Mulberry leaf alleviates streptozotocin-induced diabetic rats by attenuating NEFA signaling and modulating intestinal microflora

Abstract

Improvement of hyperglycemia through dietotherapy/herbal remedy is an effective approach to treating diabetes. In this study, mulberry leaf, famous for silkworm's special food and therapeutic value without any side effects, alleviated diabetes by attenuating NEFA signaling and modulating intestinal microflora. Mulberry leaf treatment significantly reduce fasting blood-glucose and HbA1c, ameliorate the blood lipid profile and improve insulin resistance in streptozotocin-induced diabetic rats. Mechanistically, we found that mulberry leaf inhibited NEFA signaling by reducing downstream signaling in the NEFA pathway, further verified by reduced PKC and improved cellular energy homeostasis based on restored expression of PGC-1α, AK2, OXPHOS and adiponectin. Mulberry leaf treatment also restored the phyla Bacteroidetes and Proteobacteria and class Clostridia, which were associated with insulin resistance and diabetes. Our findings reveal that mulberry leaf is an edible with therapeutic potential for diabetes and may provide a novel dietotherapy/herbal remedy to the treatment of diabetes.

Figure 1

Body weight of rats during experimental time. Acute weight loss was observed in all groups treated with STZ. Both mulberry leaf and glibenclamide could protect the animals from acute weight loss (6–10^th weeks) to a certain extent. During the 7–8^th weeks, the body weights of rats in both PCG and MTG were significantly (P < 0.01) higher than NCG. *P < 0.05 PCG compared with NCG; **P < 0.01 PCG compared with NCG; ^#P < 0.05 MTG compared with NCG; ^##P < 0.01 MTG compared with NCG. Value = mean ± SD (N = 6).

Figure 2

Antihyperglycemic effect of mulberry leaf. (a) GTT, both mulberry leaf and glibenclamide could enhance insulin sensitivity of diabetic rat, *P < 0.05 PCG compared with NCG, **P < 0.01 PCG compared with NCG, ^#P < 0.05 MTG compared with NCG, ^##P < 0.01 MTG compared with NCG. (b) Area under curve of GTT, **P < 0.01 compared with NCG. (c) FBG levels of rats, FBG of NCG maintained high level throughout the trial, however, both mulberry leaf and glibenclamide could significantly reduce FBG of diabetic rat, *P < 0.05 PCG compared with NCG, **P < 0.01 PCG compared with NCG, ^##P < 0.01 MTG compared with NCG. (d) HbA1c levels of rats, both mulberry leaf and glibenclamide treatments significantly inhibited glycation procedure of diabetic rat, *P < 0.05 compared with NCG. (e) Serum fasting insulin level of rats. (f) HOMA-IR, both mulberry leaf and glibenclamide decreased insulin resistance, *P < 0.05 compared with NCG. (g) HOMA-IS, both mulberry leaf and glibenclamide increased insulin sensitivity, *P < 0.05 compared with NCG. (h) HOMA-β, both mulberry leaf and glibenclamide improved pancreas islet β-cell function, *P < 0.05 compared with NCG. Value = mean ± SD (N = 6).

Figure 3

Anti-hyperlipidemic effect of mulberry leaf. (a) Blood lipid profile, the levels of TG, CHO and LDL were reduced with mulberry leaf treatment. (b) Enzymatic activity, AST level was reduced in NCG and restored by mulberry leaf treatment, ALP level increased in NCG and was significantly reduced with mulberry leaf treatment. (c) The level of serum NEFAs, the level of serum NEFAs was significantly elevated in diabetic rats, however, could be significantly reduced with mulberry leaf treatment. *P < 0.05 compared with NCG, **P < 0.01 compared with NCG. Value = mean ± SD (N = 6).

Figure 4

The results of RT-PCR and western blot. (a) RT-PCR, mulberry leaf decreased PKC and increased PGC-1α and AK2 in liver tissue, and increased adiponectin expression in subcutaneous fat. *P < 0.05 compared with NCG, **P < 0.01 compared with NCG. Value = mean ± SD (N = 6). (b,c) Western blot for protein in liver tissue, mulberry leaf decreased PKC and increased OXPHOS expression. β-tubulin serves as a loading control. For presentation purposes additional lanes were excised. PKC, PGC-1α, OXPHOS and β-tubulin were cropped from different parts of the same gel. These proteins were stained one by one after using Restore™ Western Blot Stripping Buffer (Thermo Scientific, USA). Full-length gels for each protein were included in Supplementary Information Fig. S2. (d,e) Western blot for protein in subcutaneous fat, mulberry leaf increased adiponectin expression in subcutaneous fat. β-tubulin serves as a loading control. For presentation purposes additional lanes were excised. Adiponectin and β-tubulin were cropped from different parts of the same gel. Full-length gels both proteins were included in Supplementary Information Fig. S3. *P < 0.05 compared with NCG, **P < 0.01 compared with NCG. Value = mean ± SD (N = 3–4).

Figure 5

Effects of mulberry leaf on the community structure of intestinal microflora. (a) NMDS plot of the Bray–Curtis similarity coefficients calculated from the 16 S rRNA sequencing data of the gut microbiota; (b) Alpha diversity of each group revealed by the Simpson index. (c) Beta diversity of each group revealed by the Bray–Curtis index. *P < 0.05 between compared groups, **P < 0.01 between compared groups. Value = mean ± SD (N = 6).

Figure 6

The structural changes of intestinal microflora affected by mulberry leaf according to the LEfSe analysis. (a) The similarity of intestinal microflora among STZ treatment groups (PCG2, MTG2, NCG2). (b) The changes of intestinal microflora between NCG3 and VCG3. (c) The changes of intestinal microflora between MTG3 and NCG3. (d) The changes of intestinal microflora between MTG3 and VCG3.