer volume (12.5 mg kidney tissue/500 L borate buffer 0.4 M). The protein concentration was determined by the Bradford method [24] (Bio Rad Protein Assay Kit; Bio Rad, Hercules, CA) using bovine serum albumin as the standard. Right kidney was fixed by immersion in formalin solution 10%, neutrally buffered, and processed for paraffin embedding according to standard procedures for histological studies. So kidney slices, with a thickness of 5 mm, were stained using Hematoxylin-Eosin (HE), Periodic Acid Schiff Stain (PAS), and Masson's Trichrome in order to analyze the presence of renal lesions related to diabetes, or any other morphological changes. Analyses were conducted by a blinded observer.

Evaluation of Renin Angiotensin System
2.5.1. Renal Renin. Active renin content was measured by determining the amount of angiotensin I (Ang I) generated in the tissue homogenates measured by high-performance liquid chromatography (HPLC), as previously described [25]. Active renin was evaluated in the absence of trypsin and to prevent further cleavage of angiotensinogen, and Ang I, a pool of enzymatic inhibitors, was added to the tissue homogenate: 50 Mm ethylenediaminetetraacetic acid (EDTA), 1 mM 1,10-phenanthroline (OPhe), 3 mM phenylmethanesulfonyl fluoride (PMSF), and 200 mM dithiothreitol (DTT). Renin activity was estimated by Ang I generation when the tissue homogenate was incubated with 10 L of 2 nmol/mL synthetic tetradecapeptide substrate (Sigma, St. Louis, MO) for 1 h at 37 ∘ C, as previously described [26]. The reaction was stopped at two times (zero and sixty minutes) by adding 10 L of 50% H 3 PO 4 . One hundred microliters of each sample were filtered and injected into the HPLC system. The released Ang I peptide was quantified by reverse-phase HPLC using an Aquapore ODS 300 column 7 m (PerkinElmer Inc, EUA) equilibrated with 0.1% phosphoric acid containing 5% acetonitrile (vol/vol). Ang I was separated by isocratic elution for 5 min, followed by a 20 min linear gradient of 5-35% acetonitrile in 0.1% phosphoric acid (vol/vol) at 1.5 mL/min. The chromatographic profile of each sample was compared with that obtained for standard samples containing angiotensinogen and Ang I at an absorbance of 214 nm. Peptide fragments were identified by elution position and quantified by integration area using repeated injections of standard peptide solution to correct for small differences in retention time (<6%) and peak height (<5%).

ACE
Activity. ACE catalytic activity was determined fluorimetrically as described by Friedland and Silverstein [27]. An aliquot of kidney homogenate (10 L) was incubated with a 200 L assay solution containing 1 mmol/L Z-Phe-His-Leu (ZPhe-HL) or 5 mmol/L HippurylHis-Leu (HHL) in 100 mmol/L borate buffer, with 300 mmol/L NaCl and 0.1 mmol/L ZnSO 4 , for 10 minutes at 37 ∘ C. The enzymatic reaction was stopped by the addition of 1.5 mL 280 mmol/L NaOH and incubated with 100 L o-phthaldialdehyde (20 mg/mL methanol) for 10 minutes. The fluorescent reaction was stopped by the addition of 200 L 3 N HCl. The liberated dipeptide HL was measured fluorimetrically (360 nm excitation and 500 nm emission) using a Hitachi fluorimeter (Hitachi F-200, Japan). The standard curve was obtained using varying concentrations of L-HL in the blank reaction mixture and it showed a linear relation between relative fluorescence and HL concentration [28].

ACE2 Activity.
Renal ACE2 activity was determined in spectrofluorimeter (Tecan, Switzerland), using the synthetic fluorogenic substrate, Mca-APK-Dnp (5 mmol/L,), as described by Pedersen et al. (2011) with some modifications [29]. Kidney samples (12 mg) were homogenized in 500 L buffer containing 75 mM Tris HCl, pH 6.5, 1 M NaCl, and 0.05 mM ZnCl 2 , added by 10 M of captopril and 1 tablet/10 mL protease inhibitor mixture (Complete, Mini; Roche Diagnostics Corp., Indianapolis, IN). Homogenates were centrifuged twice (15000 rpm, 15 minutes, 4 ∘ C) and supernatants were stored at −20 ∘ C. To each well, 5 L of a tissue sample was added, along with 50 L of buffer in the presence or the absence of ACE2 inhibitor (DX600, 10 M). Substrate was added and samples were read at 0 and 30 minutes. Arbitrary units were registered, calculations were done based on a fluorescence standard curve (OmniMMP) and the time point 0 was used as internal blank.

Angiotensin Quantification.
The extraction of angiotensins was held in Oasis HBL 3cc columns (Waters, Ireland), previously activated with methanol (5 mL), tetrahydrofuran (5 mL), hexane (5 mL), methanol (5 mL), and water (10 mL). After sample introduction, columns were washed with water (10 mL) and peptides of interest were eluted with ethanol, acetic acid, and water (90 : 4 : 6). The eluted fractions were lyophilized and resuspended in 500 L mobile phase A: 5% ACN in 0.1% orthophosphoric acid and one hundred microliters of each sample were filtered (with 0.7 mm membrane) and then injected into the HPLC system. Peptides were separated by HPLC (Shimadzu System, Japan) in reverse phase column Aquapore ODS 300 (250 mm × 4.6 mm), 7 mm, using a linear gradient from 5% to 35% of mobile phase B (95% acetonitrile in H 3 PO 4 0.1%), at a flow rate of Values represent mean ± S.E.M., = 6-11. Two-way ANOVA or two-way ANOVA for repeated measures (whether necessary) showed * < 0.05: diabetic versus control, # < 0.05: 3 copies diabetic group versus 1 copy diabetic group, $ < 0.05: versus same diabetic group at first week.
1.5 mL/min for 30 min. The chromatographic profile of each sample was compared with that obtained for standard intact and fragmented peptides (Sigma Co.) of the RAS at 214 nm absorbance. Peptides were identified according to retention time and quantified by area integration using repeated injections of standard peptide solution to correct for small differences in retention time (<6%) and peak height (<5%). The lower limit of quantification determined was approximately 3,125 pmols.

Results and Discussion
We compared morphometry and several physiologic and biochemical parameters in renal tissue of normal and diabetic mice bearing 1 and 3 copies of Ace gene to uncover the effects of Ace genotype, of diabetes, and of any interactions on the development of DN. Our results suggest that Ace locus associated with hyperglycemia disrupt RAS balance, generating a complex novel set of peptides synthesis. This includes not only a markedly increase on renal Ang II generation but also high tissue levels of bradykinin and Ang-(1-7), which in turn may have an important contribution in the development of diabetic lesions associated with increased Ace gene dosage.
As summarized in Table 1, diabetic levels of plasma glucose were present throughout the 12-week observational period in STZ diabetic mice of both Ace genotypes, but not in the untreated controls; glycemia was not affected by Ace gene copy number neither in control nor in diabetic groups. As expected, STZ treatment also produced other characteristic signs of diabetes, already described in literature [30] such as increased intake of both water and food and failure to gain weight [31]. Similar results have already been described in this animal model by Huang et al. (2001) and also show that Ace genotype does not influence these parameters [21].
To determine how Ace genotypes and diabetes affect patterns of renal morphology and function, we evaluated renal histology, kidney-to-body weight ratio, urinary volume, and also excretion of albumin, creatinine, and urea. It is well known that one of the most striking characteristics of DN is mesangial expansion, which results from accumulation of ECM proteins, decrease in filtration surface, and ultimately end stage renal disease [32][33][34]. In this study, the glomerular appearance in histology of 3 copies diabetic animals showed accelerated mesangial expansion characterized by an increase in PAS-positive relative mesangial matrix area ( Figure 1) compared with that observed in 1 copy diabetic group and also in control groups (3 copies diabetic: 0.808 > 1 copy diabetic: 0.629 > 3 copies control: 0.742 = 1 copy control: 0.692; < 0.05). These alterations in kidney morphology, worse in 3 copies mice, are similar to those seen in the initial stages of human diabetes and contrary to the data presented by Huang et al. (2001) [21]; in this study these kidney structural changes are affected by the Ace genotype. Furthermore, Masson's Trichrome staining revealed that glomerular fibrotic areas were significantly increased in 3 copies diabetic mice compared to control mice and also to 1 copy diabetic group (3 copies diabetic: 0.478 > 1 copy diabetic: 0.353 > 3 copies control: 0.308 = 1 copy control: 0.270 2 ; < 0.05). To support our findings related to damage in renal morphology induced by increased Ace dosage and diabetes, we evaluated kidney hypertrophy. STZ-induced diabetes increases kidney weight gain. This characteristic increase in kidney weight (KW) (measured by hypertrophy index mg of KW/g of body weight) Journal of Diabetes Research was found in both 1 and 3 copies Ace diabetic groups, suggesting renal injury. Renal hypertrophy was significantly higher in 3 copies versus 1 copy diabetic group, showing the important influence of Ace gene dosage on this parameter and supporting the data on renal histology. These differences related to renal morphology reflected alterations in functional capacity of this organ in diabetic animals. Both 1 and 3 copies diabetic groups presented higher urinary volume compared to controls, which consistently increased throughout the study period. Urinary excretion of creatinine and urea, also useful in estimating the extent of impairment of kidney function, was significantly lower in the diabetic groups compared to their controls. Creatinine and urea were not affected by disease duration or genotype.
On the other hand, urinary albumin excretion (UAE), the most commonly used early marker of DN [35], was significantly affected by diabetes and Ace genotype. One-and 3 copies control mice have very low levels of UAE which did not change with time. One week after induction of diabetes, neither the 1 copy nor the 3 copies mice have UAE levels higher than their controls. However, at the end of protocol not all diabetic animals presented higher UAE; in other words, only mice harboring 3 copies of Ace gene presented greater albuminuria after 12 weeks. Corroborating this result Huang et al. (2001) showed that the UAE of the 1 and 2 copies diabetic mice progresses much less rapidly than 3 copies, so 3 copies mice when diabetic develop overt albuminuria early in the course of the disease [21]. DN is thought to be a unidirectional process from microalbuminuria to end-stage renal failure; however recent studies in type 1 diabetic patients demonstrate that a large proportion of DN patients revert to normoalbuminuria and that one-third of these patients exhibit reduced renal function even in the microalbuminuria stage. This finding (higher UAE presented only by 3 copies diabetic mice) will be thoroughly discussed ahead.
To correlate the structural and functional kidney damage, induced by increased Ace gene dosage, RAS components were evaluated. Renal renin activity was estimated with an excess of substrate and is presented in Figure 2 being reduced in diabetic mice having 1 or 3 copies of the Ace gene when compared with its controls. Increased Ace gene dosage was inversely correlated with ACE activity in control mice; enzyme activity was twice as high in renal tissue from control mice harboring 1 copy of the Ace gene, as compared with 3 copies mice. Huang et al. (2001) [21], Evangelista and Krieger (2006) [36], and also Krege et al. (1997) [20] evaluated plasma renin activity in control mice harboring different copies of Ace gene and observed an inverse relationship between renin activity or renin gene transcription and Ace genotype (activity). Here we also evaluated the effect of diabetes which curiously reduced enzyme activity only in 1 copy group. This is the first report demonstrating influence of diabetes on renal renin activity in this animal model, and the reduction seems to be an important adaption of local RAS to protect the kidney from an excessive generation of Ang II in 1 copy diabetic mice. These findings argue in favor of compensatory adaptations in the RAS to maintain the homeostasis in genetically manipulated animals, and the evaluation of renal ACE activity using HHL (B) Zphe-HL as substrates (Figures 3(a) and 3(b)) also supports these data, since 1 copy control mice present lower ACE activity in the kidney (∼50%) as compared with 3 copies ones. We believe that ACE activity was not reduced in 1 copy diabetic animals, as compared with respective controls, because it is already low. In the present study, we also observed a very expressive decrease in ACE activity in renal tissue of 3 copies diabetic mice, as compared to control ones (Figures 3(a) and 3(b)). This compensatory adaptation to probably reduce the generation of Ang II has already been described in other studies using STZ-diabetic mice and also db/db [37,38].
The description of ACE2 brought important new data and perspectives to the understanding of RAS regulation, comprised by opposing axes, the classical ACE/Ang II/AT1 and AT2 receptor axis and the ACE2/ Ang-(1-7)/Mas receptor axis [39][40][41]. The role of ACE2 in the context of diabetes has been widely explored in the past few years. Different studies have shown that decreased ACE2 expression was associated with increased albuminuria [31] and pharmacological inhibition of the enzyme increased urinary albumin excretion three to fourfold in diabetic mouse models [42,43]. In the present study we observed that diabetes increased ACE2 activity only in 1 copy group, but not in renal tissue of 3 copies animals (Figure 4), and adaptation that has been already described by other groups [42][43][44][45][46][47]. We detected as presented in Figure 3 decrease of ACE activity in diabetic mice having 1 or 3 copies of the Ace gene in contrast with ACE2 activity that was increased in the same animals, suggesting that Ang-(1-7) is derived from both the metabolism of Ang I via the endopeptidase pathway and the cleavage of Ang II by the ACE2-dependent pathway [48].
Taken together, data on albuminuria and ACE2 activity reveals that one of the mechanisms involved on protection against the development of DN present in 1 copy diabetic animals is related to increased ACE2 as already described in other diabetic animal models [43,49,50]. In fact, recently studies showed that administration of ACE2 inhibitor (MLN-4760) increases albuminuria, mesangial pathologies, and fibronectin deposition in diabetic mice [38,42]. In line with these results, Elased [51] observed that urinary ACE2 excretion was positively correlated with the progression of diabetic renal injury represented by progressive albuminuria, mesangial matrix expansion, and renal fibrosis. There is evidence that deletion of ACE2 leads to the development of Ang II dependent renal damage, suggesting ACE2 as renoprotective target in diabetes, most likely a part of a mechanism to compensate for elevated Ang II levels [52][53][54][55][56].
Although some studies describe an increase in ACE2 mRNA levels and protein expression in diabetes [47], Reich et al. (2008) described a decrease in the same parameters in renal biopsies of patients with kidney disease due to diabetes [57] and Colucci et al. (2011) showed a decrease in ACE2 protein expression in the kidney from D-NOD mice [58]. We believe that these differences are related to diabetes duration, and as the progression of disease and DN, the RAS cannot compensate the imbalance on RAS anymore.
The impact and the physiological significance of the balance of renal ACE/ACE2 activities cannot be understood without assessing the tissue concentration of angiotensins. Regarding Ang I it is interesting to note that despite the significant reduction in renin activity in 3 copies control mice (compared to 1 copy) our data evidenced a greater increase in the concentration of Ang I in kidney homogenates of this group compared to 1 copy control mice (Figure 5(a) and Table 2). The induction of diabetes did not alter the level of this peptide in mice presenting 1 copy of Ace gene; however a significant reduction was observed in 3 copies mice when compared to the control group and to 1 copy  Values represent mean ± S.E.M. Two-way ANOVA showed * < 0.05: diabetic versus control. + < 0.05: 3 copies control group versus 1 copy control group. # < 0.05: 3 copies diabetic group versus 1 copy diabetic group. diabetic mice. The increase of Ang I presented by 3 copies control mice maybe related to the evidence of Ang-(1-12) being an alternate renin-independent, angiotensin-forming substrate in accordance with data presented by Ferrario et al. [59]. The possibility that Ang-(1-12) may serve as an alternate substrate for the generation of bioactive angiotensins led us to document increased expression of Ang-(1-12) in cardiac myocytes of adult spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) controls [59]. Additional studies showed the generation of Ang I, Ang II, and Ang-(1-7) from exogenous Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in the effluent of isolated hearts from Sprague-Dawley, normotensive Lewis, and mRen2 hypertensive Lewis rats, as well as WKY and SHR [59]. ACE converts Ang- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) to Ang I and Ang II in a sequential reaction consistent with the catalytic properties of the enzyme as a dipeptidyl carboxypeptidase cleaving two residues at a time [60].
As expected, concurrent to increased ACE activity presented by 3 copies control mice, the renal concentration of Ang II was significantly higher in this group compared to 1 copy control mice and to 3 copies diabetic mice ( Figure 5(b) and Table 2). Despite the reduction of Ang II induced by diabetes in both groups, the concentration of this peptide was (still) significantly higher in 3 copies mice when compared to 1 copy, so the renal content of this peptide remained 45% higher in 3 copies mice than their control group. So considering, first, as mentioned, that the content of this peptide was higher in 3 copies diabetic mice than 1 copy and, second, that there were no differences between ACE activity from these groups, these results led us to conclude that the generating of Ang II in 3 copies diabetic mice is related to nonclassical pathway. Although ACE is the most important renal carboxypeptidase that cleaves Ang I at its carboxy terminus to generate Ang II [61,62] several other described more than three decades ago [63]. Cathepsin G was discovered as an Ang II producing enzyme in human neutrophils [64] and it has been speculated that cathepsin G might be involved in mediating part of the inflammatory response in these cells [65]. Rykl and cols concluded that the angiotensin-generating activities of the fraction containing angiotensin-converting enzyme and the fraction containing cathepsin G were in the same order of magnitude, thus showing that the contribution of cathepsin G towards the production of Ang II is significant [65].
Also chymase is a potent and specific Ang II-forming enzyme in vitro [66] as well in vivo [67]. Furthermore, studies have added that chymase appears to play a role in Ang II formation in certain disease states [68][69][70], so they observed that evidences indicated that intrarenal Ang II formation is largely mediated by ACE under normal physiological conditions [61,62,71], although other non-ACE Ang II-forming enzymes have been recognized in certain types of renal disease [68,69]. Huang et al. (2003) reported that the chymase-dependent Ang II-generating system was upregulated in the human diabetic kidney and this becomes particularly strong in those with hypertension [68]. In the normal kidney, while ACE was constitutively expressed by most kidney cells, chymase was weakly expressed by mesangial cells (MC) and vascular smooth muscle cells (VSMC) only. In the diabetic kidney, while ACE expression was significantly upregulated (1-to 3-fold) by tubular epithelial cells (TEC) and infiltrating mononuclear cells, there was also markedly increased chymase expression (10-to 15fold) by both MC and VSMC, with strong deposition in the collagen-rich extracellular matrix including both diffuse and nodular glomerulosclerosis, tubulointerstitial fibrosis, and vascular sclerosis. Interestingly, while ACE expression showed no difference in patients with or without hypertension, upregulation of chymase in hypertensive patients was much stronger than that seen in those without hypertension (4-to 7-fold; < 0.001). Correlation analysis showed that, in contrast to the ACE expression, upregulation of chymase correlated significantly with the increase in BP and the severity of collagen matrix deposition within the glomerulus, tubulointerstitium, and arterial walls (all with < 0.001). In conclusion, the present study suggested that chymase, as an alternative Ang II-generating enzyme, is markedly upregulated in the diabetic kidney and may be associated with the development of diabetic/hypertensive nephropathy. In addition, differential expression of ACE and chymase in the diabetic kidney indicates that both ACE and chymase may be of equal importance for Ang II mediated DN and vascular disease. Results from this study suggest that blockade of both Ang II generating pathways may provide additional beneficial effect on DN [68].
Ang II is the final physiologically active product of RAS, and it works not only as a strong vasopressor but also as a promoter of tissue remodeling in various organs such as heart, arteries, and kidneys [72]. Many lines of evidence suggest a role for intrarenally formed Ang II in the pathogenesis of DN [73,74]. It has been shown that glucose and Ang II are able to increase the synthesis of collagen types I and IV and other matrix proteins in MC, as well as decreasing the levels of proteases involved in matrix degradation, resulting in the matrix expansion observed in DN [7]. It has been shown that increasing glucose concentrations cause proportional increases in Ang II generation in MC [75].
High glucose concentration induced an impressive elevation of Ang-(1-7) concentration as well as the increasing number of Ace gene copies. It is interesting to note that this result (higher levels of Ang-(1-7) presented by 3 copies diabetic mice) (Figure 5(c) and Table 2) has not seem to be due to activation of the classical pathway of the RAS, via hydrolysis of Ang II by ACE2, since ACE2 activity of these animals is reduced. The Ang-(1-7) can be generated by alternative routes involving the hydrolysis of Ang II by prolylcarboxypeptidase (PCP) and prolyl-endopeptidase (PEP).
Further, endopeptidases, such as neprilysin (NEP) or PEP, can also cleave Ang I to generate Ang-(1-7) [76] and, in fact, our results indicate that this maybe the production route used (synthesis of Ang-(1-7) from Ang I by hydrolysis of the NEP and PEP) since the evaluation of the concentration of this peptide (Ang I) is significantly reduced in the diabetic group 3 copies related to all other groups. We cannot exclude the processing also of Ang (1-12) specially in renal cortex to Ang-(1-7) formed by neprilysin [60].
Although Ang-(1-7) is usually described as antiproliferative and antifibrotic, opposing most of the effects of Ang II, it has been demonstrated that it can worsen renal dysfunction in experimental models triggering hypertrophy of this organ. Zimpelmann and Burns (2009) noted that the stimulation of p38 MAPK phosphorylation by Ang-(1-7) leads to release arachidonic acid and production of TGF-beta1 and extracellular matrix proteins led them to conclude that Ang-(1-7) exerts growth-stimulatory effects in human MC [13]. Simões e Silva et al. (2006) observed that end stage renal disease subjects exhibited a dramatic increase in Ang-(1-7) (25-fold higher than control values) and added that it is yet unknown if the elevation of Ang-(1-7) occurs as a compensatory mechanism that opposes the harmful renal and cardiovascular effects of Ang II or whether, due to a major unbalance in the RAS metabolism, Ang-(1-7) at supra physiologic concentrations could act as a mediator of renal dysfunction [77]. Esteban et al. (2009) showed that renal deficiency for Mas diminished renal damage in models of renal insufficiency as unilateral ureteral obstruction and ischemia/reperfusion injury while the infusion of Ang-(1-7) to wild-type mice pronounced the pathological outcome by aggravating the inflammatory response [14]. Recently, Velkoska et al. (2011) concluded that their results add to the increasing evidence that Ang-(1-7) may have deleterious cardiovascular effects in kidney failure and highlight the need for further in vivo studies of the ACE2/Ang-(1-7)/Mas receptor axis in kidney disease [78]. Then, one of the most important events observed in the present study was an impressive rise in Ang-(1-7) content induced by high-glucose exposure and increasing number of Ace gene copies, suggesting that the high concentration of this peptide may be associated to increased renal damage presented by 3 copies diabetic mice.
Bradykinin level in renal tissue ( Figure 6) did not differ from diabetic to control 1 copy mice, however when the diabetes was associated to 3 copies of Ace gene, a greater increase of this peptide was observed, according to reduced ACE activity in this group. It is well known that bradykinin is inactivated by ACE, and indeed the 3 copies diabetic animals showed a significant reduction of enzyme activity. Campbell et al. (1999) also found increased levels of bradykinin in the renal tissue of rats treated with STZ [79]. However, Colucci et al. (2011) observed an increase in bradykinin concentration, concomitant with increased ACE activity in the same tissue in NOD hyperglycemic animal model, suggesting that the increase was caused by higher activity of kallikrein, rather than by alterations in the metabolism of kinins by ACE [58,79]. Moreover, it is important to note that the increase in renal tissue bradykinin observed in 3 copies diabetic animals may also contribute to worsening renal function displayed by these animals, as well as by increasing the mortality rate, since this peptide is involved in vascular and metabolic dysfunctions induced DM [79].

Conclusion
The susceptibility to the development of DN in 3 copies animals is associated with an imbalance in activity of various enzymes (renin, ACE, and ACE2) leading to increased synthesis of Ang II and Ang-(1-7). Increased renal concentration of Ang-(1-7) appears to potentiate the deleterious effects triggered by Ang II on renal structure and function. Apart from the involvement of the RAS in the development of DN, our results also show increased bradykinin concentration only in 3 copies of the Ace gene diabetic group. Taken together, these adaptations indicate that the deleterious effects described in


PharmDB-K: Integrated Bio-Pharmacological Network Database for Traditional Korean Medicine

Despite the growing attention given to Traditional Medicine (TM) worldwide, there is no well-known, publicly available, integrated bio-pharmacological Traditional Korean Medicine (TKM) database for researchers in drug discovery. In this study, we have constructed PharmDB-K, which offers comprehensive information relating to TKM-associated drugs (compound), disease indication, and protein relationships. To explore the underlying molecular interaction of TKM, we integrated fourteen different databases, six Pharmacopoeias, and literature, and established a massive bio-pharmacological network for TKM and experimentally validated some cases predicted from the PharmDB-K analyses. Currently, PharmDB-K contains information about 262 TKMs, 7,815 drugs, 3,721 diseases, 32,373 proteins, and 1,887 side effects. One of the unique sets of information in PharmDB-K includes 400 indicator compounds used for standardization of herbal medicine. Furthermore, we are operating PharmDB-K via phExplorer (a network visualization software) and BioMart (a data federation framework) for convenient search and analysis of the TKM network. Database URL: http://pharmdb-k.org, http://biomart.i-pharm.org.

Introduction
It is known that Traditional Medicine (TM) originated in China about 3,000 years ago, and was introduced to Korea in the 6 th century [1]. Although TM began in China, it has been indigenized in Korea and developed into the unique Traditional Korean Medicine (TKM). In Korea, the TKM hospital industry continues to grow, and annual revenue of the TKM industry is expected to increase to $5.8 billion in 2015 [2]. However, TKM has not been well recognized worldwide thus far. Since TM has been brought to the attention of pharmaceutical companies for novel lead compounds, some databases for Traditional Chinese Medicine (TCM) have been developed and widely used [3][4][5][6][7][8][9]. Unfortunately, however, there is no well-known integrated bio-pharmacological TKM database for drug development.
Traditional herbal medicines consist of unpurified plant extracts or portions of plants containing several compounds. The efficacy of traditional herbal medicine depends on the compounds that it contains. Therefore, correct identification of indicator compounds in specific herbs is an essential prerequisite to relating their medical benefits with known disease indicators. As mentioned earlier, there are a number of well-known TCM databases. Traditional Chinese Medicines Information Database (TCM-ID) contains information for prescriptions, herbs, and ingredients, but there is no information about related proteins [3]. TCM@Taiwan contains information on a large number of compounds isolated from herbs, but information on related diseases and proteins is missing [4]. Traditional Chinese Medicines Integrated Database (TCMID) is the first database containing comprehensive information on interactions between compounds, proteins, and herbs, and it is likely the largest database in related fields [5]. However, since TCMID uses STICH, a resource containing known and predicted interactions from diverse organisms [10], for compound-protein interaction data without any critical filtration, it is possible that TCMID may contain unnecessary compound-protein data for drug discovery. There are also many other TCM databases such as Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), TCMGeneDIT, China Natural Products Database (CNPD), and Comprehensive Herbal Medicine Information System for Cancer (CHMIS-C) [6][7][8][9]. These TCM databases provide diverse types of information including hundreds of compounds (ingredients) for each herb. However, due to a lack of detailed information, it is difficult to recognize which compounds play major roles as indicators or active compounds.
As mentioned earlier, although TKM was started based on TCM, TKM has been developed to a unique medicinal category by acquiring region-specific medical experiences for hundreds of years. We compared our TKM-disease relationship data with TCM-ID to see the differences between TKM and TCM and found them to be not quite identical. For example, skin diseases are commonly found in two databases for indication of Isatidis Folium. Moreover, hepatitis A, hepatitis B, cholecystitis, and cholelithiasis are listed in PharmDB-K, but not in TCM-ID. Allii Bulbus has been used for anthelmintic, toxication reduction, and itching in China. However, in Korea, there are other distinct reasons for Allii Bulbus use, such as cold, snake bites, diarrhea, edema, and pain. These results suggest that TKM provides other new potentials of herbs that are not covered by TCM.
Due to the reasons mentioned above, we have developed PharmDB-K, an integrated biopharmacological network database for TKM. PharmDB-K has three unique strengths: 1) it is an integrated TKM-Drug-Protein-Disease network; 2) it contains manually curated information about indicator compounds for herbs; 3) it has diverse tools for analysis.

Integrated TKM-Drug-Protein-Disease network
Although the number of articles about TKM has been increasing, most research has focused on profiling chemicals. So, scientific knowledge for analyzing and uncovering the mechanisms of actions is still insufficient. In order to overcome this limitation, fourteen different databases (ChEMBL, CTD, DCDB, DIP, DrugBank, Entrez Gene Interactions, GAD, MATADOR, MINT, OMIM, SIDER, T3DB, Traditional Knowledge Portal, and TTD), six pharmacopoeias, and published articles were integrated to build a bio-pharmacological network that connects compounds found in herbs to known drugs, diseases, proteins, and side effects (Fig 1, S1 Table) [ [11][12][13][14][15][16][17][18][19][20][21][22][23]. For data integration in a unified format, we adopted PubChem CID for drugs, Entrez Gene ID for proteins, MeSH descriptor for diseases and side effects, and Med CD number of Korean Traditional Knowledge Portal for TKMs. [24][25][26][27] PharmDB-K consists of five kinds of nodes: TKMs, drugs, diseases, proteins, and side effects. And it is composed of eight different relationship categories: TKM-Disease, TKM-Drug, Drug-Disease, Drug-Drug, Drug-Protein, Drug-Side Effect, Disease-Protein, and Protein-Protein (Table 1). We categorized FDA approved drugs and all types of compounds including experimental compounds, indicator compounds, and ingredient compounds of herbs into the Drug node because of their potential as a new drugs. So, the TKM-Drug relationship primarily explains profiles of indicator compounds, active compounds, and chemicals from herbs (Fig 2A).
Since TKM has been developed for over a thousand years, indications for the use of TKM are described as either old disease names or symptoms that do not exactly match modern medicinal terms. This has become a big obstacle in utilizing TKM for modern drug development. To overcome this problem, we converted these symptoms and disease terms in TKM into MeSH descriptors [27]. TKM-Disease relationship information was imported from Korean Traditional Knowledge Portal that originated from traditional Korean medical books, Donguibogam (published in 1613) and Ungokbonchohak (published in 2004), and literature ( Fig 2B) [24]. At present, PharmDB-K contains 342 MeSH descriptors for herbs and 3,721 MeSH descriptors in total. Although PharmDB-K is the first integrated network database for TKM, it is not the first and biggest database for herbs. However, PharmDB-K has some unique strengths. PharmDB-K integrates seven different databases along with literature, and predicted data have been  eliminated to collect only verified compound-protein interaction data. Furthermore, the compounds isolated from herbs were manually curated and converted into PubChem CIDs based on their names and structures [26]. Thereafter, PubChem CID has been used for compoundassociated data integration to avoid mismatch problems. Taken together, we believe that PharmDB-K contains a relatively small but more reliable Drug-Protein data set, and it can also provide inferred TKM-Protein links for further research (Fig 2C).

Indicator compounds: manually curated key compounds
A pharmacopoeia is a book containing information about standards and quality specifications for medicines and is published by a national or regional authority. In certain Asian countries, pharmacopoeias also contain indicator compound information for herbal medicines. The indicator compound information is used to identify and confirm medicinal performance characteristics. Additionally, they can provide valuable information for establishing solid connections between herbal medicines and modern medicinal chemistry. This indicator compound information has been collected from pharmacopoeias of five Asian countries: China, Japan, South Korea, North Korea, and Thailand [28][29][30][31][32][33][34]. The herbs that do not have indicator compounds in these pharmacopoeias were excluded from PharmDB-K. So, PharmDB-K currently contains 250 herbs that have indicator compounds. PharmDB-K contains more than 400 indicator compounds and about 5,000 compounds isolated from herbs. The chemical information was manually curated, and chemicals without available PubChem CIDs were ignored. Additionally, compounds known (or expected) to have medicinal benefits are referred to as "active ingredients", and these data were acquired from the literature. Indicator compounds work as major active compounds in some cases. Schizandrin is one of the main dibenzocyclooctadiene lignans present in Schizandrae Fructus (Fig 3A). According to Korean and Thai Pharmacopoeias, schizandrin is an indicator compound for Schizandrae Fructus. It has been demonstrated that schizandrin reduces protein levels of TNF-alpha and IL-4 and exhibits growth inhibition effect on human breast cancer cell lines [35,36]. We evaluated the antitumor effect of schizandrin compared with three randomly selected compounds exit in Schizandrae Fructus. Among them, only schizandrin significantly suppressed the cell viability in breast cancer cells ( Fig 3B). As shown in Fig 3C and 3D, the cell viability was reduced by schizandrin in a dose-and time-dependent manner. Collectively, these data suggest that schizandrin is likely the active compound of Schizandrae Fructus as an antitumor agent.
Fig 4A illustrates another interesting example regarding roots of Scrophulariae Radix, which are used as an anti-inflammatory agent [37]. It was reported that caffeic acid is one of active compounds in Scrophulariae Radix and has an anti-inflammatory effect by suppressing NF-kB and COX-2 (PTGS2) [38,39]. According to Chinese Pharmacopoeia, caffeic acid is also an indicator compound for Malvae Semen. Although Malvae Semen is used in the treatment of edema in South Korea, its mechanism of action is still unknown [24]. The therapeutic effect of caffeic acid on edema has already been demonstrated, and there are a number of shared proteins between caffeic acid and Malvae Semen including NFKB1, NFKB2, and PTGS2 [38]. It is possible, therefore, that caffeic acid could be the active compound in Malvae Semen. To evaluate the effect of caffeic acid on edema, we investigated the role of caffeic acid in immune system. Tumor-promoting activity of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces skin edema, epidermal hyperplasia and inflammation [40]. Pretreatment of HaCaT cells (a human keratinocyte cell line) with caffeic acid attenuated TPA-induced expression of COX-2 protein in a concentration-dependent manner (Fig 4B). The expression of COX-2 is transcriptionally regulated by several transcription factors including NF-κB. We examined the effects of caffeic acid on TPA-induced activation of NF-κB in HaCaT cells. Caffeic acid treatment significantly inhibited TPA-induced DNA binding of NF-κB and nuclear translocation of its active subunit of p65/RelA (Fig 4C and 4D). In addition, caffeic acid inhibited the subsequent degradation of IκBα in TPA-stimulated HaCaT cells (Fig 4E). Moreover, as shown in Fig 4F, the upregulation of il-8 (interleukin 8) mRNA transcript by tumor necrosis factor-α (TNF-α) was significantly reduced by caffeic acid in HaCaT cells. In summary, caffeic acid inhibited the activation of NF-κB which is a major transcription factor involved in the regulation of COX-2 expression in TPA-treated HaCaT cells. In addition, we also observed that caffeic acid blocked the expression of interleukin-8, cytokine considered to play a role under inflammatory situation. Therefore, these findings support our hypothesis that caffeic acid could be the major active compound of Malvae Semen for treatment of edema. These data suggest that PharmDB-K is a useful resource for narrowing down and predicting active compounds among compounds found in herbs and for establishing hypotheses on the functional mechanisms of herbs.  HaCaT cells were pretreated with caffeic acid (50 and 100 μM) for 1 hr, and then cells were exposed to TPA (100 nM) for additional 8 hr. (C) Cells were treated with TPA (100 nM) in the presence of caffeic acid (50 and 100 μM) for 2 hr. The NF-κB DNA binding activity was assessed by the gel-shift assay. The nuclear extracts were prepared and incubated with the radiolabeled oligonucleotides containing κB consensus Inferred protein links for TKM Previously, we developed the Shared Neighborhood Scoring (SNS) algorithm to generate inferred links [41]. Unfortunately, however, the SNS algorithm could not be applied to TKM since there was only a limited amount of known data regarding the TKM-Protein relationship. The probability of a connection between two nodes showed monotonic increase with "Shared nodes count" in PharmDB [41]. Based on this observation, inferred TKM-Protein relational data were generated using the number of shared nodes between them (Fig 5A). The numbers of inferred TKM-Protein relationships were 200,481, 123,382, and 7,501, based on "shared Diseases count", "shared Drugs count", and "shared Diseases and Drugs count", respectively. We collected known TKM-Protein relation data for 16 TKMs from the literature and they were used to validate the inferred links. The result was measured by ROC curves (Fig 5B). For "shared Diseases and Drugs count" cases, "Drugs count" was assigned with a weight of 2. AUC values for "shared Diseases count", "shared Drugs count", and "shared Diseases and Drugs count" were 0.726, 0.945, and 0.965, respectively. Among the inferred TKM-Protein relation based on "shared Diseases and Drugs count", a total of 7,501 relations shared at least two nodes from two different categories (e.g., one from Disease and one from Drug) (Fig 5C). And these types of relations were used as final inferred protein links for TKMs.
PharmDB-K predicted that Ginseng Radix and Angelicae Gigantis Radix may regulate the production of IL-6 and TNF-α, pro-inflammatory cytokines that are produced by macrophages for both innate and adaptive immunity. To validate this inferred TKM-Protein relation, Raw264.7 cells were stimulated with LPS in the presence of increasing doses of extracts from Ginseng Radix and Angelicae Gigantis Radix, ginsenoside Rb1 (an indicator compound for Ginseng Radix), and decursin (an indicator compound for Angelicae Gigantis Radix). The levels of LPS-induced IL-6 and TNF-α were all significantly decreased by the addition of Ginseng Radix extracts, Angelicae Gigantis Radix extracts, and decursin in a dose-dependent manner (Fig 5D, 5E and 5G). Addition of ginsenoside Rb1 also inhibited the production of IL-6, but the production of TNF-α was not affected by the same treatment (Fig 5F). These results demonstrate that Ginseng Radix, Angelicae Gigantis Radix, and their indicator compounds, ginsenoside Rb1 and decursin, regulate the production of IL-6 and TNF-α from macrophages as PharmDB-K inferred.

Tools: phExplorer and BioMart
PharmDB-K resources are provided through a web interface (Fig 6A and 6B). PharmDB-K contains comprehensive synonym data for TKM, Drugs, Diseases, and Proteins to facilitate the search. In addition to general browsing, the option of finding the shortest path between two nodes is also available. Since the data in PharmDB-K form a highly complex network, it is neither appropriate nor informative to browse PharmDB-K in a text format. So, we are providing PharmDB-K information with two different tools, phExplorer (a network visualization software) and BioMart web service (Fig 6C and 6D) [42]. With phExplorer, users can easily browse PharmDB-K data in an interactive and dynamic manner. BioMart is a freely available data federation framework for large collaboration projects [42] and allows users to access disparate and distributed databases and to build their own analysis pipelines using a single user interface.
sequence for the analysis of NF-κB DNA binding by EMSA. (D) Nuclear proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with p65 antibody. Lamin B was used as markers of nuclear proteins. (E) The cytosolic extracts prepared from cells incubated with TPA for 3 hr in the presence or absence of caffeic acid were immunoblotted with was analyzed by Western blotting to examine the expression of IκBα. (F) HaCaT cells were treated with TNF-α (20 nM) in the absence or presence of caffeic acid (100 μM) for 24 hr and then the isolated RNA was reverse-transcribed and amplified as described in Materials and Methods. Expression of il-8 and gapdh mRNA was measured by RT-PCR.

Discussion
Although TKM provides new potentials of herbs that are not covered by TCM, TKM has not been well recognized worldwide and there is no well-known TKM database. Therefore, we developed an integrated bio-pharmacological TKM database called PharmDB-K. One of the most frequently stated challenges in the development of new TKM-based drugs is discovering active compounds and target proteins. An objective of PharmDB-K was to build a comprehensive bio-pharmacological network to explore the potential targets and indications for TKMs. PharmDB-K has several distinct advantages over existing TCM/TKM databases. By integrating bio-pharmacological databases, PharmDB-K provides 1) potential active compounds of TKM; 2) inferred links between TKM and potential target proteins. One of the most valuable information in PharmDB-K is the indicator compound information which collected from pharmacopoeias of five Asian countries. Since the indicator compound information is used to verify medicinal performance of herbs in each country, these compounds have great potential to be active compounds. Thus, PharmDB-K is able to suggest functional mechanisms of herbs from connections of the indicator compound information in TKM with known protein-disease network. This approach would be beneficial in accelerating TKM-based drug discovery. The other key information that PharmDB-K can predict is target proteins. Usually, researches on TKMs have focused on the activity measurements of some enzymes that have been known to be PharmDB-K: Integrated Network Database for Traditional Korean Medicine related to particular diseases because target proteins could not be postulated. To overcome this obstacle, PharmDB-K generated inferred TKM-Protein relationships using commonly shared compounds and diseases. It is based on the basic principle that the connection probability of a link between two different nodes is roughly proportional to the number of nodes commonly shared between them in bio-pharmacological network [41]. As shown in Fig 5, target proteins for TKMs were successfully predicted using inferred links. We believe that the systematic approach based on integrated bio-pharmacological network, such as PharmDB-K, is a promising way to uncover hidden TKM-Protein relationships and to expedite the elucidation of TKM-mediated mechanisms for a successful drug discovery. With manual curation, PharmDB-K offers more reliable and comprehensive compound and indication information for TKMs. Furthermore, phExplorer and BioMart will be very useful not only to researchers unfamiliar with databases, but also to bioinformaticians who want to carry out analyses using multiple databases. In conclusion, PharmDB-K has been designed to introduce TKM to the cutting edge drug discovery research field. We believe that PharmDB-K provides new insights on TKM-originated drug development research. We intend to continue efforts to expand our database by mining and analyzing published articles, and we plan to import prescription information in the near future to adopt combinatorial therapy concepts as well.

Materials and Methods
Cell viability assay 2,000 cells of MDA-MB-231 (purchased from ATCC) which are cultured in RPMI media containing 10% fetal bovine serum (FBS) and 1% antibiotics, were seeded in 96 well plates and incubated for 12 hr. Vanillic acid, L-Malic acid, Schizandrin and Syringin (Eleutheroside B) were purchased from Sigma. After 12 hr, they were dissolved in DMSO and treated in 5% FBScontaining media dose dependently. After 24, 48 and 72 hr, MTT reagent (5mg/ml, Sigma) was added to each well, and the plates were incubated in 37°C for 2 hr to check the cell viability. Purple-colored formazan dissolved in DMSO was analyzed spectrophotometrically at 570nm using ELISA plate reader. All the experiments were repeated three times.

Western blot analysis
HaCaT cells were kindly gifted from Dr. Zigang Dong (Hormel Institute, University of Minnesota, MN, USA) and were maintained routinely in DMEM medium supplemented with 10% fetal bovine serum and a 100 ng/ml penicillin/streptomycin/fungizone mixture at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells were incubated with TPA in the presence or absence of caffeic acid. After treatment, cell lysates were prepared according to the procedure described earlier [43]. The protein concentration was determined by using either the BCA or the BioRad protein assay kit. In some experiments, cytosolic and nuclear proteins were obtained from cells [43]. Protein samples (30-50 μg) were subjected Western blot analysis. Membranes were probed separately with antibodies against COX-2 (RB-9072-P1; Thermo Scientific, Rockford, IL), actin (sc-47778; Santa Cruz Biotechnology, CA, USA), IκBα (sc-847; Santa Cruz), p65 (clone D14E12, Cat No. 8242; Cell Signaling Technology, Beverly, MA, USA), Lamin B (sc-6216; Santa Cruz), and α-tubulin (sc-5286, Santa Cruz), and then blots were visualized according to the procedure described previously [43].

Electrophoretic mobility gel shift assay (EMSA)
The EMSA for NF-κB DNA binding was performed using a DNA-protein binding detection kit, according to the manufacturer's protocol (Gibco). The nuclear extract was prepared from cells incubated with TPA in the presence or absence of caffeic acid. The NF-kB oligonucleotide probe 5'-GAG GGG ATT CCC TTA-3' was labeled with [γ-32P] ATP. Oligonucleotide probes containing NF-κB consensus sequences were obtained from Promega (Madison, WI, USA). The transcription factor-DNA binding assay was performed as described previously [43].


Cloning and Functional Analysis of tri14 in Trichoderma brevicompactum

Corresponding Author: Xiao-Ping Yu College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang Province 310018, P.R. China Tel: +86-571-86836006 Fax: +86-571-86836006 E-mail: yxp@cjlu.edu.cn Abstract: Trichodermin, one of the trichothecenes, is a potential antifungal antibiotic and a protein synthesis inhibitor. Trichodermin is synthesized mainly by Trichoderma brevicompactum, but the biosynthetic pathway has yet been completely elucidated. Our earlier transcriptome assay on the trichodermin synthesis in T. brevicompactum 0248 identified a unique tri14 transcript, designated Tbtri14, that was co-regulated with other tri cluster genes. However, the function of this gene remains unknown. In this study, the full cDNA and DNA sequence of Tbtri14 was isolated from T. brevicompactum 0248. It was found the Tbtri14 gene encodes 364 amino acids with a 59 bp intron. For gene function study, the Tbtri14 was deleted in the 0248 strain. The silencing of Tbtri14 dramatically increased the transcription of Tbtri11, it had little influence on the expression of Tbtri4 and Tbtri5 as well as the production of trichodermin in △tri14 deletion mutants. This implied that Tbtri14 may not be directly involved in trichodermin biosynthesis. Instead, it may act as a gene regulator, such as a repressor for Tbtri11.

Introduction
Trichothecenes are a large group of sesquiterpenoidderived secondary metabolites and have mainly been synthesized by Fusarium and certain other fungal genera like Stachybotrys, Myrothecium, Trichoderma and Trichothecium (Shentu et al., 2014;Wilkins et al., 2003). The trichothecene biosynthetic pathway in Fusarium has been studied in detail and about 18 genes were found to be involved (Kimura et al., 2007). Most of the Fusarium genes directly involved in trichothecene biosynthesis are positioned at a locus designated the core trichothecene biosynthetic gene cluster (tri cluster) (Brown et al., 2004). Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA) that are synthesized mainly by T. brevicompactum and T. arundinaceum, respectively. Trichodermin and HA both have strong antifungal activities against phytopathogens such as Rhizoctonia solani and Botrytis cinerea (Moradi et al., 2012;Tijerino et al., 2011;Malmierca et al., 2012). Furthermore, HA shows extremely similar chemical structure to trichodermin. The biosynthetic pathway of trichothecenes by Trichoderma has yet been completely elucidated and the function of many genes in the Trichoderma is little known.
It was revealed that Trichoderma species also have a trichothecene biosynthetic gene cluster with orthologues of seven genes (tri3, tri4, tri6, tri10, tri11, tri12 and tri14) in the Fusarium tri cluster and tri4, tri11 are directly involved in trichothecenes biosynthesis . The tri5 gene (encoding the trichodiene synthase), located outside the cluster, is responsible for the first committed step in HA biosynthesis . It keeps unkown that other genes in tri cluster involve in trichothecenes biosynthesis or not.
The tri14 gene is located adjacent to the tri cluster in Trichoderma, it was considered as a part of the cluster because its transcription was co-regulated with the other cluster genes (Dyer et al., 2005;Brown et al., 2002). However, the function of tri14 remains unknown so far. In this study, the full sequence of Tbtri14 cDNA was cloned from T. brevicompactum 0248 and analyzed. For study of the gene function, Tbtri14 in strain 0248 was deleted. The influence on the trichodermin production as well as the expression levels of three genes directly involved in trichothecene biosynthesis Tbtri4, Tbtri5 and Tbtri11 were investigated.

Strains, Plasmids and Culture Conditions
Escherichia coli JM109 was used for plasmid construction and maintenance. The E. coli was cultivated at 37°C in Luria-Bertani (LB) medium containing 100 µg mL −1 ampicillin (Brayner et al., 2006).
T. brevicompactum 0248, isolated in our previous study and deposited in China General Microbiological Culture Collection Center (CGMCC No. 6985), was used as the original strain and transformation host. The cultivation of strain 0248 was carried out as described in the literature (Shentu et al., 2014).
The plasmid pSilent-1, kindly provided by Fungal Genetics Stock Center (FGSC) in USA, carries a hygromycin-resistant gene (hph) cassette. The plasmid pCAMBIA0380, obtained from Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, was used for ATMT.
All the primers used in this study were synthesized by Shanghai Sunny Biotechnology Co. Ltd and listed in Table 1.

Cloning and Sequencing of the tri14 Gene
Total RNA was extracted from fresh mycelia by using Spin Column Fungal Total RNA Purification Kit (Sangon, Shanghai, China). Genomic DNA of the strain 0248 was extracted by using Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). The Tri14 cDNA was amplified from the total cDNA by Polymerase Chain Reaction (PCR) with the primer 14-F/14-R and subcloned into the pMD18-T vector for sequence verification.

Construction of Recombinant Vector
A 1-kb upstream DNA fragment before the tri14 gene start codon was amplified using total DNA as template and following primers pf1 and pr2, the forward primer pf1 containing BstXI site and the reverse primer pr2 containing a 22 bp reverse complementary sequence of hph cassette. To obtain the hph expression cassette, hygromycin resistance gene was amplified by PCR using plasmid pSilent-1 as the template with the primers pf3 and pr4. Similarly, another DNA fragment downstream of the tri14 gene stop codon was amplified using total DNA as template and following primers pf5 and pr6, the forward primer pf5 containing a 22 bp overlapping sequence of hph gene and the reverse primer pr6 containing a BglII site (Fig. 1). Then, the three linearised fragments were equimolarly mixed and cyclised in a fusion PCR to generate a gene knockout fragment (Cao et al., 2014). The gene knockout fragment was cloned into the BstXI/XmaI sites of pCAMBIA0380 to generate a vector designated pCAMBIA0380-kt.

Transformation of T. brevicompactum 0248
The expression vector pCAMBIA0380-kt was introduced into A. tumefaciens AGL-1 by heat shocking at 37° for 5 min after pretreatment with liquid nitrogen for 3 min. The primer H-F/H-R was used to screen the positive cloning that was named as A. tumefaciens AP03HS. The AP03HS-mediated transformation of T. brevicompactum 0248 was then performed as described earlier (Almeida et al., 2007;dos Reis et al., 2004).

Genetic Stability Analysis of Transformants
The transformants were purified by single-spore isolation and analyzed by PCR and qRT-PCR to confirm the deletion in the Tbtri14 gene. The selected transformants were cultured in PDA plates without hygromycin B for 72 h. The fresh mycelia was picked up and inoculated into new PDA plates without hygromycin B and incubated for another 60 h. After this procedure was repeated for five times, mycelia from each transformants were inoculated in PDA plates with 100 µg mL −1 hygromycin B and their growth was observed.

Analysis of Gene Expression by using qRT-PCR
Total cDNA was extracted as described above and diluted 10 times before used. qRT-PCRs were performed in a Applied Biosystems StepOnePlus Real-Time PCR System using a SYBR ® Premix Ex Taq TM (Tli RHaseH Plus) reagent (TaKaRa, Dalian, China). The primers used in qRT-PCRs were given in Table 1. All qRT-PCRs for each gene were carried out in triplicate within a plate and the β-tubulin gene of T. brevicompactum 0248 was used as the reference gene . The quantification of the Tbtri4, Tbtri5, Tbtri11 and Tbtri14 gene expression was analyzed by the 2 -∆∆Ct method.

Analysis of Trichodermin by Using GC
The fermentation broth was separated from the mycelia by filtration using a Buchner funnel and extracted exhaustively with ethyl acetate (v/v, 1:2). The organic fractions were combined and evaporated to dryness in a vacuum at 50°C. The recovered residues were re-suspended in methanol and subjected to Gas Chromatographic (GC) analysis for the quantification of trichodermin .

Results
Sequence Analysis of Tbtri14 from T. brevicompactum 0248 The full cDNA sequence (1,092 bp) and DNA sequence (1,151 bp) of Tbtri14 from T. brevicompactum 0248 were obtained in this study. The nucleotide sequence of tri14 cDNA (Supplementary 1) and its predicated protein has been submitted to GenBank under accession no. KJ957748. It was predicted that the molecular mass of TbTRI14 is 40.11 kDa and the isoelectric point is 4.62 by using ProtParam of ExPASy. The analysis of the TbTRI14 sequence by using InterPro of EMBL-EBI indicated that there was a noncytoplasmic domain, a signal peptide and a polypeptide belonging to the calcium-dependent phosphotriesterase superfamily. A tertiary structure analysis of TbTRI14 by using CPHmodels of CBS predicted that it may be a protein with a barrel structure. The deduced protein sequence of TbTRI14 was compared with those of the Tri14 from T. arundinaceum, S. chlorohalonata and F. sporotrichioides, the identities were 99, 67 and 64%, respectively. However, the function of Tri14 proteins in these three strains was also unknown Brown et al., 2002).

Genetic Stability Analysis of Transformants
With ATMT, a total of 58 hygromycin-resistant transformants were obtained. Screened by PCR, three were identified which had the hygromycin cassette replacing the ORF of Tbtri14 (data not show), designated kt26, kt31, kt52. The genetic stability analysis of the three transformants showed that the lossof-function mutations could be passed down steadily to the next generation. The mutants were identical to each other with respect to the phenotypes observed.

Effect of Tbtri14 Deleting on the Expression Levels of Tbtri4, Tbtri5, Tbtri11
The relative expression levels of Tbtri14 and other three key genes involved in trichothecenes biosynthesis Tbtri4, Tbtri5 and Tbtri11 in △tri14 knockout mutant (kt26) and wild strain 0248 were detected. As shown in Fig. 2A, the expression of the Tbtri14 gene in the wild strain changed with time (40, 52, 64, 76 and 88 h), with the highest expression level observed at 40 h. At each time point, the Tbtri14 expression level was not detected in △tri14 knockout mutants. This indicated that the Tbtri14 gene was completely silenced in △tri14 deletion mutants.
Surprisingly, there was a significant increase in the Tbtri11 expression in △tri14 deletion mutants than that in wild strain at 40, 52, 64, 76 and 88 h (Fig. 2B). In contrast to Tbtri11, the expressions of Tbtri4 (Fig. 2C) and Tbtri5 (Fig. 2D) were hardly affected by the deleting of the Tbtri14 gene; no significant difference in the expression levels of these three genes between the transformant and wild strain was detected.

Effect of tri14 Deleting on the Production of Trichodermin
Fermentation broths from transformant kt26 and wild strain 0248 were assayed for trichodermin production. As shown in Fig. 3, the trichodermin yields produced by kt26 transformant was similar to those produced by wild strain at different time. For both transformant and wild strain, the trichodermin was rapidly synthesized from 40 to 64 h. After 64 h, no increase in the trichodermin concentrations was found.

Discussion
Transcriptome sequencing and gene expression analysis of the 0248 strain identified a unique Tbtri14 transcript that is highly similar to the Fusarium tri14 gene. Furthermore, Tbtri14 was shown to have a significant differential expression under the trichodermin-producing condition. The cDNA and DNA sequence of Tbtri14 was cloned and analyzed in this study. Subsequently, the function of the Tbtri14 gene was also researched in the 0248 strain.
It was found that while the expression levels of Tbtri14 in the three △tri14 deletion mutant was completely silenced, the yields of trichodermin were comparable between the mutant and the wild strain (Fig.  3). This indicated that Tbtri14 may be indirectly involved in the trichodermin synthesis in T. brevicompactum. Similar to this finding, deletion of the Fstri14 gene in F. sporotrichioides was found not to change the synthesis of T-2 toxin that also belongs to the trichothecenes family, which implied that Fstri14 may not be required for the T-2 toxin synthesis in F. sporotrichioides (Brown et al., 2002). The study of the tri14 function in F. graminearum showed that Fgtri14 was required for the wild-type levels of virulence on wheat, but not for the synthesis of deoxynivalenol (another trichothecene mycotoxin) in cracked corn culture.
With the deleting of Tbtri14, the expression levels of Tbtri11 were significantly increased in the transformant. This implied that the expression of Tbtri14 may repress the transcription of Tbtri11 in T. brevicompactum 0248. This result was consistent with the earlier assumption that the Fgtri14 gene in F. graminearum may act as a regulator of the deoxynivalenol synthesis by affecting the transcription of other relevant genes (Dyer et al., 2005).
The tri11 gene in T. arundinaceum (Tatri11) has been shown to encode a requisite enzyme, TaTRI11, in the HA biosynthetic pathway; it catalyzes the C-4 hydroxylation of trichothecene . As we know that both trichodermin and HA can be synthesized by the Trichoderma species and their chemical structure are extremely similar (Degenkolb et al., 2008). The only difference is the side group at the C-4 position; trichodermin has an acetyl group while HA has an octa-2,4,6-trienedioic acid (Malmierca et al., 2012). Thus, it is very likely that the trichodermin in T. brevicompactum shares the similar biosynthetic pathway with the HA in T. arundinaceum. In other words, the Tbtri11-encoded TbTRI11 may also catalyze the C-4 hydroxylation of trichothecene in the biosynthetic pathway of trichodermin, as does the TaTRI11 in HA biosynthesis. However, with the significant enhancement of the Tbtri11 expression in the T. brevicompactum transformant, the yield of trichodermin was not improved. This implied that the expression of Tbtri11 is probably not a rate-limiting step in trichodermin biosynthesis. Other tri cluster genes (e.g., Tbtri3, Tbtri4 or Tbtri5), of which the expression was not affected by the deleting of Tbtri14, may play critical roles in trichodermin biosynthesis. However, it was recently reported that overexpression of Tatri4 in T. arundinaceum did not enhance the HA biosynthesis either (Malmierca et al., 2012). In contrast, overexpression of Tbtri5 in T. brevicompactum IBT 40841 resulted in an increased production of trichodermin. These results indicated that Tbtri5 is most likely a critical gene that limits the trichodermin synthesis in Trichoderma (Tijerino et al., 2011).

Conclusion
Tbtri14 may not be required for trichodermin biosynthesis in T. brevicompactum 0248. Instead, it may act as a regulator of other relevant genes, such as a repressor for Tbtri11. The results obtained from this research provide new information for future study on the biosynthesis pathway of trichodermin or other trichothecenes.


s2fieldsofstudy: Biology Medicine

text:
Moderate exercise training is more effective than resveratrol supplementation for ameliorating lipid metabolic complication in skeletal muscle of high fat diet-induced obese mice

[Purpose] The aim of this study was to compare the effectiveness of moderate exercise training or resveratrol supplementation with a low fat diet on lipid metabolism in the skeletal muscle of high fat diet-induced obese mice. [Methods] C57BL/6J mice (5 weeks old, n = 30) were fed a high fat diet (45% fat) for 8 weeks first to make them obese. Afterward, all the mice were fed a low fat diet during 8 weeks of intervention with moderate exercise training and resveratrol supplementation. Before the intervention, the mice were separated into 3 groups: low-fat diet control (HLC; n = 10), low fat diet with resveratrol (HLR; n = 10) or low fat diet with exercise (HLE n = 10). The exercise group (HLE) performed treadmill running for 30-60 min/day at 10-22 m/min, 0% grade, 5 times/week for 8 weeks, while the resveratrol group (HLR) received a daily dose of resveratrol (10 mg/kg of body weight), 5 days/week for 8 weeks. [Results] Body weight was significantly reduced in HLE. Further, the lipogenesis marker SREBP and the inflammatory cytokine TNF-α were significant reduced in HLE. However, there was no significant effect from resveratrol supplementation with a low fat diet. Taken together, exercise training with a low fat diet has the positive effect of ameliorating lipid disturbance in the skeletal muscle of high fat diet-induced obese mice. [Conclusion] These findings suggest that exercise training with a low fat diet is most effective to improve lipid metabolism by reducing lipogenesis and inflammation in the skeletal muscle of high fat diet-induced obese mice.

INTRODUCTION *
Recently, the polyphenol resveratrol (3,5,4-trihydroxystilbene) was spotlighted as a health functional food throughout the world. Resveratrol is known to increase the expression of enzymes related to nitric oxide synthesis, reduce blood pressure and protect heart function by inhibiting platelet aggregation [1][2][3]. Further, previous studies reported that resveratrol delayed or protected against neuro degenerative disease and stroke induced brain damage [4,5]. Other studies showed that resveratrol prevents aging and increases the life span by activating sirtuin, which regulates gene transcription, DNA repair, cell signaling and apoptosis as its key role in promoting longevity [6,7].
Ungvari et al. [8] reported that resveratrol improves endurance capacity by increasing mitochondria number, reducing reactive oxygen species (ROS) and ameliorating lipid metabolism. This result was supported by the finding that resveratrolmediated activation of AMPK increased intercellular NAD+ level [9,10] and decreased PGC-1α (peroxisome proliferator activated receptor-γ coactivator-1α) acetylation through deacetylation of SIRT-1 (silent mating type information regulation 2 homolog) [11,12]. In addition, resveratrol contributes to changing the type of muscle twitch fiber and PGC-1α activation by improving the function of mitochondria biogenesis [13]. Surprisingly, the maximal oxygen consumption was significantly increased and average running distance was about 2 times as long in the resveratrol supplementation group compared to the control group [13]. In obese research, it was reported that resveratrol supple-  [14]. In human studies, it was shown that resveratrol supplementation (150 mg/kg) in obese individuals significantly increased citrate synthase activity but not in the control group. However, there was no significant difference in mitochondria biogenesis and aerobics capacity [14]. Excessive nutrient-induced accumulated fat is known to result in chronic low inflammation, especially the inflammatory cytokines TNF-α (tumor necrosis factor-α) and interleukin-6 (IL-6), which are higher in obese individuals than in normal weight individuals [15,16]. Also, it has been reported that these inflammatory cytokines inhibit lipid oxidation in skeletal muscle, but increase lipid synthesis. Borst et al. reported that adipose tissue-induced TNF-α increased the enzyme activity related to lipid synthesis such as SREBP-1c (sterol regulatory element binding protein-c), ACC (acetyl-CoA carboxylase) and FAS (fatty acid synthase) [17].
On the other hand, previous studies on diet intervention for obese-induced metabolic complications reported that low fat and nutrient balanced diets improved pro-inflammatory milieu through the reduction of CRP, IL-6, IL-18, TNF-α, insulin sensitivity, endothelial function, metabolic syndrome, Type 2 DM and morbidity of CHD (coronary heart disease) [23,24].
Similarly, Calder et al. showed that a low fat diet was a key factor for positive changes in weight loss, lipid metabolism and inflammatory cytokine response [25]. Further, it was suggested that exercise intervention was more effective than low fat diet treatment for reducing hepatic steatosis, but both exercise and a low fat diet were effective in improving the anti-inflammatory response and metabolic outcomes [26,27].
Nonetheless, there are few studies comparing the effects of exercise training and resveratrol supplementation. In addition, there is little research to our knowledge, comparing the effects of exercise training and resveratrol supplementation using a low fat diet as a base in high fat diet-induced obese mice. The effect of a low fat diet has been well documented and a low fat diet can be easily applied as a treatment for obese-induced metabolic complications. Also, in previous rodent studies, there were positive effects observed with resveratrol dosage (25-30 mg/kg/day or 400 mg/kg/day). These doses for mice would be equivalent to 2,100 or 28,000 mg/kg/day for humans (70 kg). Thus, these doses are unlikely to be obtained from natural or functional foods [9,13].
We hypothesized that if a low fat diet were combined with either exercise training or resveratrol supplementation, the efficacy of resveratrol as an exercise mimicker would be clearly identified. Consequently, we investigated the effect of either moderate exercise training or resveratrol supplementation with a low fat diet on lipid metabolism in the skeletal muscle of high fat diet-induced obese mice.

Animals care and diet
Male C57BL/6 mice (5-weeks-old) were purchased from Raonbio (Seoul, Korea) and housed in standard cages placed in a room at 22 ± 2.0℃, 55 ± 10% relative humidity, and a 12 hour-light/12 hour-dark cycle. All mice consumed a commercial diet and tap water ad libitum for 1 week. Mice were randomly assigned to three groups: (1) HFD for 8 weeks and low-fat diet for 8 weeks as the control (HLC; n = 10); (2) HFD for 8 weeks and low-fat diet for 8 weeks with resveratrol (HLR; n = 10); (3) HFD for 8 weeks and low-fat diet for 8 weeks with exercise (HLE; n = 10). The mice were weighed every week during the experimental period. Commercially available dried resveratrol (Sigma Aldrich, St Louis, MO) was used, dissolved in dimethyl sulfoxide. The control (HLC) and exercise (HLE) groups were orally administered with 100 μl of dimethyl sulfoxide as a vehicle. The resveratrol group was orally gavaged with resveratrol (10 mg/kg body weight) dissolved in dimethyl sulfoxide. This dose is similar to the amount that humans consume from natural or functional foods. Each treatment was administered once per day, 5days/week for 8 weeks [27,28].

Exercise protocol
Exercise training was performed on a motor treadmill at moderate intensity for 8 weeks, 5 days/week for 30-60  Warm-up for all exercises was conducted for 5~10 minutes at the speed of 8-10 m/min and the main exercise was performed at the speed of 10-22 m/min for 30~60 minutes, following the principle of cumulative overload. This exercise intensity corresponds to 65-70% of maximal oxygen uptake [29]. To control for any stress associated with the training protocol, animals in the control and resveratrol groups were exposed to the same noise and handling as the exercise groups.

Tissue preparation
The animals were sacrificed 24 hours after the 8 th week of exercise training. Skeletal muscle (gastrocnemius muscle) tissue was stored at -70℃ until analysis. This study used the gastrocnemius muscle because it was determined that aerobic exercise for 8 weeks can affect the muscles.

RNA extraction and quantitative RT-PCR
Total RNA was isolated from the skeletal muscle tissue of each mouse using Trizol (Invitrogen, Carlsbad, CA, USA). Quantitative real-time RT-PCR was conducted using the Biorad CFX96(Biorad, USA) PCR system. The mRNA levels were normalized to the β-actin mRNA levels.

Statistical methods
The data was analyzed using SPSS ver. 20.0 (SPSS, Inc., Chicago, IL, USA) to obtain means ± standard deviations. To investigate the effects of moderate exercise and resveratrol intake on body weight, inflammation, and lipid metabolism in skeletal muscle, all the groups were compared by a one-way ANOVA. The differences were considered statistically significant at α = 0.05. Fig. 1 shows body weight in the low fat diet, resveratrol supplementation and moderate exercise training groups following consumption of a low fat diet for 8 weeks after 8 weeks of a high fat diet was used to induce obesity in mice. The body weight of HLE was significantly reduced compared to HLC and HLR (p < 0.05, Fig. 1). The TNF-α mRNA expression of HLE was significantly reduced compared to HLC and HLR (p < 0.05, Fig. 2).    Fig. 3 shows lipolysis markers mRNA expression in low fat diet, resveratrol supplementation and moderate exercise training groups following consumption of a low fat diet for 8 weeks after 8 weeks of a high fat was used to induce obesity in mice. Fig. 4 shows the mRNA expression of lipogenesis markers in low fat diet, resveratrol supplementation and moderate exercise training groups following consumption of a low fat diet for 8 weeks after 8 weeks of a high fat diet was used to induce obesity in mice.

Lipogenesis mRNA expression
SREBP mRNA expression of HLE was significantly reduced compared to HLC and HLR (p < 0.05, Fig. 4).

DISCUSSION
Since calorie restriction is known to increase life span, the popularity of one meal per day, low calorie consumption and interval fasting diets has increased.
A previous research reported that the effects of calorie restriction are caused by a protein called sirtuin [30]. Sirtuin is activated by NAD+-dependent deacetylases during the fasting condition in the brain, liver and kidney [31]. Further, it has been shown that sirtuin regulates metabolism and gene expression by stimulating the activation of SIRT-1 deacetylation. It even recovers damaged genes by activating PGC-1α, inhibiting weight gain and aging [6,11,13]. These results were supported by studies reporting a reduction in body weight changes and increased life span in the artificially sirtuinactivated group compared to the control group. However, after treatment with high carbohydrate or high fat diets, the positive effects of sirtuin vanished [6,30,31].
It seems that the reason why resveratrol research has been spotlighted may be related to the increment of SIRT-1. Interest in resveratrol supplementation increased because the effects coincided with those of calorie restriction [6]. Also, research interest in resveratrol was dramatically increased after a few studies reported that resveratrol supplementation could mimic the effects of exercise such as body weight reduction, mitochondria biogenesis improvement, endurance performance enhancement and energy consumption increment [5,7].
Body weight in this study was significantly reduced by moderate exercise training but not by resveratrol supplementation. This result contrasted with a previous study that reported significant weight reduction following resveratrol supplementation (200-400 mg/kg/day) in obese mice [13]. However, our previous results also showed no significant effect on body weight. In this research, 10 mg/kg each of quercetin and resveratrol were injected into high fat dietinduced obese mice for 8 weeks. The body weight of the group was then compared to the moderate exercise training group. Body weight was significantly reduced only in the exercise group, which coincides with other studies [28,32,33]. Further, it seems that the dose of resveratrol was not enough to reduce body weight.
It seems that even if the effect of polyphenols such as quercetin and resveratrol depends on the amount used, intervention with moderate exercise training is more effective in reducing body weight.
Adipocyte-produced inflammatory cytokines such as IL-6 and TNF-α have been reported to inhibit lipid oxidation but to increase lipid synthesis in skeletal muscles. Especially, TNF-α activates SREBP-1c, ACC and FAS, which are related to lipid synthesis. However, ACOX and CPT-1 were inhibited, which deteriorated insulin sensitivity [17,18].
In this research, TNF-α gene expression was significantly decreased in HLE compared to HLC and HLR, but there was no significant difference in IL-6 and MCP-1 gene expression. These results show that resveratrol supplementation had no positive effects on inflammatory cytokine production compared to moderate exercise training. Further, CPT-1 and ACOX were not significantly different but were slightly increased only in the HLE group. There was no change in the HLR group. The lipogenesis markers ACC, FAS and SREBP were slightly decreased in HLE, but only SREBP was significantly lower in HLE. In contrast, there was no significant difference in HLR. This result shows that moderate exercise training might inhibit lipid synthesis and promote lipid oxidation in the skeletal muscle of high fat diet-induced obese mice.
In addition, in the review of our previous research that compared the effects of moderate exercise training and quercetin supplementation for 8 weeks in 12-week high fat diet-induced obese mice, SREBP, FAS and ACC were significantly decreased in the exercise group compared to the polyphenol supplementation group. At the same time, LEPTIN and ACOX in exercise group were positively changed [33].
In a similar experiment, we analyzed the effects of either resveratrol supplementation or moderate exercise training on inflammation cytokines, mitochondria biogenesis, lipolysis and lipogenesis markers in the soleus and tibialis anterior muscles after exhaustive exercise in high fat diet-induced obese mice. We found that IL-6, TNF-α and MCP-1 were significantly reduced and so were MCD, ACC and SREBP-1 [32].
In previous studies of obese animals, polyphenol compounds such as resveratrol and quercetin appeared to mimic the effects of calorie restriction or exercise training. However, the dosage, duration and side effects of polyphenols should be studied further [5]. The effects of resveratrol with a low fat diet did not exceed the effects of only a low fat diet in this study. On the other hand, the effects of moderate exercise training with a low fat diet were much higher compared to either a low fat diet alone or resveratrol with a low fat diet in terms of improving lipid metabolism in the skeletal muscles of high fat diet-induced obese mice. This research evaluated only the resveratrol dose (10 mg/kg) similar to the amount that humans can obtain from natural or functional foods. The effect of resveratrol supplementation was not enough to reduce body weight, whereas moderate exercise training had a positive effect.
In conclusion, moderate exercise training combined with a low fat diet was more effective than resveratrol supplementation with a low fat diet in ameliorating lipid metabolic complications in the skeletal muscles of high fat diet-induced obese mice.