Exploring the Effects of Cyanidin-3‑<i>O</i>‑Glucoside on Type 2 Diabetes Mellitus: Insights into Gut Microbiome Modulation and Potential Antidiabetic Benefits

Berries and their functional components have been put forward as an alternative to pharmacological treatments of type 2 diabetes mellitus (T2DM), and more attention has been paid to the gut microbiome in the pathophysiology of T2DM. Thus, we tried to examine the metabolic impact of red bayberry-derived cyanidin-3-O-glucoside (C3G) and investigate whether the antidiabetic effects of C3G were associated with the gut microbiome. As a result, C3G administration was found to reduce blood glucose levels of diabetic db/db mice, accompanied by increased levels of glucagon-like peptide (GLP-1) and insulin. Moreover, 16S rRNA analysis showed that the dominant microbiota modulated by C3G were pivotal in the glucose metabolism. Furthermore, the modulation of C3G on metabolic activities of gut bacteria leads to an increase in intestinal levels of key metabolites, particularly short-chain fatty acids. This contribution helps in promoting the secretion of GLP-1, which in turn increases insulin release with the purpose of reducing blood glucose levels. Overall, these findings may offer new thoughts concerning C3G against metabolic disorders in T2DM

Nucleotide sequences and positions of primers used in polymerase chain reactions.

<p>F and R indicate the forward and reverse directions, respectively. The underlined regions represent the adscititious recognition sequences of restriction endonucleases.</p

Semiquantitative RT-PCR analysis of cyclin K expression in <i>Artemia</i>.

<p>(<b>A</b>) Cyclin K expression during <i>Artemia</i> development: postdiapause development stages (0, 4 and 8 h) represent postdiapause cysts incubated for 0, 4 and 8 h, respectively. Larval and post-larval development stages (0.2, 0.5 and 1.0 cm) represent larva of body length 0.2, 0.5 and 1.0 cm, respectively. ANE: adults without eggs. (<b>B</b>) Cyclin K expression in cephalothorax (ce) and ovisacs (ov) of both oviparous and ovoviviparous animals. 0 h is as the same sample as 0 h in (A) and represents postdiapause cysts incubated for 0 h. (<b>C</b>) Expression of cyclin K and Rpb1 in oocytes and embryos of both oviparous and ovoviviparous developmental pathways. EO, early oocytes; LO, late oocytes; 1dE, 3dE and 4dE represent embryos having entered the uterus for 1 day, 3 days and 4 days, respectively. (<b>D</b>) Expression of cyclin K and Rpb1 during the hatching process of diapause embryos (which includes the diapause embryo, postdiapause embryo, 0- to 14-h incubated embryos and nauplius); α-tubulin was used as a loading control.</p

Knockdown of cyclin K in developing embryos.

<p>(<b>A</b>) Semiquantitative RT-PCR analysis of cyclin K mRNA levels in control (GFPi) and test (cycKi) groups. (<b>B</b>) Western Blot analysis of cyclin K protein levels in control (GFPi) and test (cycKi) groups. (<b>C</b>) Morphology of adult <i>Artemia</i>: cyclin K RNAi (a) and GFP RNAi (b); Offspring produced by cyclin K RNAi (c) and GFP RNAi (d) <i>Artemia</i>; Trypan Blue staining of embryos entering utrus for four days reproduced by cyclin K RNAi (e) and GFP RNAi (f) <i>Artemia</i>. The bars in (a) and (b) represent 30 mm. The bars in (c) represent 215 µm. The bars in (d–f) represent 150 µm. (<b>D</b>) DAPI staining of early development embryos. The samples are eggs having entered the uterus for 12, 24, 32 and 48 h respectively. The upper two panels are GFP RNAi groups (GFPi); the lower two panels are cyclin K RNAi groups (RNAi). (<b>E</b>) TUNEL assay of embryos at the same stages of development as shown in D, 8-µm frozen sections were prepared and DNA strand breaks were detected by the TUNEL assay. Arrows indicated positive signals in cyclin K RNAi embryos. The bars in both (D) and (E) indicate 35 µm.</p

The template activity of cyclin K mRNA is repressed in encysted embryos.

<p>Poly(A)-containing mRNA was purified from postdiapause and 8-h incubated (8 h) embryos using oligo(dT)-Cellulose, and <i>in vitro</i> translation was performed using each purified mRNA as a template. (<b>A</b>) Cyclin K was detected in each purified mRNA sample by Northern blotting. (<b>B</b>) Detection of cyclin K <i>in vitro</i> translation product by Western blotting. NS: nonspecific bands. CK⁻, DEPC-treated water was used as template for the <i>in vitro</i> translation control. The molecular weight was shown on the left.</p

Involvement of PHB1 in the degradation of yolk platelets.

<p>A, SDS-PAGE analysis of isolated yolk platelet lysates. Proteins were stained by Coomassie brilliant blue R-250. 0 h, 0 h after incubation; 6 h, 6 h after incubation; 12 h, 12 h after incubation; 24 h, 24 h after incubation. B, Yolk platelets were isolated by density centrifugation and probed with an anti-PHB1 antibody. An anti-VGN antibody was used as the control. 0 h, 0 h after incubation of cysts; 6 h, 6 h after incubation of cysts; 12 h, 12 h after incubation of cysts; 24 h, 24 h after incubation of nauplii. C. Yolk protein degradation during embryonic development. 0 h, 0 h after incubation of cysts; 6 h, 6 h after incubation of cysts; 12 h, 12 h after incubation of cysts; 24 h, 24 h after incubation D, Western blot analysis of yolk protein degradation in PHB1 knockdown nauplii. E, TEM analysis showed aberrant yolk platelet degradation in the PHB1 knockdown group (magnification 1250×, scale bar = 1 µm). F, Immunoprecipitation with an anti-PHB1 antibody using isolated yolk platelet lysates (12 h after incubation of cysts). The precipitates were then probed with an anti-ubiquitin antibody.</p

Sequence comparison with other species and phylogenetic analysis of <i>Artemia</i> cyclin K.

<p>Alignment of <i>Artemia</i> cyclin K with human (NP_001092872.1), mouse (NP_033962.2), <i>Xenopus</i> (NP_001089373.1), zebrafish (NP_001157251.1) and <i>Drosophila</i> (NP_788082.1). Numbers on the right side refer to the amino-acid position. Asterisks indicate conserved residues in all sequences; a single dot indicates semi-conserved substitutions and a double dot indicates conserved substitutions. The sequences underlined refer to the two cyclin boxes in <i>Artemia</i> cyclin K.</p

Cyclin K protein expression and phosphorylation of RNAP II in different developmental phases and their subcellular localization in <i>Artemia</i> embryos.

<p>(<b>A</b>) Cyclin K protein expression and CTD Ser2 phosphorylation of RNAP II in the two developmental pathways. EO, early oocytes; LO, late oocytes; 1dE, 3dE and 4dE represent embryos entering uterus for 1 day, 3 days and 4 days, respectively. (<b>B</b>) Cyclin K protein expression and CTD Ser2 phosphorylation of RNAP II during the hatching process of diapause embryos (includes diapause embryo, postdiapause embryo, 0- to 14-h incubated embryos and nauplius); α-tubulin was used as a loading control. (<b>C</b>) Supernatant (S) and pellet (P) fractions were prepared using buffer K (pH 6.0 or 8.0) from 14-h incubated embryos. Cyclin K and CTD Ser2 phosphorylation of RNAP II were detected by Western blotting. Tubulin and H3 were also examined to indicate the purity of the different extracts. (<b>D</b>) Immunofluorescence staining of nuclei from 14-h incubated embryos confirmed that cyclin K co-localizes with phosphorylated RNAP II in nuclei. a, cyclin K; b, phosphorylation of CTD Ser2; c, DAPI stain. The bars represent 10 µm. (<b>E</b>) Cyclin K and its associated factors (anti-cyclin K immunoprecipitates) were affinity purified from the 14-h incubated embryos and analysed by western blot. Another polyclonal antibody produced in rabbit (anti-SGEG2a) was used in a parallel procedure for control (con). The input loading quantity was 1/100 of the total supernatants.</p

Sequence and phylogenetic analysis of ArPHB1.

<p>A, Schematic view of PHB1 architecture. B, Amino acid sequence alignment of ArPHB1 with PHB1 from other species. The sequences used in this alignment and their Genbank/EMBL/DDBJ or SWISS-PROT database accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109152#pone.0109152.s006" target="_blank">Table S2</a>. Gaps inserted to maximize alignment are denoted by hyphens. The amino acid position numbers are shown to the right of the sequences. The phosphorylation sites against which the antibody was generated is marked by P. Conserved domains are marked by lines below sequences (the N-terminal hydrophobic domain by yellow, the PHB domain by black, the coiled-coil domain by blue, and the nuclear export sequence by red). The alignment was performed by MEGA5 using the ClustalW method. C, A phylogenetic tree of the amino acid sequences of prohibitins. The sequences used in this analysis and their GenBank/EMBL/DDBJ or SWISS-PROT database accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109152#pone.0109152.s005" target="_blank">Table S1</a>. The phylogenetic tree was constructed using the Neighbor-joining method. Bootstrap percentage values for 1000 replicate analysis are shown at branching points. The bar at the bottom shows the branch length, and it corresponds to the mean number of differences (0.05) per residue along each branch.</p

ArPHB1 knockdown resulted in aberrant mitochondrial function.

<p>A, mitochondrial morphology by TEM (magnification, 24000×; scale bar = 0.5 µm). Mitochondria are denoted by red arrows. B, Real-time PCR of mitochondrial genes (ATP6, ATP synthase F0 subunit 6; ND5, NADH dehydrogenase 5; and Cyto B, cytochrome b). Results were normalized to α-tubulin. Means ± SD are plotted. P<0.05. The primers used for real-time PCR are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109152#pone.0109152.s005" target="_blank">Table S1</a>. C, Western blot analysis of ATPase β subunit.</p