Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR

민한솔,김진일,김영진,윤미숙,Richard E. Pratley,이용호 한국통합생물학회 2017 Animal cells and systems Vol.21 No.2

Obesity is associated with increased risk of Alzheimer’s disease. Previous studies have demonstrated that amyloid-beta precursor protein (APP) is expressed in subcutaneous adipose tissue (SAT), upregulated with obesity, and correlates with insulin resistance and adipose tissue inflammation. APP is alternatively spliced into several isoforms, which may be indicative of the pathogenesis of APP-related diseases, but the accurate quantification has been difficult to standardize and reproduce. In light of this, we developed isoform-specific absolute cDNA standards for absolute quantitative real-time PCR (AQ-PCR), and measured transcript copy numbers for three major APP isoforms (APP770, APP751, and APP695), in SAT from C57BL/6 mice fed either a normal or highfat diet. Expression of all three major APP isoforms was increased in diet-induced obese mice. Transcript copy numbers of APP770 and APP695 correlated with plasma insulin and CCL2 gene expression. The ratios of APP770 and APP751 to APP695 gradually decreased with aging, and correlated with plasma glucose levels. In addition, APP770 was significantly decreased in thiazolidinedione-treated mice. We describe quantification of APP isoform transcripts by AQ-PCR, which allows for direct comparison of gene copy number across isoforms, between experiments, and across studies conducted by independent research groups, which relative quantitative PCR does not allow. Our results suggest a possible role of differential expression of APP isoforms in the development of obesity-related insulin resistance and adipose tissue inflammation. In addition, it is important to determine if altered ratios of APP isoforms in SAT contribute to higher circulating Aβ peptides and increased risk of abnormalities in obesity.

Laboratory Bioassay for Canola (Brassica napus) Allelopathy

Md. Asaduzzaman,Min An,James Edward Pratley,David John Luckett,Deirdre Lemerle 한국작물학회 2014 Journal of crop science and biotechnology Vol.17 No.4

Crop allelopathy provides a viable alternative in managing resistant weed populations. A successful demonstration of an allelopathicinteraction is needed to be separated from resource competition. A reliable laboratory screening technique can test this chemicalinteraction between a donor and a receiver species. However, the standardization of any screening tactic for a new species is reallychallenging. In this study we optimized the simple root exudates bioassay, Equal Compartment Agar Method (ECAM), for canolathrough basic features including sowing patterns, density, and distance between canola and annual ryegrass. We established that allof the above features have major roles on canola seedling interference ability (allelopathy + competition). Due to the combined effectof allelopathy and competition, the zigzag canola sowing pattern enhanced the inhibitory effects of canola seedlings more than othersowing patterns, i.e. circular, parallel, and cone. The sowing distance of canola played a major role on root growth of annual ryegrassin that the nearer the receiver annual ryegrass plants to the donor canola seedlings, the greater were the toxic effects. Canola sowingdistance at 1, 2, and 3 cm inhibited the root growth of ryegrass by 50, 46, and 36%, respectively. Results also showed that rootgrowth of ryegrass decreased with increased canola densities (0 – 40 seedlings/beaker). Furthermore, the added carbon experimentshowed that the inhibitory effects of canola were diluted by added carbon in agar growth medium, indicating canola root exudatesacted directly on the root growth of annual ryegrass. This study suggests parameters such as canola sowing pattern, density, and sowingdistance between canola and test weed species need to be considered for designing canola allelopathy bioassay through ECAMunder laboratory conditions

Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR

Min, Hansol,Kim, Jinil,Kim, Young-Jin,Yoon, Mi-Sook,Pratley, Richard E.,Lee, Yong-Ho ZOOLOGICAL SOCIETY OF KOREA 2017 ANIMAL CELLS AND SYSTEMS Vol.21 No.2

<P><B>ABSTRACT</B></P><P>Obesity is associated with increased risk of Alzheimer’s disease. Previous studies have demonstrated that amyloid-beta precursor protein (APP) is expressed in subcutaneous adipose tissue (SAT), upregulated with obesity, and correlates with insulin resistance and adipose tissue inflammation. APP is alternatively spliced into several isoforms, which may be indicative of the pathogenesis of APP-related diseases, but the accurate quantification has been difficult to standardize and reproduce. In light of this, we developed isoform-specific absolute cDNA standards for absolute quantitative real-time PCR (AQ-PCR), and measured transcript copy numbers for three major APP isoforms (APP770, APP751, and APP695), in SAT from C57BL/6 mice fed either a normal or high-fat diet. Expression of all three major APP isoforms was increased in diet-induced obese mice. Transcript copy numbers of APP770 and APP695 correlated with plasma insulin and CCL2 gene expression. The ratios of APP770 and APP751 to APP695 gradually decreased with aging, and correlated with plasma glucose levels. In addition, APP770 was significantly decreased in thiazolidinedione-treated mice. We describe quantification of APP isoform transcripts by AQ-PCR, which allows for direct comparison of gene copy number across isoforms, between experiments, and across studies conducted by independent research groups, which relative quantitative PCR does not allow. Our results suggest a possible role of differential expression of APP isoforms in the development of obesity-related insulin resistance and adipose tissue inflammation. In addition, it is important to determine if altered ratios of APP isoforms in SAT contribute to higher circulating Aβ peptides and increased risk of abnormalities in obesity.</P>

Dysregulation of Cannabinoid CB1 Receptor Expression in Subcutaneous Adipocytes of Obese Individuals

Lee, Yong-Ho,Tharp, William G.,Dixon, Anne E.,Spaulding, Laurie,Trost, Susanne,Nair, Saraswathy,Permana, Paska A.,Pratley, Ridhard E. The Korean Society for Integrative Biology 2009 Animal cells and systems Vol.13 No.4

The endocannabinoid system (ECS) plays a key role in the regulation of appetite, body weight and metabolism. We undertook the present study to further clarify the regulation of the cannabinoid CB1 receptor (CB1, CNR1) in human adipose tissue in obesity. CB1 receptor mRNA expression was ~1.6-fold (p<0.004) and 1.9-fold higher (P<0.05) in subcutaneous adipocytes from obese compared to non-obese subjects in microarray and quantitative real-time PCR studies, respectively. Higher CB1 receptor mRNA expression levels in both adipose tissue (~1.2 fold, P<0.05) and adipocytes (~2 fold, P<0.01) were observed in samples from visceral compared to subcutaneous depots collected from 22 obese individuals. Immunofluorescence confocal microscopy demonstrated the presence of CB1 receptor on adipocytes and also adipose tissue macrophages. These data indicate that adipocyte CB1 receptor is up-regulated in human obesity and visceral adipose tissue and also suggest a potential role for the ECS in modulating immune/inflammation as well as fat metabolism in adipose tissue.

Dysregulation of Cannabinoid CB1 Receptor Expression in Subcutaneous Adipocytes of Obese Individuals

이용호,William G. Tharp,Anne E. Dixon,Laurie Spaulding,Susanne Trost,Saraswathy Nair,Paska A. Permana,Richard E. Pratley 한국통합생물학회 2009 Animal cells and systems Vol.13 No.4

The endocannabinoid system (ECS) plays a key role in the regulation of appetite, body weight and metabolism. We undertook the present study to further clarify the regulation of the cannabinoid CB1 receptor (CB1, CNR1) in human adipose tissue in obesity. CB1 receptor mRNA expression was ~1.6-fold (P<0.004) and 1.9-fold higher (P<0.05) in subcutaneous adipocytes from obese compared to non-obese subjects in microarray and quantitative real-time PCR studies, respectively. Higher CB1 receptor mRNA expression levels in both adipose tissue (~1.2 fold, P<0.05) and adipocytes (~2 fold, P<0.01) were observed in samples from visceral compared to subcutaneous depots collected from 22 obese individuals. Immunofluorescence confocal microscopy demonstrated the presence of CB1 receptor on adipocytes and also adipose tissue macrophages. These data indicate that adipocyte CB1 receptor is up-regulated in human obesity and visceral adipose tissue and also suggest a potential role for the ECS in modulating immune/inflammation as well as fat metabolism in adipose tissue. The endocannabinoid system (ECS) plays a key role in the regulation of appetite, body weight and metabolism. We undertook the present study to further clarify the regulation of the cannabinoid CB1 receptor (CB1, CNR1) in human adipose tissue in obesity. CB1 receptor mRNA expression was ~1.6-fold (P<0.004) and 1.9-fold higher (P<0.05) in subcutaneous adipocytes from obese compared to non-obese subjects in microarray and quantitative real-time PCR studies, respectively. Higher CB1 receptor mRNA expression levels in both adipose tissue (~1.2 fold, P<0.05) and adipocytes (~2 fold, P<0.01) were observed in samples from visceral compared to subcutaneous depots collected from 22 obese individuals. Immunofluorescence confocal microscopy demonstrated the presence of CB1 receptor on adipocytes and also adipose tissue macrophages. These data indicate that adipocyte CB1 receptor is up-regulated in human obesity and visceral adipose tissue and also suggest a potential role for the ECS in modulating immune/inflammation as well as fat metabolism in adipose tissue.