Selenium-Enriched and Ordinary Black Teas Regulate the Metabolism of Glucose and Lipid and Intestinal Flora of Hyperglycemic Mice

Black tea is one of the six major tea categories and has a variety of bioactivities. However, little is known about its comprehensive evaluation of hypoglycemic effects and potential mechanisms. In this study, we investigated the in vivo hypoglycemic activity and potential mechanism for aqueous extracts of ordinary black tea (BT) and selenium-enriched black tea (Se-BT) by using an established high-fat diet together with streptozotocin (STZ)-induced hyperglycemic mouse model. Additionally, we also explored their α-glucosidase inhibition activity. The results show that both BT and Se-BT had a favorable glycosidase inhibitory activity. Moreover, the intervention of BT and Se-BT could regulate the mRNA expression and the level of serum parameters related to glucose and lipid metabolisms. Accordingly, they could activate the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) signaling pathway and alleviate insulin resistance (IR) and hyperglycemia. Moreover, supplementation of BT and Se-BT increased the richness and diversity of intestinal flora and altered the abundance of beneficial and harmful bacteria. Both BT and Se-BT could regulate glucose metabolism, alleviate tissue damage, and restore intestinal flora dysbiosis, suggesting that they could be used as a natural functional food for preventing hyperglycemia.


Introduction
Type 2 diabetes (T2D) is the most common form of diabetes and IR is one of its main features [1]. IR is the decreased capability of insulin to uptake and utilize glucose in its primary target tissues, such as skeletal muscle, adipose tissue, and liver [2]. In addition, diabetes is also associated with dyslipidemia, one of the major risks for cardiovascular disease [3]. The study shows that hyperglycemia, body weight (BW), and dyslipidemia in diabetic mice had improved after specific dietary interventions [4]. However, in this process, the composition and structure of intestinal microbiota changed with the adjustment of dietary structure [5].
The gut microbiota plays an important role in supporting the development of the immune system, protecting the body from pathogenic microorganisms, and exogenous metabolism of carbohydrates [6]. An unbalanced diet like excessive intake of sugar and fat can lead to disruption of the stability and diversity of the gut flora [4]. Accumulative studies so far have shown that hyperglycemia is inextricably linked to gut microbiota and metabolism [7,8]. T2D is associated with some symptoms such as IR, glucose, and lipid metabolism disorders, and gut microbiota dysbiosis [1]. Besides, there are also great differences in the human gut microbiota between people with T2D and those without diabetes [9]. Therefore, modifying the gut microbiota by adjusting the dietary structure is an effective way to lower blood glucose (BG).
In recent years, studies have consistently shown that tea and its active components can regulate the structure and function of the intestinal flora, and then have become a hot option for treatment of the metabolic syndrome. Some in vivo and in vitro experiments have shown that green and dark teas have a favorable hypoglycemic effect by alleviating metabolic disorders and improving gut microbiota dysbiosis [7,10]. Previous studies have shown that tea and tea polysaccharides can reduce BG by enhancing the expression of the PI3K/Akt signaling pathway [7,11]. The PI3K/Akt pathway plays an important role in the metabolic effects of insulin, which can maintain the stability of BG by improving hepatic insulin sensitivity and accelerating glucose transport and metabolism [12]. Teas rich in organic selenium are more active and may have a better hypoglycemic effect [13]. Taken together, tea may play a promising role in preventing and ameliorating hyperglycemia by regulating intestinal flora and metabolic disorders and stimulating PI3K/Akt signaling pathway. However, existing reports do not clarify the mechanism of selenium-enriched tea and ordinary tea in lowering blood glucose and their relationship and differences.
In this work, we investigated the effects of BT and Se-BT on BG, metabolic disorders, and the structure and composition of intestinal flora in the high-fat diet together with streptozotocin-induced hyperglycemic mice. Our focus is on investigating the hypoglycemic activity of BT and Se-BT, exploring the potential hypoglycemic mechanism, and providing a theoretical basis for applying tea for special populations.

Materials and Methods
This part is presented as supplementary materials.

Results and Discussion
Chemical Composition and α-Glucosidase Inhibition As shown in Table S2, the contents of tea polysaccharides, catechin, and epicatechin (EC) were higher in Se-BT than in BT, and BT had higher contents of tea polyphenols and total nitrogen, with significant differences. These results suggest that both Se-BT and BT have high nutrient contents and may play a role in regulating BG [1,10]. In the α-glucosidase inhibition assay, Se-BT exhibited higher inhibition activity, which may be related to the high content of tea polysaccharides in Se-BT [1].

Effects of BT and Se-BT on Hypoglycemic Activity In vivo
To evaluate the effects of Se-BT and BT on hyperglycemia, we established the hyperglycemic mouse model induced by a high-fat diet together with STZ. The BW of mouse was significantly decreased after the Se-BT intervention compared MC group, whereas BT did not play a notably role (Fig. 1A). Additionally, BT and Se-BT had similar effects in reducing BG (Fig. 1B). Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) show that BT and Se-BT could markedly change the decrease of insulin sensitivity and glucose tolerance inducted by high-fat diet and STZ  Asterisk indicates a significance compared with the control group, *, p < 0.05; **, p < 0.01, ***, p < 0.001; ns, not significant that hyperglycemia led to a large number of vacuoles in the liver tissue, and both hepatocytes and adipocytes appeared enlarged and swollen. Accordingly, BT and Se-BT significantly ameliorated these damages. Likewise, Se-BT showed a better improvement effect. Previous research [14] show a significant improvement in OGTT in diabetic patients after ingestion of sucrose drinks containing BT polyphenols, but no significant difference in insulin tolerance. It may not be the tea polyphenols but other components that improve IR. Therefore, we speculate that the difference in the content of tea polyphenols and polysaccharides may be one of the factors for the distinction in hypoglycemic activity of BT and Se-BT. Additionally, our previous study shows that the binding of Se to polysaccharides led to the change in the structure of polysaccharides, which would affect their hypoglycemic activity [15]. Previous research [4] reveals that Pluchea indica (L.) Less tea could inhibit lipid and carbohydrate accumulation in adipocytes, changing fat cell morphology, indicating that the hypoglycemic effect of BT and Se-BT may be related to lipid metabolism.
Dyslipidemia and metabolic disorders often occur in patients with hyperglycemia [5]. Accordingly, we evaluated serum factor levels of lipid metabolism and oxidative stressrelated. Figure 2A-F shows that hyperglycemia significantly induced the decreased levels of high density liptein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and the activity of superoxide dismutase (SOD), and considerably enhanced the levels of total cholesterol (TC) and triglycerides (TG) and glycosylated serum protein (GSP). As expected, Se-BT and BT treatments noticeably decreased the TG level and significantly increased HDL-C level. Differently, Se-BT notably decreased TC and LDL-C levels, while BT significantly reduced GSP levels. However, the effect on SOD activity was limited. Furthermore, IR can cause hepatic damage and inflammation [16]. To reflect the degree of liver damage caused by hyperglycemia, we measured the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. The results showed that AST was increased in the MC group, but not as significant as ALT (Fig. 2G-H), indicating the formed hepatic damage and inflammation [16]. As expected, Se-BT and BT treatments reversed this trend. Excessive sugar and fat diets can cause disruptions in lipid and glucose metabolism, finally the hyperglycemia [4]. TC and TG reflect the degree of lipid metabolism, and HDL-C and LDL-C are involved in the mediation of cholesterol transport [17]. The level of GSP indicates the average BG concentration over a period of time and is an important indicator of BG level [18]. Our previous study showed that Liubao tea intervention regulated TC, TG, HDL-C, LDL-C, AST and ALT levels, and then alleviated metabolic disorders caused by hyperglycemia [7]. Gao et al. [16] found that BT decreased TG and ALT levels but had p-Akt and Akt protein (O-P). Asterisk indicates a significance compared with the control group, *, p < 0.05; **, p < 0.01, ***, p < 0.001; ns, not significant little effects on TC, HDL-C, and LDL-C levels. This may be related to the different sources, processing methods and distribution of active compounds in tea. Accordingly, BT and Se-BT could alleviate hyperglycemia by improving the proinflammation and regulating glucose and lipid metabolism.
We measured some BG-related mRNA levels and protein expression. As can be seen in Fig. 2I-N, the mRNA level of phosphatase and tensin homolog (PTEN) of mice in MC group was significantly increased compared with NC group, while the mRNA levels of insulin receptor substrate 1 (IRS-1), recombinant glucose transporter 2 (GLUT-2), Akt, pancreatic duodenal homeobox 1 (PDX-1) and PI3K were considerably declined. As expected, the intervention of BT or Se-BT could significantly prevent this phenomenon. Notably, Se-BT is more effective in mRNA levels of PTEN, PI3K and Akt. The PI3K/Akt pathway is closely associated with lipid metabolism and glucose homeostasis, and further T2D-associated metabolic syndrome is also closely related to its imbalanced expression [19]. IRS-1, located upstream of the PI3K-Akt pathway, is an insulin receptor substrate and shows a great significance in insulin signal transduction [20]. However, IR can lead to a blocked binding of insulin  [20]. For PI3K-Akt transportation, PTEN and PDX-1, located in the core region of the pathway, can inhibit and activate insulinsignaling transportation, respectively [19,21]. Notably, PTEN inhibits the activity of Akt [19]. An experiment [11] in diabetic mice shows that tea polysaccharides could activate the PI3K/Akt signaling pathway, transport GLUT-4 to the membrane surface and enhance glucose uptake. In addition, Liubao tea could activate the PI3K-Akt-peroxisome proliferators-activated receptors (PPARs)-GLUT2 cascade signaling pathway to improve metabolic disorders and IR [7]. As seen in Fig. 2O-P, the BT and Se-BT upregulated the expression of p-Akt/Akt protein compared with the control group, and the Se-BT had a significant effect [7]. This indicates that the Akt signaling pathway was activated with the intervention of BT. Accordingly, we hypothesize that BT could regulate glucose metabolism and IR by activating PI3K/Akt signaling pathway.

Effects of BT and Se-BT on the Gut Microbial
The diversity and stability of the intestinal flora can be disrupted by excessive ingestion of sugar and fat, resulting in intestinal inflammation, IR, and eventually, type 2 diabetes [22]. The Chao index, which indicates the bacterial richness, was significantly increased by the interventions of BT and Se-BT (Fig. 3A). Besides, the Shannon index, which indicates species and community richness, was markedly lower in MC group than in the BT and Se-BT groups (Fig. 3B). In the PLS-DA analysis (beta-diversity), BT group more closely resembling the NC group (Fig. 3C). This suggests the effectiveness of BT and Se-BT in preventing hyperglycemia and the strong link between the regulation of gut microbiota and symptom improvement.
To further confirm the importance of changing dietary habits in regulating hyperglycemia, we analyzed the abundance of dominant species at the genus and phylum levels. At the phylum level, the ratio of Firmicutes/Bacteroidetes was reduced after intervention with BT and Se-BT, and the effect of Se-BT was more pronounced (Fig. S2). Many members of Firmicutes and Bacteroidetes can encode carbohydrate-active enzymes [23], thus we initially speculated that BT or Se-BT might enhance glucose metabolism by improving the abundance of Firmicutes and Bacteroidetes. At the genus level, the top 10 genera were Lactobacillus, Allobaculum, Unspecified Clostridiales, Unspeci-fied_S24_7, Turicibacter, Unspecified Lachnospiraceae, Bacteroides, Ruminococcus, Ruminococcus_1, Oscillospira (Fig. 3D-M). Allobaculum produced short-chain fatty acids (SCFAs) and alleviated diabetes and obesity by reducing the migration of endotoxins into the bloodstream [24]. SCFA can affect host glucose metabolism by activating intestinal gluconeogenesis through a complementary mechanism, including acetic acid, propionic acid, butyric acid, etc. In mice with diet-induced obesity, SCFA supplementation improved IR [25]. Similarly, in a study of Fig. 4 Spearman correlation analysis *, p < 0.05; **, p < 0.01, ***, p < 0.001; ns, not significant glucose metabolism in diabetic rats, Lachnospiraceae was significantly decreased in treated rats and was significantly positively correlated with 2 h-glucose, isobutyric acid and isovaleric acid, and negatively correlated with butyric acid [26]. The main fermentation products of bacteria are acetic acid, butyric acid and succinic acid, while the main fermentation products of lactic acid bacteria are lactic acid, etc. [1]. Ruminococcus_1 was significantly increased in patients with T2D and was a conditionally pathogenic bacterium [27]. Turicibacter is a genus associated with disorders of glucolipid metabolism and also with intestinal butyric acid, which increases insulin secretion and sensitivity and has significant anti-obesity and anti-inflammatory effect [28]. Recent evidence [24] suggests that the abundance of Roseburia, Blautia, Allobaculum, Alistipes and Turicibacter was increased in mice fed wih a high-fat diet after Berberine intervention, and these microbiota showed a positive anti-inflammatory effect.
Based on Spearman correlation analysis (Fig. 4), 15 of the top 50 OTUs correlated with at least one parameter associated with hyperglycemia. Among them, Enterococcus, Klebsiella, Akkermansia, Sutterella, Prevotella, Paraprevotella, Weissella showed significant negative correlations with hyperglycemia-related parameters. While Ruminococcus, Coprobacillus, Odoribacter, Turicibacter, Allobaculum, Adlercreutzia, Clostridium showed significant positive correlation with hyperglycemia-related parameters. Coprobacillus is an important butyric acid producer with high abundance in obese animals or humans [3]. Odoribacter is a common, short-chain fatty acid producing member [29]. Both of them can affect intestinal function and mediate related intestinal diseases [29]. This further proves that black tea extract may play a crucial role in alleviating hyperglycemia and IR by affecting the secretion of SCFA and altering the composition of gut microbes. However, it remains unclear how SCFA regulates glucose and lipid metabolism and alleviates hyperglycemia. Further research is needed to explore in hypoglycemic mechanism of BT.

Conclusion
In this study, black tea exerted hypoglycemic effects by regulating insulin resistance and glucose metabolism through activation of PI3K/Akt pathway. Through intestinal flora analysis, we hypothesized that black tea could play an important role in alleviating hyperglycemia and insulin resistance by affecting the secretion of short-chain fatty acids and altering the composition of intestinal microorganisms. Our findings serve as a catalyst for further investigation of the hypoglycemic effects of tea.