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Find out more about progress in countries and regions
Governments play a fundamental role in protecting the health of women and children through improving policies and programmes that support and enhance breastfeeding. The Global Breastfeeding Collective (the Collective), led by WHO and UNICEF, has identified seven policy action priorities, each with an indicator and a set target to be achieved by 2030.
The Global Breastfeeding Collective has developed a country scorecard to track progress on seven policy actions and on breastfeeding practices within countries. It examines these indicators at the national and global levels. The Scorecard is designed to encourage and document progress on the promotion, protection and support of breastfeeding. Each action has a progress indicator and target – to be met by 2030 – which serve as a call for action for policymakers worldwide to make breastfeeding support a collective national priority.
The Global Breastfeeding Scorecard was first introduced by the Collective in 2017 and has been updated annually ever since.
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Q: Transpose rows into columns using PowerShell I having a hard time,again, trying to transpose row into columns.
The original code comes from @Mathias R. Jessen, Powershell transpose rows into columns but now I need to add some additionals columns in the csv file. I would like to have (I don't mind about the order)
EndTime,ActualStartTime,"MachineName1", "MachineName2",  MachineNameX",ElapsedTime,FinalJobStatus
 #1.csv
 #MachineName, TotalDataSizeBytes, ActualStartTime,EndTime,ElapsedTime,FinalJobStatus
 #SERVER1, 322349304901, 28/02/2016 23:00:03, 29/03/2016 23:33:23, 222,OK
 #SERVER1, 322349304902, 26/02/2016 23:00:03, 27/03/2016 23:33:24, 222,OK
 #SERVER2, 322349304903, 28/02/2016 23:00:01, 29/03/2016 23:33:25, 11, OK
 #SERVER2, 322349304904, 26/02/2016 23:00:01, 27/03/2016 23:33:26, 122,OK
 #

 #2.csv (Desired output)
 #EndTime","ActualStartTime","SERVER1","SERVER2",ElapsedTime,FinalJobStatus
 #"29/03/2016 23:33:23","28/02/2016 23:00:03","322349304901",222,OK
 #"27/03/2016 23:33:24","26/02/2016 23:00:03","322349304902",222,OK
 #"29/03/2016 23:33:25","28/02/2016 23:00:01",,"322349304903",11,OK
 #"27/03/2016 23:33:26","26/02/2016 23:00:01",,"322349304904",122,OK

$Rows = Import-Csv c:\_Scripts\1.csv
$MachineNames = $Rows |Select-Object -ExpandProperty MachineName |Sort -Unique

$ConsolidatedRows = $Rows |Group-Object EndTime |ForEach-Object {
    $NewRowProperties = @{ EndTime = [DateTime]::Parse($_.Name) }
    foreach($Row in $_.Group)
   {
    $NewRowProperties.Add($Row.MachineName,$Row.TotalDataSizeBytes)
   }
   New-Object psobject -Property $NewRowProperties
}
$ConsolidatedRows |Select-Object @("EndTime";$MachineNames) |
 Sort-Object EndTime |Export-Csv c:\_Scripts\2.csv -NoTypeInformation

Edit1. I have succeeded:

A: $Rows = Import-Csv c:\_Scripts\1.csv
$MachineNames = $Rows |Select-Object -ExpandProperty MachineName |Sort -Unique
$ConsolidatedRows =""
$NewRowProperties =""

$ConsolidatedRows = $Rows |Group-Object EndTime |ForEach-Object {
$NewRowProperties = @{ EndTime = [DateTime]::Parse($_.Name) }
foreach($Row in $_.Group)
{
$NewRowProperties.Add("ActualStartTime",$Row.ActualStartTime)
$NewRowProperties.Add("ElapsedTime",$Row.ElapsedTime)
$NewRowProperties.Add($Row.MachineName,$Row.TotalDataSizeBytes)
$NewRowProperties.Add("FinalJobStatus",$Row.FinalJobStatus)
}
New-Object psobject -Property $NewRowProperties

}

$ConsolidatedRows |Ft
$ConsolidatedRows |Select-Object @("EndTime";$MachineNames;"ActualStartTime";"ElapsedTime";"FinalJobStatus")
|Sort-Object EndTime |Export-Csv c:\_Scripts\2.csv -NoTypeInformation

redpajama_set_name: RedPajamaStackExchange


text:
filter your search All ContentAll JournalsDiabetes
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Research Design and Methods
Metabolism| April 16 2021
l-Theanine Activates the Browning of White Adipose Tissue Through the AMPK/α-Ketoglutarate/Prdm16 Axis and Ameliorates Diet-Induced Obesity in Mice
Wan-Qiu Peng;
Wan-Qiu Peng
1Key Laboratory of Metabolism and Molecular Medicine of the Ministry of Education, Department of Biochemistry and Molecular Biology of School of Basic Medical Sciences and Department of Endocrinology and Metabolism of Zhongshan Hospital, Fudan University, Shanghai, China
Gang Xiao;
Gang Xiao
Bai-Yu Li;
Bai-Yu Li
Ying-Ying Guo;
Ying-Ying Guo
Liang Guo;
Liang Guo
2School of Kinesiology, Shanghai University of Sport, Shanghai, China
Corresponding authors: Qi-Qun Tang, qqtang@shmu.edu.cn, and Liang Guo, guoliang@sus.edu.cn
Qi-Qun Tang
Diabetes 2021;70(7):1458–1472
https://doi.org/10.2337/db20-1210
Wan-Qiu Peng, Gang Xiao, Bai-Yu Li, Ying-Ying Guo, Liang Guo, Qi-Qun Tang; l-Theanine Activates the Browning of White Adipose Tissue Through the AMPK/α-Ketoglutarate/Prdm16 Axis and Ameliorates Diet-Induced Obesity in Mice. Diabetes 1 July 2021; 70 (7): 1458–1472. https://doi.org/10.2337/db20-1210
l-Theanine is a nonprotein amino acid with much beneficial efficacy. We found that intraperitoneal treatment of the mice with l-theanine (100 mg/kg/day) enhanced adaptive thermogenesis and induced the browning of inguinal white adipose tissue (iWAT) with elevated expression of Prdm16, Ucp1, and other thermogenic genes. Meanwhile, administration of the mice with l-theanine increased energy expenditure. In vitro studies indicated that l-theanine induced the development of brown-like features in adipocytes. The shRNA-mediated depletion of Prdm16 blunted the role of l-theanine in promoting the brown-like phenotypes in adipocytes and in the iWAT of mice. l-theanine treatment enhanced AMPKα phosphorylation both in adipocytes and iWAT. Knockdown of AMPKα abolished l-theanine–induced upregulation of Prdm16 and adipocyte browning. l-Theanine increased the α-ketoglutarate (α-KG) level in adipocytes, which may increase the transcription of Prdm16 by inducing active DNA demethylation on its promoter. AMPK activation was required for l-theanine–induced increase of α-KG and DNA demethylation on the Prdm16 promoter. Moreover, intraperitoneal administration with l-theanine ameliorated obesity, improved glucose tolerance and insulin sensitivity, and reduced plasma triglyceride, total cholesterol, and free fatty acids in the high-fat diet–fed mice. Our results suggest a potential role of l-theanine in combating diet-induced obesity in mice, which may involve l-theanine–induced browning of WAT.
Obesity has become a global epidemic and is a major risk factor associated with several metabolic syndromes, such as type 2 diabetes, insulin resistance, heart disease, stroke, hyperglycemia, hypertension, and cancer (1,2). Adipose tissue is a key metabolic organ. Adipose tissue composition is always in a dynamic process of change. Adipocytes play critical roles in whole-body energy metabolism and homeostasis, which can be classified into three types. White adipocytes store excess energy in lipid droplets (3), while beige and classical brown adipocytes are characterized by their unique ability to transform mitochondrial energy into heat via uncoupling protein 1 (Ucp1) (4).
Mounting evidence suggests that browning of white adipose tissue (WAT) is of great benefit to human metabolic health. It can not only burn fat, but also enhance the insulin sensitivity of the body and reduce the fibrosis and inflammatory response of WAT, which plays an important role in maintaining the metabolic homeostasis. Compound supplementation that promotes browning of white adipocytes provides a therapeutic option to ameliorate obesity.
Natural products have been used for medical purposes for a long time. Many kinds of teas are consumed widely for health care needs. Growing studies suggest that drinking green tea reduces the risk of obesity and related disorders (5). Green tea is enriched with many secondary metabolites, such as catechins, caffeine, and l-theanine (6). For a long time, attention was significantly focused on catechins and caffeine for their metabolic benefits. However, in recent years, there is increasing attention on the health benefits of l-theanine. l-Theanine accounts for 1–3% of the dry weight of tea, which varies according to growing environment, variety, collection, and production process of the tea (7). l-Theanine was reported to improve sleep quality, emotional state, and cognitive performance and to combat cancer, cardiovascular diseases, and the common cold (8). However, its role in adipose tissue metabolism and antiobesity effects are poorly understood.
In this study, we showed that an l-theanine intervention significantly induced the browning of subcutaneous WAT in mice. The molecular mechanism underlying the above role of l-theanine was dissected both in vitro and in vivo. We have demonstrated that l-theanine could regulate a thermogenic program in WAT, and these effects are AMPK/α-ketoglutarate (α-KG)/Prdm16 axis–dependent. Our data clarified a previously unknown role of l-theanine in increasing energy expenditure, which might have a prospective application in improving metabolism and combating obesity-related metabolic diseases.
Cell Culture and Reagents
C3H10T1/2 cells were obtained from the ATCC (Manassas, VA). For the ex vivo system, iWAT from wild-type C57BL/6 mice was fractionated with digestion buffer. Primary iWAT stromal vascular fractions (SVFs) were fractionated by collagenase digestion and plated in culture according to the methods described previously (9). C3H10T1/2 cells and primary SVFs were cultured in complete DMEM with 10% FCS at 37 degrees and 5% CO2. Adipocytes were differentiated with an adipogenic cocktail (0.5 mmol/L isobutylmethylxanthine, 1 μmol/L rosiglitazone, 1 μg/mL insulin, and 1 μmol/L dexamethasone) in medium containing 10% (v/v) FBS (Invitrogen). After 2 days, the medium was exchanged for DMEM supplemented with 10% (v/v) FBS, 1 μg/mL insulin, and 1 μmol/L rosiglitazone for another 2 days. Media was changed every other day starting from the 4th day postdifferentiation with DMEM containing 10% (v/v) FBS. Cells were treated with l-theanine and infected with adenoviruses from the 6th day, and cell cultures were harvested 48 h later. Insulin, dexamethasone, and isobutylmethylxanthine were purchased from Sigma-Aldrich. Rosiglitazone was purchased from Cayman Chemical. The primary antibodies used in this study are as follows: anti-Ucp1 (ab209483; Abcam); anti-PGC1α (ab54481; Abcam); anti-Prdm16 (ab106410; Abcam); anti-AMPK (detecting AMPKα1, 5832; Cell Signaling Technology); anti–phospho-AMPK (Thr-172 of AMPK, 2535; Cell Signaling Technology); HSP-90 (4874; Cell Signaling Technology); 5-hydroxymethylcytosine (5-hmc) (A4001; Zymo Research); 5-methylcytosine antibody (5-mc) (A3001; Zymo Research); and anti–β-actin (sc-47778; Santa Cruz Biotechnology). l-Theanine was purchased from Shandong Topscience Biotech Co., Ltd. (T2800) and dissolved in saline. Hoechst was from US Everbright Inc. (H4047), and MitoTracker Green (MTG) was from Beyotime Biotechnology (C1048). Oil Red O stock solution (0.25 g of Oil Red O [Sigma-Aldrich] in 50 mL of isopropanol) was diluted with water (3:2 v/v), followed by filtration. After staining, samples were washed three times in water.
Our animal protocol has been reviewed and approved by the Animal Care and Use Committee of the Fudan University Shanghai Medical College (20180302–010). Mice were maintained under a 12:12-h light/dark cycle at room temperature (25°C) with free access to food and water. For the cold tolerance test, mice were placed in a temperature-controlled chamber (Friocell; MMM Group, München, Germany). Body temperature was measured using an animal electronic thermometer (ALT-ET03; Shanghai Alcott Biotech Co. Ltd.). For high-fat feeding studies, male C57BL/6J mice were put on the diet beginning at 6 weeks of age and continuing for up to 16 weeks. Blood and various tissue samples were collected. Animals were given l-theanine or saline by intraperitoneal (i.p.) injection for 7 days at room temperature during chow diet or for 10 consecutive weeks during high-fat diet (HFD). For metabolic studies, mice were housed and monitored individually in metabolic cages (Columbia Instruments International) with free access to regular chow and drinking water for 48 h. The animals were acclimated to the system for 24 h, and measurement of VO2 and VCO2 was performed during the next 24 h. Each cage was monitored for metabolic parameters (including oxygen consumption and carbon dioxide production) at 25-min intervals throughout the 48-h period. Parameters of oxygen consumption (in milliliters per kilogram per hour), carbon dioxide production (in milliliters per kilogram per hour), heat (in kilocalories per hour), and respiratory exchange ratio (RER) (as VCO2/VO2) were calculated for each mouse divided by its body weight. Body fat and lean mass were measured using an NMR analyzer (Minispec LF90II; Bruker Optics). The serum total triglyceride (TG) and total cholesterol (TC) were measured using a Fully Automatic Biochemical Analyzer (Roche) according to the manufacturer's protocol. The plasma free fatty acid (FFA) levels of the mice were measured by using the FFA assay kit from Abcam.
Glucose Tolerance Test and Insulin Tolerance Test
For the glucose tolerance test (GTT), mice were subjected to fasting at 6:00 p.m. On the next day at 10:00 a.m., GTT was conducted through i.p. injection of glucose at 2 g/kg body weight. For the insulin tolerance test (ITT), mice were subjected to fasting for 5 h from 7:00 a.m. to 12:00 p.m., after which ITT was performed by i.p. injection of human insulin (0.5 units/kg body weight). Glucose level was measured in tail blood at the indicated time after glucose or insulin administration using a glucometer (Accu-Chek; Roche).
Hematoxylin and Eosin Staining and Immunohistochemistry
For histology, adipose tissues were fixed with neutral-buffered formalin and embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed as described previously (10). The anti-Ucp1 (ab209483; Abcam) antibody was used for IHC. Images were captured using a charge-coupled device camera, and representative images are shown.
RNA Extraction and Quantitative PCR
Total RNA from adipose and adipocyte samples was isolated using TRIzol (Invitrogen) reagent according to the manufacturer's instructions and reverse-transcribed with SuperScript III Reverse Transcriptase (Invitrogen). cDNA was reverse-transcribed from 1 μg of RNA. Quantitative PCR (QPCR) was performed with SYBR Green QPCR Master Mix (Applied Biosystems, Carlsbad, CA) using a Prism 7500 instrument (Applied Biosystems), with 18S rRNA or 36b4 as an endogenous control. All primer sequences are listed in Supplementary Table 1. Expression levels were calculated according to the relative 2−ΔΔCt method (11).
Western Blotting Analysis
Whole-cell protein lysates were prepared using lysis buffer containing 1% SDS, 50 mmol/L Tris-HCl (pH 6.8), 10 mmol/L dithiothreitol, 10% glycerol, 0.002% bromophenol blue, 1 mmol/L sodium fluoride (Sigma-Aldrich), 1 mmol/L sodium orthovanadate (Sigma-Aldrich), and protease inhibitor cocktail (Complete Mini EDTA-free; Roche Applied Science). Protein was resolved using Tris-glycine gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking with 5% nonfat dried milk in Tris-buffered saline with Tween, the membranes were incubated with the primary antibodies and secondary antibodies and visualized with horseradish peroxidase-coupled secondary antibodies.
Oxygen Consumption Assays
Cultured adipocytes were detached from plates by trypsin. Cells or minced tissues were then resuspended in 1 mL Dulbecco's PBS (25 mmol/L glucose, 1 mmol/L pyruvate, and 2% BSA). Cellular respiration was measured with a Clark-type oxygen electrode (Oxygraph+ system; Hansatech Instruments Ltd.). Data were normalized by total protein content or tissue weight.
MTG Staining
Cultured cells were incubated in DMEM with MTG (final concentration 200 nmol/L; C1048; Beyotime Biotechnology) for 20 min in a 37°C incubator and then washed two times in PBS. The cells were observed and photographed under an Axioskop 2 microscope (Carl Zeiss) with a DP70 charged-coupled device system (Olympus). Cell nuclei were labeled with Hoechst.
Lactate Dehydrogenase Activity
Cytotoxicity was determined in a colorimetric assay based on the measurement of lactate dehydrogenase (LDH) released from the C3H10T1/2 cells into the supernatant according to the method (12). The LDH release activity was determined using an LDH cytotoxicity assay kit (Beyotime Biotechnology) according to the manufacturer's instructions.
Analysis of mtDNA Content by Quantitative Real-time PCR
Cells and tissues were homogenized and digested with proteinase K in a lysis buffer for DNA extraction by using the DNeasy Blood and Tissue Kit (Qiagen). The ratio of mtDNA to nuclear DNA reflects the concentration of mitochondria per cell (13). The Prism 7500 instrument (Applied Biosystems) was used to amplify nuclear DNA (forward, 5′-GCCAGCCTCTCCTGATTTTAGTGT-3′ and reverse,5′-GGGAACACAAAAGACCTCTTCTGG-3′) and mtDNA (forward, 5′-CCGCAAGGGAAAGATGAAAGA-3′ and reverse, 5′-TCGTTTGGTTTCGGGGTTTC-3′). The results were calculated from the difference in the threshold cycle (ΔCT) values for mtDNA and nuclear-specific genes.
Construction of Adenoviral Expression Vectors and Infection
Adenoviral expression vector pBLOCK-iT (Invitrogen) encoding shRNA against LacZ, Prdm16, and AMPKα was constructed according to the manufacturer's protocol. The targets of shRNA were listed as: shRNA against LacZ (shLacZ), 5′-CTACACAAATCAGCGATTT-3′; shRNA against Prdm16 (shPrdm16), 5′-AGTGACTTTGAGGATATCA-3′; shRNA against AMPKα1 (shAMPKα1), 5′-GCACACCCTGGATGAATTAAA-3′; and shRNA against AMPKα2 (shAMPKα2), 5′-GCTGAGAACCACTCCCTTTCT-3′. Adenovirus vectors were amplified and purified with Sartorius Adenovirus Purification kits. Viral titers were determined by the 50% tissue culture infectious dose method using 293A cells. For in vivo studies, 109 plaque-forming units/mouse of adenoviruses harboring shLacZ or shPrdm16 were diluted to 200 μL with PBS, and then 100 μL of adenovirus solution was injected to the subcutaneous fat pads at each side, twice a week as indicated. For in vitro studies, adenoviruses harboring shLacZ, shPrdm16, or shAMPKα at a multiplicity of infection of 70 were added to the cell culture medium. Then, cells were cultured with the viruses for 48 h and then replaced with fresh culture medium. Experiments were performed 48 h after adenovirus infection.
α-KG Assay
The α-KG contents in iWAT and the adipocytes derived from C3H10T1/2 cells were analyzed using liquid chromatography-tandem mass spectrometry. C3H10T1/2 cells were cultured in 2 mL medium on 3.5-cm dishes and induced into mature adipocyte. Cells were collected on day 8 of differentiation and then lysed with precooled 800 μL of 80% methanol/water (v/v) for 20 min at 4°C. Half of the cell lysis solution was vacuum freeze dried and redissolved in 200 μL of H2O. For measuring the α-KG in iWAT, up to 50 mg adipose tissue was weighted and homogenized in 1 mL precooled 80% methanol/water (v/v). The supernatants were collected by centrifugation at 12,000 rpm 4°C for 10 min. The samples were firstly separated by a UPLC system using a Waters Acquity UPLC LC-20AB (ZIC-HILIC; Merck KGaA) (2.1 × 150 mm, 5 μm) with a two-solvent system (A: 5 mL 1 mol/L ammonium acetate and 2 mL ammonia in 1 L double-distilled H2O; and B: 100% acetonitrile) and a flow rate of 0.2 mL/min and then analyzed by API 4000 Qtrap Mass Spectrograph (AB Sciex API 4000 Qtrap; Thermo Fisher Scientific).
Ten-Eleven Translocation Hydroxylase Activity Assay
C3H10T1/2 cells nuclei were isolated by using Nuclear and Cytoplasmic Extraction Reagents (P0027; Beyotime Biotechnology). Ten-eleven translocation hydroxylase (TET) activity was determined by using the TET Hydroxylase Activity Quantification Kit (ab156913, Fluorometric; Abcam) according to the manufacturer's instructions.
Chromatin immunoprecipitation (ChIP) was performed as previously described (14). Briefly, C3H10T1/2 cells were fixed, lysed, and sonicated to obtain 200–1,000-bp DNA fragments. Soluble chromatin was precleared by using 30 μL of Protein G Plus/Protein A-Agarose suspension (Calbiochem) saturated with salmon sperm (1 mg/mL) and then incubated with 1 μg anti-Prdm16 or PGC1α antibody. Bound DNA–protein complexes were eluted, cross-links were reversed, and samples were phenol-chloroform extracted, ethanol precipitated, and used for QPCR with the primers listed in Supplementary Table 2.
Hydroxymethylated and Methylated DNA Immunoprecipitation
According to the method described previously (15), genomic DNA of C3H10T1/2 cells at day 8 of differentiation was extracted by phenol-chloroform method. Genomic DNA (10 μg) was diluted in 300 μL TE buffer and sonicated by 4°C Waterbath Sonicator for 12–16 cycles 30-s on/30-s off, until the size of DNA was 300–1,000 bp. The sonicated DNA was purified, and 10% of the sonicated DNA was used as input sample. The sonicated DNA (3 μg) was diluted in 450 μL TE buffer, 10 min at 95°C, and then immediately cooled on ice along with adding 50 μL 10 times IP buffer (100 mmol/L Na-phosphate, pH 7.0, 1.4 mol/L NaCl, and 0.5% Triton X-100). Then, 5-hmc (A4001; Zymo Research) or 5-mc antibody (A3001; Zymo Research) was added to the DNA solution with rotation overnight at 4°C. The DNA–antibody mixture was further incubated with preblocked Pierce magnetic protein A/G (88802; Thermo Scientific) for 2 h. The beads–antibody–DNA complex was washed three times with one time IP buffer and responded in 250 μL digestion buffer (50 mmol/L Tris HCl, pH 8, 10 mmol/L EDTA, and 0.5% SDS) and then digested with 7 μL proteinase K (10 mg/mL) by incubating at 50°C for 3 h. The purified DNA and input samples were quantized by QPCR analysis using the primers listed in Supplementary Table 2. Relative enrichment folds of indicated DNA regions were determined after normalization to jnput, and 36b4 promoter was used as a negative control.
GraphPad Prism 8 (GraphPad Software) was used for statistical analysis. Only wounded animals were excluded from the analyses (n = 6 in this study). Data are presented as mean ± SD. Comparisons between groups were made using unpaired two-tailed Student t tests. For comparison of three or more independent groups with only one variable, one-way analyses of variance plus Bonferroni post hoc tests were performed. For comparison of two or more independent groups with two variables, two-way ANOVA plus Bonferroni post hoc tests were carried out. The statistical analyses were also indicated in the legends of each figure, and P < 0.05 was considered significant. All experiments were repeated at least three times, and representative data are shown.
Data and Resource Availability
The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. The resource generated during and/or analyzed during the current study is available from the corresponding author on reasonable request.
l-Theanine Promotes the Browning of Subcutaneous WAT in Mice
The metabolic effects of l-theanine were evaluated by i.p. administration of l-theanine into normal chow diet (NCD)–fed C57BL6 male mice. To examine the thermogenic activity, the 8-week-old mice were treated with l-theanine or saline as a control once every day for 7 days.
After cold exposure (4°C) for 6 h, l-theanine–treated mice had a better capacity to maintain body temperature compared with controls (Fig. 1A). Mice treated with l-theanine showed less body weight gains as compared with saline-treated control mice (Supplementary Fig. 1A). Adipose tissue oxygen consumption rate (OCR) is usually associated with mitochondrial energy metabolism. l-Theanine significantly increased the OCR of WAT compared with the control group (Fig. 1B), which means enhanced mitochondrial function.
l-Theanine treatment promotes subcutaneous fat browning and adaptive thermogenesis in NCD-fed mice. The 8-week-old C57BL6 NCD-fed male mice were administered l-theanine i.p. (100 mg/kg/mouse, once a day) or saline as a control for 7 days. A: Rectal temperature of the mice was recorded at the indicated time points after cold exposure (4°C). B: Basal OCR of iWAT and eWAT tissues in mice after 6-h cold exposure was measured by Clark-type electrode oxygraph and shown. C: Relative mtDNA copy number of iWAT and eWAT in mice after 6-h cold exposure. Data were normalized to the saline group. D: Representative H&E staining images of BAT, eWAT, and iWAT of the mice after 6-h cold exposure. Scale bars, 40 μm. E: The mRNA expression of the indicated genes in iWAT after 6 h of cold exposure. n = 6. F: Immunoblotting for indicated proteins in iWAT after 6-h cold exposure. For statistical analysis, two-way ANOVA and Bonferroni post hoc tests were performed in A, and unpaired two-tailed Student t tests were performed in B, C, and E. All groups were compared with the saline group. All values are represented as means with error bars representing SD. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with saline group. n = 6 for each group. ATP syn, ATP synthase; P-AMPK, phosphorylated AMPK.
As is demonstrated in Fig. 1C, the abundance of mitochondria in WAT was quantified by mtDNA copy number, which was increased by l-theanine treatment. Histological examination of the subcutaneous (iWAT) and epididymal WAT (eWAT) revealed smaller adipocytes, which presented brown-like transformation of the WAT (Fig. 1D). We next isolated RNA from the three different fat pads—eWAT, iWAT, and brown adipose tissue (BAT)—and compared mRNA expression levels between l-theanine –treated mice and saline-treated mice. Expression levels of genes involved in controlling energy expenditure and the thermogenic program, including Prdm16, PGC1α, Ucp1, mtTFA, Dio2, and Cpt1b, were examined in iWAT, eWAT, and BAT. Although to different extents, l-theanine induced the expression of the above genes in iWAT (Fig. 1E), eWAT (Supplementary Fig. 1C), and BAT (Supplementary Fig. 1D), with its most significant effect in iWAT (Fig. 1E). Besides Ucp1 and other classical thermogenic-related marker genes, such as Dio2 and mtTFA, fatty acid oxidation–related gene expression was also significantly increased in iWAT (Fig. 1E). Importantly, Western blot analysis shows that l-theanine could obviously promote the protein expression of Prdm16, Ucp1, and PGC1α and the phosphorylation of AMPK in iWAT (Fig. 1F and Supplementary Fig. 1B). In BAT, Prdm16 protein level and the phosphorylation of AMPK were slightly increased by l-theanine treatment (Supplementary Fig. 1E and F). Our data consistently showed that l-theanine could promote the browning of WAT and thermogenic capacity in mice.
l-Theanine Increases Energy Consumption in NCD-Fed Mice
As the above results show, l-theanine promotes the browning of WAT during cold exposure. With this in mind, we next investigated the metabolic effect of l-theanine in mice using a comprehensive laboratory animal monitoring system. As shown in Fig. 2A–D, basal oxygen consumption and carbon dioxide release rates were significantly higher in mice administered l-theanine compared with control subjects through a 12-h light/dark cycle. As shown in Fig. 2E, the l-theanine–treated mice showed a higher consumption in the whole-body energy through a 12-h light/dark cycle. The RER was calculated from VCO2 and VO2. Higher RER values suggest higher carbohydrate consumption, and lower RER values suggest more fat is used as the source of energy (16). As shown in Fig. 2F, l-theanine treatment decreased RER of the mice, demonstrating a tendency to fatty acid oxidation (17). Taken together, these results indicate that l-theanine promotes the whole-body energy expenditure and increase fatty acid combustion in NCD-fed mice.
l-Theanine increases energy expenditure in mice. Mice were treated as indicated in Fig. 1. Energy expenditure was evaluated by measurement of oxygen consumption (VO2) as shown in A and B, carbon dioxide release (VCO2) as shown in C and D, and heat production of the mice as shown in E. The above parameters were measured in mice placed in a metabolic cage during a 12:12-h light/dark cycle. Energy expenditure expressed as kcal/h per animal. F: RER was calculated using equations described in Research Design and Methods. The adjacent bar graphs represent the average values for each group (B for A and D for C) (n = 6). Error bars represent SD, and significant differences compared with saline groups are indicated by *P < 0.05, **P < 0.01 (Student t test).
l-Theanine Induces a Brown Adipocyte-like Feature in Adipocytes In Vitro
Because the browning of subcutaneous WAT in vivo is complicated, we examined whether l-theanine induces adipocyte browning directly. To better investigate the role of l-theanine in the browning of adipocytes and its underlying mechanism, we used the C3H10T1/2 cell line, which is a mesenchymal stem cell line and can be induced to become mature adipocytes in vitro. We treated the adipocytes derived from C3H10T1/2 cells with l-theanine at different concentrations of 0 μmol/L, 10 μmol/L, 50 μmol/L, and 100 μmol/L. To evaluate the possible cytotoxicity of l-theanine, we assessed LDH leakage after 24 h of exposure to l-theanine. Exposure of the adipocytes to 10μmol/L, 50 μmol/L, and 100 μmol/L of l-theanine did not influence the leakage rate of LDH (Fig. 3A), which suggests that l-theanine was not toxic to C3H10T1/2 at the above three concentrations. We then measured C3H10T1/2 OCR using a Clark-type electrode oxygraph (Fig. 3B). The OCR of 50 μmol/L and 100 μmol/L l-theanine–treated cells was significantly greater than that of controls. Increase of mitochondrial contents was also observed in l-theanine–treated cells (Fig. 3C). MTG staining showed that l-theanine increased mitochondrial abundance (Fig. 3D), which was consistent with the results of the above mtDNA content analysis. l-Theanine treatment of the adipocytes upregulated the mRNA levels of the genes involved in the activation of energy expenditure and thermogenesis, including Prdm16, PGC1α, Ucp1, mtTFA, and Cpt1b (Fig. 3E) in the l-theanine dose-dependent manner. Western blot analysis further confirmed that Prdm16 and Ucp1 protein were significantly upregulated by l-theanine at the concentrations of 50 μmol/L and 100 μmol/L (Fig. 3F and G). Similar effects of l-theanine were also observed in primary iWAT adipocytes (Supplementary Fig. 2). The lipid droplets in adipocytes were stained by Oil Red O, and the result was shown in Fig. 3H. The adipocytes derived from C3H10T1/2 cells treated with l-theanine manifested diminishing tendency of lipid droplet size, as indicated by Oil Red O staining, which is consistent with the greater energy expenditure caused by l-theanine treatment (Fig. 3H). All of the above phenotypes present enhanced acquisition of brown-like adipocyte features. These results demonstrate that l-theanine could directly activate the browning of adipocytes in vitro.
l-Theanine induces a brown adipocyte-like phenotype in the adipocytes derived from C3H10T1/2 cells in vitro. The adipocytes were treated with l-theanine at the indicated dose or with saline as a control. After 48 h of treatment, cells were harvested for analyses. A: Relative LDH release from C3H10T1/2 exposed to saline or three different concentrations of l-theanine (10 μmol/L, 50 μmol/L, and 100 μmol/L). B: Basal OCR of C3H10T1/2 was measured by Clark-type electrode oxygraph and shown. C: mtDNA copy number of C3H10T1/2. Data were normalized to the saline group. D: Cells were stained with MTG and Hoechst, and representative images are shown. Scale bar, 100 μm. Staining was performed after 48 h of treatment with saline or 50 μmol/L l-theanine. E: The mRNA levels of the indicated genes were determined by using RT-qPCR. Data were normalized to the saline group. F: Cell lysates were then subjected to Western blotting by using the indicated antibodies. Hsp90 serves as an internal control. G: Quantification of Western blotting was done by using ImageJ and shown. H: Oil Red O staining of differentiated adipocytes. Scale bar, 50 μm. For statistical analysis, two-way ANOVA and Bonferroni post hoc tests were performed in A–C, and unpaired two-tailed Student t tests were performed in E and G. All groups were compared with the saline group. All values are represented as means with error bars representing SD. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with saline group. n = 5 for each group. ATP syn, ATP synthase; P-AMPK, phosphorylated AMPK.
Knockdown of Prdm16 Blunts the Role of l-Theanine in Promoting the Brown Adipocyte-like Features in the Adipocytes Derived From C3H10T1/2 Cells
Prdm16 transcriptional complex is a dominant activator of brown/beige adipocyte development (18). The above results show that Prdm16 expression was obviously induced by l-theanine treatment both in vivo and in vitro. The binding of Prdm16 to the enhancer and transcription start site region of Ucp1 and Dio2 was significantly enhanced by l-theanine treatment (Fig. 4A). PGC1α is a key regulator of adipose tissue energy metabolism, especially when involved in mitochondrial biosynthesis and thermogenesis (19). As shown in Fig. 4B, l-theanine–induced binding of PGC1α to the above regions is less obvious. Next, we constructed adenovirus harboring shLacZ or shPrdm16 to infect the adipocytes. The l-theanine–mediated upregulation of browning gene expression was blunted upon the knockdown of Prdm16 (Fig. 4C). Consistently, the induction of Ucp1 protein expression by l-theanine was abolished in shPrdm16-treated cells (Fig. 4D and Supplementary Fig. 3). Oxygen electrode experiments and MTG staining showed that the effects of l-theanine treatment on promoting mitochondria respiration and increasing mitochondrial abundance were eliminated by the knockdown of Prdm16 (Fig. 4E and F, respectively). These results indicate that Prdm16 plays an essential role in l-theanine–mediated browning of adipocytes.
Prdm16 plays an indispensable role in l-theanine–induced browning in the adipocytes derived from C3H10T1/2 cells. A and B: ChIP QPCR analysis for Prdm16 and PGC1α binding to the enhancer or promoter regions of the indicated genes in cultured adipocytes. The adipocytes were treated with saline or 50 μmol/L l-theanine for 48 h. Then, Prdm16 and PGC1α were processed by ChIP from C3H10T1/2 and analyzed for their binding at the indicated regions. Data were normalized to saline group. Shown is a representative ChIP QPCR. C–F: C3H10T1/2 cells were infected with shLacZ or shPrdm16 adenoviruses as indicated, and medium was changed 48 h later. Then, the cells infected with adenovirus were treated with saline or 50 μmol/L l-theanine for 48 h. The following experiments were then performed. C: The mRNA levels of the indicated genes. D: Representative Western blot images of the indicated proteins. E: OCR in cells. F: Cells were stained with MTG and Hoechst, and representative images are shown. Scale bar, 100 μm. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 as compared with saline group in A and B or compared with shLacZ+saline group in C and E by Student t test. TSS, transcription start site.
Adenovirus-Mediated Specific Knockdown of Prdm16 in iWAT Eliminates l-Theanine–Induced Browning of iWAT
Prdm16 promotes browning of iWAT by activating a brown fat-like gene program (20). Given the importance of Prdm16 in l-theanine–mediated browning of C3H10T1/2 and in order to clarify the role of Prdm16 in l-theanine effects on browning of WAT and energy expenditure in vivo, mice were subjected to subcutaneous injection at the iWAT with adenovirus harboring shLacZ or shPrdm16 and then were administered l-theanine or saline for 1 week (Fig. 5A). We isolated the SVF and mature adipocyte fraction from iWAT and performed RT-qPCR to examine the expression level of Prdm16. As shown in Supplementary Fig. 4, the expression level of Prdm16 in the mature adipocyte fraction was significantly downregulated by shPrdm16. It should be noted that Prdm16 expression level was also knocked down by Prdm16 in SVF, which indicates that the adenovirus can infect not only mature adipocytes, but also other cell types in iWAT. We measured the rectal temperature of mice every hour during the 6 h of cold exposure. The results show that mice administered shPrdm16 adenovirus at the iWAT displayed impaired cold tolerance compared with the mice injected with shLacZ adenovirus (Fig. 5B). l-Theanine treatment significantly elevated rectal temperature of shLacZ-treated mice at cold temperatures, whereas it did not obviously impact that of shPrdm16-treated mice. Moreover, l-theanine treatment increased the mRNA levels of browning markers such as Prdm16, Ucp1, Cidea, and Dio2 in iWAT of control mice, whereas it had no obvious effect on the above genes' expression in the iWAT of shPrdm16-treated mice (Fig. 5C). Gene expression levels were also examined in eWAT (Supplementary Fig. 5A). Knockdown of Prdm16 in iWAT did not affect the expression level of Prdm16 in eWAT (shLacZ+Saline vs. shPrdm16+Saline). In the control shRNA group (shLacZ group), treatment of the mice with l-theanine increased the expression of browning marker genes in eWAT, such as Prdm16, UCP1, Cidea, and Cox8b (shLacZ+Saline vs. shLacZ+l-theanine). However, knockdown of Prdm16 in iWAT blunted the ability of l-theanine to promote the expression of browning marker genes in eWAT (shPrdm16+Saline vs. shPrdm16+l-theanine). There may be an l-theanine–triggered signal from iWAT to eWAT through some endocrine factors to enhance the browning gene expression in eWAT, which could be impaired by the knockdown of Prdm16 in iWAT. Further studies are needed to investigate the above hypothesis. Meanwhile, l-theanine failed to promote adipose tissue oxygen consumption in the iWAT of shPrdm16-treated mice (Fig. 5D). Similarly, the Prdm16 and Ucp1 protein expression levels were upregulated upon l-theanine treatment in shLacZ-treated mice, whereas they stayed unchanged in the iWAT of shPrdm16-treated mice (Fig. 5E and Supplementary Fig. 5B). H&E staining of the inguinal WATs showed that l-theanine decreased adipocyte size and increased Ucp1 IHC-positive areas in shLacZ control groups. However, these effects were abolished in shPrdm16 groups (Fig. 5F). Therefore, we conclude that Prdm16 expression is essential for the l-theanine–mediated browning effect on the iWAT and proadaptive thermogenesis in mice.
Prdm16 plays an indispensable role in l-theanine–induced iWAT browning in mice. Eight-week-old male WT mice were injected subcutaneously (i.h.) into iWAT with adenovirus of shLacZ or shPrdm16 twice a week for 2 weeks. The mice were i.p. injected with saline or l-theanine in the 2nd week for 7 days (d). Then, the following experiments were performed. A: Schematic description of animal experiments. B: Rectal temperature of the mice was recorded at the indicated time points after cold exposure (4°C). C: The mRNA levels of the indicated genes. n = 6. D: OCR of iWAT was measured by Clark-type electrode oxygraph and shown. E: Western blot analysis of the protein levels in iWAT by using the indicated antibodies. Hsp90 serves as an internal control. F: H&E staining (HE) and IHC for UCP1 protein in iWAT sections of the mice. Scale bars, 50 μm. n = 6. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 compared with shLacZ+saline group by Student t test.
AMPK Activation Is Indispensable for l-Theanine–Induced Browning of Adipocytes
AMPK is a crucial cellular energy sensor that has recently been demonstrated to be important in regulating the metabolic activity of brown-like adipocytes (21). It was found that the phosphorylation of AMPK was activated by l-theanine in mice livers (22). We therefore investigated the role of l-theanine in the activation of AMPK in adipocytes. Our results demonstrated that l-theanine induced the phosphorylation of AMPK both in the adipocytes (Fig. 3F) and in iWAT (Fig. 1F) of the mice. Therefore, we sought to explore if l-theanine promotes the browning of adipocytes via activating the AMPK signaling pathway. The shRNA-mediated knockdown of the AMPKα subunit was performed in the adipocytes. As shown in Fig. 6A, knocking down of AMPK blunted the role of l-theanine in inducing the mRNA expression of Prdm16 and Ucp1. Western blotting showed that knockdown of AMPK reduced the expression of Ucp1 and Prdm16 promoted by l-theanine (Fig. 6B and C and Supplementary Fig. 6). As shown in Fig. 6D, l-theanine promoted a significant increase in oxygen consumption in C3H10T1/2, and this increase was completely abrogated by AMPK knockdown in C3H10T1/2. Enhanced mitochondrial abundance by l-theanine was also abolished in C3H10T1/2 infected with shAMPK adenovirus (Fig. 6E). As downregulation of AMPK signaling blunted the role of l-theanine in inducing the expression of Prdm16, it is suggested that AMPK and Prdm16 were both involved in l-theanine–mediated browning of adipocytes, and Prdm16 could be a downstream target of AMPK. These data demonstrate a potential l-theanine/AMPK/Prdm16 pathway activation in l-theanine–mediated browning of adipocytes.
AMPK activation is necessary for l-theanine to promote adipocyte browning. C3H10T1/2 cells were infected with shLacZ or shAMPKα adenoviruses as indicated, and medium was changed 48 h later. Then, the cells infected with adenovirus were treated with saline or 50 μmol/L l-theanine for 48 h. The following experiments were then performed. A: The mRNA levels of the indicated genes. B: Cell lysates were then subjected to Western blotting by using the indicated antibodies. Hsp90 serves as an internal control. C: Statistical analysis of Western blotting gray-level results for B. D: OCR in cells was measured. E: Cells were stained with MTG and Hoechst, and representative images are shown. Scale bar, 100 μm. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 compared with shLacZ+saline group by Student t test.
l-Theanine Increases the Cellular Content of Tricarboxylic Acid Metabolite α-KG and Decreases the DNA Methylation Level of Prdm16 Promoter in C3H10T1/2
As Prdm16 plays an important role in l-theanine–mediated browning of adipocytes, the mechanism of how Prdm16 expression is regulated was then investigated. DNA methylation is a complicated process and plays essential roles in regulating gene expression. Based on a methylated and hydroxymethylated DNA immunoprecipitation procedure, we further studied DNA methylation (5-mc) and hydroxymethylation (5-hmc) in the Prdm16 promoter in three different regions of Prdm16 promoter. As shown in Fig. 7A and B, l-theanine significantly decreased 5-mc level and increased 5-hmc level in Prdm16 promoter. The tricarboxylic acid cycle (TCA) metabolite α-KG is known to facilitate DNA demethylation. The α-KG concentration was determined by liquid chromatography-tandem mass spectrometry and showed a significant elevation in l-theanine–treated adipocytes (Fig. 7C). Moreover, the content of α-KG was increased in the iWAT of l-theanine–treated mice (Fig. 7D).
l-Theanine increases α-KG in adipocytes and epigenetically decreases the DNA methylation of Prdm16 promoter. A: The adipocytes were treated with saline or 50 μmol/L l-theanine for 48 h. Then, a methylated and hydroxymethylated DNA immunoprecipitation procedure was performed according to protocol. The a, b, and c primers were used to determine relative enrichment folds of three different regions in the Prdm16 promoter after normalization to inputs, and a 36b4 promoter region was used as a negative control. The enrichment level of 5-mc in Prdm16 promoter is shown. B: Cells were treated as in A, and the enrichment level of 5-hmc in Prdm16 promoter is shown. n = 3. C: Cells were treated as in A, and the intracellular α-KG level was determined. D: Mice were treated as in Fig. 1, and the α-KG level in iWAT was measured. E: Cells were treated as in A, and the activity of TETs was measured. F: Cells were treated as in Fig. 6, and the intracellular α-KG level was determined. n = 5. Cells were treated as in F, and the enrichment level of 5-mc (G) and 5-hmc (H) on the Prdm16 promoter (using primers a as described in A) was examined. n = 3. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with saline group or shLacZ+saline group by Student t test or one-way ANOVA followed by Dunnett multiple comparisons.
Given that the TETs, including TET1, 2, and 3, catalyze active DNA demethylation, the effect of l-theanine on TET activity was examined. It was found that l-theanine significantly enhanced TET activity in the adipocytes derived from C3H10T1/2 cells (Fig. 7E). This result suggests that l-theanine–induced DNA demethylation on the Prdm16 promoter may be mediated by enhanced TET activity. Further, we found that l-theanine–mediated elevation of α-KG concentration was significantly blunted in shAMPK-treated cells compared with the corresponding control group (Fig. 7F). In addition, knocking down of AMPK abolished the role of l-theanine in decreasing 5-mc level and increasing 5-hmc level on the Prdm16 promoter (Fig. 7G and H). This suggests that AMPK is an essential factor in α-KG/Prdm16 axis and is an upstream regulator of α-KG. AMPK signaling–mediated elevation of α-KG promotes Prdm16 promoter DNA demethylation, thereby stimulating Prdm16 expression. Overall, our data suggest that l-theanine inhibits the DNA methylation of the Prdm16 promoter and facilitates Prdm16 expression through the AMPK–α-KG pathway.
Antiobesity Effects of l-Theanine in HFD-Induced Obese Mice
Because l-theanine promotes browning of adipose tissue, we aimed to investigate the potential antiobesity effects of l-theanine. C57BL6 male mice were fed with HFD for 16 weeks. From the 7th week of HFD feeding, mice were injected i.p. with saline or l-theanine (100 mg/kg), once a day for the next 10 consecutive weeks (Fig. 8A). l-Theanine did not affect the amount of food intake in mice (Supplementary Fig. 7A). As shown in Fig. 8B, l-theanine reduced the weight gain of mice induced by HFD, which is associated with the reduced fat mass content (Fig. 8C). In addition, we weighed three types adipose tissue. l-Theanine significantly reduced tissue weight in iWAT and eWAT, while brown fat content did not statistically differ between the two groups (Fig. 8D). Obesity is closely associated with elevation of lipids in plasma. The mice plasma was collected and analyzed for TG fatty acid, TC, and FFA. Plasma TG, TC, and FFA levels were reduced in mice treated with l-theanine compared with saline-treated mice (Fig. 8E). HFD-induced obesity is also closely related to glucose intolerance and insulin resistance. We next evaluated whether l-theanine treatment ameliorates glucose intolerance and insulin resistance by conducting i.p. GTT and ITT (IP-GTT and IP-ITT, respectively). l-Theanine–treated mice exhibited an improved metabolic profile in glucose tolerance and insulin sensitivity (Fig. 8F–I). We isolated the iWAT from these mice. Proteins and mRNAs were extracted from tissues in the two groups for QPCR and Western blotting analysis. QPCR revealed significantly increased Prdm16, Ucp1, and other browning-related gene expression in the l-theanine–treated mice (Fig. 8J). Western blotting analysis demonstrated that the expression of Prdm16 and Ucp1 were significantly elevated in the l-theanine–treated group compared with the saline control group (Fig. 8K and Supplementary Fig. 7B).
l-Theanine reduces obesity in diet-induced obese mice. A: Schematic description of animal experiments. C57BL6 male mice were fed with HFD for 16 weeks. From the 7th week of HFD feeding, mice were injected i.p. with saline or l-theanine (100 mg/kg) once a day for the next 10 consecutive weeks. B: Body weight of mice after treatment of saline or l-theanine. C: Body composition of the mice after 10 weeks of l-theanine or saline treatment. D: Tissue weight (normalized to body weight) of iWAT, eWAT, and BAT after 10 weeks of saline or l-theanine treatment. E: Plasma TG, TC, and FFA. F: Glucose concentrations during an IP-GTT. G: Area under the curve analysis of IP-GTT. H: Glucose concentrations during an IP-ITT. I: Area under the curve analysis of IP-ITT. J: The mRNA levels of the indicated genes in iWAT. K: Tissue lysates were subjected to Western blotting by using the indicated antibodies. Hsp90 serves as an internal control. L: H&E (HE) and Ucp1 IHC staining of iWAT. Scale bars, 40 μm. For statistical analysis in B–J, data were compared between the saline group and the l-theanine group. All values are represented as means with error bars representing SD. *P < 0.05; **P < 0.01; ***P < 0.001. n = 6 for each group. 6w, 6 weeks; ATP syn, ATP synthase; P-AMPK, phosphorylated AMPK.
There was also an upregulation of thermogenic genes in eWAT, as shown in Supplementary Fig. 7C. There was no expression level difference in most of the thermogenic genes except Dio2 in BAT (Supplementary Fig. 7D). H&E staining revealed that HFD-fed mice treated by l-theanine had smaller adipocytes in iWAT, and IHC staining of iWAT showed higher expression of Ucp1 (Fig. 8L). As shown in Supplementary Fig. 7E, visceral adipocyte sizes were reduced in mice administered l-theanine, and the adipocyte sizes in BAT were also slightly reduced. Furthermore, metabolic cage studies and an OCR test of iWAT were performed at the 9th week (9 weeks of HFD feeding with l-theanine treatment in the last 3 weeks). As shown in Supplementary Fig. 7F, 3 weeks of l-theanine treatment enhanced the OCR of iWAT in HFD-fed mice. In addition, metabolic cage studies indicated that 3 weeks of l-theanine treatment significantly promoted the basal oxygen consumption (Supplementary Fig. 7G) and energy expenditure (Supplementary Fig. 7H) of the whole body and mildly decreased RER (Supplementary Fig. 7I). These data suggest that l-theanine–mediated resistance to diet-induced obesity is driven by increased energy expenditure and increased iWAT thermogenesis. Overall, our results demonstrate that l-theanine activates the browning of WAT and contributes to ameliorating diet-induced obesity and related disorders in mice.
l-Theanine is a nonprotein amino acid mainly found in tea plants. l-Theanine is an important taste- and health-related component in tea and is used as an indicator to evaluate tea functions (23). l-Theanine has multifaceted health benefits (24) and is considered as an index of tea quality (25). However, the role and mechanism of l-theanine in treating obesity and metabolic diseases is still poorly investigated. In this study, we proposed that l-theanine promotes the browning of WAT, which contributes to the amelioration of obesity, the improvement of insulin sensitivity, and the reduction of plasma lipids in HFD-fed mice.
In this article, we found that NCD-fed mice i.p. administered l-theanine showed an enhanced cold tolerance capacity compared with control mice. Our study further demonstrates that l-theanine activates the thermogenic program of adipose tissue in NCD-fed mice, with the most significant effects on iWAT (Fig. 1). l-Theanine significantly increases energy consumption in NCD-fed mice compared with the control group (Fig. 2). Moreover, using primary iWAT adipocytes and the adipocytes derived from C3H10T1/2 cells as an in vitro cell culture model, we found that l-theanine directly induced the browning of adipocytes in vitro (Fig. 3 and Supplementary Fig. 2). Prdm16 was a critical transcriptional regulator (26) and an essential modulator in the downstream of l-theanine because knockdown of Prdm16 in the adipocytes blunted the role of l-theanine in the induction of a brown-like program in adipocytes (Fig. 4). Similarly, knockdown of Prdm16 in iWAT inhibited l-theanine–mediated brown-like fat formation in mice (Fig. 5). AMPKα phosphorylation played an important role in l-theanine–induced browning of adipocytes. We demonstrated that the l-theanine–induced browning of adipocytes, including elevated Prdm16 expression, was blocked by AMPKα knockdown (Fig. 6). We show a potential l-theanine/AMPK/Prdm16 pathway activation in l-theanine–induced browning of adipocytes (Fig. 7). We found that l-theanine significantly reduced the 5-mc level and increased the 5-hmC level in the Prdm16 promoter. Furthermore, l-theanine increased the concentration of C3H10T1/2 intracellular α-KG, which was blocked by the knockdown of AMPKα. α-KG is known to facilitate DNA demethylation. Our results suggest that l-theanine activates AMPK signaling to elevate α-KG concentration in adipocytes, which leads to DNA demethylation and decreases the DNA methylation level on the Prdm16 promoter, thereby facilitating Prdm16 expression and ultimately promoting adipocyte browning (Fig. 7). Furthermore, HFD-fed mice administered l-theanine exhibited improved glucose and insulin sensitivity and reduced plasma TG, TC, and FFA levels compared with saline-treated mice. (Fig. 8).
Browning of adipose tissue was inducible by exercise, cold exposure, and fasting in WAT, which has substantial metabolic benefits (27). Compared with classical brown adipocytes, beige adipocytes have a better inducibility toward thermogenic gene expression, especially Ucp1, and therefore a higher thermogenic capacity in response to stimulus (28). Accordingly, thermogenesis, which means dispersing energy in the form of heat, will contribute to maintaining metabolic homeostasis through enhancing energy expenditure. This process can be achieved by the activation of Ucp1, an uncoupling hallmark mitochondria inner membrane protein abundantly expressed in BAT. Ucp1 induces uncoupling respiration and mitochondrial ATP synthesis by reducing the proton gradient across the mitochondrial inner membrane (29). Ucp1 ablation is reported to induce obesity and abolish thermogenesis at thermoneutrality (30). Our data suggest a functional role of l-theanine in inducing the browning of white adipocytes, which help to improve thermogenic capacity and combat obesity in mice.
AMPK plays an important role in energy metabolism and mitochondrial biogenesis (31,32). However, the mechanisms by which AMPK regulates metabolism are not completely understood. Many studies have shown that activating AMPK can improve metabolic rate and help to resist obesity. For instance, metformin promotes browning of adipose tissue and prevents fatty liver (33). Taurine activates AMPK to promote browning of WAT and combat obesity in mice (34). Obesity and glucose metabolic disorder are closely associated with a decrease in AMPK activity (35). Our results identified that the master regulator AMPK is involved in the l-theanine–mediated WAT thermogenic program.
The structure of l-theanine is similar to that of l-glutamine. l-Glutamine is conditionally essential and the most abundant amino acid in the body. It acts as an important energy source and supplies carbons to the TCA cycle to produce ATP. Therefore, insufficiency of l-glutamine could cause an energy shortage and lead to AMPK activation (36). It has been reported that l-theanine could compete with l-glutamine in binding to the l-glutamine transporter on the cell surface, which is responsible for the incorporation of extracellular L-glutamine (37). In this way, l-theanine could inhibit the uptake of l-glutamine by the cells. Therefore, l-theanine treatment of the adipocytes may inhibit the uptake of l-glutamine and decrease the intracellular l-glutamine level, which would lead to an energy shortage and activate AMPK phosphorylation. Further studies are needed to investigate the above hypothesis.
Our data revealed a functional role of l-theanine in the activation of thermogenic program in adipocytes, which is shown to be involved in the increased energy expenditure and antiobesity function. Indeed, our results show that l-theanine could activate differentiated C3H10T1/2 and iWAT SVF adipocytes to express more thermogenic-related genes and exhibit brown adipocyte-like phenotypes. We demonstrated that transdifferentiation of adipocytes could be induced by l-theanine in vitro. l-Theanine could upregulate the expression of many browning-associated genes. The most prominent shift in this process was Prdm16. Notably, Prdm16 is a critical regulator of browning and the thermogenic gene expression program (20,38). Raising the Prdm16 level ameliorates fibrosis of aged mice and promotes beige adipocyte activation (39). Thus, Prdm16 plays an important role in l-theanine–mediated browning of white adipocytes.
C3H10T1/2 and iWAT exhibit a significant increase in α-KG concentration after l-theanine treatment, which could facilitate DNA demethylation of Prdm16. Beneficial effects of α-KG on extending the life span of adult worms have been reported (40). α-KG is a product of the TCA cycle and produced from isocitrate by isocitrate dehydrogenase, which can also be produced from glutamate by glutamate dehydrogenase. It functions as a cofactor of TETs to catalyze DNA demethylation. Inhibition of the AMPK/α-KG signaling pathway suppressed BAT development, which contributes to obesity (41). Our results reveal an l-theanine/AMPK/α-KG/Prdm16 axis that induces the brown-like features in white adipocytes.
In conclusion, our results show a previously unknown role for l-theanine in regulating adipose tissue and adipocyte metabolism. We identified that l-theanine promotes the browning of adipose tissue in NCD- and HFD-fed mice, which ameliorates obesity, improves glucose and insulin tolerance, and reduces levels of plasma lipids (Supplementary Fig. 8). Thus, l-theanine may provide an approach to help combat obesity and obesity-associated disorders based on its positive role in WAT browning.
Funding. This study was supported by the National Key R&D Program of China (2018YFA0800401 to Q.-Q.T.) and National Natural Science Foundation of China grants (32070751 and 31871435 to L.G. and 81730021 and 32070760 to Q.-Q.T.).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. W.-Q.P. and L.G. were involved in study design, conducted the experiments, analyzed the data, and drafted the paper. G.X., B.-Y.L., and Y.-Y.G. performed the experiments. L.G. and Q.-Q.T. designed and supervised the study and wrote the paper. W.-Q.P. and L.G. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
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Şanlier
L-theanine, unique amino acid of tea, and its metabolism, health effects, and safety
Crit Rev Food Sci Nutr
Ahammed
Stimulation in primary and secondary metabolism by elevated carbon dioxide alters green tea quality in Camellia sinensis L
Kissig
Ishibashi
PRDM16 represses the type I interferon response in adipocytes to promote mitochondrial and thermogenic programing
EMBO J
Bléher
Meshko
Cacciapuoti
Egr1 loss-of-function promotes beige adipocyte differentiation and activation specifically in inguinal subcutaneous white adipose tissue
Vegiopoulos
Müller-Decker
Strzoda
Cyclooxygenase-2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes
Kajimura
Promoting brown and beige adipocyte biogenesis through the PRDM16 pathway
Int J Obes Suppl
Golozoubova
Nedergaard
UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality
AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation
Proc Natl Acad Sci USA
Cantó
Gerhart-Hines
AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity
Jhun
Metformin prevents fatty liver and improves balance of white/brown adipose in an obesity mouse model by inducing FGF21
Mediators Inflamm
Peng
Taurine-mediated browning of white adipose tissue is involved in its anti-obesity effect in mice
Ruderman
Prentki
AMPK, insulin resistance, and the metabolic syndrome
The ability to utilise ammonia as nitrogen source is cell type specific and intricately linked to GDH, AMPK and mTORC1
An L-glutamine transporter isoform for neurogenesis facilitated by L-theanine
Neurochem Res
Transcriptional control of brown fat determination by PRDM16
Trefely
A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate
The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR
AMPK/α-ketoglutarate axis dynamically mediates DNA demethylation in the Prdm16 promoter and brown adipogenesis
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/content/license.
Butyrate-Producing Bacteria and Insulin Homeostasis: The Microbiome and Insulin Longitudinal Evaluation Study (MILES)
Management of Latent Autoimmune Diabetes in Adults: A Consensus Statement From an International Expert Panel
The Antipsychotic Dopamine 2 Receptor Antagonist Diphenylbutylpiperidines Improve Glycemia in Experimental Obesity by Inhibiting Succinyl-CoA:3-Ketoacid CoA Transferase
Differentiation of Diabetes by Pathophysiology, Natural History, and Prognosis
Metabolic Endotoxemia Initiates Obesity and Insulin Resistance

redpajama_set_name: RedPajamaCommonCrawl


text:
Al Kaszniak: 8-27-2011: Zen Brain: Trauma, Stress, Loss, and Happiness (Part 7)
Al Kaszniak, PhD, Sensei
Note: This episode is a lecture.
Al Kaszniak received his Ph.D. in clinical and developmental psychology from the University of Illinois at Chicago in 1976, and completed an internship in clinical neuropsychology at Rush-Presbyterian-St. Luke's Medical Center in Chicago. He is currently Head of the Department of Psychology, Director of Clinical Neuropsychology, Director of the Arizona Alzheimer's Consortium Education Core, and a professor in the departments of psychology, neurology, and psychiatry at The University of Arizona. His research, published in over 150 journal articles, chapters and books (including edited volumes on consciousness and science), has been supported by grants from the NIH, NIMH, and several private foundations. His work has focused on the neuropsychology of Alzheimer's disease and other age-related neurological disorders, memory self-monitoring, the biological bases of emotion, and emotion response and regulation in long-term Zen and mindfulness meditators.
Series Summary
Buddhism is a path to liberation from suffering, and among the most pervasive universal triggers of suffering are trauma, stress, and loss, including bereavement. Fundamental to Buddhist teaching, and the path of Zen, is the recognition that freedom from suffering can be found through practice realization of the fundamental nature of the mental continuum as ever-changing, interdependent, and without any fixed, unchanging self at its core.
Recently, scientific studies of human resilience following trauma and loss, response to stressful events, and the consequences of meditation training have begun to provide third-person evidence that converges with the first-person experience of Zen practice.
In this retreat, prominent scientists and Zen practitioners will explore Buddhist, neuroscientific, and clinical science perspectives on trauma, stress, loss, and the human potential for resilience and happiness. Talks, discussions, and explorations with participants are embedded within Zazen practice throughout each day.
To access the entire series, please click on the link below:
Zen Brain Aug 2011 Series: Trauma, Stress, Loss, and Happiness: All 10 Parts
Al Kaszniak received his Ph.D. in clinical and developmental psychology from the University of Illinois in 1976, and completed an internship in clinical neuropsychology at Rush Medical Center in Chicago. He is presently Emeritus Professor of Psychology, Faculty Advisory Board Member of the Center for Compassion Studies, and Pedagogy Fellow…


text:
Pundits Say Results in Governor's Races Carry Warning on Spending
By Peter Schmidt
Although gubernatorial candidates regarded as having strong records and stands on education fared well over all in last week's elections, observers said the poll results also carried a warning that voters are likely to balk at costly education reforms.
In contrast to the Congressional races, in which almost all incumbents were re-elected, the nation's 36 gubernatorial elections were marked by considerable turmoil. Six incumbents went down in defeat, at least eight open seats changed partisan hands, and two former Republicans running as independents gained office.
The only contest still to be decided late last week was in Arizona, where the major-party contenders apparently will have to meet again in a run-off election.
The national teachers' unions, which mounted major efforts to influence the gubernatorial races this year, pronounced themselves satisfied with the results. Eighteen of 31 candidates endorsed by state affiliates of the National Education Assoion were successful, as were 13 of 22 contenders backed by American Federation of Teachers' affiliates.
And, although voters tossed out two educationally prominent Democratic Governors, James J. Blanchard of Michigan and Rudy Perpich of Minnesota, they also chose several other candidates who previously had gained the approval of educators in their roles as governors or members of the Congress.
"This was a very serious election," observed Rachelle Horowitz, political director of the A.F.T. "The people who just did negative spots did not do well. It was those people who seriously talked about issues, who talked about education, who talked about the economy, who did well."
But Steven D. Henriksen, who followed the gubernatorial elections as a consultant to the Education Commission of the States, injected a note of caution.
"If there is one characteristic, one theme, that runs through many of the gubernatorial races," he said, "it's that voters are increasingly stingy with their money."
"In the coming two years," Mr. Henriksen warned, "educators will have to be very careful in terms of phrasing their requests for spending--not only in terms of voter attitudes, but in terms of the predispositions of the new governors not to support any tax increases."
With the economy hovering on the brink of a recession that could have a potentially devastating impact on state governments, few candidates found much political mileage this year in proposing costly new initiatives for schools. In the campaigns where education became an important issue, the debate frequently centered on how the state can pay for current programs, rather than how it can start others. (See Education Week, Oct. 31, 1990.)
Mr. Henriksen also argued that economic unease and resistance to taxation appeared to have been a key factor contributing to the defeat of Mr. Blanchard of Michigan by his Republican challenger, State Senator John M. Engler.
Similar factors were at work, he said, in Democratic victories over two Republican incumbents: Gov. Mike Hayden of Kansas, who lost to State Treasurer Joan Finney; and Gov. Kay A. Orr of Nebraska, who was beaten by Ben Nelson.
And in the pivotal California race, an anti-tax platform appeared to have helped the Republican standard-bearer, U.S. Senator Pete Wilson, hold off his Democratic opponent, Dianne Feinstein, and thus keep that crucial governorship in GOP hands.
In the one state where education spending appeared to be the central issue, however, the electorate of Illinois swung in favor of a Republican candidate who resisted cutting a tax that aids the schools.
Secretary of State Jim Edgar narrowly defeated Attorney General Neil Hartigan, the Democratic nominee, after Mr. Hartigan gained considerable ground on him by promising to end an income-tax surcharge that provides more than $350 million a year for schools.
Mr. Edgar said he would ask the legislature to extend the 20 percent tax surcharge that it had approved two years ago, but Mr. Hartigan sought to mobilize a wave of anti-tax sentiment by promising to let the surcharge die. Instead, he promised to generate funding for education by trimming budgetary fat and committing portions of state revenue growth to schools.
Mr. Henriksen noted that there were other key factors in determining the result, such as Mr. Hartigan's relatively poor relations with Chicago's traditionally Democratic black community.
But Randy J. Barnette, press secretary of the Illinois Education Association, which supported Mr. Edgar, offered a different explanation.
The Republican's victory, Mr. Barnette argued, shows "you can be honest with people and tell them you intend to put more money in education."
Mr. Edgar's victory was bittersweet for the N.E.A., though, for it meant that a Republican governor would be holding veto power when legislative districts are redrawn under the 1990 Census, which appears likely to deprive the state of two U.S. House seats.
Over all, however, the N.E.A. fared well in its goal of making sure that Democratic governors have a key role in redistricting states and thus determining their party's influence in the House, asserted Mickey Ibarra, political-advocacy manager for the union's office of governmental relations. (See Education Week, Oct. 10, 1990.)
Republicans handed the governorship over to Democrats Lawton Chiles in Florida, which stands to gain four House seats, and Ann Richards in Texas, which stands to gain three seats.
Although Republicans retained the governorship of the biggest redistricting prize--California, predicted to gain seven seats--Democrats control both houses of the legislature.
Governor Perpich, a former chairman of the E.C.S. who received national attention for his support of Minnesota's pioneering open-enrollrogram, was among the most prominent election losers with close ties to education.
In addition to Governor Blanchard, their ranks also included Paul R. Hubbert of Alabama, head of the state teachers' union and the unsuccessful Democratic candidate against the Republican incumbent, Guy Hunt, and John R. Silber, Democrat of Massachusetts. Mr. Silber, on leave from his post as president of Boston University, was defeated by William F. Weld, a Republican, after a series of controversial remarks angered many in the state.
The list of gubernatorial winners with close ties to education included:
Lowell P. Weicker Jr. of Connecticut, a former U.S. Senator who was regarded as one of the GOP's leading Congressional supporters of education. He ran as an independent, defeating U.S. Representatives John G. Rowland, a Republican, and Bruce A. Morrison, a Democrat.
Mr. Chiles of Florida, also a former U.S. Senator, who as chairman of the Senate Budget Committee during the 1980's staunchly defended education programs against budget cuts. Mr. Chiles defeated the one-term Republican incumbent, Gov. Bob Martinez.
Four Governors who were very active last year in the national education summit: Carroll A. Campbell Jr., Republican of South Carolina; Bill Clinton, Democrat of Arkansas; Roy Romer, Democrat of Colorado, who serves as chairman of the National Governors' Association panel on educational goals; and Terry E. Branstad, Republican of Iowa.


Centre for European Reform: Whatever happened to the G20?
Whatever happened to the G20?
George W Bush convened the first G20 summit in Washington in November 2008, at the height of the global financial and economic crisis. At two further summits in 2009, G20 leaders pledged to co-ordinate their economic stimulus packages (as well as exit strategies), avoid protectionism, address global imbalances, triple the resources of the IMF, and work out stricter rules for banks, hedge funds and other financial players.
The G20 was hailed as the body that would prevent the global economy from hurtling into another great depression. It would also allow the world's top economies, including for the first time the big emerging markets, to co-ordinate their policies in such a way as to make future crises less likely.
"Whatever happens, the G20 is already a winner", wrote Martin Wolf in the Financial Times at the time of the G20 Pittsburgh summit in September 2009. "The fact that it has become central to global policymaking may prove a more important legacy of this crisis than any specific agreements it reaches."
Less than 18 months after the initial Washington summit, however, the G20 has almost disappeared from public view. As growth has returned in most countries, the sense of urgency to 'fix' the world economy has started to fade. The new body's legitimacy is already being questioned. That is unfortunate because the G20 still has a daunting to-do list. The risk now is that the debate about what the G20 should do is superseded by one about what it should look like.
Critics are right that the G20 is unwieldy. Once the representatives from international organisations such as the IMF and the WTO, as well as regional groupings such as ASEAN, are added, the total number of leaders and top officials at G20 summits is closer to 30. The easiest way to cut the G20 down to size would be to reduce Europe's over-representation: France, Germany, Italy and the UK are members. Spain, which holds the rotating EU presidency, will once again attend the next G20 summit. So will the presidents of the European Commission, the European Council and the European Central Bank (and now Jean-Claude Juncker, who heads the eurogroup, wants to come, too). The fact that one third of G20 participants hail from Europe and only two from Africa reduces the legitimacy of this body in the eyes of many poorer countries.
The Europeans will one day have to streamline their representation. (Meanwhile, Pascal Lamy suggests how the Europeans can make less of a nuisance of themselves, see 'Too many Europeans in G20: If you must hog the seats, could you at least talk less?'.) Perhaps other emerging economies will be added instead. But this is not the time to open the Pandora's box of who should be allowed to attend G20 summits. If the group is to regain momentum and authority, it needs to first and foremost deliver on its promises.
On trade, G20 governments have not fully lived up to their pledge to refrain from protectionism. In the 12 months following the Washington summit, the countries represented there adopted 179 policies that harmed foreign trade, investment or workers, according to Global Trade Alert. The overall damage, however, has been limited and the pace with which G20 countries have imposed new tariffs and anti-dumping actions has slowed in the last six months. World leaders had also instructed their trade negotiators to finish the WTO's Doha round by the end of 2009. But multilateral trade talks remain stuck.
The G20's report card is similarly mixed when it comes to re-regulating financial markets. Despite the promise to work out new rules together, several G20 members have announced measures without consulting their partners, for example the US administration's Volcker rule or the EU's alternative investment directive. Nevertheless, the newly established Financial Stability Board (consisting of G20 finance ministers, central bankers and regulators) has helped to forge a broad consensus on what needs to be done in terms of capital and liquidity ratios, bankers' pay and so on. At the next G20 summit in June in Toronto, governments are likely to back plans for a new levy on banks.
Least progress has been achieved on global imbalances. The G20 has been sidelined, while the real discussions about exchange rate policies and trade balances have taken place between Washington and Beijing. Whether the Americans manage to put global imbalances on the Toronto agenda despite Chinese opposition is a serious test for the new forum.
To achieve results, the G20 leaders need to do two things. First, they need to concentrate on unfinished business and resist the temptation, or the pressure, to take on new tasks. The G20 has rightly rejected suggestions that it should discuss geo-political issues, such as Iran's nuclear programme. It should avoid being saddled with global climate change discussions. South Korea's idea of adding development and poverty reduction to the agenda of the G20 summit in Seoul in November is harder to dismiss. Such a broadening of the agenda would keep emerging economies interested and show that the G20 agenda does not merely reflect rich countries' interests.
Second, for the G20 to make a difference, leaders need to focus on the urgent but unexciting task of integrating the G20 into the existing systems of global governance. The G20, like the G7/8 before it, is a process, not an organisation. It cannot take legally binding decisions. It does not have a permanent secretariat. But it does have a signalling function that can galvanise governments and other international organisations to act. It did so, for example, by adopting a strong stance on tax havens at the London summit in April 2009. In March 2010, Angel Gurria, secretary-general of the OECD, said (at the GMF Brussels Forum) that on taxation and transparency, there has been "more progress in the last 12 months than in the previous 12 years, because of a clear mandate from the G20". But not all institutional links function that smoothly. Officials in the UN and other venerable bodies resent that the G20 is hogging the limelight. Yet it is these bodies that will have to implement G20 decisions.
Katinka Barysch is deputy director of the Centre for European Reform.


text:
WHEELS & BRAKES, INC. v. CAPITAL FORD TRUCK SALES, INC.
(307 SE2d 13)
DEEN, Presiding Judge.
Action on policy. DeKalb State Court. Before Judge Seeliger.
Capital Ford Truck Sales, Inc. (Capital), a truck dealer and appellee here, brought an action against appellant Wheels and Brakes, Inc. (Wheels), to recover a truck, or the value thereof, which had been stolen from Wheels' premises in September 1980 while undergoing modifications ordered by the customer to whom Capital had contracted to sell it. Prior to filing suit Capital had unsuccessfully made a formal demand on Wheels and had then sought recovery from Wheels' liability insurance carrier, Continental Insurance Co. (Continental), which denied the claim. Capital then recovered $39,414.80 from its own insurer, American Road Insurance Co. (American), according to the terms of the insurance policy issued by the latter. By a contemporaneous oral agreement memorialized by a letter that is a part of the record, Capital and American agreed that Capital would retain the cause of action against Wheels and that American would not be subrogated thereto.
Wheels answered Capital's complaint, denying liability and counterclaiming for $24,008.31 which it alleged was owed it on open account. After Continental was dismissed as a party to the action, Wheels moved for severance of its counterclaim and summary judgment on the counterclaim. The trial court denied both motions but found that $18,978.92 of Wheels' claim was undisputed by Capital.
At trial the court sustained Wheels' motions for partial directed verdicts on the issues of loss of use, exemplary damages and attorney fees, and the jury awarded $55,545.00 (representing the truck's value plus interest) to Capital and $18,000.00 (representing the balance due on the open account) to Wheels. Upon the court's instruction, the verdict was reformed to reflect the set-off and to award $37,445.00 [sic] to Capital. After its motions for judgment notwithstanding the verdict and for a new trial were denied, Wheels appealed from the judgment, enumerating the following errors: (1) the trial court's failure to grant appellant's motion to try the counterclaim separately and to hold a bifurcated trial on the issues of liability and damages; (2) the jury instruction that appellee might be awarded pre-judgment interest on the truck's fair market value; (3) the insufficiency of the evidence to support the verdict and judgment; (4) the court's failure to admit into evidence that portion of the insurance contract and proof of loss that had been negated by the memorialized oral agreement between Capital and American; (5) the court's permitting Capital's counsel, over objection, to question Wheels' employee concerning other thefts in the vicinity of Wheels' premises; and (6) the denial of appellant's motions for judgment notwithstanding the verdict and for a new trial. Held:
1. Severance is largely a matter of discretion for the trial judge, and absent clear and manifest abuse of that discretion, it will not be interfered with on appeal. OCGA 9-11-42 (Code Ann. 81A-142); Lansky v. Goldstein, 141 Ga. App. 345 (233 SE2d 437) (1977); Sollek v. Laseter, 124 Ga. App. 131 (183 SE2d 86) (1971). Where, as in the instant case, the issues were not complex and were so closely related that essentially the same evidence would be presented in the trial of the original complaint and the counterclaim, judicial economy would dictate that they should be tried together. Lincoln Land Co. v. Palfery, 130 Ga. App. 407 (203 SE2d 597) (1973). The court has similar discretion regarding whether to order a bifurcated trial for trying the issues of liability and damages. Cline v. Kehs, 146 Ga. App. 350 (246 SE2d 329) (1978). The factual situations in the cases cited by appellant in support of his contention are readily distinguishable from that in the instant case. This enumeration is without merit.
2. The court did not err in instructing the jury regarding the awarding of interest from the time of the theft until trial. The rule is that interest must be awarded on a liquidated sum from the time the liability arises, OCGA 7-4-15 (Code Ann. 57-110); and that the jury may award interest until the time of recovery "in all cases where an amount ascertained would be the damages at the time of the breach." OCGA 13-6-13 (Code Ann. 20-1408). Moreover, it is proper for the court to instruct the jury that where damages are unliquidated, interest at the legal rate may be awarded at the jury's discretion. B. G. Sanders & Assoc., Inc. v. Castellow, 154 Ga. App. 433 (268 SE2d 695), cert. denied, (1980); Norair Eng'r. Corp. v. St. Joseph's Hosp., 147 Ga. App. 595 (249 SE2d 642), cert. denied, (1978). In the challenged jury instruction the court stated, "If you find that such sum is a liquidated sum, you may also award the legal rate of 7% . . ." (Emphasis supplied.) Since the question of damages is properly the province of the jury, its determination should not be interfered with on appeal "unless the damages are either so small or so excessive as to justify the inference of gross mistake or undue bias." OCGA 13-6-4 (Code Ann. 20-1411).
In the case at bar sufficient evidence was adduced to permit calculation of the amount of the damages, and hence the amount of interest, should the jury find in its discretion that an award of interest was appropriate. Capital's president, with nearly twenty years' experience in the truck business, testified as to original cost and replacement cost (both considerably more than the amount awarded), and the original invoice for the truck was entered into evidence. See OCGA 24-9-66 (Code Ann. 38-1709). This enumeration is also devoid of merit.
3. Having determined in Division 2, supra, that the evidence was sufficient to authorize the verdict and judgment, we likewise find appellant's third enumeration without merit.
4. Appellant correctly states the rule that once an insured has settled a claim with his insurer, the latter, under the ordinary insurance contract, is subrogated to the insured's claims and can pursue an action to recover for the sums paid to the insured. See, e.g., Bryant v. Atlanta Gas Light Co., 149 Ga. App. 126 (253 SE2d 807 (1979). It is a basic tenet of contract law, however, that the parties may by contract modify or otherwise alter their obligations. See, e.g., 17 AmJur2d 924, Contracts, 459. By so doing in this case, the parties rendered inoperative the provision of the insurance contract that appellant contends should have been admitted into evidence. To have done as appellant wished would have injected irrelevant and potentially confusing data into the case, since the agreement between Capital and its insurer concerned only relationships and obligations between themselves and was not binding on a third party. See 17 AmJur2d 710 et seq., 294, 297. To insist, as appellant does, that a superseded provision of appellee's insurance contract should have been admitted into evidence, and thereby obliquely to contend that Capital was not the real party in interest, is specious. See OCGA 9-11-17 (Code Ann. 81A-117). This enumeration is without merit.
5. The trial court erred in permitting appellee's attorney to question appellant's employee regarding thefts from other businesses in the area. We recognize that, as appellee argues, such questions were intended to establish whether appellant was or should have been on notice regarding the possibility of thefts from its premises. Since the burden of proving ordinary diligence, the standard of care required of a bailee for hire, is upon the bailee once his failure to return the bailor's property has been established, the evidence appellee sought to elicit was relevant. Rhodes v. Duarte, 142 Ga. App. 885 (237 SE2d 212) (1977).
We cannot agree, however, that questions posed by appellee's counsel regarding a prior theft from Wheels' premises and Wheels' physical security arrangements constituted an adequate foundation for introduction of the series of questions about thefts from neighboring businesses. Even on cross-examination, these questions were patently improper in this case, as absolutely no foundation had been established for them. They were not merely leading questions. Rather, under the guise of a question, the attorney was in fact endeavoring to testify that a theft had actually occurred at the places and times stated. " 'A question assuming a fact not proved or admitted is improper and should be excluded.' " Kamman v. Seabolt, 149 Ga. App. 167, 170 (253 SE2d 842) (1949). The fact that appellee's attorney had a police report in his hand while asking these questions does not make the questions proper, nor does the fact that no objection was made to a similar question of another witness make these questions proper over objection.
Since these questions could have a prejudicial effect on the jury in this bailment case, they were harmful and should have been excluded.
Michael V. Elsberry, for appellee.
M. David Merritt, H. A. Stephens, Jr., for appellant.
DECIDED JUNE 28, 1983 -- REHEARING DENIED JULY 22, 1983.
BROOKS et al. v. H & H CREEK, INC. et al. (223 Ga. App. 635) (478 SE2d 451) (1996)
SOUTHERN GUARANTY INSURANCE COMPANY v. NIXON. (194 Ga. App. 398) (390 SE2d 638) (1990)
PARKS et al. v. CONSOLIDATED FREIGHTWAYS et al. (187 Ga. App. 576) (370 SE2d 827) (1988)
BURGESS et at. v. GORLIN et at.; FIRST GEORGIA BANK v. GORLIN et at.; GORLIN et at. v. HALPERN et at. (184 Ga. App. 10) (360 SE2d 729) (1987)
EASTERN AIR LINES, INC. v. FULTON COUNTY. (183 Ga. App. 891) (360 SE2d 425) (1987)
VITNER et al. v. FUNK. (182 Ga. App. 39) (354 SE2d 666) (1987)
BLUE CROSS OF GEORGIA/COLUMBUS, INC. et al. v. WHATLEY. (180 Ga. App. 93) (348 SE2d 459) (1986)
HARRELL v. GOMEZ et al. (174 Ga. App. 8) (329 SE2d 302) (1985)
DEPARTMENT OF TRANSPORTATION v. DEFOOR et al. (two cases). (173 Ga. App. 218) (325 SE2d 863) (1984)


text:
Exhibition: Thirteen Ways of Looking
Covering up the Cracks: The Return of Wallpaper
Lindsey Mendick, 'The Yellow Wallpaper', 2020, installation detail, Eastside Projects, Birmingham. Courtesy: the artist and Eastside Projects; photograph: Stuart Whipps
https://www.frieze.com/article/covering-cracks-return-wallpaper
Feature News
National Portfolio Organisations: A Response from Director Craig Ashley
New Art West Midlands' director Craig Ashley reflects on yesterday's announcement from Arts Council England about investment to the region's visual arts organisations through their National Portfolio for 2018-22.
ESP & Axisweb: Lunch and Pitch
What would you do with £200, do you have a great idea that you want to share with other people? Have you got an artwork that needs a boost, an idea that needs to be realised? Deadline: Wednesday 26 September 2018.
Win for Eastside Projects
Eastside Projects has been announced as part of the winning team to design the new market of Birmingham as part of the £1.5 billion Birmingham Smithfield regeneration.
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New Art West Midlands is funded by Arts Council England and we are hosted by our lead partner Birmingham City University. We are supported by our project partners Coventry University, University of Wolverhampton and University of Worcester. New Art West Midlands Exhibition 2017 is led by Birmingham Museums Trust with support from participating host venues. It is funded by Arts Council England alongside Birmingham City University, Coventry University, Hereford College of Arts, Staffordshire University, University of Wolverhampton and University of Worcester. New Art West Midlands Engine is led by The New Art Gallery Walsall. It is funded by Arts Council England, and delivered with support from Walsall Council and our programme partners.
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text:
There are ten scheduled monuments in Coventry. In the United Kingdom, a scheduled monument is a "nationally important" archaeological site or historic building that has been given protection against unauthorised change by being placed on a list (or "schedule") by the Secretary of State for Digital, Culture, Media and Sport; English Heritage takes the leading role in identifying such sites. Monuments are defined in the Ancient Monuments and Archaeological Areas Act 1979 and the National Heritage Act 1983. Scheduled monuments—sometimes referred to as scheduled ancient monuments—can also be protected through listed building procedures, and English Heritage considers listed building status to be a better way of protecting buildings and standing structures. A scheduled monument that is later determined to "no longer merit scheduling" can be descheduled.

Coventry is an ancient city and a metropolitan borough in the West Midlands of England. The city's history dates back to at least the 11th century (CE), and by the 14th century, it was a thriving centre of commerce. Like several of the other monuments in the city, Coventry's city walls were erected towards the end of the 14th and the beginning of the 15th centuries, though city walls served little defensive purpose by that time and were largely a status symbol.

The oldest monuments on this list—Caludon Castle and St Mary's Priory and Cathedral—were built in the 11th century. Both are now ruins. Coventry's most modern scheduled monument is Vignoles Bridge—a single-span iron footbridge over the River Sherbourne, made in 1835 and moved to its current location in 1969.

Monuments

See also
History of Coventry
Grade I listed buildings in Coventry
Grade II* listed buildings in Coventry

Notes

References

Archaeological sites in the West Midlands (county)
Scheduled
Coventry
Scheduled

redpajama_set_name: RedPajamaWikipedia


White christmas? Not in Switzerland!
temperatures were no longer allowing snow.
degrees celsius which is far below the average medium.
down to the flatlands isn't what one is expecting towards June!
So hopefully the weather will change soon again!
Finally… the winter seems back!
the snow last and how long is the timespan until the next thaw time?
special, happens often at my place" – But is yours only 550m above sea level?
February to be that cold, however it is uncommon after such a warm January.
storm called "Andrea" brought rain and heavy winds.
So it is winter… finally! Every year the people are hoping for snow for christmas here in Bern.
This year it's a bit late as the last two days were the only days where we got 10cm of snow!
should usually know how to prepare for it and try to reduce delay times as good as possible.
Last year, deep winter, this year, not?
temperatures are rather mild for November. So hopefully winter will still come.
Why deep autumn makes depressive!
sleep all day long. Also it's the time where the most projects run.
Autumn and winter at the same time?
polar air to the south over Europe and temperatures have dropped by 15 to 25 °C.
situated villages such as Davos for example.


Justice Secretary and International Anti-Corruption Champion, Kenneth Clarke, has welcomed UN International Anti-Corruption Day.
'As the UK Government's lead Minister on International Anti-Corruption I am delighted to support UN International Anti-Corruption Day.
'We are working hard to tackle corruption both at home and abroad. Through the Bribery Act, the UK has sent a unequivocal message that we intend to take a strong stand on corruption and that bribes should not play any part in winning business. This significant step has been reflected in our improved ranking on Transparency International's Annual Corruptions Index, reflecting the UK's status as one of the least corrupt countries in the world.


Chick-fil-A has retired their calendar program after 2018. 🙁 Those silly Chick-fil-A cows are always badgering us to "eat mor chikin", and you can comply with their demands in 2018 with the 2018 "Steers of Yesteryear" Chick-fil-A Calendar.
Chick-Fil-A's October 2017 Cow Calendar Mystery Offer is… Any size Coca-Cola beverage or a Dasani bottled water. Present your 2017 Chick-Fil-A Cow Calendar Card or your Chick-Fil-A One app at check-out to get your freebie!
When I traveled for work we frequented a Chick-fil-A that employed a young man with Downs syndrome and wed often see him doing tasks like refilling the condiments and bussing trays.
December – Chick-fil-A chicken sandwich, grilled chicken sandwich or spicy chicken sandwich The calendar is a really fun and inexpensive gift for any Chick-fil-A fanatic. If your family dines there often, it's worth it to pick up a few extra copies just to get the freebies all year-long — just recycle the unwanted calendars.