Significantly hypoglycemic effect of a novel functional bread rich in mulberry bark and improving the related functions of liver, pancreas, and kidney, on T2D mice

Abstract To develop a novel functional food with lowering blood glucose for diabetics, the mixed bread containing mulberry branch bark powder (MBBP) was used for the oral administration of the type 2 diabetic (T2D) mice induced by streptozocin (STZ), high‐fat and high‐sugar diet for 7 weeks. 5%, 10%, and 15% MBBP bread diets had a significatively positive influence on the biochemical indicators, histological examination, and immunohistochemistry observations in T2D mice. The 15% MBBP‐rich bread diet evidently retarded loss weight of T2D mice, and decreased in FBG by about 55% and in glycosylated hemoglobin (HbA1c) levels by about 30%. Its glucose tolerance and serum insulin levels were very close to normal level. The abnormal lipid metabolism and insulin‐related index of T2D mice in both the 10% and 15% MBBP bread diet groups were partly reversed. The Western blotting results showed that the expression levels of key proteins in PI3K/AKT signaling pathway were decreased and expression levels of immunoproteins PPARγ, TNF‐α, P65, and IL6 were increased. In general, oral MBBP bread diets effectively restored some functions and repaired damage to the pancreas, liver, and kidney in T2D mice. Therefore, MBBP is potential to develop into a novel functional food additive with significantly hypoglycemic effect.

life. Hence, people with diabetes often feel hungry. In view of the above two situations, we want to develop a new type of food that can show better both in large quantities and in the treatment of diabetes.
Mulberry (Morus L.) has been a traditional Chinese medicine since ancient China. The annual yield of mulberry branches is quite large, but mulberry branches utilization rate is low. In recent years, plenty of studies have shown that mulberry branches contain many bioactive components including alkaloids, phenols, polysaccharides, and flavonoids (Qiu et al., 2016), mulberry branches also have rich medicinal value including hypoglycemic (Wu et al., 2005;Ye et al., 2002;Wang et al., 2014), hypolipidemic (He et al., 2007), and antioxidant activities (Mazimba et al., 2011)  Diabetics cannot consume too much carbohydrates. In clinical treatment, diabetics usually are allowed to keep a low carbohydrate diet which means carbohydrate intake should between 30-200 g/ day (Wang et al., 2018). This causes diabetes to feel hungry at times.
Man has a long history of making and eating bread all over the world (Samuel, 1996). Bread is made from wheat flour and contains a lot of carbohydrates. Therefore, diabetes is not ought to eat a lot of bread. Now, researchers have been studying bread that has a hypoglycemic effect (Waghmare and Arya, 2014).
Our laboratory has studied the effects of mulberry branch bark powder (MBBP) both as Chinese medicine and as food supplement on diabetes (Liu et al., 2016). The ethanol extract of MBBP could regulate the mRNA expression of glycometabolism genes to regulate sugar metabolism and reduce the blood glucose level in T2D mice. It could also increase the mRNA expression of the genes those could help decrease the insulin resistance in T2D mice (Liu et al., 2014).
Through the results of metabolic pathways by GC-MS in T2D mice, oral administration of MBBP could ameliorate the hyperglycemia and hyperlipidemia symptoms on unitary level and have a dosagedependent effect on restoring the abnormal metabolic state to a near normal level (Qiu and Zhang, 2019). In this experiment, MBBP was added into bread to make MBBP-bread, and then fed to T2D mice induced by combined STZ with high fat and high glucose diet.
Finally, therapeutic effects of MBBP-bread on mice with type 2 diabetes were going to be explored.

| MBBP and MBBP-bread preparation
The Ramulus mori branches of the mulberry (HuSang 32,M. multicaulis L.) were provided by the Mulberry Garden of Soochow University, Suzhou, China, in November 2018. These barks which were peeled from mulberry branches, then were placed in the oven and dried at 100°C for 2 hr. Pulverized into powder twice to make sure it could pass through a 100-mesh sieve. According to Table 1, bread mechan (Philips, HD9016) was used to make normal bread and MBBP-bread.
The bread and standard laboratory fodder were put into high-speed functional grinder to powder, bread powder and standard laboratory fodder powder were mixed with water (bread powder: fodder powder = 1:1) to make a 3-4 cm cylindrical rod. Normal bread fodder, 5% MBBP-bread fodder, 10% MBBP-bread fodder, and 15% MBBPbread fodder were under test (as in Figure 1, main nutritions were shown in Table 2).

| Animals experiment
ICR male mice (3-4 weeks) were provided from the Experimental Animal Centre of Soochow University. The mice were raised in standard experimental conditions of humidity of 50%-80%, temperature of 18-25°C, and 12-hr light/dark cycle. Mice acclimation for 3 days, the mice could free gain normal fodder and purified water. After 3 days of acclimation, all mice (n = 60) were randomly divided into three groups: control group (n = 10), normal group (n = 10), and type 2 diabetes model group (n = 40). The control group and the normal group mice were fed with normal fodder. Mice in type 2 diabetes model group received high-fat and high-sugar diet for 4 weeks, high-fat and high-sugar diet was composed with Finally, all the mice were divided into control group, normal group, T2D model group, 5%, 10%, and 15% MBBP-bread fodder group.
The mice in control group were fed with SLF for 7 weeks, the mice in normal group and the model group were fed with normal bread fodder for 7 weeks. The other three groups were fed with different dose MBBP-bread fodder for 7 weeks (Table 3). The blood glucose and weight of mice were measured from the first to seventh weeks.
Blood, pancreas, liver, and kidney were collected for the follow-up experiments. Whole animal research processes were in accordance with guidelines by the Animal Ethics Committee at Soochow University.

| Oral glucose tolerance test
The glucose tolerance of mice was measured at sixth week. All mice were fasted for 12 hr; FBG was determined by tail-vein sampling using a glucose meter (0 min). Mice received an intraperitoneal injection of glucose (1 g/kg BW). FBG was determined at appropriate time intervals (30, 60, 90, and 120 min).

| Serum Insulin and Glycosylated Hemoglobin (HbA1c) determination
The serum insulin of mice was analyzed by assay kit (Nanjing Jiancheng Bioengineering Institute). HbA1c was analyzed by assay kit (Lai Er Bio-Tech).

F I G U R E 1
The appearance (a) and section (b) of normal bread and MBBPbreads

| Blood lipid determination
The ADVIA 1,800 Chemistry System (Siemens Ltd.) was used for appraisal of the total cholesterol (CHOL), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) in serum.

| Assays of CREA, UREA, ALT & AST
The creatinine (CREA) and serum urea (UREA) in kidney of mice were analyzed by assay kit. CREA assay kit was purchased from Jin-Pin Chemical Technology (Shanghai) Co., Ltd. UREA assay kit was purchased from Nanjing Jin Yibai Biological Technology Co., Ltd. We analyzed glutamic-pyruvic transaminase ALT and AST (glutamic oxalacetic transaminase) of mice by assay kit (Nanjing Jiancheng Bioengineering Institute).

| Histopathology
The mice were dissected; pancreas, liver, and kidney were collected.
The tissue samples were soaked at solution of formalin (10.0%) for 1 week. Samples were dehydrated by acetone. After being embedded in paraffin, samples were cut into 3 µm thickness slices. Samples were colored by hematoxylin and eosin (H&E), and were observed under an optical microscope.

| Immunofluorescence assay
Pancreas were collected from the mice. The tissue samples were soaked at solution of formalin (10.0%) for 1 week. Samples were dehydrated by using acetone. After being embedded in paraffin, samples were cut into 6µm thickness slices. The primary antibody was incubated with tissues, then second antibody combined with primary antibody. The samples were observed under a fluorescence microscope (DM6000B, Leica Microsystems Inc).

| Western blotting analysis
Western blotting was operated according to methods described by Afrin (Afrin et al., 2016). In the liver samples, all protein concentration was measured by BCA assay kit (Beyotime). To analyze protein levels, the protein extracts which were same quality were separated by using 7.5%-15% SDS polyacrylamide gel electrophoresis, then transferred protein from electrophoresis membranes to nitrocellulose membranes. Nitrocellulose membranes were blocked by 5% Bovine Serum Albumin (BSA). Antibodies and reagents were supplied from KeyGEN Tech CO., LTD. incubated overnight at 4℃. Then, the membranes were washed with TBST for three times, each for 5 min. The secondary antibodies which were bound to chemiluminescence agents were incubated for 1.5 h. After that, the membranes were washed again with TBST for 4 times, each for 5 min. G:BOX che-miXR5 was used for imaging.

| Statistical analysis
The experimental data were expressed as the Mean ± SD and were analyzed with Origin 8.5 software. The one-way ANOVA was used to evaluate the difference between multiple groups, with p < .05 considered as significant and p < .01 as very significant.

| Body weight
Diabetes induced weight loss is a common clinical symptom.
Therefore, the weight changes were measured in T2D mice during treatment for 7 weeks. In Figure 2a, mice in the control and the normal group continuously gained weight. The mice in the model group kept losing weight; weight loss was 24.14%. There was a significant change in body weight between the three treatment groups.
In consideration of effects on mice weight recovery, 15% MBBPbread diet group was best compared with model group. The results showed that MBBP-bread could effectively restore the weight loss of T2D mice.

| FBG
High blood glucose is the most typical characteristic of diabetes; at present, the main diabetes drugs on the market have good blood glucose control ability. Therefore, the FBG of mice was examined for 7 weeks. In Figure 2b, the FBGs of the control group and the normal group remained between 6.85 and 7.75 mmol/L, while model group maintained between 22.21 and 27.76 mmol/L and its blood glucose significantly increased compared with normal group (p < .01). When the mice were orally administered MBBP-bread for 7 weeks, blood glucose in 5%, 10%, and 15% diet group decreased significantly compared to the model group (p < .05 or p < .01), blood glucose levels fell 23.78%, 28.79%, and 54.66%, respectively. The results showed that MBBP-bread diets could effectively decreased the blood glucose levels of T2DM mice.

| Oral glucose tolerance
Diabetics have a disordered blood glucose metabolism, and blood glucose regulation is impaired. Oral glucose tolerance test (OGTT) can help researchers to understand the body's ability to regulate glucose level. Therefore, the glucose tolerance was measured in the mice to assess the effect of MBBP bread on blood glucose regulation. In Figure 3, glucose tolerance of control and normal group were ordinary, glucose tolerance of model group severely worse compared to normal group (p < .05 or p < .01). When the mice were orally administered MBBP-breads for 7 weeks, the glucose tolerance of mice treated with MBBP-bread diets recovered to different degrees. The 15% MBBP-bread diet group was the best glucose tolerance from 17.2 mmol/L at 0.5 hr decreasing to 5.7 mmol/L at 2 hr compared to model group (p < .01). The results showed that MBBP-bread could effectively restore glucose tolerance of T2D mice.

| Serum insulin
At present, the pathogenesis of type 2 diabetes is mainly divided into two aspects: One is the deficiency of insulin secretion, and the other is the insulin resistance. Hyperinsulinemia and hyperglycemia are also common when insulin resistance occurs (Nakagomi et al., 2013).
Therefore, the serum insulin content of the mice was measured. As shown in Figure 4, serum insulin levels were low in the control group and the normal group, ~22 and ~ 8 mUI/L, respectively. The level of the model group was as high as ~ 296 mUI/L, which was significantly higher than that of the normal group (p < .01). When the mice were orally administered MBBP-bread diets for 7 weeks, serum insulin in

| HbA1c
HbA1c is important to a large extent, because it gives clinicians an objective assessment of the effects of glucose control over the past period of time (Goldstein et al., 1986). In addition, the effect of HbA1c has also been proved by clinical diabetes control and complications tests (Kilpatrick et al., 1998). As shown in Figure 5, the HbA1c in the control group was basically the same as that in the normal group, there was no significant difference between the two groups.
The content of HbA1c in the model group was double (6.65%) that of the normal group, and there was a significant difference (p < .01).
When these T2D mice were orally administered MBBP-bread diets for 7 weeks, the HbA1c of mice treated with MBBP-bread diet decreased in different degrees, HbA1c of 15% MBBP-bread diet group decreased to 3.52% significantly compared to model group (p < .01), close to the HbA1c level (2.88%) in both normal and control groups.
The results showed that high concentration of MBBP-bread could effectively decreased HbA1c of T2D mice.

| Blood lipid
Besides the disorder of blood glucose metabolism in vivo, diabetic patients are also accompanied by the disorder of lipid metabolism in vivo. With the deepening of research, glucose metabolism and lipid metabolism jointly influence the development of diabetes in many aspects. Early in the onset of diabetes, patients with diabetes have a disorder of lipid metabolism. With the deepening of the disease, the abnormality of lipid metabolism is more significant (Turner et al., 1998). According to prior investigations, the main causes of death related to type 2 diabetes are cardiovascular and cerebrovascular complications, and thus, measurement of blood lipids in patients with type 2 diabetes is a critical factor in assessing their risk of cardiovascular and cerebrovascular complications (Li et al., 2012). As shown in Table 4, the TG, CHOL, HDL-C, and LDL-C of control group were basically the same as that of normal group, the TG, CHOL, HDL, and LDL conditions of model group were severely worse compared with normal group (p < .05 or p < .01). When the mice were orally administered MBBP-bread diet for 7 weeks, the TG, CHOL, HDL, and LDL of mice treated with MBBP-bread diet decreased in different degree, the TG, CHOL, HDL, and LDL of 15% MBBP-bread diet group decreased significantly compared to model group (p < .05). The results showed that MBBP-bread has lipid regulation ability to T2D mice in certain degree.

| Histopathological observation of pancreas
Damage to islet β-cells affects the production and secretion of insulin, which in turn causes the body's internal substance and energy metabolism disorders and insulin resistance (Kayaniyil et al., 2010). As shown in Figure 6, pathological changes were not observed in the islets of mice in the control group and normal group. The islet injury of mice in the model group was severe, such as pancreas was small, number of islet cells decreased greatly, cell vacuolation was serious. When the mice were orally administered MBBP-bread diet for 7 weeks, the pancreas islets of mice treated with MBBP bread gradually recovered, the distribution was even and compact, and the structure was more complete. These results showed that MBBP bread effectively restored the islet injury of T2D mice.

| Insulin expression
One of the important causes of diabetes is the islet damage in the pancreas, which leads to the disorder of insulin secretion and thus causing diabetes (Norquay et al., 2009). As shown in Figure 7, large amounts of insulin (green fluorescence) were secreted in the islets of the control and normal group. Insulin secretion in the pancreas islets was minimal in model group mice. When the mice were orally administered MBBP-bread diet for 7 weeks, with the increase of addition, the green fluorescence intensity in the islets gradually became stronger and the structure of the islets became intact, indicating that the number of islets β cells increased and showed a certain dose effect. The results showed that MBBP-bread effectively restored insulin expression of T2D mice.

F I G U R E 5
Effects of MBBP-breads on HbA1c in T2D mice. ## p < .01: versus normal group. ** p < .01: versus model group

| PDX-1 expression
Pancreatic duodenal homeobox-1(PDX-1) plays a key role in the development of pancreatic and islet cells (Mckinnon and Docherty, 2001). Its absence is closely related to the development of type 2 diabetes. As shown in Figure 8, large amounts of PDX-1 (red fluorescence) were secreted in the islets of the control and normal group.
The secretion of PDX-1 in pancreatic of model group decreased significantly compared to normal group. When the mice were orally administered MBBP-bread diet for 7 weeks, the amount of PDX-1 in the islets of mice treated with MBBP-bread diet increased gradually, and the distribution was consistent with islet structure. The results showed that MBBP-bread effectively decreased PDX-1 expression of T2DM mice.

| Liver function
Liver disease is one of the important causes of death in type 2 diabetes (Tolman et al., 2007). Liver function is an important index to evaluate liver injury. The determination of liver function is usually done by detecting the content of glutamic-pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST) in the blood.
As shown in Figure 9, the ALT and AST in serum of control group were basically the same as that of normal group. The ALT and AST of model group increased significantly compared to normal group (p < .01). When the mice were orally administered MBBP-bread diet for 7 weeks, the ALT and AST of mice treated with MBBP-bread diet decreased in certain degree. In particular, the ALT of 10% and 15% MBBP-bread diet groups declined significantly compared to model group (p < .05). The results showed that MBBP-bread effectively restored liver function of T2DM mice.

| Liver histopathological observation
To further evaluate the effect of MBBP-bread on the liver of T2D mice, the histopathological sections of liver were observed. As shown in Figure

| Signaling pathway
Liver is an important organ of metabolism of glucose for glucose metabolism of liver cells by controlling of insulin and insulin signaling pathways in the cell; the PI3K/AKT insulin signaling pathway is critical for glucose absorbtion and transportation (Li et al., 2015). When the insulin signaling pathway in the liver is damaged, the body's expression of some key proteins changes (Klover and Mooney, 2004).

| Hepatic glycometabolism
Liver plays its important role in glucose metabolism, the regulation of glucose of liver cells is through gluconeogenesis and glycolysis two ways to achieve dynamic equilibrium of glucose, the two ways play an important role in maintaining the stability of the blood glucose concentration. The expression of gluconeogenesis key enzyme phosphoenol pyruvate carboxyl kinase (PEPCK), glucose-6phosphatase (G6pase), and glycolysis key enzyme glucose kinase (GLK) was determined. As shown in Figure 12, there was little difference in protein expression between the control group and the nor- compared to model group (p < .05 or p < .01), close to the expression in the normal group. The results showed that MBBP-bread could effectively decreased the gluconeogenesis effect, increased the glycolysis effect, so that T2D mice blood glucose decreased.

| Lipid metabolism and inflammatory factors
Peroxisome proliferators activated receptor (PPAR) is primary in the regulation of lipid metabolism; PPARγ is one of PPARs subunits.
Inflammation also causes insulin resistance. The expression of PPAR and inflammatory factors P65, TNFα, and IL-6 was measured. As shown in Figure 13, there was little difference in protein expression between the control group and the normal group, the protein ex-

| Histopathological observation
To further evaluate the effect of MBBP-bread on the kidney of T2DM mice, the histopathological sections of mice kidney were observed. As shown in Figure 14, pathological changes were not observed in the kidney of mice in the control group and normal group.
The kidney injury of mice in the model group was severe including glomerular volume increases, cell vacuolization, and cell population decline. When the mice were orally administered MBBP-bread diet for 7 weeks, with the increase of MBBP dose, the kidney injury of mice treated with MBBP-bread diet improved gradually, 15% MBBPbread diet group's kidneys almost recovered. The results showed that MBBP-bread effectively restored kidney injury of T2D mice.

| Kidney function
Diabetic nephropathy is a complication of diabetes, kidney function is an important index to evaluate kidney injury, and the determination of kidney function is usually done by detecting the content of CREA and UREA. As shown in Table 5, there was little difference in CREA and UREA between the control group and the normal group, the CREA and UREA of model group increased compared to normal group (p < .05). When the mice were orally administered MBBPbread diet for 7 weeks, the CREA and UREA of mice treated with MBBP-bread diet decreased in certain degree, close to the expression in the normal group. The results showed that MBBP-bread could decrease CREA and UREA in certain degree.

| Kidney antioxidant
Researchers have shown that antioxidant drugs can significantly delay the progress of diabetic nephropathy by protecting the activity of antioxidant enzymes and inhibiting lipid peroxidation in the kidneys of T2DM mice (Ha et al., 1999). The kidney T-SOD, GSH-Px, and MDA of mice were measured. As shown in Table 6

| D ISCUSS I ON
In this paper, the therapeutic effect of MBBP-bread on T2D mice was investigated. It is well known that diabetes is a chronic meta- Dyslipidemia is a common symptom in diabetes mellitus, which can cause lots of diabetes complications, including coronary heart disease, hypertension, and atherosclerosis (Mitra et al., 1995). Liver is also a main place for insulin resistance since its role in glucose and lipid metabolism. Glucose metabolism in the liver is regulated to insulin and its insulin signaling pathway. Insulin resistance in the liver is characterized by impaired insulin signaling pathways in the liver cells (Cordero-Herrera et al., 2015). So it is of great value to evaluate the liver injury of diabetes mellitus. First liver function index is associated with liver injury, alanine transaminase (ALT) and aspartate aminotransferase (AST) are major indicators of liver function. The results showed that the MBBP-bread could significantly decrease ALT and AST level in the T2D mice, which proved that MBBP-bread effectively restored the T2D mice liver function. The HE staining results showed that the liver cells of T2D mice had complete structure and homogeneous cytoplasm after MBBP-bread treatment. So that MBBP-bread could effectively repair liver damage in T2D mice. Besides, we investigated the internal mechanism of the effect of MBBP-bread on liver of T2D mice.
PI3K/AKT is a golden insulin signaling pathway, and the substrate is GSK3β, which is the key to glycogen synthesis. p-AKT inactivated its phosphorylation and GS activity increased, which in turn increased glycogen synthesis and decreased blood glucose. In the model group, the expression levels of IR, IRS, PI3K, p-AKT, p-GSK3β, and GS decreased.
After the treatment with MBBP-bread, the expression levels of these proteins were significantly increased. By activated PI3K/AKT insulin signaling pathway, GLUT4 was also significantly up-regulated. This suggested that the MBBP-bread could transfer glucose to the liver by activating GLUT4 and improve the blood glucose regulation of T2D mice.
The regulation of blood glucose in liver is mainly through two metabolic pathways of glycosylation and glycolysis. We studied the expression of two key enzymes PCK and G6pase in glycosylation, and GLK in glycosylation. The results showed the expression of PCK and G6pase was increased while GLK was decreased in the diabetic model group. After MBBP-bread treatment, the situation reversed.
The results showed that the MBBP-bread could effectively reduce the liver's gluconeogenesis and increase the glycolysis to decrease the blood glucose.
In diabetic patients, lipid metabolism is disordered, PPARγ also plays an important role in the regulation of lipid metabolism, with decreased expression in insulin resistance. In study of T2D mice, both the expression of PPARγ was down-regulated in liver, and both the expression of PPARγ was up-regulated after the treatment  TA B L E 6 Effect of MBBP-breads on antioxidant abilities of kidney in T2D mice of MBBP-bread. The results showed that the MBBP-bread could effectively improve the lipid metabolism in the liver of T2DM mice.
Meanwhile, the inflammatory factors in the liver of each group were also analyzed. Results showed that the MBBP-bread effectively alleviated the inflammatory response in the liver of T2D mice.
Diabetic nephropathy is a common complication of diabetes.
We measured CREA and UREA in serum, which are both important for diagnosing early renal disease in clinic. The results showed that MBBP-bread could effectively reduce the content of CREA and UREA in the serum and improve the renal function of T2D mice. The rise of oxidative stress is common in diabetic patients, and oxidative stress injury is important to promote diabetic nephropathy. We

ACK N OWLED G M ENTS
The authors gratefully acknowledge the earmarked fund (CARS-

18-ZJ0502) from the China Agriculture Research System (CARS) and
a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China.

CO N FLI C T O F I NTE R E S T
The authors would like to declare that they have no conflict of interest.

E TH I C A L A PPROVA L
Ethics approval was not required for this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.