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Transcriptome profiling reveals the antihyperglycemic mechanism of pelargonidin-3-O-glucoside extracted from wild raspberry
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Transcriptome profiling reveals the antihyperglycemic mechanism of pelargonidin-3-O-glucoside extracted from wild raspberry Hongming Sua, Tao Baoa, Lianghua Xiea, Yang Xua, Wei Chena,b,
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a
Department of Food Science and Nutrition, National Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou 310058, China b Ningbo Research Institute, Zhejiang University, Ningbo 315100, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Raspberry Pelargonidin-3-O-glucoside Diabetes RNA-sequence Insulin resistance
Emerging evidence suggests that anthocyanins prevent type 2 diabetes (T2D), however, its mechanisms remain elusive. Herein, we sought to elucidate the antihyperglycemic mechanism of pelargonidin-3-O-glucoside (Pg3G) extracted from wild raspberry based on RNA-sequence. Our study showed that Pg3G ameliorated glucose intolerance and insulin resistance in db/db mice. Besides, Pg3G improved serum lipid profiles and attenuated hepatic dysfunction. RNA-sequence analysis showed that Pg3G contributed to 301 genes upregulated and 269 genes downregulated. Further GO, KEGG and GSEA analysis indicated that Pg3G led to differential gene expressions enriched in glucose and lipid metabolism by upregulation of Elovl7, C1ql3, Cybb, Cytip, Src, Pdk4 and downregulation of Fasn, Aacs, Srebp1c, Mlxipl, Sorbs3, Gck, Slc2a4. RT-PCR validation of selected genes exhibited a positive correlation between RNA-sequence and RT-PCR results. Taken together, this study demonstrated that Pg3G attenuated T2D by regulating glucose and lipid metabolism, which implicates the potential for T2D therapeutics.
1. Introduction The incidence of type 2 diabetes (T2D) is increased rapidly in recent decades both in developing and developed countries, which affected approximately 425 million people worldwide (Disease, Injury, & Prevalence, 2016). The high prevalence of T2D dramatically increases the economic burden. Emerging evidence indicates that both genetic factors and unhealthy lifestyles including the reduced level of exercise, long-time sedentary lifestyle and high fat dietary intake result in T2D (DeFronzo et al., 2015). T2D, in general, is characterized by hyperglycemia, insulin resistance and the lack of insulin release, which is associated with an increased risk of several metabolic complications, such as diabetic nephropathy, diabetic retinopathy and diabetic neuropathy (DeFronzo et al., 2015). Metformin, acarbose and rosiglitazone are FDA-approved agents that are available for pharmaceutical management of diabetes (American Diabetes, 2018). However, long-time use of those drugs may cause undesirable side-effects (Rines, Sharabi, Tavares, & Puigserver, 2016). Therefore, it is urgently needed to develop novel anti-diabetic agents. Recently, natural products derived from daily consumed food or traditional Chinese medicine (TCM) display a beneficial effect on glucose metabolism. Those phytochemicals have achieved increasing
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attentions (Martel et al., 2017). For example, natural polyphenols such as myricetin, catechin as well as resveratrol showed promises to prevent T2D (Li et al., 2017; Sung et al., 2017; Yan, Zhao, Suo, Liu, & Zhao, 2012). In this regard, phytochemicals could be an important natural reservoir to exploit anti-diabetic agents and develop novel therapeutics. Anthocyanins have the demonstrated ability to protect against a myriad of human diseases including diabetes and obesity (Xie, Su, Sun, Zheng, & Chen, 2018). The effects of anthocyanin on diabetes have been summarized in our previous published review papers (Gowd, Jia, & Chen, 2017; Xie et al., 2018). The mechanism of anthocyanins underlying the anti-diabetic action could be attributed to enzymatic activation or inhibition, increased insulin sensitivity and insulin secretion as well as reduced hepatic gluconeogenesis and increased glycolysis (Rines et al., 2016). The chemical structure of disparate anthocyanin explains varied activity and mechanism. For example, our recent study reported a purified natural anthocyanin prevented postprandial hyperglycemia by inhibiting α-glucosidase (Xu, Xie, Xie, Liu, & Chen, 2018), suggesting a plausible mechanism underling anthocyanins’ anti-diabetic action. Although increasing evidence indicates the protective role of anthocyanins against diabetes and diabetes-associated complications, it is still largely unknown regarding the molecular mechanisms of anthocyanins (e.g. pelargonidin-3-O-glucoside) against diabetes.
Corresponding author at: Department of Food Science and Nutrition, Zhejiang University, No. 866 Yuhangtang Road, Xihu District, Hangzhou 310058, China. E-mail address: [email protected] (W. Chen).
https://doi.org/10.1016/j.jff.2019.103657 Received 16 August 2019; Received in revised form 22 October 2019; Accepted 28 October 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Hongming Su, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103657
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liquid nitrogen. All samples were preserved at −80 °C for further investigation.
Raspberry was reportedly rich in anthocyanins, which are responsible for exerting the health-promoting effects (Xie et al., 2018). In our preliminary study, we collected wild raspberry (Rubus hirsutus Thunb.) from the local area in Zhejiang Province, China. One major anthocyanin pelargonidin-3-O-glucoside (Pg3G) identified was predominantly enriched in wild raspberry according to LC-MS/MS and NMR analyses (H. Su et al., 2019). Since anthocyanins show the potential for anti-diabetic application, in the present study we investigated the hyperglycemia-lowering effect of Pg3G on db/db diabetic mice. RNA-sequence (RNA-seq) analysis has been widely used to study the gene expression profiles involved in a specific disease phenotype and facilitates the identification of signature genes. To elucidate the underlying mechanism, thus we employed RNA-seq analysis to identify the differential gene expressions (DEGs) and illustrate the alterations of DEGs corresponding functional pathways in terms of GO, KEGG and GSEA analyses. This study may constitute a novel insight into the application of Pg3G for the prevention and treatment of T2D.
2.3. Glucose tolerance test (GTT) and insulin tolerance tests (ITT) GTT and ITT were determined according to a previous report (Liu, Lee, Salazar Hernandez, Mazitschek, & Ozcan, 2015). In the eighth week, the mice fasted for 12 h, and GTT was performed after 0.5 g/kg glucose was administered intraperitoneally. Blood glucose levels were measured by collecting blood from the tail vein before glucose administration and 30, 60 and 120 min after glucose administration (Roche Septi-Chek system). At the end of the eighth week, ITT was performed after mice fasted for 6 h. Insulin (2 IU/kg) was administered intraperitoneally. Blood glucose levels were measured by collecting blood from the tail vein before insulin administration and 30, 60 and 120 min after the administration. 2.4. Biochemical analysis
2. Materials and methods The blood samples were collected and centrifuged at 5000 rpm for 10 min, then serum was collected for biochemical analysis. Serum glucose, free fatty acids (FFAs), cholesterol, triglyceride (TG), lowdensity lipoprotein cholesterol (LDL-c), aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were measured by Hitachi automatic biochemistry analyzer.
2.1. Preparation of Pg3G from wild raspberry Pg3G was extracted from wild raspberry according to our previously established methods (Xu, Hu, Li, Sun, & Chen, 2018; Xu, Xie, et al., 2018). Briefly, anthocyanins fraction was extracted from wild raspberry fruits using 70% ethanol for 12 h and then filtered through three layers of gauze. The collected filtrate was evaporated at 45 °C and then purified by AB-8 macroporous resin column. The elution solution was lyophilized to yield raspberry anthocyanins fraction. The raspberry anthocyanins fraction was further purified by HSCCC (TBE-300A, Tauto Biotechnique Company, Shanghai, China) using a two-phase solvent system of tert-butyl methyl ether - n-butanol - acetonitrile - water (2:2:1:5, v/v/v/v, acidified with 0.1% trifluoroacetic acid). The structure of purified Pg3G was confirmed by HPLC-ESI–MS (Agilent 1200 series and 6430 triple quadrupole mass spectrometers, USA) and NMR (Bruker AVANCETM III spectrometer (14.1 T)), respectively. The purity of Pg3G was 98.6%. The ESI–MS and NMR results were indicated in Supplementary Materials and methods.
2.5. RNA extraction and sequencing Hepatic total RNA was prepared from control and Pg3G (150 mg/kg BW) treated mice (n = 3 per group) using TRIzol following the manufacturer’s protocol (Life Technologies). The RNA quality was determined using Agilent 2100 Bioanalyzer. Then mRNAs were isolated from total RNA with oligo(dT) method and the cDNA fragments were linked with adapters. The libraries were sequenced with an Illumina HiSeq Platform. 2.6. RNA-seq analysis The low-quality reads (More than 20% of the bases qualities are lower than 10), reads with adaptors and reads with unknown bases (N bases more than 5%) were filtered to get the clean reads using SOAPnuke. Those clean reads were mapped onto the reference genome using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts). The mapped clean reads were mapped to the reference gene using Bowtie2 (v2.2.5) and the gene expression levels were calculated with RSEM (v1.2.12). We detected DEGs with DEseq2. DEseq2 is based on the negative binomial distribution. Finally, we identified DEGs with DEseq2 between samples (Fold Change ≥ 1.00 and Adjusted P-value ≤ 0.05). Gene Ontology (GO) and KEGG pathway analyses were performed using phyper, a function of R. False discovery rate (FDR) was calculated and FDR less than 0.01 are defined as significantly enriched. The diagrams were drawn with ggplot2, a function of R. Gene set enrichment analysis was performed based on the GO gene sets and KEGG gene sets using GSEA 2.0 with default parameters (permutation type: gene_set. Collapse dataset to gene symbols: false).
2.2. Animal experiments The Animal experiments were conducted according to the guidelines on the use and care of laboratory animals in China (GB/T 358922018 and GB/T 35823-2018). All animal experimental procedures were performed in the Animal Experiment Center of Zhejiang Chinese Medical University (Hangzhou, China). The animal protocol was approved by the laboratory animal management and ethics committee of Zhejiang Chinese Medical University (201610087). 36 male Leprdb mutation (db/db) mice with C57BL/6J background, aged six weeks, were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). The temperature in the cage was maintained with constant temperature (23 °C) and humidity under a 12 h/ 12 h light/dark cycle. The mice were fed a standardized diet (kcal %: 10% fat, 20% protein, and 70% carbohydrate). All mice have free access to diet and sterilized water. After one week of acclimatization, the mice were divided into three groups (n = 12 per group): (1) db/db mice were treated with vehicle (ultra-purified water); (2) db/db mice were orally gavaged with 50 mg/kg BW per mice Pg3G; (3) db/db mice were orally gavaged with 150 mg/kg BW per mice Pg3G. The doses of Pg3G were according to a previous report with modifications (Mirshekar, Roghani, Khalili, Baluchnejadmojarad, & Arab Moazzen, 2010). After fasting for 12 h overnight, the fasting blood glucose (FBG) levels were determined every week using Roche Septi-Chek system by collecting blood from the tail vein of mice. After eight weeks, the mice were sacrificed, following 12 h fasting. Blood samples were collected by cardiac puncture. Liver samples were collected, weighed and snap-frozen by
2.7. Quantitative real-time PCR The quantitative real-time PCR (RT-PCR) analysis was performed to validate the results from RNA-seq as previously described (Su et al., 2018). Total RNA was isolated from the liver using Trizol and then pooled for the RT-PCR analysis. cDNA was synthesized from total RNA using the PrimeScript RT reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was carried out in the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). SYBR Green 2
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(Roche, Germany) was used as a fluorescent dye for quantitative PCR analysis. The primers were synthesized by GENEWIZ (Suzhou, China) and the primer sequences were indicated in Supplementary Table 1. A relative gene-expression quantification method was used to calculate the fold change of mRNA expression. GAPDH was set as an internal reference for normalization.
sensitivity in db/db diabetic mice. In addition, 150 mg/kg BW Pg3G administration showed a better glucose-lowering effect than 50 mg/kg BW Pg3G administration in terms of FBG levels, GTT and ITT. 3.2. Pg3G ameliorated serum biochemical profile As shown in Fig. 2A, B and D, Pg3G (50 and 150 mg/kg BW) administration led to a significant decrease of serum FFAs, cholesterol and LDL-c compared with those in the control. Nonetheless, Pg3G had no impact on the level of serum triglyceride (Fig. 2C). In addition, this study showed that 150 mg/kg BW Pg3G administration significantly reduced serum AST, ALT, LDH and ALP levels (Fig. 2F–H), in comparison with those in the control, suggesting the improvement of hepatic dysfunction involved in T2D.
2.8. Statistical analysis Data are expressed as the mean ± SEM. Significant differences were evaluated by two-tailed Student’s t-test between two groups or one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test between multiple groups. p < 0.05 was considered to be a significant difference. All statistical analysis was performed using GraphPad Prism V.7.0a (GraphPad Software, USA).
3.3. Pg3G contributed to differential gene expressions (DEGs) in the liver 3. Results The liver plays a crucial role in the regulation of glucose metabolism. Since 150 mg/kg BW Pg3G displayed a better antihyperglycemic effect than 50 mg/kg BW Pg3G, we therefore further examined the effect of Pg3G 150 mg/kg BW on hepatic gene transcriptional profile. High throughput RNA-seq analysis was adopted to identify the DEGs. The classification of raw reads from each group was indicated in Fig. 3A–F. We obtained an average of 44.47 million clean reads from each group. Total clean reads Q30% in each group was higher than 95%, which indicated a good sequencing quality (Supplementary Table 2). The total gene mapping ratio was beyond 69.67%. The gene expression levels were quantified and expressed as FPKM. Fig. 3G indicated the distribution of gene expression in each mouse. Those results indicated that the quality of all libraries was good and suitable for subsequent analysis. The total gene number on average in control mice was 14,590, and
3.1. Pg3G attenuated glucose intolerance and insulin resistance In the present study, the results of fasting blood glucose (FBG) levels within 8 weeks indicated that both concentrations of Pg3G caused a remarkable FBG reduction (Fig. 1A), suggesting a long-term beneficial role of Pg3G against hyperglycemia in T2D. Pg3G administration contributed to a significant improvement of glucose tolerance compared to the control group (Fig. 1B). The area under the curve (AUC) of GTT in Pg3G treated mice was significantly lower than that in the control (Fig. 1C). ITT assay indicated that Pg3G administration significantly reduced blood glucose level after insulin injection compared with the control (Fig. 1D). The AUC of ITT in Pg3G treated mice was remarkably decreased compared with that in the control (Fig. 1E). Together, these results indicated that Pg3G improved glucose tolerance and insulin
Fig. 1. Pg3G exerted glucose-lowering effect on db/db diabetic mice. Db/db mice were orally gavaged with vehicle or Pg3G (50 and 150 mg/kg BW per day) for 8 weeks. (A) The fasting blood glucose was determined every week for overall 8 weeks. (B) Glucose tolerance test (GTT) was performed at the eighth week. (C) The area under the curve (AUC) of GTT was calculated. (D) At the end of the eighth week, insulin tolerance test (ITT) was performed. (E) The AUC of ITT was calculated. Control, db/db mice orally administered with vehicle; Pg3G 50, db/db mice orally administered with 50 mg/kg BW Pg3G; Pg3G 150, db/db mice orally administered with 150 mg/kg BW Pg3G. Data represent means ± s.e.m., n = 10 per group, *p < 0.05 versus control group. 3
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Fig. 2. Pg3G improved serum biochemical profile. (A) serum FFAs, (B) cholesterol, (C) triglyceride, (D) LDL-c, (E) AST, (F) ALT, (G) LDH and (H) ALP levels were determined (n = 10). Control, db/db mice orally administered with vehicle; Pg3G 50, db/db mice orally administered with 50 mg/kg BW Pg3G; Pg3G 150, db/db mice orally administered with 150 mg/kg BW Pg3G. Data represent means ± s.e.m., *p < 0.05 versus control group.
KEGG pathway. The top three DEGs number of upregulated genes were included in PI3K-Akt signaling pathway, focal adhesion and metabolic pathways. The top three DEGs number of downregulated genes were mainly included in metabolic pathways, arachidonic acid metabolism and chemical carcinogenesis (Fig. 4D).
that number in Pg3G treated mice was 14,959. As shown in Fig. 3H and Supplementary Table 3, the DEGs analysis indicated that Pg3G administration contributed to 301 genes up-regulated and 269 genes down-regulated compared with those in the control. In addition, 15,808 genes were not changed upon Pg3G treatment. These results indicated that Pg3G administration resulted in hepatic DEGs.
3.6. Gene set enrichment analysis (GSEA) 3.4. GO analysis Gene set enrichment analysis (GSEA) determines whether a predefined set of genes shows a significant difference (Subramanian et al., 2005). To further interpret the effect of Pg3G on functional gene expression, GSEA was performed based on the GO gene sets and KEGG gene sets, respectively. According to GSEA in terms of GO gene sets, Pg3G administration contributed to a positive enrichment of mitochondrial respiratory chain, oxidation-reduction process and lipid metabolic process (Fig. 5A–C and Supplementary Figs. 1–3). Conversely, Pg3G administration led to negative enrichment of response to lipopolysaccharide and inflammatory response (Fig. 5D–E and Supplementary Figs. 4 and 5). According to GSEA in terms of KEGG gene sets, Pg3G administration resulted in a positive enrichment of KEGG pathway of energy metabolism oxidative phosphorylation, digestive system bile secretion (Fig. 5F and G, and Supplementary Figs. 6 and 7). Moreover, Pg3G administration led to a negative enrichment, in the KEGG pathway, of endocrine and metabolic disease age-rage signaling in diabetic complications, PI3K-Akt-signaling pathway and TNF signaling pathway (Fig. 5H–J and Supplementary Figs. 8–10).
To further analyze the role of DEGs upon Pg3G administration, we performed GO analysis to ascertain the function of DEGs. The results of GO were divided into biological process, cellular component and molecular function. According to GO analysis, the top three high-enriched GO term in biological process were response to chemical, biological process and response to organic substance. The top three significantly enriched GO term in cellular component were extracellular region, extracellular region part and extracellular matrix. The top three highenriched GO term in molecular function were monooxygenase activity, aromatase activity and heme binding (Fig. 4A). The DEGs of the most enriched pathway in each GOTerm include cellular process, cell and binding (Fig. 4B). 3.5. KEGG analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to elucidate the function of DEGs. As shown in Fig. 4C and Supplementary Table 4, the top five significantly enriched KEGG pathway included ECM-receptor interaction, Focal adhesion, Arachidonic acid metabolism, Leishmaniasis and Phagosome. PI3K-Akt signaling pathway and metabolic pathways, which were involved in the regulation of glucose metabolism, were also significantly enriched
3.7. Pg3G altered hepatic gene expressions involved in glucose and lipid metabolism The overall up and down-regulated genes were listed in 4
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Fig. 3. Pg3G contributed to differential gene expressions (DEGs) in the liver. (A-F) Bar graphs show the classification of raw reads in each mouse. The raw read contains N base, adapter, low-quality reads and clean reads. (G) The distribution of the gene expression in each sample. The gene expression level was expressed as log10 FPKM. (H) Volcano plot of DEGs between control and Pg3G treated group. C or Control, db/db mice orally administered with vehicle; P or Pg3G, db/db mice orally administered with 150 mg/kg BW Pg3G. Data represent means ± s.e.m., n = 3, *p < 0.05 versus control group.
have been found to prevent T2D through multiple molecular mechanisms (Martel et al., 2017). Anthocyanin-rich fruits not only conferred protection against hyperglycemia, insulin resistance and diabetic associated complications, but also exhibited therapeutic effects on metabolic damage (Gowd et al., 2017; Skates et al., 2018). In the present study, we identified a purified anthocyanin Pg3G derived from wild raspberry prevented hyperglycemia and insulin resistance on db/db diabetic mice, and further elucidated the anti-diabetic mechanism of Pg3G using RNA-seq. The dosages (50 and 150 mg/kg per day) of Pg3G used was according to previous report with slight modification (Mirshekar, Roghani, Khalili, & Baluchnejadmojarad, 2011), which were equivalent to 5.5 and 16.5 mg/kg Pg3G per day consumption by humans in terms of human and mice dosage conversion. Our preliminary study indicated that 100 g of wild raspberry contains approximately 33.465 mg Pg3G, which is achievable in humans through regular wild raspberry consumption. The development of metabolic diseases such as diabetes and obesity can be elucidated through the study of transcriptome (Christodoulou et al., 2019). RNA-seq is a powerful technology that enables the identification of signature genes responsible for the pathogenesis of diabetes (Ackermann, Wang, Schug, Naji, & Kaestner, 2016; Gong, Chen, Zhang, Chen, & Li, 2017). It has been identified that diabetes is associated with DEGs in alpha and beta cell in terms of RNA-seq (Ackermann et al., 2016; Neelankal John, Ram, & Jiang, 2018). Recently, RNA-seq analysis was employed to investigate the protective effect and mechanism of natural products against diabetes (Cao, Sun, Zou, Li, & Xu, 2017; Jiang et al., 2018; H. Su et al., 2018). For example, RNA-seq analysis identified that mulberry leaf extract prevented diabetes in mice by influencing the insulin receptor substrate (IRS) signaling pathway (Ge et al., 2018). A genome-wide representation of metformin hepatic response has been characterized which plays a critical role in glucose homeostasis (Luizon et al., 2016). To the best of our knowledge, this study for
Supplementary Table 3. The RNA-seq analysis showed that Pg3G enhanced the hepatic gene expression involved in lipid and glucose metabolism, including elongation of very long-chain fatty acids protein 7 (Elovl7), adiponectin (C1ql3), NADPH oxidase (Cybb), cytohesin-interacting protein (Cytip), tyrosine-protein kinase Src (Src) and pyruvate dehydrogenase kinase isozyme 4 (Pdk4) when compared with those in the control group. In addition, Pg3G administration resulted in the down-regulation of hepatic genes associated with lipogenesis including fatty acid synthase (Fasn), acetoacetyl-CoA synthetase (Aacs), sterol regulatory element-binding transcription factor 1 (Srebp1c). Furthermore, the gene expressions responsible for glucose metabolism including carbohydrate-responsive element-binding protein isoform X5 (Mlxipl), sorbin and SH3 domain containing 1 (Sorbs3), glucokinase isoform 1 (Gck) and solute carrier family 2, member 4 (Slc2a4) were significantly decreased by Pg3G treatment compared with those in the control group. 3.8. RT-PCR validation of DGEs In the present study, 16 DEGs, including Tlr12, Serpine1, Elovl7, Cybb, Cytip, Pygb, Src, Pdk4, Fasn, Aacs, Glt1d1, Mlxpl, Sorbs3, Slc2a4, Cux2 and Eci3, were selected for further validation by RT-PCR. The results from RT-PCR were similar to those from RNA-seq (Fig. 6A and B). Correlation analysis showed a correlation coefficient of 0.949, indicating a highly positive linear correlation between RT-PCR and RNAseq results (Fig. 6C). 4. Discussion Millions of people are suffered from T2D worldwide. It is important to develop nutritional and pharmaceutical strategies to prevent and treat T2D. In recent decades, numerous natural bioactive components 5
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Fig. 4. GO enrichment and KEGG pathway analysis of DEGs. (A) GO enrichment of all DEGs in control versus Pg3G treated group. The blue bar represented molecular function; the yellow bar represented cellular component; the pink bar represented biological process, respectively. (B) GO classification of up and down-regulated genes. (C) The size of the point indicated the number of DEGs enriched in the pathway. The blue color indicated the significance of enrichment. The deeper blue color indicated more significant enrichment. (D) KEGG pathway functional enrichment result of up and down-regulated genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the first time used high throughput RNA-seq to investigate the alteration of gene expression profile and its associated pathway underlying the effect of Pg3G on T2D. Emerging evidence indicates that pelargonidin derivatives or pelargonidin-rich extracts exhibited anti-diabetic effect (Roy, Pal, & Chakraborti, 2017). In addition, chronic administration of pelargonidin alleviated diabetes-associated complications including learning and memory disturbances (Mirshekar et al., 2011). The underlying mechanism of action of pelargonidin derivatives could be attributed to the regulation of insulin secretion and hepatic glucose uptake as well as inhibition of enzymes involved in glucose metabolism (Jayaprakasam, Vareed, Olson, & Nair, 2005; Karkute et al., 2018; Luna-Vital & Gonzalez de Mejia, 2018; Zhang et al., 2019). Although previous studies indicated the beneficial role of pelargonidin against hyperglycemia in different diabetic models (Mirshekar et al., 2010; Roy, Sen, & Chakraborti, 2008; Samadder, Tarafdar, Abraham, Ghosh, & KhudaBukhsh, 2017), it is still unknown the effect of a glycosylated pelargonidin Pg3G on diabetic mice. In the present study, Pg3G exhibited profound effects against glucose intolerance and insulin resistance in diabetic mice. Moreover, Pg3G not only modified the serum lipid profiles, but also improved hepatic function. Those results confirmed the beneficial role of Pg3G against T2D and its associated metabolic
disorders. The liver plays a critical role in maintaining whole-body glucose homeostasis (Rines et al., 2016). Therefore, we investigated the DEGs and their associated pathway by RNA-seq. Based on the GO classification of DEGs, we found that Pg3G significantly enriched monooxygenase activity involved in molecular function, extracellular region involved in cellular components, and response to chemical involved in biological process. These GOterms are linked to diabetes and its associated complications (Wang et al., 2019; Wang et al., 2018; Xiao et al., 2018). Based on the KEGG classification of DEGs, we found ECM-receptor interaction was significantly enriched in the KEGG pathway. Although the previous study has indicated the association of ECM-receptor interaction pathway and diabetes (Xu, Hu, et al., 2018), the precise mechanism regarding Pg3G on regulating ECM-receptor interaction pathway warrants further investigation. Moreover, several pathways including metabolic pathway and PI3K-Akt signaling pathway were significantly enriched in the present study. Targeting metabolic pathway could be a potential strategy to prevent diabetes (Rines et al., 2016). Several natural products showed the antihyperglycemic effect by regulating PI3K-Akt signaling pathway (Liao et al., 2019; Mazibuko-Mbeje et al., 2019). Therefore, Pg3G may prevent T2D through regulating metabolic pathway and PI3K-Akt signaling 6
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Fig. 5. GSEA analysis of DEGs. (A-E) GSEA evaluated the live gene expression linked to the indicated processes in terms of GO gene sets between control and Pg3G treated mice. (F-J) GSEA evaluated the live gene expression linked to the indicated processes in terms of KEGG gene sets between control and Pg3G treated mice.
phosphorylation and digestive system bile secretion), negatively enriched the gene sets involved in inflammation (response to lipopolysaccharide, inflammatory response, PI3K-Akt pathway and TNF signaling pathway), suggesting that Pg3G could reduce diabetic associated inflammatory response. In the present study, the RNA-seq study reveals that Pg3G treatment upregulated the genes involved in glucose metabolism. Especially, Pg3G dramatically up-regulated the gene expression of adiponectin (C1ql3). Accumulating evidence uncovered the antidiabetic effects of
pathway. To further interpreted the gene expression profiles, we performed GSEA to elucidate the functional difference between control and Pg3Gtreated mice. GSEA identifies classes of genes that are over-represented in a large gene set, which may have an association with disease phenotypes (Subramanian et al., 2005). Based on the GSEA results, it can be concluded that Pg3G positively enriched the gene sets involved in glucose metabolism (mitochondrial respiratory chain, oxidation-reduction process, lipid metabolic process, metabolism oxidative
Fig. 6. RNA validation of selected DEGs. (A) 16 selected DEGs, including Tlr12, Serpine1, Elovl7, Cybb, Cytip, Pygb, Src, Pdk4, Fasn, Aacs, Glt1d1, Mlxpl, Sorbs3, Slc2a4, Cux2 and Eci3, in terms of fold change was measured by RNA-seq. (B) 16 selected DEGs, including Tlr12, Serpine1, Elovl7, Cybb, Cytip, Pygb, Src, Pdk4, Fasn, Aacs, Glt1d1, Mlxpl, Sorbs3, Slc2a4, Cux2 and Eci3, in terms of fold change was measured by RT-PCR. (C) Correlation analysis between RT-PCR and RNA-seq results (n = 3). 7
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Declaration of Competing Interest
adiponectin (Tsuduki, Kikuchi, Kimura, Nakagawa, & Miyazawa, 2013; Xu & Sweeney, 2015). A previous study indicated that adiponectin ameliorated insulin resistance by stimulating autophagy and reducing oxidative stress (Liu et al., 2015), suggesting a critical role of adiponectin in diabetes. Pdk4 encodes pyruvate dehydrogenase kinase, which are located in the mitochondrial matrix of eukaryotes that converts pyruvate to acetyl-coA (Sivasankar, George, & Sriram, 2018). AcetylcoA is then transported into the mitochondria to produce energy. In the current study, the upregulation of Pdk4 may contribute to the generation of acetyl-coA and energy production, which facilitated the reduction and conversion of glucose. In addition, we observed a significant down-regulation of Gck and Slc2a4. Gck encodes glucokinase, which facilitates the phosphorylation of glucose to glucose-6-phosphate. Slc2a4 also called Glut4, is an insulin-regulated glucose transporter. However, Pg3G downregulated Gck and Slc2a4 in the hepatic tissue, suggesting the alternative roles of Gck and Slc2a4 involved in regulating glucose metabolism. To gain more insight into the mechanism of Pg3G on T2D, we evaluated the genes involved in lipid metabolism. Srebp1c regulates the expression of target gene Fasn involved in fatty acids synthesis (Su, Feng, Zheng, & Chen, 2016). We observed a significant downregulation of lipogenesis-related genes including Srebp1c and Fasn, suggesting a role of Pg3G in preventing T2D associated metabolic disorder. Taken together, these results indicated that Pg3G has the potential to regulate glucose and lipid metabolism, thereby attenuating T2D and T2D associated metabolic dysfunction. Accumulating evidence indicates that gut microbiota plays a profound role in regulating glucose homeostasis (Tilg & Moschen, 2014). Previous studies showed that berry anthocyanins were extensively metabolized by digestive enzymes and gut microbiota, which led to the production of anthocyanin metabolites (Kay, Pereira-Caro, Ludwig, Clifford, & Crozier, 2017). In addition to the intact anthocyanins (glycosides), several anthocyanin metabolites such as protocatechuic, vanillic acid and ferulic acid were detected in circulation. These anthocyanin metabolites were responsible for the health-promoting effects of raspberry (Mullen, Edwards, Serafini, & Crozier, 2008; Warner et al., 2017). It has been confirmed that Pg3G was converted to several pelargonidin metabolites (e.g., pelargonidin 3-O-glucuronide) by lactase phlorizen hydrolase or β-glucosidase in the small intestine (Nemeth et al., 2003). Moreover, gut microbiota was involved in metabolizing Pg3G and produced Pg3G metabolites (Faria, Fernandes, Norberto, Mateus, & Calhau, 2014). Our recent study indicated that Pg3G modified the gut microbiota composition by increasing the abundance of Prevotella and elevating Bacteroidetes/Firmicutes ratio (Su et al., 2019), suggesting an important role of Pg3G on modulating gut microbiota. However, it is still possible that Pg3G metabolites mediate the antihyperglycemic effect of raspberry. In summary, this study indicated that Pg3G derived from wild raspberry can ameliorate T2D and modulate diabetes-associated metabolic disorders. In addition, RNA-seq analysis suggested that Pg3G exerted an antihyperglycemic effect partially through regulating the genes involved in glucose and lipid metabolism by upregulation of Elovl7, C1ql3, Cybb, Cytip, Src, Pdk4, and downregulation of Fasn, Aacs, Srebp1c, Mlxipl, Sorbs3, Gck, Slc2a4. GO and KEGG pathway analysis revealed that most of the DEGs were enriched in glucose metabolism and inflammation, suggesting a protective role of Pg3G against T2D presumably through regulating the inflammatory signaling pathway. It is necessary to investigate the detailed mechanism based on RNA-seq results, which is our ongoing study. The present study suggested that Pg3G could be a novel candidate agent and wild raspberry could be functional foods for the nutritional or pharmaceutical intervention of T2D.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Grants from National Key R&D Program of China (2018YFA0903202), Zhejiang Provincial Natural Science Foundation of China (LR18C200002), and National Natural Science Foundation of China (21876152). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103657. References Ackermann, A. M., Wang, Z., Schug, J., Naji, A., & Kaestner, K. H. (2016). Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Molecular Metabolism, 5(3), 233–244. American Diabetes, A. (2018). 5. Prevention or delay of type 2 diabetes: Standards of medical care in diabetes-2018. Diabetes Care, 41(Suppl 1), S51–S54. Cao, Y., Sun, Y., Zou, S., Li, M., & Xu, X. (2017). 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Ethics statement All relevant ethical safeguards have been met in relation to patient or subject protection, or animal experimentation. 8
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Transcriptome profiling reveals the antihyperglycemic mechanism of pelargonidin-3-O-glucoside extracted from wild raspberry Hongming Sua, Tao Baoa, Lianghua Xiea, Yang Xua, Wei Chena,b,
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a
Department of Food Science and Nutrition, National Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou 310058, China b Ningbo Research Institute, Zhejiang University, Ningbo 315100, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Raspberry Pelargonidin-3-O-glucoside Diabetes RNA-sequence Insulin resistance
Emerging evidence suggests that anthocyanins prevent type 2 diabetes (T2D), however, its mechanisms remain elusive. Herein, we sought to elucidate the antihyperglycemic mechanism of pelargonidin-3-O-glucoside (Pg3G) extracted from wild raspberry based on RNA-sequence. Our study showed that Pg3G ameliorated glucose intolerance and insulin resistance in db/db mice. Besides, Pg3G improved serum lipid profiles and attenuated hepatic dysfunction. RNA-sequence analysis showed that Pg3G contributed to 301 genes upregulated and 269 genes downregulated. Further GO, KEGG and GSEA analysis indicated that Pg3G led to differential gene expressions enriched in glucose and lipid metabolism by upregulation of Elovl7, C1ql3, Cybb, Cytip, Src, Pdk4 and downregulation of Fasn, Aacs, Srebp1c, Mlxipl, Sorbs3, Gck, Slc2a4. RT-PCR validation of selected genes exhibited a positive correlation between RNA-sequence and RT-PCR results. Taken together, this study demonstrated that Pg3G attenuated T2D by regulating glucose and lipid metabolism, which implicates the potential for T2D therapeutics.
1. Introduction The incidence of type 2 diabetes (T2D) is increased rapidly in recent decades both in developing and developed countries, which affected approximately 425 million people worldwide (Disease, Injury, & Prevalence, 2016). The high prevalence of T2D dramatically increases the economic burden. Emerging evidence indicates that both genetic factors and unhealthy lifestyles including the reduced level of exercise, long-time sedentary lifestyle and high fat dietary intake result in T2D (DeFronzo et al., 2015). T2D, in general, is characterized by hyperglycemia, insulin resistance and the lack of insulin release, which is associated with an increased risk of several metabolic complications, such as diabetic nephropathy, diabetic retinopathy and diabetic neuropathy (DeFronzo et al., 2015). Metformin, acarbose and rosiglitazone are FDA-approved agents that are available for pharmaceutical management of diabetes (American Diabetes, 2018). However, long-time use of those drugs may cause undesirable side-effects (Rines, Sharabi, Tavares, & Puigserver, 2016). Therefore, it is urgently needed to develop novel anti-diabetic agents. Recently, natural products derived from daily consumed food or traditional Chinese medicine (TCM) display a beneficial effect on glucose metabolism. Those phytochemicals have achieved increasing
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attentions (Martel et al., 2017). For example, natural polyphenols such as myricetin, catechin as well as resveratrol showed promises to prevent T2D (Li et al., 2017; Sung et al., 2017; Yan, Zhao, Suo, Liu, & Zhao, 2012). In this regard, phytochemicals could be an important natural reservoir to exploit anti-diabetic agents and develop novel therapeutics. Anthocyanins have the demonstrated ability to protect against a myriad of human diseases including diabetes and obesity (Xie, Su, Sun, Zheng, & Chen, 2018). The effects of anthocyanin on diabetes have been summarized in our previous published review papers (Gowd, Jia, & Chen, 2017; Xie et al., 2018). The mechanism of anthocyanins underlying the anti-diabetic action could be attributed to enzymatic activation or inhibition, increased insulin sensitivity and insulin secretion as well as reduced hepatic gluconeogenesis and increased glycolysis (Rines et al., 2016). The chemical structure of disparate anthocyanin explains varied activity and mechanism. For example, our recent study reported a purified natural anthocyanin prevented postprandial hyperglycemia by inhibiting α-glucosidase (Xu, Xie, Xie, Liu, & Chen, 2018), suggesting a plausible mechanism underling anthocyanins’ anti-diabetic action. Although increasing evidence indicates the protective role of anthocyanins against diabetes and diabetes-associated complications, it is still largely unknown regarding the molecular mechanisms of anthocyanins (e.g. pelargonidin-3-O-glucoside) against diabetes.
Corresponding author at: Department of Food Science and Nutrition, Zhejiang University, No. 866 Yuhangtang Road, Xihu District, Hangzhou 310058, China. E-mail address: [email protected] (W. Chen).
https://doi.org/10.1016/j.jff.2019.103657 Received 16 August 2019; Received in revised form 22 October 2019; Accepted 28 October 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Hongming Su, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103657
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liquid nitrogen. All samples were preserved at −80 °C for further investigation.
Raspberry was reportedly rich in anthocyanins, which are responsible for exerting the health-promoting effects (Xie et al., 2018). In our preliminary study, we collected wild raspberry (Rubus hirsutus Thunb.) from the local area in Zhejiang Province, China. One major anthocyanin pelargonidin-3-O-glucoside (Pg3G) identified was predominantly enriched in wild raspberry according to LC-MS/MS and NMR analyses (H. Su et al., 2019). Since anthocyanins show the potential for anti-diabetic application, in the present study we investigated the hyperglycemia-lowering effect of Pg3G on db/db diabetic mice. RNA-sequence (RNA-seq) analysis has been widely used to study the gene expression profiles involved in a specific disease phenotype and facilitates the identification of signature genes. To elucidate the underlying mechanism, thus we employed RNA-seq analysis to identify the differential gene expressions (DEGs) and illustrate the alterations of DEGs corresponding functional pathways in terms of GO, KEGG and GSEA analyses. This study may constitute a novel insight into the application of Pg3G for the prevention and treatment of T2D.
2.3. Glucose tolerance test (GTT) and insulin tolerance tests (ITT) GTT and ITT were determined according to a previous report (Liu, Lee, Salazar Hernandez, Mazitschek, & Ozcan, 2015). In the eighth week, the mice fasted for 12 h, and GTT was performed after 0.5 g/kg glucose was administered intraperitoneally. Blood glucose levels were measured by collecting blood from the tail vein before glucose administration and 30, 60 and 120 min after glucose administration (Roche Septi-Chek system). At the end of the eighth week, ITT was performed after mice fasted for 6 h. Insulin (2 IU/kg) was administered intraperitoneally. Blood glucose levels were measured by collecting blood from the tail vein before insulin administration and 30, 60 and 120 min after the administration. 2.4. Biochemical analysis
2. Materials and methods The blood samples were collected and centrifuged at 5000 rpm for 10 min, then serum was collected for biochemical analysis. Serum glucose, free fatty acids (FFAs), cholesterol, triglyceride (TG), lowdensity lipoprotein cholesterol (LDL-c), aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were measured by Hitachi automatic biochemistry analyzer.
2.1. Preparation of Pg3G from wild raspberry Pg3G was extracted from wild raspberry according to our previously established methods (Xu, Hu, Li, Sun, & Chen, 2018; Xu, Xie, et al., 2018). Briefly, anthocyanins fraction was extracted from wild raspberry fruits using 70% ethanol for 12 h and then filtered through three layers of gauze. The collected filtrate was evaporated at 45 °C and then purified by AB-8 macroporous resin column. The elution solution was lyophilized to yield raspberry anthocyanins fraction. The raspberry anthocyanins fraction was further purified by HSCCC (TBE-300A, Tauto Biotechnique Company, Shanghai, China) using a two-phase solvent system of tert-butyl methyl ether - n-butanol - acetonitrile - water (2:2:1:5, v/v/v/v, acidified with 0.1% trifluoroacetic acid). The structure of purified Pg3G was confirmed by HPLC-ESI–MS (Agilent 1200 series and 6430 triple quadrupole mass spectrometers, USA) and NMR (Bruker AVANCETM III spectrometer (14.1 T)), respectively. The purity of Pg3G was 98.6%. The ESI–MS and NMR results were indicated in Supplementary Materials and methods.
2.5. RNA extraction and sequencing Hepatic total RNA was prepared from control and Pg3G (150 mg/kg BW) treated mice (n = 3 per group) using TRIzol following the manufacturer’s protocol (Life Technologies). The RNA quality was determined using Agilent 2100 Bioanalyzer. Then mRNAs were isolated from total RNA with oligo(dT) method and the cDNA fragments were linked with adapters. The libraries were sequenced with an Illumina HiSeq Platform. 2.6. RNA-seq analysis The low-quality reads (More than 20% of the bases qualities are lower than 10), reads with adaptors and reads with unknown bases (N bases more than 5%) were filtered to get the clean reads using SOAPnuke. Those clean reads were mapped onto the reference genome using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts). The mapped clean reads were mapped to the reference gene using Bowtie2 (v2.2.5) and the gene expression levels were calculated with RSEM (v1.2.12). We detected DEGs with DEseq2. DEseq2 is based on the negative binomial distribution. Finally, we identified DEGs with DEseq2 between samples (Fold Change ≥ 1.00 and Adjusted P-value ≤ 0.05). Gene Ontology (GO) and KEGG pathway analyses were performed using phyper, a function of R. False discovery rate (FDR) was calculated and FDR less than 0.01 are defined as significantly enriched. The diagrams were drawn with ggplot2, a function of R. Gene set enrichment analysis was performed based on the GO gene sets and KEGG gene sets using GSEA 2.0 with default parameters (permutation type: gene_set. Collapse dataset to gene symbols: false).
2.2. Animal experiments The Animal experiments were conducted according to the guidelines on the use and care of laboratory animals in China (GB/T 358922018 and GB/T 35823-2018). All animal experimental procedures were performed in the Animal Experiment Center of Zhejiang Chinese Medical University (Hangzhou, China). The animal protocol was approved by the laboratory animal management and ethics committee of Zhejiang Chinese Medical University (201610087). 36 male Leprdb mutation (db/db) mice with C57BL/6J background, aged six weeks, were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). The temperature in the cage was maintained with constant temperature (23 °C) and humidity under a 12 h/ 12 h light/dark cycle. The mice were fed a standardized diet (kcal %: 10% fat, 20% protein, and 70% carbohydrate). All mice have free access to diet and sterilized water. After one week of acclimatization, the mice were divided into three groups (n = 12 per group): (1) db/db mice were treated with vehicle (ultra-purified water); (2) db/db mice were orally gavaged with 50 mg/kg BW per mice Pg3G; (3) db/db mice were orally gavaged with 150 mg/kg BW per mice Pg3G. The doses of Pg3G were according to a previous report with modifications (Mirshekar, Roghani, Khalili, Baluchnejadmojarad, & Arab Moazzen, 2010). After fasting for 12 h overnight, the fasting blood glucose (FBG) levels were determined every week using Roche Septi-Chek system by collecting blood from the tail vein of mice. After eight weeks, the mice were sacrificed, following 12 h fasting. Blood samples were collected by cardiac puncture. Liver samples were collected, weighed and snap-frozen by
2.7. Quantitative real-time PCR The quantitative real-time PCR (RT-PCR) analysis was performed to validate the results from RNA-seq as previously described (Su et al., 2018). Total RNA was isolated from the liver using Trizol and then pooled for the RT-PCR analysis. cDNA was synthesized from total RNA using the PrimeScript RT reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was carried out in the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). SYBR Green 2
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(Roche, Germany) was used as a fluorescent dye for quantitative PCR analysis. The primers were synthesized by GENEWIZ (Suzhou, China) and the primer sequences were indicated in Supplementary Table 1. A relative gene-expression quantification method was used to calculate the fold change of mRNA expression. GAPDH was set as an internal reference for normalization.
sensitivity in db/db diabetic mice. In addition, 150 mg/kg BW Pg3G administration showed a better glucose-lowering effect than 50 mg/kg BW Pg3G administration in terms of FBG levels, GTT and ITT. 3.2. Pg3G ameliorated serum biochemical profile As shown in Fig. 2A, B and D, Pg3G (50 and 150 mg/kg BW) administration led to a significant decrease of serum FFAs, cholesterol and LDL-c compared with those in the control. Nonetheless, Pg3G had no impact on the level of serum triglyceride (Fig. 2C). In addition, this study showed that 150 mg/kg BW Pg3G administration significantly reduced serum AST, ALT, LDH and ALP levels (Fig. 2F–H), in comparison with those in the control, suggesting the improvement of hepatic dysfunction involved in T2D.
2.8. Statistical analysis Data are expressed as the mean ± SEM. Significant differences were evaluated by two-tailed Student’s t-test between two groups or one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test between multiple groups. p < 0.05 was considered to be a significant difference. All statistical analysis was performed using GraphPad Prism V.7.0a (GraphPad Software, USA).
3.3. Pg3G contributed to differential gene expressions (DEGs) in the liver 3. Results The liver plays a crucial role in the regulation of glucose metabolism. Since 150 mg/kg BW Pg3G displayed a better antihyperglycemic effect than 50 mg/kg BW Pg3G, we therefore further examined the effect of Pg3G 150 mg/kg BW on hepatic gene transcriptional profile. High throughput RNA-seq analysis was adopted to identify the DEGs. The classification of raw reads from each group was indicated in Fig. 3A–F. We obtained an average of 44.47 million clean reads from each group. Total clean reads Q30% in each group was higher than 95%, which indicated a good sequencing quality (Supplementary Table 2). The total gene mapping ratio was beyond 69.67%. The gene expression levels were quantified and expressed as FPKM. Fig. 3G indicated the distribution of gene expression in each mouse. Those results indicated that the quality of all libraries was good and suitable for subsequent analysis. The total gene number on average in control mice was 14,590, and
3.1. Pg3G attenuated glucose intolerance and insulin resistance In the present study, the results of fasting blood glucose (FBG) levels within 8 weeks indicated that both concentrations of Pg3G caused a remarkable FBG reduction (Fig. 1A), suggesting a long-term beneficial role of Pg3G against hyperglycemia in T2D. Pg3G administration contributed to a significant improvement of glucose tolerance compared to the control group (Fig. 1B). The area under the curve (AUC) of GTT in Pg3G treated mice was significantly lower than that in the control (Fig. 1C). ITT assay indicated that Pg3G administration significantly reduced blood glucose level after insulin injection compared with the control (Fig. 1D). The AUC of ITT in Pg3G treated mice was remarkably decreased compared with that in the control (Fig. 1E). Together, these results indicated that Pg3G improved glucose tolerance and insulin
Fig. 1. Pg3G exerted glucose-lowering effect on db/db diabetic mice. Db/db mice were orally gavaged with vehicle or Pg3G (50 and 150 mg/kg BW per day) for 8 weeks. (A) The fasting blood glucose was determined every week for overall 8 weeks. (B) Glucose tolerance test (GTT) was performed at the eighth week. (C) The area under the curve (AUC) of GTT was calculated. (D) At the end of the eighth week, insulin tolerance test (ITT) was performed. (E) The AUC of ITT was calculated. Control, db/db mice orally administered with vehicle; Pg3G 50, db/db mice orally administered with 50 mg/kg BW Pg3G; Pg3G 150, db/db mice orally administered with 150 mg/kg BW Pg3G. Data represent means ± s.e.m., n = 10 per group, *p < 0.05 versus control group. 3
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Fig. 2. Pg3G improved serum biochemical profile. (A) serum FFAs, (B) cholesterol, (C) triglyceride, (D) LDL-c, (E) AST, (F) ALT, (G) LDH and (H) ALP levels were determined (n = 10). Control, db/db mice orally administered with vehicle; Pg3G 50, db/db mice orally administered with 50 mg/kg BW Pg3G; Pg3G 150, db/db mice orally administered with 150 mg/kg BW Pg3G. Data represent means ± s.e.m., *p < 0.05 versus control group.
KEGG pathway. The top three DEGs number of upregulated genes were included in PI3K-Akt signaling pathway, focal adhesion and metabolic pathways. The top three DEGs number of downregulated genes were mainly included in metabolic pathways, arachidonic acid metabolism and chemical carcinogenesis (Fig. 4D).
that number in Pg3G treated mice was 14,959. As shown in Fig. 3H and Supplementary Table 3, the DEGs analysis indicated that Pg3G administration contributed to 301 genes up-regulated and 269 genes down-regulated compared with those in the control. In addition, 15,808 genes were not changed upon Pg3G treatment. These results indicated that Pg3G administration resulted in hepatic DEGs.
3.6. Gene set enrichment analysis (GSEA) 3.4. GO analysis Gene set enrichment analysis (GSEA) determines whether a predefined set of genes shows a significant difference (Subramanian et al., 2005). To further interpret the effect of Pg3G on functional gene expression, GSEA was performed based on the GO gene sets and KEGG gene sets, respectively. According to GSEA in terms of GO gene sets, Pg3G administration contributed to a positive enrichment of mitochondrial respiratory chain, oxidation-reduction process and lipid metabolic process (Fig. 5A–C and Supplementary Figs. 1–3). Conversely, Pg3G administration led to negative enrichment of response to lipopolysaccharide and inflammatory response (Fig. 5D–E and Supplementary Figs. 4 and 5). According to GSEA in terms of KEGG gene sets, Pg3G administration resulted in a positive enrichment of KEGG pathway of energy metabolism oxidative phosphorylation, digestive system bile secretion (Fig. 5F and G, and Supplementary Figs. 6 and 7). Moreover, Pg3G administration led to a negative enrichment, in the KEGG pathway, of endocrine and metabolic disease age-rage signaling in diabetic complications, PI3K-Akt-signaling pathway and TNF signaling pathway (Fig. 5H–J and Supplementary Figs. 8–10).
To further analyze the role of DEGs upon Pg3G administration, we performed GO analysis to ascertain the function of DEGs. The results of GO were divided into biological process, cellular component and molecular function. According to GO analysis, the top three high-enriched GO term in biological process were response to chemical, biological process and response to organic substance. The top three significantly enriched GO term in cellular component were extracellular region, extracellular region part and extracellular matrix. The top three highenriched GO term in molecular function were monooxygenase activity, aromatase activity and heme binding (Fig. 4A). The DEGs of the most enriched pathway in each GOTerm include cellular process, cell and binding (Fig. 4B). 3.5. KEGG analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to elucidate the function of DEGs. As shown in Fig. 4C and Supplementary Table 4, the top five significantly enriched KEGG pathway included ECM-receptor interaction, Focal adhesion, Arachidonic acid metabolism, Leishmaniasis and Phagosome. PI3K-Akt signaling pathway and metabolic pathways, which were involved in the regulation of glucose metabolism, were also significantly enriched
3.7. Pg3G altered hepatic gene expressions involved in glucose and lipid metabolism The overall up and down-regulated genes were listed in 4
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Fig. 3. Pg3G contributed to differential gene expressions (DEGs) in the liver. (A-F) Bar graphs show the classification of raw reads in each mouse. The raw read contains N base, adapter, low-quality reads and clean reads. (G) The distribution of the gene expression in each sample. The gene expression level was expressed as log10 FPKM. (H) Volcano plot of DEGs between control and Pg3G treated group. C or Control, db/db mice orally administered with vehicle; P or Pg3G, db/db mice orally administered with 150 mg/kg BW Pg3G. Data represent means ± s.e.m., n = 3, *p < 0.05 versus control group.
have been found to prevent T2D through multiple molecular mechanisms (Martel et al., 2017). Anthocyanin-rich fruits not only conferred protection against hyperglycemia, insulin resistance and diabetic associated complications, but also exhibited therapeutic effects on metabolic damage (Gowd et al., 2017; Skates et al., 2018). In the present study, we identified a purified anthocyanin Pg3G derived from wild raspberry prevented hyperglycemia and insulin resistance on db/db diabetic mice, and further elucidated the anti-diabetic mechanism of Pg3G using RNA-seq. The dosages (50 and 150 mg/kg per day) of Pg3G used was according to previous report with slight modification (Mirshekar, Roghani, Khalili, & Baluchnejadmojarad, 2011), which were equivalent to 5.5 and 16.5 mg/kg Pg3G per day consumption by humans in terms of human and mice dosage conversion. Our preliminary study indicated that 100 g of wild raspberry contains approximately 33.465 mg Pg3G, which is achievable in humans through regular wild raspberry consumption. The development of metabolic diseases such as diabetes and obesity can be elucidated through the study of transcriptome (Christodoulou et al., 2019). RNA-seq is a powerful technology that enables the identification of signature genes responsible for the pathogenesis of diabetes (Ackermann, Wang, Schug, Naji, & Kaestner, 2016; Gong, Chen, Zhang, Chen, & Li, 2017). It has been identified that diabetes is associated with DEGs in alpha and beta cell in terms of RNA-seq (Ackermann et al., 2016; Neelankal John, Ram, & Jiang, 2018). Recently, RNA-seq analysis was employed to investigate the protective effect and mechanism of natural products against diabetes (Cao, Sun, Zou, Li, & Xu, 2017; Jiang et al., 2018; H. Su et al., 2018). For example, RNA-seq analysis identified that mulberry leaf extract prevented diabetes in mice by influencing the insulin receptor substrate (IRS) signaling pathway (Ge et al., 2018). A genome-wide representation of metformin hepatic response has been characterized which plays a critical role in glucose homeostasis (Luizon et al., 2016). To the best of our knowledge, this study for
Supplementary Table 3. The RNA-seq analysis showed that Pg3G enhanced the hepatic gene expression involved in lipid and glucose metabolism, including elongation of very long-chain fatty acids protein 7 (Elovl7), adiponectin (C1ql3), NADPH oxidase (Cybb), cytohesin-interacting protein (Cytip), tyrosine-protein kinase Src (Src) and pyruvate dehydrogenase kinase isozyme 4 (Pdk4) when compared with those in the control group. In addition, Pg3G administration resulted in the down-regulation of hepatic genes associated with lipogenesis including fatty acid synthase (Fasn), acetoacetyl-CoA synthetase (Aacs), sterol regulatory element-binding transcription factor 1 (Srebp1c). Furthermore, the gene expressions responsible for glucose metabolism including carbohydrate-responsive element-binding protein isoform X5 (Mlxipl), sorbin and SH3 domain containing 1 (Sorbs3), glucokinase isoform 1 (Gck) and solute carrier family 2, member 4 (Slc2a4) were significantly decreased by Pg3G treatment compared with those in the control group. 3.8. RT-PCR validation of DGEs In the present study, 16 DEGs, including Tlr12, Serpine1, Elovl7, Cybb, Cytip, Pygb, Src, Pdk4, Fasn, Aacs, Glt1d1, Mlxpl, Sorbs3, Slc2a4, Cux2 and Eci3, were selected for further validation by RT-PCR. The results from RT-PCR were similar to those from RNA-seq (Fig. 6A and B). Correlation analysis showed a correlation coefficient of 0.949, indicating a highly positive linear correlation between RT-PCR and RNAseq results (Fig. 6C). 4. Discussion Millions of people are suffered from T2D worldwide. It is important to develop nutritional and pharmaceutical strategies to prevent and treat T2D. In recent decades, numerous natural bioactive components 5
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Fig. 4. GO enrichment and KEGG pathway analysis of DEGs. (A) GO enrichment of all DEGs in control versus Pg3G treated group. The blue bar represented molecular function; the yellow bar represented cellular component; the pink bar represented biological process, respectively. (B) GO classification of up and down-regulated genes. (C) The size of the point indicated the number of DEGs enriched in the pathway. The blue color indicated the significance of enrichment. The deeper blue color indicated more significant enrichment. (D) KEGG pathway functional enrichment result of up and down-regulated genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the first time used high throughput RNA-seq to investigate the alteration of gene expression profile and its associated pathway underlying the effect of Pg3G on T2D. Emerging evidence indicates that pelargonidin derivatives or pelargonidin-rich extracts exhibited anti-diabetic effect (Roy, Pal, & Chakraborti, 2017). In addition, chronic administration of pelargonidin alleviated diabetes-associated complications including learning and memory disturbances (Mirshekar et al., 2011). The underlying mechanism of action of pelargonidin derivatives could be attributed to the regulation of insulin secretion and hepatic glucose uptake as well as inhibition of enzymes involved in glucose metabolism (Jayaprakasam, Vareed, Olson, & Nair, 2005; Karkute et al., 2018; Luna-Vital & Gonzalez de Mejia, 2018; Zhang et al., 2019). Although previous studies indicated the beneficial role of pelargonidin against hyperglycemia in different diabetic models (Mirshekar et al., 2010; Roy, Sen, & Chakraborti, 2008; Samadder, Tarafdar, Abraham, Ghosh, & KhudaBukhsh, 2017), it is still unknown the effect of a glycosylated pelargonidin Pg3G on diabetic mice. In the present study, Pg3G exhibited profound effects against glucose intolerance and insulin resistance in diabetic mice. Moreover, Pg3G not only modified the serum lipid profiles, but also improved hepatic function. Those results confirmed the beneficial role of Pg3G against T2D and its associated metabolic
disorders. The liver plays a critical role in maintaining whole-body glucose homeostasis (Rines et al., 2016). Therefore, we investigated the DEGs and their associated pathway by RNA-seq. Based on the GO classification of DEGs, we found that Pg3G significantly enriched monooxygenase activity involved in molecular function, extracellular region involved in cellular components, and response to chemical involved in biological process. These GOterms are linked to diabetes and its associated complications (Wang et al., 2019; Wang et al., 2018; Xiao et al., 2018). Based on the KEGG classification of DEGs, we found ECM-receptor interaction was significantly enriched in the KEGG pathway. Although the previous study has indicated the association of ECM-receptor interaction pathway and diabetes (Xu, Hu, et al., 2018), the precise mechanism regarding Pg3G on regulating ECM-receptor interaction pathway warrants further investigation. Moreover, several pathways including metabolic pathway and PI3K-Akt signaling pathway were significantly enriched in the present study. Targeting metabolic pathway could be a potential strategy to prevent diabetes (Rines et al., 2016). Several natural products showed the antihyperglycemic effect by regulating PI3K-Akt signaling pathway (Liao et al., 2019; Mazibuko-Mbeje et al., 2019). Therefore, Pg3G may prevent T2D through regulating metabolic pathway and PI3K-Akt signaling 6
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Fig. 5. GSEA analysis of DEGs. (A-E) GSEA evaluated the live gene expression linked to the indicated processes in terms of GO gene sets between control and Pg3G treated mice. (F-J) GSEA evaluated the live gene expression linked to the indicated processes in terms of KEGG gene sets between control and Pg3G treated mice.
phosphorylation and digestive system bile secretion), negatively enriched the gene sets involved in inflammation (response to lipopolysaccharide, inflammatory response, PI3K-Akt pathway and TNF signaling pathway), suggesting that Pg3G could reduce diabetic associated inflammatory response. In the present study, the RNA-seq study reveals that Pg3G treatment upregulated the genes involved in glucose metabolism. Especially, Pg3G dramatically up-regulated the gene expression of adiponectin (C1ql3). Accumulating evidence uncovered the antidiabetic effects of
pathway. To further interpreted the gene expression profiles, we performed GSEA to elucidate the functional difference between control and Pg3Gtreated mice. GSEA identifies classes of genes that are over-represented in a large gene set, which may have an association with disease phenotypes (Subramanian et al., 2005). Based on the GSEA results, it can be concluded that Pg3G positively enriched the gene sets involved in glucose metabolism (mitochondrial respiratory chain, oxidation-reduction process, lipid metabolic process, metabolism oxidative
Fig. 6. RNA validation of selected DEGs. (A) 16 selected DEGs, including Tlr12, Serpine1, Elovl7, Cybb, Cytip, Pygb, Src, Pdk4, Fasn, Aacs, Glt1d1, Mlxpl, Sorbs3, Slc2a4, Cux2 and Eci3, in terms of fold change was measured by RNA-seq. (B) 16 selected DEGs, including Tlr12, Serpine1, Elovl7, Cybb, Cytip, Pygb, Src, Pdk4, Fasn, Aacs, Glt1d1, Mlxpl, Sorbs3, Slc2a4, Cux2 and Eci3, in terms of fold change was measured by RT-PCR. (C) Correlation analysis between RT-PCR and RNA-seq results (n = 3). 7
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Declaration of Competing Interest
adiponectin (Tsuduki, Kikuchi, Kimura, Nakagawa, & Miyazawa, 2013; Xu & Sweeney, 2015). A previous study indicated that adiponectin ameliorated insulin resistance by stimulating autophagy and reducing oxidative stress (Liu et al., 2015), suggesting a critical role of adiponectin in diabetes. Pdk4 encodes pyruvate dehydrogenase kinase, which are located in the mitochondrial matrix of eukaryotes that converts pyruvate to acetyl-coA (Sivasankar, George, & Sriram, 2018). AcetylcoA is then transported into the mitochondria to produce energy. In the current study, the upregulation of Pdk4 may contribute to the generation of acetyl-coA and energy production, which facilitated the reduction and conversion of glucose. In addition, we observed a significant down-regulation of Gck and Slc2a4. Gck encodes glucokinase, which facilitates the phosphorylation of glucose to glucose-6-phosphate. Slc2a4 also called Glut4, is an insulin-regulated glucose transporter. However, Pg3G downregulated Gck and Slc2a4 in the hepatic tissue, suggesting the alternative roles of Gck and Slc2a4 involved in regulating glucose metabolism. To gain more insight into the mechanism of Pg3G on T2D, we evaluated the genes involved in lipid metabolism. Srebp1c regulates the expression of target gene Fasn involved in fatty acids synthesis (Su, Feng, Zheng, & Chen, 2016). We observed a significant downregulation of lipogenesis-related genes including Srebp1c and Fasn, suggesting a role of Pg3G in preventing T2D associated metabolic disorder. Taken together, these results indicated that Pg3G has the potential to regulate glucose and lipid metabolism, thereby attenuating T2D and T2D associated metabolic dysfunction. Accumulating evidence indicates that gut microbiota plays a profound role in regulating glucose homeostasis (Tilg & Moschen, 2014). Previous studies showed that berry anthocyanins were extensively metabolized by digestive enzymes and gut microbiota, which led to the production of anthocyanin metabolites (Kay, Pereira-Caro, Ludwig, Clifford, & Crozier, 2017). In addition to the intact anthocyanins (glycosides), several anthocyanin metabolites such as protocatechuic, vanillic acid and ferulic acid were detected in circulation. These anthocyanin metabolites were responsible for the health-promoting effects of raspberry (Mullen, Edwards, Serafini, & Crozier, 2008; Warner et al., 2017). It has been confirmed that Pg3G was converted to several pelargonidin metabolites (e.g., pelargonidin 3-O-glucuronide) by lactase phlorizen hydrolase or β-glucosidase in the small intestine (Nemeth et al., 2003). Moreover, gut microbiota was involved in metabolizing Pg3G and produced Pg3G metabolites (Faria, Fernandes, Norberto, Mateus, & Calhau, 2014). Our recent study indicated that Pg3G modified the gut microbiota composition by increasing the abundance of Prevotella and elevating Bacteroidetes/Firmicutes ratio (Su et al., 2019), suggesting an important role of Pg3G on modulating gut microbiota. However, it is still possible that Pg3G metabolites mediate the antihyperglycemic effect of raspberry. In summary, this study indicated that Pg3G derived from wild raspberry can ameliorate T2D and modulate diabetes-associated metabolic disorders. In addition, RNA-seq analysis suggested that Pg3G exerted an antihyperglycemic effect partially through regulating the genes involved in glucose and lipid metabolism by upregulation of Elovl7, C1ql3, Cybb, Cytip, Src, Pdk4, and downregulation of Fasn, Aacs, Srebp1c, Mlxipl, Sorbs3, Gck, Slc2a4. GO and KEGG pathway analysis revealed that most of the DEGs were enriched in glucose metabolism and inflammation, suggesting a protective role of Pg3G against T2D presumably through regulating the inflammatory signaling pathway. It is necessary to investigate the detailed mechanism based on RNA-seq results, which is our ongoing study. The present study suggested that Pg3G could be a novel candidate agent and wild raspberry could be functional foods for the nutritional or pharmaceutical intervention of T2D.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by Grants from National Key R&D Program of China (2018YFA0903202), Zhejiang Provincial Natural Science Foundation of China (LR18C200002), and National Natural Science Foundation of China (21876152). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103657. References Ackermann, A. M., Wang, Z., Schug, J., Naji, A., & Kaestner, K. H. (2016). Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Molecular Metabolism, 5(3), 233–244. American Diabetes, A. (2018). 5. Prevention or delay of type 2 diabetes: Standards of medical care in diabetes-2018. Diabetes Care, 41(Suppl 1), S51–S54. Cao, Y., Sun, Y., Zou, S., Li, M., & Xu, X. (2017). 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