Mechanisms of Dangua Fang (丹瓜方) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics

Abstract

OBJECTIVE:

To explore the mechanism of Dangua Fang (丹瓜方, DGR) in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.

METHODS:

Sprague-Dawley rats with normal glucose levels were randomly divided into three groups, including a conventional diet control group (Group A), high-fat-high-sugar diet model group (Group B), and DGR group (Group C, high-fat-high-sugar diet containing 20.5 g DGR). After 10 weeks of intervention, the fasting blood glucose (FBG), 2 h blood glucose [PBG; using the oral glucose tolerance test (OGTT)], hemoglobin A1c (HbA1c), plasma total cholesterol (TC), and triglycerides (TG) were tested, and the livers of rats were removed to calculate the liver index. Then, hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis followed by database search and bioinformatics analysis. Finally, cell experiments were used to verify the results of phosphoproteomics. Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4 (MAP4k4) and phosphorylated adducin 1 (ADD1) were detected using western blotting.

RESULTS:

DGR effectively reduced PBG, TG, and the liver index (P < 0.05), and significantly decreased HbA1c, TC, and hepatic portal TG (P < 0.01), showed significant hematoxylin and eosin (HE) staining, red oil O staining, and Masson staining of liver tissue. The total spectrum was 805 334, matched spectrum was 260 471, accounting for accounting 32.3%, peptides were 19 995, modified peptides were 14 671, identified proteins were 4601, quantifiable proteins were 4417, identified sites were 15 749, and quantified sites were 14659. Based on the threshold of expression fold change ( > 1.2), DGR up-regulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins, and down-regulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins, which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism. Therefore, DGR improved biological tissue processes, including information storage and processing, cellular processes and signaling, and metabolism. The metabolic functions regulated by DGR mainly include energy production and conversion, carbohydrate transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport, and catabolism. In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies.

CONCLUSION:

DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders, thereby regulating glycolipid metabolism through a multi-target and multi-method process.

1. INTRODUCTION

Glycolipid metabolism disorder accompanied by complex pathophysiological changes involves multiple levels, pathways, and targets in the human body, and is especially suitable for study with omics in bioinformatics.¹ Phosphorylation is a key reaction of protein and enzymatic function, sugar metabolism, and energy storage and release, and plays an important role in the process of cell signal transmission. Chinese medicine compound (CMC) has the characteristics of multiple components and can play an intervening role in multiple pathways and with multiple targets. Therefore, protein phosphorylation studies may profoundly reveal the mechanism of CMC to improve glucose and lipid metabolism. In recent years, there were few reports on mechanistic studies of CMC regulating glycolipid metabolism based on phosphoproteomics. Dangua Fang (丹瓜方, DGR) is an in-hospital preparation used by the People's Hospital of Fujian Province to treat glucose and lipid metabolism diseases. It has been government approved for the treatment of glycolipid metabolism diseases and awarded a national invention patent certificate. Randomized controlled clinical studies have shown that the addition of this formula to conventional intensive treatment can effectively improve the glycolipid metabolism of patients with type 2 diabetes as well as significantly reduce the risk of new coronary heart disease and decrease all-cause endpoint events.² In the past, we have conducted preliminary studies on the mechanism of DGR improving glycolipid metabolism using transcriptomics.³ In this study, we have systematically analyzed the mechanism of DGR regulation of glycolipid metabolism from the perspective of phosphorylation modification.

2. METHODS

2.1. Animals

Specific-pathogen free male Sprague-dawley rats [(311 ± 11) g] were provided by Shanghai Slark Experimental Animal Co., Ltd., animal License was SCXK (Shanghai) 2017-0005. The animals were housed in separative ventilated cages and entrained to a 12 h/12 h light-dark cycle at (22 ± 1) ℃ and (50% ± 5%) relative humidity. Free access to food and water was given.

2.2. Drugs and reagents

Arcylamide/Bis-Arcylamide (lot No. A2792-100 mL), Tris-base (lot No. V900483-500 g), N,N,N',N'- Tetramethylethylenediamine (TEMED; lot No. T9281-100 mL), iodoacetamide (IAM; lot No. V900335-5 g), DL-Dithiothreitol (DTT; lot No. D916-5 g), Urea (lot No. V900119-500 g), Triethy-lammonium bicarbonate buffer (TEAB; lot No. 17902-100 mL), Trichloroacetic acid (TCA; lot No. G8200-500 g), and Trifluoroacetic acid (TFA) (lot No. 302031-100ML) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Glycine (lot No. 0167-1 kg) and sodium dodecyl sulfate (SDS; lot No. M107-500 g) were purchased from Amresco (Amresco Inc., Solon, Ohio, USA). Trizol reagent (lot No. 15596018), methyl alcohol (lot No. A452-4), protein marker (lot No. 26619), water (H2O) (lot No. W5-4), and acetonitrile (CAN) (lot No. A998-4) were purchased from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA). Trypsin (lot No. V5111) was purchased from Promega (Promega, Madison, WI, USA). Protease inhibitor CocktailⅢ (lot No. 535140-1 mL) and protease inhibitor CocktailⅤ (lot No. 539137-1 mL) were purchased from Merck Millipore (MilliporeSigma, Burlington, MA, USA). Phospho-general control non-derepressible 2 (GCN2) (Thr899) antibody (lot No. AF8154) was purchased from Affinity Biosciences (Changzhou, China). Anti-sterol-regulatory element binding protein 1(SREBP1) (phospho S439; lot No. ab138663) was purchased from Abcam (London, UK), ammonium persulfate (APS; lot No. A600072-0025) was purchased from Sangon Biotech (Shanghai, China). The Bicinchoninic acid (BCA) kit (lot No. P0011-1) was purchased from Beyotime Biotechnology (Shanghai, China). The Gross permanent optimization-participatiory action planning (GPO-PAP) enzymatic triglyceride test kit (lot No. A110-1-1) was purchased from Nanjing Jiancheng Bioengineering institute (Nanjing, China). Beta actin monoclonal antibody (lot No. 66009-1-1 g) was purchased from Wuhan Sanying Proteintech Group, Inc. (Wuhan China). The oral solution of DGR (lot No. 150707) was purchased from the Pharmacy Department in the People's Hospital of Fujian Province (Fuzhou, China).

DGR consists of Danshen (Radix Salvia Miltiorrhizae), Banxia (Rhizoma Pinelliae), Chisuo (Radix Paeoniae Rubra), Gualou (Fructus et Semen Trichosanthis), Xiebai (Bulbus Allii Macrostemonis), and Jiangcan (Bombyx Batryticatus) in equal parts. The liquid for gavage of DGR was produced by the hospital at 1∶1 concentration using a standard production process. During the experiment, the solution was modified to the required concentration. The medicine underwent in-hospital preparation and has been awarded a national invention patent.

2.3. Equipment

The research was performed at the Science Laboratory Animal Center of Fujian University of Traditional Chinese Medicine. The following instruments were used: Mini-PROTEAN® Tetra Vertical Electro-phoresis Cell (Mini Trans-Blot cell), Microplate Reader (IMARK), and PowerPac™ Basic Power Supply (PowerPac Basic) from Bio-Rad Laboratories (Bio-Rad, Hercules, CA, USA). The 300Extend C18 column (770450-902, Agilent, Santa Clara, CA, USA), Analytical Column and Acclaim™ PepMap™ 100 C18 (164568, ThermoFisher Scientific, Waltham, MA, USA), SpeedVac Concentrator (SPD111V, ThermoFisher Scientific), NanoDrop® ND-1000 Micro UV spectrophotometer (ND-1000, NanoDrop Technologies, Waltham, MA, USA), Heraeus Multifuge X1R High speed refrigerated centrifuge (Multifuge X1 X1R,Thermo Scientific Co., Ltd., Waltham, MA, USA), Amicon Ultra (Amicon Ultra-50, Ultracel-3k and Amicon Ultra-500, Ultracel-10k, Merck Millipore, Burlington, MA, USA), high-intensity ultrasonic processor (JY92-ⅡN, Scientz, Linbo, China), shaker (TS-200, Qilinbeier, Shanghai, China), high pH reverse-phase HPLC (L3000, RIGOL Technologies, Beijing, China), Dk-8d electric constant temperature water tank (Senxin Laboratory Instrument Co., Ltd., Shanghai, China); high-intensity ultrasonic processor (JY92-ⅡN, Scientz, Linbo, China), centrifuge (Velocity 18R, Dynamica Scientific Ltd., London, UK), Centrifuge (Velocity 18R, Dynamica Scientific Ltd., London, UK), timsTOF Pro (timsTOF Pro, Bruker, Saarbrucken, Germany), and TECAN Continuous Wavelength Multifunctional Enzyme-labeled Instrument (Infinite M200 Pro, TECAN, Vienna, Austria) was used.

2.4. Grouping and administration

After 1 week of adaptation, rats were tested for oral glucose tolerance using the using the oral glucose tolerance test (OGTT) twice. Normal rats (18) with fasting blood glucose (FBG) < 6.1 mmol/L and OGTT 2 h blood glucose (PBG) < 7.8 mmol/L were selected. Then, the rats were stratified using fasting body mass (FBM) and, based on the average PBG, randomly divided into the control group (group A) and model group at 1∶2. The baseline levels of FBM, FBG, and PBG between the groups had no statistical differences. Rats in group A were fed conventional chow provided by the Laboratory Animal Center, Fujian Institute of Medical Sciences, China, and treated with high-temperature disinfection and irradiation. All rats in model group were fed a high-fat-high-sugar diet that contained refined lard (10%), cholesterol (2%), pig bile salt (0.3%), sucrose (20%), and conventional feed (67.7%) for 4 weeks. Thereafter, the oral glucose tolerance and FBM of modal rats were measured again, and they were divided into groups B and C at 1∶1 using the method described above. These were continuously fed a high-fat-high-sugar diet. Additionally, rats in group C were fed DGR liquid according to 20.5 g·kg^-1·d^-1, which is equivalent to the usage for a 60-kg person, once daily. Both groups were provided with the same amount of clean tap water daily. Activity status and water and food intake were observed and recorded daily. After 10 weeks of intervention, rats were fasted with free access to water for 12 h and euthanized for the analysis of the blood and liver.

All animal procedures were performed according to the Chinese Law of Animal Care Guidelines and departmental guidelines, and our protocols were approved by the Animal Care Committee of Fujian University of Traditional Chinese Medicine [Fujian Chinese Medicine (2019); Ethical Review No. 037].

2.5. Routine testing and detection

FBG and PBG were assessed using Optium glucose meters (Abbott, glucose dihydrogen method); glycosylated hemoglobin (HbA1) was quantitated using the In2it (1) A 1c test cartridge (Bio-Rad, boronic acids affinity chromatography). Plasma total cholesterol (TC) and triglyceride (TG) levels were assessed using a Beckman DXC800 automatic biochemical analyzer (end assay). Samples of the hepatic hilar region tissue (80 mg) were removed from the same section, homogenized, and the TG content was determined using the GPO-PAP enzymatic method. The liver index (LI) was calculated as follows: liver mass (LM) divided by final FBM.

2.6. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis

Middle lobe liver specimens (100 g) were obtained from each rat, and two specimens from each group were grinded with liquid nitrogen. Three samples containing six rat livers from each group were therefore obtained for protein extraction and phosphorylation-modified non-labeled quantitative omics analysis. After trypsin digestion, on-machine inspection was performed according to the manufacturer’s instructions. Therefore, the technical process was conducted in the following order: protein extraction → trypsin digestion → affinity enrichment → LC-MS/MS analysis → database search → bioinformatics analysis.

2.7. Database search

The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). MS/MS data were searched against the Rattus uniprot database (v.1.5.2.8 http://www.maxquant.org/) concatenated with a reverse decoy database. Trypsin/P was specified as the cleavage enzyme, allowing up to four missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as the fixed modification and acetylation modification and oxidation on Met were specified as the variable modifications. FDR was adjusted to < 1% and the minimum score for modified peptides was set to > 40.

2.8. Bioinformatics analysis

Identified protein domain functional description was annotated using InterProScan, a sequence analysis application, based on the protein sequence alignment method, and the InterPro domain database (v.5.14-53.0 http://www.ebi.ac.uk/interpro/) was used. Wolf-psort (v.0.2 http://www.genscript.com/psort/wolf_psort.html), a subcellular localization predication software was used to predict subcellular localization. The Gene Ontology (GO) annotation proteome was derived from the UniProt- Gene Ontology Annotation (GOA) database (http://www.ebi.ac.uk/GOA/). First, the identified protein IDs were converted to the UniProt IDs and then mapped to GO IDs using the protein ID. When identified proteins could not be annotated using the UniProt-GOA database, the InterProScan software (v.5.14-53.0 http://www.ebi.ac.uk/interpro/) was used to annotate the GO functionality of the protein using the protein sequence alignment method. Then, proteins were classified using GO annotation based on three categories: biological processes (BP), cellular components (CC), and molecular function (MF). For each category, a two-tailed Fisher’s exact test was performed to test the enrichment of the differentially expressed proteins against all identified proteins. The GO with a corrected P-value < 0.05 was considered significant. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (v.2.0 http://www.genome.jp/kaas-bin/kaas_main) was used to identify enriched pathways through a two-tailed Fisher’s exact test to test the enrichment of the differentially expressed proteins against all identified proteins. The pathway with a corrected P value < 0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website.

For further hierarchical clustering based on differentially modified protein functional classification, we first collated all the categories obtained after enrichment with their P values, then filtered for those categories which were enriched in at least one of the clusters with P value < 0.05. This filtered P value matrix was transformed using the function χ = −log10 (P value). Finally, these χ values were Z-transformed for each functional category. These Z scores were then clustered using one-way hierarchical clustering (Euclidean distance, average linkage clustering) with Genesis software. Cluster membership was visualized through a heat map using the “heatmap.2” function from the “gplots” R-package (v.2.0.3; https://cran.r-project.org/web/packages/cluster/).

2.9. Verification the discovery of phosphoproteomics through cell experiments

Cell experiment methods and findings from previous studies were studies and applied. human derived hepatoma cells were used and divided into four groups, including the control (conventional culture, no intervention), model (without drug intervention after modelling), 5% DGR (5% medium-dose medicated serum + 10% blank serum after modelling), and 10% DGR (10% medium-dose medicated serum + 5 % blank serum) groups. The high-fat (palmitic acid, 0.125 mmol/L) and high-glucose (25 mmol/L) cell model was used in this study. Levels of phosphorylated mitogen-activated protein kinase kinase kinase kinase 4 (MAP4k4, also known as Phospho-general control non-derepressible 2 (p-GCN2) and phosphorylated adducin 1 (ADD1, also known as p-SREBP1) were detected using Western blotting, according to the instructions of the kit.

2.10. Statistical analysis

The GraphPad Prism 5.0 statistical software (GraphPad Software, San Diego, CA, USA) was used to construct graphs and SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used for data analysis. All measurement data of normal distribution were represented as mean ± standard deviation ($\bar{x}±s$), and the least-significant difference (LSD) test was used for comparison between groups. In instances where the variances were not uniform, the logarithmic transformation was followed by LSD comparison. Statistical significance was set at P < 0.05.

3. RESULTS

3.1. Glycolipid metabolism

There were significant deviations in the PBG, TG, hepatic portal TG, TC, and liver index values of the model group compared with that of the control group (P < 0.01, Table 1). There was also clear deviation in HbA1c between the two groups (P < 0.05). Compared with the model group, levels of PBG, HbA1c, TG, hepatic portal TG, TC, and the liver index were significantly decreased (P < 0.01 or P < 0.05) in the DGR group (Table 1).

Table 1.

Comparison of glycolipid metabolism index between the groups ($\bar{x}±s$)

Group	n	FBG (mmol/L)	PBG (mmol/L)	HbA1c (%)	TG (mmol/L)	Hepatic portal TG (mmol/L)	TC (mmol/L)	Liver index (%)
A	6	6.05±0.30	6.58±0.43	6.05±0.35	1.07±0.22	0.22±0.06	2.35±0.32	2.39±0.20
B	6	6.26±0.43	8.00±0.80a	6.48±0.30c	1.80±0.20a	0.40±0.14a	5.99±0.68a	4.37±0.34a
C	6	6.06±0.56	7.38±0.19b	5.87±0.31d	1.49±0.19b	0.19±0.13d	5.06±0.41d	3.98±0.29b

Notes: A: group A; B: group B; C: group C. Rats in group A were fed conventional chow. Rats in groups B and C were fed a high-fat-high-sugar diet for 4 weeks. Then, rats in group C were administered DGR liquid and rats in group B were administered the same amount of clean water for another 10 weeks. HbA1c: glycosylated hemoglobin A1c. FBG: fasting blood glucose; PBG: OGTT 2 h blood glucose. TC: total cholesterol. TG: triglycerides. DGR: Dangua Fang. Data were expressed as the mean ± standard deviation (n = 6). Compared with the control group: ^aP < 0.01, ^cP < 0.05; model group: ^bP < 0.05, ^dP < 0.01.

3.2. Liver pathological staining

The hepatic pathology is shown in supplementary Figure 1. After the removal of blood, the livers of rats in the control group were dark brown, those of rats in the model group was bright maroon, and those of rats in the DGR group was brown. Hematoxylin and eosin staining of hepatic tissue in the control group rats showed clear lobules, and cords and sinusoids were arranged in a radial pattern. Hepatic cords and hepatic sinusoids in the model group rats were arranged disorderly and loose radial structures with vacuolation of hepatocytes and inflammatory cell infiltration was observed. Hepatic cords and sinusoids in the DGR group rats appeared radial in structure with clear intercellular spaces and without inflammatory cell infiltration. Masson staining of the livers of control group rats showed fiber structure only in the manifold area. Liver tissue of model group rats exhibited extensive changes in reticular fibrosis, but liver tissues from rats in the DGR group showed changes in fibrosis in the manifold area. Sporadic fat staining using oil red O was observed in the livers of control group rats. Fat staining of liver tissues in model group rats showed extensive steatosis in more than 70% of the tissue area, but staining in DGR group rats was clearly reduced.

3.3. Analysis of the general characteristics of DGR regulation of the phosphorylation modification of protein in rats with glycolipid metabolism disorders

In the MS/MS spectrum analysis, the total spectrum obtained was 805 334, matched spectrum was 260 471, accounting spectrum was 32.3%, peptides were 19 995, modified peptides were 14 671, identified proteins were 4601, quantifiable proteins were 4417, identified sites were 15 749, and quantified sites were 14 659. Compared with the model group, DGR up-regulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins, and down-regulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins, based on the threshold of expression fold change (> 1.2). The numbers of differentially modified modification sites and modified proteins after they were filtered with a threshold value of expression fold change are summarized in Table 2. These results show that there may several abnormal phosphorylation sites that were adjusted.

Table 2.

Differentially modified modification sites (modified proteins) summary (filtered with threshold value of expression fold change)

Site comparison	Regulated type	Fold change >1.2	Fold change >1.3	Fold change >1.5	Fold change >2
B vs A	up-regulated	1171 (777)	1105 (745)	896 (607)	487 (332)
	down-regulated	788 (570)	728 (525)	536 (390)	245 (196)
C vs B	up-regulated	228 (204)	200 (179)	145 (130)	70 (63)
	down-regulated	432 (358)	358 (302)	180 (164)	35 (32)

Notes: A: group A; B: group B; C: group C. Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg^-1·d^-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang.

The analysis of motif models of phosphorylation showed that 91 motif logos in S.motif.mode and 17 in T.motif.mode were found. Typically, the enhanced amino acid next to the S.motif.mode phosphorylation site are proline, followed by Serine and Arginine. Moreover, Aspartic acid and Glutamate mainly appeared downstream and Lysine mainly upstream (supplementary Figure 2B). The enhanced amino acid next to the T.motif.model phosphorylation site are also typically proline, followed by Arginine and Serine (supplementary Figure 2A).

It was discovered from the motif enrichment heat map of all the amino acids upstream and downstream of the identified phosphorylation modification sites that if the threonine of a protein was phosphorylated, then all the proline sites in each of the six upstream and downstream positions of this threonine would exhibit up-regulated phosphorylation. Additionally, cysteine, phenylalanine, isoleucine, leucine, and tryptophan would exhibit down-regulated phosphorylation (supplementary Figure 2A). Correspondingly, if the serine of a protein was phosphorylated, each proline and arginine in the six upstream sites of the serine, and each aspartic acid and proline in the six downstream sites would exhibit up-regulated phosphorylation, and each cysteine, pheny-lalanine, isoleucine, valine, tryptophan, and tyrosine in the six upstream sites of the serine, as well as each cysteine, valine, tryptophan, and tyrosine in the six downstream sites would exhibit down-regulated phos-phorylation (supplementary Figure 2B). By comparing supplementary Figure 2C to 2D and 2E to 2F, the overall regulatory effect of DGR on protein phosphorylation modification is shown (supplementary Figure 2C-2F).

3.4. Functional classification and subcellular structure location

The corresponding proteins of the differential phosphorylation modification sites were classified based on BP, CC, and MF from different perspectives (Figure 1A, 1B). Figure 1A shows that a large number of abnormally phosphorylated proteins in the model group were found compared to the control group, which involved all aspects of BP, CC, and MF. Figure 1B shows that DGR regulated phosphorylated proteins involved in various aspects of BP, such as cellular processes, biological regulation, single-organism processes, metabolic processes, response to stimuli, CC organization, multicellular organismal processes, developmental processes, localization, signaling and immune system processes, and multi-organism processes.

Figure 1. Functional classification and subcellular structure location.

A, B: GO functional classification; C, D: Distribution of the subcellular structure of the corresponding proteins of the differential phosphorylation modification sites. A, C: group B vs group A; B, D: group C vs group B. Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg^-1·d^-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang; GO: Gene Ontology.

For CC, the phosphorylated proteins regulated by DGR involved the cell, organelle, membrane, macromolecular complex, membrane-enclosed lumen, extracellular region, and cell junction. Based on the directed acyclic graph corresponding differential phosphorylation modification sites, this was the basis for DGR improving cell function and up-regulating energy metabolism. For MF, DGR regulated phosphorylated proteins involved in binding, catalytic activity, the MF regulator, nucleic acid binding transcription, transcription factor activity, structural molecule activity, signal transducer activity, and transporter activity.

Regarding the subcellular structure location of the corresponding proteins of the differential phosphorylation modification site (Figure 1C, 1D) involving the above functions, they were widely located in the nucleus, cytoplasm, and cell membrane system, and several were also located in the mitochondria. These formed the basis of DGR regulation of cell energy metabolism. In addition, some proteins located outside the cell can transmit regulatory information to the cell, thereby improving cell function.

3.5. GO Function enrichment of the corresponding proteins of the differential phosphorylation modification site regulated by DGR

In this study, our interested proteins were those corresponding to the differential phosphorylation modification sites regulated by DGR. For BP, the interested proteins were highly significantly enriched with down-regulation of the gene expression, RNA metabolism, biosynthetic process of cellular and macromolecule, and significantly affected the processes of nucleic acid and RAN metabolism and cellular macromolecule biosynthesis (Figure 2A), which are beneficial for suppressing excessive cell proliferation caused by excess energy.

Figure 2. Analysis of GO function enrichments involving in the corresponding proteins of the differential phosphorylation modification site regulated by DGR (group C vs group B).

A, B: biological processes; C, D: molecular function; G: cellular components. A, D: down-regulated; B, E: up-regulated; C, F, G: the directed acyclic graph. The circle in figures represent the GO classification where the differentially modified proteins are located. Red represents the highly significant (P < 0.01) enriched GO classification of the differentially modified protein, yellow represents the significantly enriched GO classification of the differentially modified protein (P < 0.05), and blue represents the non-significantly enriched GO category. The line with arrows represents the upper and lower levels of GO classification, and the size of the circle represents the degree of enrichment (fold enrichment). Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg^-1·d^-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang; GO: Gene Ontology.

Additionally, the interested proteins were significantly enriched also in up-regulation of the development of the gland and hepatobiliary system, the transportation of the lipid, organic hydroxy compounds, amides, peptides, and drugs, metabolic processes of hexose and lipids, regeneration of the tissue and skeletal muscle, activity of ATPase, and assembly of mitochondrion (Figure 2B), which are beneficial for improving energy metabolism, enhancing the function of tissues and cells, and improving quality of life.

By up-regulating or down-regulating the various cell functions above, DGR ultimately positively regulated production of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) as well as hemopoiesis involving multicellular organismal, developmental, and immune system processes. Cell activation, ATPase activity, and muscle relaxation was also positively regulated. Moreover, DGR improved tissue regeneration involved in tissue development, growth and wound healing, and N-acyltransferase activity, and regulated amide transport and peptide transport related with organic substance transport or nitrogen compound transport, as shown in the acyclic graph of supplementary Figure 3A.

For MF, the regulatory effect of DGR showed the same characteristics as for BP. DGR significantly down-regulated the binding of the nucleic acids, RNA and DNA, including regulatory region nucleic acids, transcription regulatory region DNA, double-stranded DNA, sequence-specific double-stranded DNA, and sequence-specific DNA, also reduced the binding of proteins related to transcription factor activity. DGR down-regulated a variety of MF, including lamin binding, activating transcription factor binding, N-acyltransferase activity, transcription cofactor activity, transcription coactivator activity, and ARF guanyl-nucleotide exchange factor activity (Figure 2C), which restored functionality to the normal state.

DGR up-regulated MF that were beneficial to restore cell vitality and promote energy metabolism, including increasing ubiquitin-like protein-specific protease activity, ubiquitinyl hydrolase activity, thiol-dependent ubiquitinyl hydrolase activity, structural constituents of cytoskeleton, structural molecule activity, cysteine-type peptidase activity, amino acid binding, ion channel binding, and chaperone binding, thus improving lipid transporter, ATPase regulator, MAP kinase kinase kinase, and ATPase activator activities (Figure 2D). These findings suggest that DGR corrected the disorder of glycolipid metabolism and increased the conversion of glycolipids into energy.

The acyclic graph of MF shows that DGR treatment up-regulated the activities of the ATPase activator, ATPase regulator, N-acetyltransferase, and MAP kinase kinase kinase, and also improved RNA binding, double-stranded DNA binding, and structural constituents of the cytoskeleton (supplementary Figure 3B).

Previous studies have shown that DGR intervention promotes the formation of cell pseudopodia, effectively resists the depolymerization of microfilament micro-tubules by colchicine,⁶ increases the ratio of NAD/ NADH, optimizes the function of the respiratory chain,⁴ and enhances cell activity,⁵ which ware consistent with the findings in this study.

For CC, the corresponding proteins of the differential phosphorylation modification sites affected by DGR treatment were extremely significantly enriched in the calcium channel complex, asymmetric neuron to neuron synapses, myosin complex of the cytoskeleton, small nuclear ribonucleoprotein complex, and sections of the nucleoplasm. Proteins in the histone methyltransferase, the transmembrane transporter, ATPase dependent transmembrane transport, and ATPase complexes were also significantly enriched (supplementary Figure 3C). These functions were also observed in previous studies.⁵ The modulation of CC by DGR corresponds to the improvement of the MF and BP described above.

Then, we further analyzed the changes in the level of up-regulation or down-regulation of the corresponding proteins at the differential phosphorylation modification sites involved in the above-mentioned GO function enrichment analysis (Table 3).

Table 3.

Quantity contrast of the corresponding proteins of the differential phosphorylation modification sites involving glycolipid metabolism in the GO function enrichment analysis

COG/KOG category	Category description	No. of proteins of B vs A			No. of proteins of C vs B
		Up	Down		Up	Down
Information storage and processing	[J] Translation, ribosomal structure, and biogenesis	13	11		4	6
	[A] RNA processing and modification	11	13		6	11
	[K] Transcription	26	19		5	16
	[L] Replication, recombination, and repair	7	1			1
	[B] Chromatin structure and dynamics	11	2			4
Cellular processes and signaling	[D] Cell cycle control, cell division, and chromosome partitioning	15	9		2	3
	[V] Defense mechanisms	6	3		1
	[Y] Nuclear structure	1	4			1
	[T] Signal transduction mechanisms	105	36		11	21
	[N] Cell motility	2	1			2
	[Z] Cytoskeleton	29	8		3	5
	[W] Extracellular structures	3	1		1
	[U] Intracellular trafficking, secretion, and vesicular transport	23	13		3	7
	[O]Posttranslational modification, protein turnover, and chaperones	18	10		11	6
Metabolism	[C] Energy production and conversion	4	8		1	3
	[G] Carbohydrate transport and metabolism	4	9		4
	[E] Amino acid transport and metabolism	2	17		1
	[F] Nucleotide transport and metabolism	4	2		1
	[H] Coenzyme transport and metabolism		1
	[I] Lipid transport and metabolism	14	14		2	3
	[P] Inorganic ion transport and metabolism	12	5		1	1
	[Q] Secondary metabolites biosynthesis, transport, and catabolism	1	5		2
Poorly characterized	[R] General function prediction only	55	38		14	12
	[S] Function unknown	19	14		5	6

Notes: the differential phosphorylation modification sites involving glycolipid metabolism in the GO function enrichment analysis of group C vs group B. Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg^-1·d^-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang; GO: Gene Ontology; COG: Clusters of Orthologous Groups ofproteins; KOG: euKaryotic Ortholog Groups.

3.6. Analysis of DGR-regulated phosphorylation sites

Furthermore, we analyzed the differential phos-phorylation modification sites involved in the functional proteins relating to glycolipid metabolism, as shown in Figure 3. With the control group as the standard, 75 pathological phosphorylation sites involving 64 proteins in group B were corrected toward normal when they appeared in group C Among these, there were 39 proteins wherein all pathological phosphorylation sites were corrected, involving 42 position amino acids. Among the 75 corrected phosphorylation sites, 64 were serine sites, 10 were threonine sites, and 1 was a tyrosine site (Figure 3), which involved corresponding proteins. These results suggest that the phosphorylation of serine, threonine, and tyrosine may be the key target of DGR regulating glycolipid metabolism.

Figure 3. Corrected pathological phosphorylation modification sites and corresponding amino acids and their proteins (C vs B).

A: proteins with modification sites that were all down-regulated; B: proteins with modification sites that were partially down-regulated; C: proteins with modification sites that were all up-regulated; D: proteins with modification sites that were partially up-regulated. Rats in group A (n = 7) were fed conventional chow. Rats in groups B (n = 7) and C (n = 7) were fed high-fat-high-sugar diet lasting for 4 weeks. Then, DGR liquid was administered according to 20.5 g·kg^-1·d^-1 to rats in group C and the same amount of clean water was administered to rats in group B until the end of the experiment. DGR: Dangua Fang.

The phosphorylated proteins were mapped using STRING software (https://cn.string-db.org/)to obtain a protein interaction map. The map shows that these proteins are extensively involved in complex metabolic regulation and with various stresses, ubiquitination, and nuclear factors (supplementary Figure 4).

3.7. Phosphorylation verification using Western blotting

The phosphorylation levels of p-GCN2 and p-SREBP1 were verified using Western blotting (Figure 4). The increased phosphorylation levels of p-GCN2 and p- SREBP1 in the model group were compared with those in control group (P < 0.05). After intervention with DGR-medicated serum, the level of phosphorylation of p-GCN2 decreased significantly in the 5%DGR and 10%DGR groups compared with that in model group (P < 0.05 or P < 0.01). Phosphorylation of p-SREBP1 decreased significantly in the 10%DGP group compared with that in the model group (P < 0.05), but that in the 5%DGP group did not reach statistical significance (P = 0.216). These results were consistent with those of the phosphoproteomics analysis.

Figure 4. Phosphorylation levels of p-GCN2 and p-SREBP1 in cell experiment detected using western blot.

A, C: western blot; western blots in A are control, 5%DGR, model, 10%DGR. in proper order and in C are control, model, 5%DGR, 10%DGR. B, D: quantitative comparison of western blot detection results. Control: conventional culture without intervention; Model: without drug intervention after modelling; 5%DGR: 5% medium-dose medicated serum + 10% blank serum after modelling; 10%DGR: 10% medium-dose medicated serum + 5 % blank serum. DGR: Dangua Fang; p-GCN2: phospho-general control non-derepressible 2 (n = 4); p-SREBP 1: phospho- sterol-regulatory element binding protein 1 (n = 6). Compared with control group: ^aP < 0.05; compared with model group: ^bP < 0.05, ^cP < 0.01.

4. DISCUSSION

Post-translational modification (PTM) of proteins, including phosphorylation, glycosylation, acetylation, and ubiquitination, are related to the occurrence and development of diseases and have been widely considered in the study of the therapeutic mechanism of Chinese herb medicine. Phosphorylation modification plays an important role in the processes of glucose and lipid metabolism.

Based on the variation in phosphate amino acid residues, phosphorylated proteins can be divided into 4 categories: O-phosphate, N-phosphate, acyl phosphate, and S-phosphate proteins. O-phosphate proteins are formed by the phosphorylation of hydroxyl amino acids. In this phosphorylation reaction, a strong negatively charged phosphate group is added to the amino acid side chain of the protein, thereby changing the configuration, activity, and interaction performance of the protein with other molecules. These changes play an important role in the regulation of many BP, including signal transduction, gene expression, and cell division. Serine, threonine, and tyrosine are all hydroxyl amino acids commonly observed in this role.

The phosphorylation modifications regulated by DGR occur on these hydroxyl amino acids, and are most prominent for serine and threonine. In the present study, DGR-regulated phosphorylation modifications involved in up-regulating 228 phosphorylation sites relating to 204 corresponding function proteins, and in down-regulating the modification of 358 phosphorylation sites relating to 358 corresponding functional proteins. DGR regulates the MF of proteins by affecting the phosphorylation of hydroxyl amino acids, thereby improving the BP of glucose and lipid metabolism. In this study, it was found that DGR regulates the phosphorylation modification of proteins relating to glycolipid metabolism, mainly involving 75 pathological phosphorylation sites and the expression of 64 corresponding proteins. These modifications act as key therapeutical effects in this multi-method system. First, DGR promotes the transfer of glycolipids and accelerates the catabolism of glycolipids. Second, DGR up-regulates oxidative phosphorylation and improves respiratory chain function and ATPase activity. Third, DGR up-regulates ubiquitinyl hydrotase activity and plays a role in deubiquitination, which helps regulate cell cycle progression, apoptosis, transcriptional regulation, DNA repair, and immune response. Fourth, DGR maintains the normal occurrence of cell functions by protecting the structural constituents of the cytoskeleton. Fifth, down-regulation of nucleic acids (DNA and RNA) binding function helps to inhibit the excessive proliferation of cells, thereby preventing complications in diabetes.

In vitro verification showed that the phosphorylation of MAP4k4 and ADD1 is significantly regulated by DGR. The protein encoded by the Mapk4k gene is a member of the serine/threonine protein kinase family. This kinase had been shown to specifically activate MAPK8/JNK and mediate the TNF-ɑ signaling pathway, possibly play a role in the response to environmental stress and cytokines such as TNF-alpha, appear to act upstream of the JUN N-terminal pathway, and phosphorylates SMAD1 on Thr-322. Map4k4 plays an important role in the development of fatty liver,⁷ pancreatic islet function,⁸ and the pathogenesis of diabetes.⁹

ADD1 is a member of a family of cytoskeletal proteins encoded by three genes (alpha, beta, and gamma) and acts as a heterodimer of the related alpha, beta, or gamma subunits. The protein encoded by this gene represents the alpha subunit. Alpha- and beta-ADD exhibit a protease-resistant N-terminal region and a protease-sensitive hydrophilic C-terminal region. ADD binds with high affinity to Ca ⁽²⁺⁾/calmodulin and is a substrate for protein kinases A and C. ADD1 plays important roles in a variety of physiological and pathological processes, including glycolipid decomposition, energy metabolism, and hypertension through salt sensitivity.¹⁰

In conclusion, this study shows that DGR can improve the function of the corresponding proteins by regulating the phosphorylation of hydroxyl amino acids, thereby effectively preventing and treating glycolipid metabolism diseases in through a multi-method and multi-target process (supplementary Figure 5).

5. ACKNOWLEDGMENTS

We heartfully appreciate the Experimental Animal Center and Research Center of the Fujian University of Traditional Chinese Medicine for providing support and assistance in this work. LIN Yuhua, CHEN Jinchuan, SU Yongxin, JIANG Zhenye, RUAN Yi, RUAN Yanyan, and LI Xiaolin, all are graduate students of the Fujian University of Traditional Chinese Medicine, participated experiment work. Phosphoproteomics analysis was conducted by Hangzhou PTM BIOLABS. Their contributions were important constituents for this study.

6. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://www.journaltcm.cn.