Commiphora myrrha stimulates insulin secretion from ß-cells through activation of atypical protein kinase C and mitogen-activated protein kinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plant-based remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse ß-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. AIM OF THE STUDY: To investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from ß-cells were examined. METHODS: The effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKCζ and ERK1/2 were also assessed using the Wes™ capillary-based protein electrophoresis. RESULTS: Ca2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5-2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31-8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLCß (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKCζ inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, Wes™ capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKCζ and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. CONCLUSIONS: Our data indicate that CM directly stimulates insulin secretion through activating known downstream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKCζ, which in turn phosphorylates and activates ERK1/2.

Commiphora myrrha stimulates insulin secretion from mouse and human islets of Langerhans.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally plant-based remedies such as Commiphora myrrha (CM) have been used as an ayurvedic medicine to treat diabetes mellitus in some region of Arabia and Africa. Previous reports have shown that CM reduced blood glucose levels and increased insulin concentrations in animal models of diabetes in vivo. However, the exact mechanisms by which CM improved glycemic control in these animals are not fully understood. We hypothesized that CM may have a direct insulinotropic activity on ß-cells to increase insulin secretion. AIM OF THE STUDY: The direct effects of CM were investigated using MIN6 ß-cells and isolated mouse and human islets in static and perifusion insulin secretion experiments. Isolated mouse and human islets were used to investigate the rate and pattern of CM-induced insulin secretion. MATERIALS AND METHODS: The effect of CM on insulin secretion was assessed by static and perifusion experiments using MIN6 cells, a mouse-derived ß-cell line, and primary mouse and human islets. The effects of CM on cell viability and membrane integrity of MIN6 cells and mouse islets were assessed using an ATP viability assay and a trypan blue exclusion test. The mRNA expression profiles of preproinsulin and Pdx1, a major ß-cell transcription factor, were determined by quantitative RT-PCR following chronic exposure to CM. RESULTS: Exposing MIN6 cells to a CM resin solution (0.5-10 mg/ml) caused a concentration-dependent increase in insulin secretion in a static setting. Similarly, incubating mouse islets to CM (0.1-10 mg/ml) resulted in stimulation of insulin secretion in a concentration-dependent manner. CM concentrations at ≤ 2 mg/ml were not associated with reduction in cell viability nor with reduction in cell membrane integrity. However, higher concentrations of CM were accompanied with marked uptake of trypan blue dye and cell death. In a perifusion setting, CM (2 mg/ml) caused rapid and reversible increases in insulin secretion from both mouse and human islets at both sub-stimulatory and stimulatory glucose levels. The stimulatory effect of CM on insulin secretion did not change the total insulin content of ß-cells nor the mRNA expression of preproinsulin and Pdx1. CONCLUSIONS: These data indicate that aqueous CM resin solution has a direct stimulatory effect on ß-cells without compromising plasma membrane integrity. CM stimulates insulin secretion from MIN6 cells, a mouse-derived ß-cell line, and isolated primary mouse and human islets in vitro at both sub-stimulatory and stimulatory glucose concentrations. The mechanism by which CM may induce insulin secretion is most likely due to a stimulation of insulin granules release rather than insulin synthesis.

Costus pictus extracts stimulate insulin secretion from mouse and human islets of Langerhans in vitro.

Plant-derived extracts have been used as folk remedies for Type 2 diabetes mellitus (T2DM) for many centuries, and offer the potential of cheap and readily available alternatives to conventional pharmaceuticals in developing countries. Extracts of Costus pictus (CP), a plant belonging to the Costaceae family, are reported to have antidiabetic activity in vivo. The exact molecular mode of action(s) of CP is unclear but the antihyperglycemic effect seen in animal studies was associated with dramatic increases in insulin secretion so in our study we have measured the effect of aqueous CP extract on insulin secretion in vitro from the MIN6 ß-cell line and isolated mouse and human islets. Our data demonstrate that CP has a direct stimulatory effect on insulin secretion at basal but not stimulatory glucose concentrations which was not associated with compromised membrane integrity or decrease ß-cell viability. Single cell calcium microfluorimetry measurements showed that CP caused elevations in ß-cell intracellular Ca(2+) concentrations ([Ca(2+)](i)), an effect which was completely abolished by the removal of extracellular Ca(2+) or blockade of voltage-gated Ca(2+) channels (VGCC). These in vitro observations suggest that one mode of action of CP is through stimulating insulin secretion which may be mediated, in part, by the ability of CP to increase [Ca(2+)](i) levels through VGCC. CP extracts may provide an affordable and inexpensive alternative for treating patients with T2DM.