Antioxidant rich flavonoids from Oreocnide integrifolia enhance glucose uptake and insulin secretion and protects pancreatic β-cells from streptozotocin insult

Abstract

Background: Insulin deficiency is the prime basis of all diabetic manifestations and agents that can bring about insulin secretion would be of pivotal significance for cure of diabetes. To test this hypothesis, we carried out bioactivity guided fractionation of Oreocnide integrifolia (Urticaceae); a folklore plant consumed for ameliorating diabetic symptoms using experimental models.

Methods: We carried out bioassay guided fractionation using RINmF and C2C12 cell line for glucose stimulated insulin secretion (GSIS) and glucose uptake potential of fractions. Further, the bioactive fraction was challenged for its GSIS in cultured mouse islets with basal (4.5 mM) and stimulated (16.7 mM) levels of glucose concentrations. The Flavonoid rich fraction (FRF) was exposed to 2 mM streptozotocin stress and the anti-ROS/RNS potential was evaluated. Additionally, the bioactive fraction was assessed for its antidiabetic and anti-apoptotic property in-vivo using multidose streptozotocin induced diabetes in BALB/c mice.

Results: The results suggested FRF to be the most active fraction as assessed by GSIS in RINm5F cells and its ability for glucose uptake in C2C12 cells. FRF displayed significant potential in terms of increasing intracellular calcium and cAMP levels even in presence of a phosphodiesterase inhibitor, IBMX in cultured pancreatic islets. FRF depicted a dose-dependent reversal of all the cytotoxic manifestations except peroxynitrite and NO formation when subjected in-vitro along with STZ. Further scrutinization of FRF for its in-vivo antidiabetic property demonstrated improved glycemic indices and decreased pancreatic β-cell apoptosis.

Conclusions: Overall, the flavonoid mixture has shown to have significant insulin secretogogue, insulinomimetic and cytoprotective effects and can be evaluated for clinical trials as a therapeutant in the management of diabetic manifestations.

Figure 1

Effect of different fractions of Oreocnide integrifolia on glucose stimulated insulin secretion. RINm5F cells were either cultured in basal (4.5 mM) or stimulated (16.7 mM) glucose concentrations in presence of fractions. Insulin secretion was quantified using Rat Insulin ELISA kit. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001: when control was compared with rest of the groups; α = p < 0.01, β = p < 0.01, δ = p < 0.01: when 4.5 mM glucose group was compared to rest of the groups and ➊ = p < 0.01, ➋ = p < 0.01, ➌ = p < 0.01: when 16.7 mM glucose group was compared to rest of the groups.

Figure 2

Effect of different fractions of Oreocnide integrifolia on glucose uptake in C2C12 cells. Differentiated C2C12 cells were incubated with or without 100 nM of insulin along with various fractions. Glucose uptake was measured by using 2NBDG fluorescent probe. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when insulin treated group was compared to rest of the groups.

Figure 3

(A). Represents effect of flavonoidal fraction on glucose stimulated insulin secretion in RINm5F cells. RINm5F cells were either cultured in basal (4.5 mM) or stimulated (16.7 mM) glucose concentrations in presence of fractions. Insulin secretion was quantified using Rat Insulin ELISA kit. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when 4.5 mM glucose group was compared to FRF 10, 50, 100, 250 and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when 16.7 mM glucose group was compared to FRF 10, 50, 100, 250 group.(B) Represents effect of flavonoid fraction on glucose uptake in C2C12 cells. Differentiated C2C12 cells were incubated with or without 100 nM of insulin along with flavonoidal fraction. Glucose uptake was measured by using 2NBDG fluorescent probe. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01: Insulin was compared to rest of the groups. (C) Represents effect of flavonoidal fraction on insulin secretion on isolated mouse islets. Islets were either cultured in basal (4.5 mM) or stimulated (16.7 mM) glucose concentrations in presence of flavonoidal fraction. Insulin secretion was quantified using Mouse Insulin ELISA kit. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when 4.5 mM glucose group was compared to FRF 10, 50, 100, 250 groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when 16.7 mM glucose group was compared to FRF 10, 50, 100, 250. (D) Represents effect of flavonoidal fraction on cAMP levels in mouse islets. Cultured islets were exposed to 11.1 mM for 60 min in the presence of phosphodiesterase inhibitor (1 mM IBMX) along with flavonoidal fraction. Values are expressed as ± SEM in triplicates where α = p < 0.01, β = p < 0.01, δ = p < 0.01 when 16.7 mM glucose group was compared to its 10, 50, 100, 250 groups and ➊ = p < 0.01, ➋ = p < 0.01, ➌ = p < 0.01 when 11.1 mM glucose + IBMX group was compared to its 10, 50, 100, 250 groups.

Figure 4

(A) Represents effect of flavonoidal fraction on intracellular calcium levels in mouse islets. Intracellular calcium levels were measured using fluorescent probe fura-2AM. Values are expressed as ± SEM in triplicates where α = p < 0.01, β = p < 0.01, δ = p < 0.01 when 16.7 mM glucose group was compared to its 10, 50, 100, 250 groups and ➊ = p < 0.01, ➋ = p < 0.01, ➌ = p < 0.01 when 11.1 mM glucose + IBMX group was compared to its 10, 50, 100, 250 groups. (B) Represents effect of flavonoidal fraction on MTT activity in mouse islets. Cultured islets were exposed to pancreatic β- cell specific toxin, streptozotocin (2 mM) along with flavonoidal fraction at various concentrations and assessed for cytotoxicity. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 where diabetic group was compared to rest of the groups. (C) Represents effect of flavonoidal fraction on intracellular ROS activity in mouse islets. Intracellular ROS was assessed by fluorescent probe DCFH-DA. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when diabetic group was compared to rest of the groups.

Figure 5

(A) Represents effect of flavonoidal fraction on peroxynitrite levels in mouse islets exposed to streptozotocin. Peroxynitrite levels were quantified by fluorescent probe dihydrorhodamine 123. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when diabetic group was compared to rest of the groups (B) Represents effect of flavonoidal fraction on NO levels in mouse islets exposed to streptozotocin. Nitric oxide levels were quantified by fluorescent probe DAF-FM. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when diabetic group was compared to rest of the groups (C) Represents effect of flavonoidal fraction on mitochondrial membrane potential (MMP) in mouse islets exposed to streptozotocin. MMP levels were quantified by fluorescent probe Rhodamine 123. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups and α = p < 0.01, β = p < 0.01, δ = p < 0.01 when diabetic group was compared to rest of the groups (D) Represents effect of flavonoidal fraction on lipid peroxidation levels (LPO) in mouse islets exposed to streptozotocin. Lipid peroxidation was measured by using thiobarbituric acid reagent. Values are expressed as ± SEM in triplicates where • = p < 0.01, ■ = p < 0.001, ♦ = p < 0.0001 when control group was compared with rest of the groups.

Figure 6

Images represent haemetoxylin and eosin stained pancreas sections of (A) control mice showing intact islet histoarchitecture, (B) diabetic mice showing islet cell destruction and wider intracellular spaces due to streptozotocin toxicity (C-E) diabetic animals treated with flavonoidal fraction at 100, 250 and 500 mg/kg bodyweight demonstrating ameliorating effects and improved islet integrity (Magnification 200×) (F) Quantification of islet cell apoptosis in multiple dose streptozotocin mouse treated with various concentrations of flavonoidal fraction (G) Images represent confocal optical slices of immunostained pancreas of control, diabetic and diabetic treated with various concentrations of flavonoidal fraction. Guinea Pig anti insulin (red) and ApoBrdu-dUTP (green) were used as primary antibodies. DAPI (Blue) was used to visualize nuclei. The slides were visualized by Laser Scanning Confocal Microscope (LSM 510 META, ZEISS, Germany). Scale bar represents 100 μm).