Anti-diabetic effect of anthocyanin cyanidin-3-O-glucoside: data from insulin resistant hepatocyte and diabetic mouse

Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycemia driven largely by decreased insulin sensitivity and insulin resistance (IR). Insulin signaling begins with receptor activation and phosphorylation of substrates such as IRS-2, which propagates downstream pathways to enhance cellular insulin responsiveness. PTP1B acts as a negative regulator of insulin signaling, in part by inhibiting IRS-2 phosphorylation. Given the limitations and side effects of many chemical antidiabetic drugs, natural products like anthocyanins are of growing interest. Anthocyanins exert anti-inflammatory, antioxidant, anti-obesity, and anti-diabetic effects. Prior work suggests anthocyanins can reverse IR via PI3K/AKT activation and GLUT4 modulation. This study investigates whether cyanidin-3-O-glucoside (C3G) from red bayberry can improve glucose metabolism by alleviating IR in hepatocytes and in diabetic db/db mice, and explores PTP1B/IRS-2 signaling as a potential mechanism.

Evidence from multiple studies indicates anthocyanins benefit glucose metabolism and insulin sensitivity: mulberry anthocyanin extract improved IR via PI3K/AKT in HepG2 cells and db/db mice; purple corn anthocyanins ameliorated TNF-α–induced IR by activating insulin signaling and promoting GLUT4 translocation in 3T3-L1 adipocytes; aronia melanocarpa anthocyanins enhanced glucose uptake and glycogen synthesis in C2C12 and HepG2 IR models by modulating GLUT4 and increasing the p-GSK-3β(Ser9)/GSK-3β ratio, with SOCS3 implicated as a target. Red bayberry is rich in anthocyanins and is used in traditional Chinese medicine, supporting investigation of its principal anthocyanin, C3G, in IR and T2DM contexts.

Anthocyanin extraction and identification: Fresh red bayberry fruits underwent ultrasonic extraction in anhydrous ethanol (4× volume, 90 min), filtration, solvent removal at 49°C, ethyl acetate partitioning, collection of aqueous phase, D-101 macroporous resin column purification (elution with 1% formic acid in 80% methanol, 2 mL/min), concentration, lyophilization, and storage at −80°C. Components were analyzed by UPLC (Thermo UltiMate 3000) with detection at 520 nm using a water/formic acid (A) and acetonitrile/0.1% formic acid (B) gradient; C3G was inferred as the main component. C3G standard (Solarbio SC8740) was used for treatments. Cell culture and IR induction: HepG2 and L02 hepatocyte lines were cultured to ~80% confluence and exposed to 0.2 mM palmitic acid (PA) plus 30 mM glucose for 24 h to induce IR; controls received 5 mM glucose. IR cells were then treated with C3G at 0, 20, 40, or 80 µg/mL for 24 h (cell viability established safe below 100 µg/mL by MTT). Assays: Cell viability assessed by MTT (0.5 mg/mL, 4 h). Glucose consumption measured from media using a glucose detection kit, normalized by MTT-derived viability; insulin sensitivity index (%) = (glucose consumption in C3G+insulin − C3G without insulin)/(control with insulin − control without insulin) ×100. Glucose uptake: 0.1 mM 2-NBDG incubation (30 min, 37°C), imaging and quantification by fluorescence microscopy and Image-Pro Plus. Glycogen synthesis: alkali digestion (30% KOH), ethanol precipitation, anthrone colorimetry (620 nm), normalized to cell mass; glycogen staining via kit and light microscopy. qRT-PCR: total RNA isolation, cDNA synthesis, TB Green-based qPCR with primers for PTP1B and GAPDH. Western blot: RIPA lysis, BCA quantification, SDS-PAGE (12%), PVDF transfer, 10% milk blocking, primary antibodies (PTP1B; p-IRS-2 Tyr612; GAPDH), HRP secondary, ECL detection; densitometry in ImageJ. Statistics: data as mean±SD; one-way ANOVA, Duncan’s test, t-test; p<0.05 significant. Animals: Four-week-old male C57BL/6J and db/db mice (C57BL/6J background) housed SPF, 23±3°C, 12 h light/dark, chow diet, water ad libitum. Randomized to: (a) C57BL/6J + water (n=6), (b) db/db + water (n=6), (c) db/db + C3G (n=6). Daily gavage with C3G 150 mg/kg/day or water for 6 weeks; fasting blood glucose monitored. OGTT: 12 h fast, 1.5 g/kg oral glucose; tail blood glucose at 0, 30, 60, 90, 120, 150 min; AUC calculated. HOMA-IR: serum insulin (ELISA) and glucose measured; HOMA-IR = glucose (mmol/L) × insulin (mU)/22.5. Animal procedures approved by Zhejiang Chinese Medical University Animal Ethics Committee (20201103-08).

- UPLC profiling indicated cyanidin-3-O-glucoside (C3G) is the main anthocyanin in red bayberry extract. C3G was non-cytotoxic to HepG2 and L02 cells below 100 µg/mL (MTT). - IR was successfully induced in HepG2 and L02 cells by 0.2 mM PA + 30 mM glucose for 24 h, evidenced by decreased glucose consumption, reduced 2-NBDG uptake, and diminished glycogen content versus control. - C3G treatment (20–80 µg/mL, 24 h) significantly increased glucose consumption and 2-NBDG uptake in IR HepG2 and L02 cells (p<0.05), and restored glycogen synthesis/content (p<0.05). - C3G significantly improved the insulin sensitivity index in both IR hepatocyte models (p<0.05). - Mechanistically, IR elevated PTP1B mRNA and protein levels; C3G dose-dependently suppressed PTP1B expression (qRT-PCR and Western blot) and increased phosphorylation of IRS-2 at Tyr612, consistent with enhanced insulin signaling. - In vivo, db/db mice gavaged with C3G (150 mg/kg/day) for 6 weeks showed significantly reduced fasting blood glucose compared with db/db controls. OGTT profiles improved with lower glucose AUC, and HOMA-IR scores were reduced in the C3G group versus db/db + water (p<0.05), indicating improved glucose tolerance and insulin resistance.

The study tested the hypothesis that cyanidin-3-O-glucoside (C3G) ameliorates insulin resistance and associated glucose metabolism disorders. In two hepatocyte models of IR, C3G improved key metabolic endpoints: glucose consumption, uptake, and glycogen synthesis, and enhanced insulin sensitivity, indicating a direct effect on hepatocyte insulin responsiveness. Mechanistic assays showed that C3G suppressed the IR-associated increase in PTP1B, a negative regulator of insulin signaling, while increasing IRS-2 phosphorylation at Tyr612, consistent with restoration of proximal insulin signaling. These molecular changes align with literature implicating PTP1B as a target to enhance insulin action and IRS-2 as a critical substrate in hepatic insulin signaling. In db/db mice, C3G reduced fasting glycemia, improved OGTT AUC, and lowered HOMA-IR, supporting translation of in vitro findings to an in vivo T2DM model. Together, the data suggest that dietary C3G can mitigate hepatic IR and improve systemic glucose homeostasis, potentially offering a natural product–based adjunct for T2DM management.

C3G, the principal anthocyanin from red bayberry, alleviates insulin resistance in vitro and in vivo. In hepatocytes, C3G increases glucose consumption and uptake, restores glycogen synthesis, and enhances insulin sensitivity, associated with downregulation of PTP1B and increased IRS-2 phosphorylation. In db/db mice, oral C3G for 6 weeks lowers fasting blood glucose, improves glucose tolerance, and reduces HOMA-IR. These findings support C3G as a dietary bioactive with potential to correct glucose metabolic disturbances linked to IR in T2DM. Future work should include insulin tolerance tests, hyperinsulinemic–euglycemic clamps, and broader molecular profiling to validate and extend these results.

- OGTT and related figures did not include the C57BL/6J control group for direct comparison with db/db + water, limiting conclusions about the magnitude of glucose intolerance in controls versus db/db mice. - Reliance on OGTT and HOMA-IR alone in vivo does not definitively establish improved insulin sensitivity; insulin tolerance tests and hyperinsulinemic–euglycemic clamp studies are needed. - Additional molecular assessments of IR pathways in vivo would strengthen mechanistic conclusions. - Dosage translation from in vitro to in vivo was based on prior literature rather than pharmacokinetic equivalence; direct exposure measurements were not reported.