*2.5. SR, CR and Combined Extracts Suppressed Hepatic Glucose Output by Inhibiting Gluconeogenesis and Glycogenolysis as Well as Promoting Glycolysis and Glycogenesis*

Gluconeogenesis and glycogenolysis are two major pathways for endogenous glucose production [24,25]. PEPCK, FBPase, G6Pase, and GP are the rate-limiting enzymes controlling gluconeogenesis and glycogenolysis in the liver, respectively. The activities of these enzymes were increased in T2DM rats compared to normal rats but the increased activities were remarkably decreased to a range from 96.4% to 71.1% by SR and CR, especially by their combined extracts (Figure 6).

Glycolysis is regarded as a feeder pathway that prepares glucose for further catabolism and energy production. Besides, glucose storage in the form of glycogen could suppress hepatic glucose output to maintain blood glucose homeostasis. GK, PFK, PK, and GS are the rate-limiting enzymes controlling glycolysis and glycogen synthesis, respectively [26]. However, the activities of GK, PFK, PK, and GS were decreased in T2DM rats. After treatment, the decreased activities were increased to a range from 111.7% to 141.5% by SR and CR, especially their combined extracts (Figure 6).

#### *2.6. Intervention Effects of SR, CR and Combined Extracts on the Metabolic Profiling of T2DM Rats*

In this study, UPLC-Q-TOF-MS/MS was applied to generate metabolic profiles of plasma and urine from normal and T2DM rats before and after oral administration of SR, CR, LSC, and HSC in the negative and positive ESI modes. The metabolomics profiles of N and M groups were separated by PCA analysis and orthogonal partial least squares discriminant analysis (OPLS-DA). To investigate the amelioration of SR, CR, LSC, and HSC for curing T2DM, another PCA model was built. The variations of plasma and urine metabolic profiling of SR, CR, LSC, and HSC were restored to the levels of the normal group (Figures 7 and 8). Furthermore, the relative quantities of nine potential biomarkers in plasma and five in urine were significantly regulated by LSC and HSC. There were no obvious effects on the levels of xanthosine and N-acetyl-L-tyrosine for the SR group. The levels of xanthosine and hippuric acid were not regulated by the CR group. It was interesting to note that the levels of xanthosine were neither affected by SR nor CR, but by their combination. The contents of the potential biomarkers in Table S2 were considered as biomarkers for the effects of treatment.

The metabolic pathway was established by importing the potential metabolites into the web-based database MetPA. The pathway impact value calculated from pathway to topology analysis with MetPA above 0.1 was screened out as the potential target pathway. Here in Figure 9, glycerophospholipid metabolism with the impact values of 0.18 were filtered out as the most important metabolic pathways. Moreover, a correlation network of potential metabolites related to T2DM was exhibited in Figure 10. Our results also indicated that administration of LSC and HSC could more effectively modify these biomarkers and their related metabolic pathways including purine metabolism in which SR and CR could not be involved in T2DM rats.

**Figure 6.** Activities of glucokinase (GK), phosphofructokinase (PFK), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase), glycogen synthase (GS) and glycogen synthase (GP) in livers of the normal group (N), model group (M), and groups gavaged with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC). The values were shown as mean ± SD. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. normal group; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. model group. Data were analyzed by One-way-ANOVA.

**Figure 7.** PCA model results between normal (N) and T2DM (M) rats in negative mode ((**A**) 2-D plot of plasma; (**B**) 2-D plot of urine). 3D PLS-DA scores plot of LC–MS spectral data ((**C**) plasma; (**D**) urine). S-plot of OPLS-DA model for M vs. N group ((**E**) plasma; (**F**) urine). PCA analytical results from N and M rats treated with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC) at negative mode ((**G**) plasma; (**H**) urine).

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**Figure 8.** PCA model results between normal (N) and T2DM (M) rats in positive mode ((**A**) 2-D plot of plasma; (**B**) 2-D plot of urine). 3D PLS-DA scores plot of LC-MS spectral data ((**C**) plasma; (**D**) urine). S-plot of OPLS-DA model for M vs. N group ((**E**) plasma; (**F**) urine). PCA analytical results from N and M rats treated with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC) at positive mode ((**G**) plasma; (**H**) urine).

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**Figure 9.** Metabolic pathways involved in potential markers in plasma and urine. (**A**) Scutellaria Radix group (SR); (**B**) Coptidis Rhizome group (CR); (**C**) metformin group (P), Low dose of combined extracts group (LSC) and High dose of combined extracts group (HSC). (**1**) Glycerophospholipid metabolism, (**2**) primary bile acid biosynthesis, (**3**) pyrimidine metabolism, (**4**) linolenic acid metabolism, (**5**) alpha-linolenic acid metabolism, (**6**) arachidonic acid metabolism, (**7**) purine metabolism.

**Figure 10.** Correlation networks of main potential biomarkers in response to T2DM rats. Compared with the normal group, the red arrows represent the decrease of the contents of endogenous metabolites, while the green arrows represent the increase of the contents of endogenous metabolites.

#### **3. Discussion**

SR and CR, especially their combined extract, have been clinically used to treat T2DM for thousands of years; however, their compatibility mechanism remains unknown. Thus, in this study, the amelioration of SR, CR and their combined extracts on T2DM was first investigated, and their compatibility mechanism was further unraveled by the metabolomics and MAPK/PI3K/Akt signaling pathway. Importantly, the induction of the T2DM animal model is widely desired. The genetically modified mice and STZ-induced T2DM rat models have ever been established, however, neither these models nor stable animal models are appropriate because of ignoring dietary factors. Therefore, a suitable and stable T2DM rat model using HFD along with low dose of STZ was successfully established in our study. The levels of FBG, TG, TC, LDL-C, and FFA were remarkably increased, while the level of HDL-C was notably decreased in T2DM rats, which was similar to human T2DM sharing common symptoms (Figure 1).

Additionally, accumulating studies indicated that chronic inflammation was a major contributor to insulin resistance and T2DM [27–30]. Pro-inflammatory cytokines, particularly TNF-α, could enhance the serine phosphorylation of insulin receptor substrate-1, which induced insulin resistance. Some studies have shown glucose lowering effects of specific TNF-α or IL-1β inhibitors [31,32]. As expected, SR and CR could markedly ameliorate inflammation, insulin resistance, hyperglycemia, and hyperlipemia of T2DM rats. Furthermore, their compatibility (LSC and HSC) was more effective than single drug (Figures 1 and 3).

MAPK signaling pathway played a crucial role in inflammation [33]. Especially, NF-κB was a principle factor in regulating inflammatory mediators during the inflammatory process. The stimulation of NF-κB increased the levels of downstream inflammatory factors such as TNF-α, IL-1β, and IL-6, which promoted the insulin resistance in the liver. mRNA expressions of key targets in

MAPK pathway were significantly decreased after treatment. Additionally, protein expression of p-P65 was also decreased. Current data elucidated that LSC and HSC could exert better anti-inflammatory effects via blocking of the MAPK pathway (Figure 4).

The amelioration of inflammation might contribute to activating insulin signaling in liver. Remarkably, LSC and HSC inhibited inflammation in liver, but it was tempting to know whether this action was responsible for improving the insulin signaling pathway. The levels of Glut2, p-PI-3K, and p-Akt are major indexes that reflect the activity of the insulin signaling pathway, play a vital role in insulin-activated glucose uptake in liver, and inhibit glucose release from hepatocytes. As expected, LSC and HSC markedly up-regulated the mRNA expressions of IRS1, PI3K, Akt2, and Glut2, while protein expressions of p-PI3K, p-Akt, and Glut2 were also enhanced. These data suggested that LSC and HSC could overcome the impairment in PI3K/Akt insulin signaling pathway, contributing to the improvement of glucose disposal in T2DM rats (Figure 5).

Subsequently, the influence of activated insulin signaling pathway on glycogenolysis, glycogen synthesis, gluconeogenesis, and glucose metabolism in liver was investigated. The liver is crucial for the maintenance of normal glucose homeostasis—it produces glucose during fasting and stores glucose postprandially. Insulin could inhibit glycogenolysis, stimulate glycogen synthesis, reduce gluconeogenesis, and increase glucose metabolism. However, these hepatic processes were dysregulated in T2DM because the liver became insulin resistant. Gluconeogenesis and glycogenolysis constituted two major pathways for endogenous glucose production. Gluconeogenesis involved the production of glucose from non-carbohydrate precursors such as glycerol, glycogenic amino acid, and lactate during non-availability of dietary glucose, or glycogen stored in the liver was degraded to glucose by glycogenolysis. PEPCK, FBPase, and G6Pase acted irreversibly in the gluconeogenesis pathway. FBPase inhibitors and a decrease in transcription of PEPCK and G6Pase genes could reduce excessive endogenous glucose production in T2DM [34,35]. GP regulated hepatic glycogenolysis and GP inhibitor could lower blood glucose [36]. GS modulated hepatic glycogen synthesis and GSK-3 specific inhibitor could enhance glycogen synthesis by increasing GS activity [37]. GK mainly expressed in hepatocytes had effects on regulating blood glucose level in vivo [38]. Previous study also found that the ability of glucose metabolism in T2DM was reduced when the syntheses of PFK and PK were decreased [39]. In this work, owing to the amelioration of the insulin signaling pathway, PEPCK, FBPase, G6Pase, and GP activities were notably decreased, while prominent increased activities of GK, PFK, PK, and GS were detected after treatment. The above data indicated that the activities of key enzymes related to glycometabolism were modulated to notably lower FBG by SR and CR, especially by their compatibility (Figure 6).

As T2DM was a metabolic disease, the effects of SR, CR, LSC, and HSC on metabolic disorders in T2DM rats were further investigated to depict more insight into their compatibility mechanism. Metabolite profiles are closely related to the development of T2DM [40–43]. Among the most recent investigations on metabolic dysfunctions and plasma metabolites in T2DM, plasma level of bile acids have been confirmed to be closely associated with T2DM [44,45]. Moreover, metabolites of plasma and urine could reflect the changes of systemic physiology. Therefore, based on the comparative analysis of plasma and urine metabolomics in normal and T2DM rats, potential biomarkers and the correlated pathways could be discovered, which would be helpful to clarify the compatibility mechanism of SR and CR in improving metabolic disturbance of T2DM rats. In this study, plasma metabolomics of T2DM rats suggested that cholic acid and deoxycholic acid (secondary bile salts) levels were obviously increased, while the glycocholic acid (conjugated primary bile salts) level was dramatically decreased. Bile acids are major components of bile formed from cholesterol through various enzymatic reactions in hepatocytes. Primary bile acids are synthesized mainly by conjugation with taurine or glycine through the terminal side-chain carboxylic group presenting in the bile acid structure. Bile salt hydrolases, which are produced by many members of gut microbiota, could remove the conjugated amino acid from the primary bile salt to cholate and deoxycholate. Bile acids could exert effects on glucose metabolism via activation of bile acid receptors and G-protein-coupled membrane

bound receptors (TGR5) [46–51]. TGR5 was expressed in cells of the hematopoietic system such as monocytes and macrophages and conferred anti-inflammatory properties in vitro and in vivo, decreasing cytokine production in monocytes, macrophages and kupffer cells [52,53]. Recent works revealed that the immune-suppressive actions of TGR5 in macrophages contributed to the prevention of the T2DM process [48]. Thus, we speculated that glycocholic acid might be an endogenous TGR5 ligand, while cholic acid and deoxycholic acid were ligand inhibitors. So glycocholic acid could suppress inflammation by TGR5 activation. Both P38 and ERK were significantly stimulated by deoxycholic acid in a prior study [54]. Supplied with deoxycholic acid, serum glucose and triglyceride levels were increased in mice [55]. After treatment, the levels of cholic acid and deoxycholic acid were markedly decreased and glycocholic acid was notably increased. Furthermore, the combination of SR and CR was more effective than the single drug (Table S2).

Additionally, the glycerophospholipid metabolism was the most important metabolic pathway. The decreased levels of glycerophospholipid metabolites including LysoPE(0:0/18:0), LyspPC(16:0), LysoPE(20:2(11Z,14Z)/0:0), LysoPC(18:2(9z,12z)), PC(16:1(9z)/18:1(11z)), and LysoPC(16:1(9z)) indicated a marked perturbation in the glycerophospholipid metabolism of T2DM rats. PCs, LysoPCs, LysoPEs were largely involved in the pathogenesis of inflammatory and metabolic diseases such as diabetes [56]. PCs and LysoPCs could block ERK, P38 activation and NF-ƙB translocation to the nucleus in peritoneal macrophages, which had an intrinsic anti-inflammatory property [57–59]. Many studies also indicated that levels of lysoPCs are closely associated with oxidative stress and inflammation [60,61]. LysoPCs containing an unsaturated fatty acyl group such as C20:4 and C22:6 were reported to show potent anti-inflammatory activity in in vivo and in vitro models [62,63]. Lower levels of LysoPCs were found to be predictors for T2DM in earlier studies [64] and treatment with LysoPCs could result in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes [65]. Thus, the modification of SR, CR, LSC, and HSC on LysoPCs, LysoPEs and PCs levels might contribute to the amelioration of inflammation and insulin resistance. SR and CR combined therapy exhibited better efficacy (Table S2).

By the analysis of urine metabolomics, purine metabolism was notably modified after combined therapy, however, the single drug showed no effect; purine metabolism was terribly dysregulated which was associated with T2DM [66]. Xanthosine, a nucleoside derived from the purine base xanthine and ribose, was elevated in T2DM rats and catabolism of xanthosine could ultimately lead to the production of high uric acid which was linked to diabetic complications [67,68]. Interestingly, xanthosine was affected by neither SR nor CR, but by their combination. Additionally, hippuric acid, which could be converted from dietetic aromatic compounds by gut microbes [69], was significantly decreased in urine of T2DM rats. This might be due to disturbed gut microbiota metabolism under diabetic status [70]. As reported, concentrations of hippuric acid were lower in HFD-induced hyperlipidemic rats than in normal rats [71]. D-glucurono-6,3-lactone, participating in ascorbate and aldarate metabolism, was increased in T2DM rats and was downregulated after treatment of SR, CR and their combined extracts. Pyrimidine metabolism was also disturbed and increased metabolite of deoxyuridine in the urine of T2DM rats was identified. After 30 days therapeutic intervention, these changed trends in T2DM rats were reversed by SR and CR, particularly by their combined extracts (Table S2).

### **4. Materials and Methods**

#### *4.1. Chemicals, Reagents, and Materials*

Acetonitrile (HPLC grade) was bought from TEDIA (Fairfield, OH, USA). Formic acid was supplied by Merck KGaA (Darmstadt, Germany). Metformin was purchased from Merck Serono (Shanghai, China). SR and CR were purchased from Nanjing Guo-yao Pharm (Nanjing, Jiangsu, China). Streptozotocin (STZ) was obtained from Sigma chemical (St Louis, MO, USA).
