Dios reversed sustained transcriptional changes in KK-Ay
diabetic mice.
To study the mechanism of Dios on
KK-Ay diabetic mice, the liver samples were collected for transcription
analysis. We calculated FPKM for all samples and performed PCA analysis
(Fig. 3A) and correlation analysis
(Fig. 3B), which indicates that our samples have good repeatability. To
identify gene sets with a statistically significant difference in
hepatocytes, we screened the differentially expressed genes (DEGs)
between Control vs Model and Model vs Dios by the following criteria:
|logFC| >1, FDR < 0.05 and p
value < 0.05. We found 4182 DEGs between model and control
(Fig.3C), of those DEGs, 1967 genes were up-regulated and 2215 genes
were down-regulated. Besides, a total of 173 DEGs between Model and Dios
were revealed. Among them, 101 genes were up-regulated and 72 genes were
down-regulated respectively. We used hierarchical cluster analysis (Fig.
3D-3E) to compare the DEGs in three groups, which shows that the
similarity of expression patterns of three biological replicates. To
identify the biological features of DEGs, we performed significant GO
analysis by the clusterProfiler R package, and the biological processes
analysis revealed that the DEGs between Model and Dios group (Fig 3F)
were associated with glucose metabolism including fructose metabolic
process, carbohydrate metabolic process, carboxylic acid biosynthetic
process. Among significant GO terms of down-regulated genes (Fig 3G), it
was obvious that most GO terms were associated with organic anion
transport, carboxylic acid transport, organic acid transport and lipid
transport. To further explore the potential mechanism of DEGs between
Model and Dios group, KEGG pathway analysis was performed using the
clusterProfiler R package. The results of KEGG analysis revealed that
DEGs were mainly associated with glucose metabolism including
glycolysis/gluconeogenesis, biosynthesis of amino acids, fructose and
mannose metabolism (Fig 3H). Interestingly, we found that pathways
involved in glucose metabolism were the most enriched up-regulated
pathways (Fig 3I), which is consistent with
the reactome functional enrichment
analysis (Fig 3J). Those results
revealed that the potential mechanism of Dios ameliorated T2DM may be
through regulating glucose metabolism.
Dios
treatment ameliorated
HFD-induced glucose metabolism disorder inKK-Ay diabetic mice.
To
clarify the potential mechanism of Dios in KK-Ay diabetic mice, the
glucose metabolism and insulin resistance-related signaling proteins in
liver and skeletal muscle were observed by western blot analysis. Our
data showed that a continuous HFD diet can lead to severe insulin
resistance in the liver (Fig. 4A-4C) and skeletal muscle (Fig. 4D-4F).
However, treatment with Dios and Met
could significantly ameliorate insulin resistance by activating the
IRS/PI3K/AKT signaling pathway. Once AKT is activated, it participates
in glucose metabolism related pathways caused by insulin. We found that
Dios promoted glycogen synthesis in
liver and skeletal muscle by phosphorylating GSK-3β and activating GS.
Besides, the level of
phosphorylation AS160 and Glut4 were
significantly up-regulated in skeletal muscle by Dios treatment, which
suggests that Dios promote glucose transport.
Dios treatment reverses
HFD-induced gut dysbiosis in KK-Ay diabetic mice.
To further explore the effects of Dios on gut microbiota composition,
the 16S bacterial rRNA genes composing the V4 hypervariable regions in
the faeces of KK-Ay mice were performed. After removing low-quality
sequences, a total of 72292 tags were generated. The remaining clean
tags were clustered into OTUs (Operational Taxonomic Units) based on
97% similarity. UniFrac distance-based principal coordinate analysis
(PCoA) revealed a relative clustering of
gut microbiota within each group
(Fig 5A). Remarkably, the microbes in Dios and Met groups were more
closely clustered relative to ND groups, which is an indication that
Dios treatment induced similar microbial composition changes.
Unweighted Pair-group Method with
Arithmetic Mean (UPGMA) indicates a
statistically significant separation between Model group and H-Dios, Met
groups (Fig 5B). Notably, Firmicutes and Bacteroidetes are
the most abundant microbiota at the phylum level. Compared with the CK
group, the content of the phylum Firmicutes in the Model group
significantly increased and theBacteroidetes abundance significantly increased. However, a
remarkable reversal of this pattern was observed following Dios and Met
treatment (Fig 5C-5F). An increasedFirmicutes/Bacteroidetes ratio
is a commonly used indicator of
diabetic mice as reported in a previous study, as shown in Fig 5D, the
increased ratio of the phylum Firmicutes /Bacteroideteswere observed in the diabetic model group. On the contrary,
Dios and Met treatment remarkably
suppressed the ratio of the phylum Firmicutes/Bacteroidetes . At
the same time, we interestingly found that the ratio ofActinobacteria phylum was
significantly higher in the H-Dios group than in the other groups. Next,
we further analyzed the relative
abundances of the gut microbiota at
the
species level, as illustrated in
Fig 5G-5K, there were several bacteria decreased in the diabetic Model
group, which were
markedly
increased by Dios treatment, includingAerococcus_viridans (70
fold), C._glu (155 fold),
Combined, these data suggested that Dios treatment may effectively
reverse gut microbiota dysbiosis in diabetic mice.
C.
glu and Dios treatmentreducedHFD-induced body weight and
blood glucose gain in KK-Ay diabetic mice.
Recent
studies have shown that the ability of the gut microbiota to modulate
obesity can be transferred to other animals. To explore whether the gut
microbiota of Dios-treated animals may improve the condition of KK-Ay
diabetic mice, we selected the highest abundance bacterium(C.glu ) for further study. After treatment with Dios andC.glu for 4 weeks, the
related indexes were measured. As shown in Fig. 6A-6B, it is noteworthy
that the body weight and blood glucose of
Dios, C.glu and Dios+C.
glu groups were significantly reduced compared to the Model group at
the end of the feeding period, similar to the results shown above (Fig
1A and 1B). In addition, our results
further showed that the weight gain (Fig. 6C), liver index (Fig. 6D) and
adipose index (Fig. 6E) of Dios,C.glu and Dios+C. glu groups were significantly lower than
that of KK-Ay diabetic mice. On the other hand, Expression of TC(Fig
6F), TG (Fig 6G), LDL-C (Fig 6H) was also increased and HDL-C (Fig 6I)
and insulin (Fig 6J) decreased in KK-Ay diabetic mice compared with NC
group at the end of transplantation. Surprisingly,
Dios, C.glu and Dios+C.
glu treatment significantly
decreased the serum levels of TC,
TG, LDL-C and increased the serum level of HDL-C and insulin.
Furthermore, the liver histopathology also produced significant changes
in less vacuolation and lipid droplets when treatment with
Dios,C.glu and Dios+C. glu. compared with KK-Ay diabetic mice
(Fig 6K).