Momordica charantia and Its Novel Polypeptide Regulate Glucose Homeostasis in Mice via Binding to Insulin Receptor

Momordica charantia (MC) has been used as an alternative therapy for diabetes mellitus. This study analyzed and elucidated therapeutic targets contributing to the hypoglycemic effect of aqueous extract of MC seeds (MCSE) by transcriptomic analysis. Protein ingredients aimed at the hypoglycemic target were further identified by proteomic, docking, and receptor-binding assays. The data showed that MSCE (1 g/kg) significantly lowered the blood glucose level in normal and diabetic mice. Moreover, MCSE primarily regulated the insulin signaling pathway in muscles and adipose tissues, suggesting that MCSE might target insulin receptor (IR), stimulate the IR-downstream pathway, and subsequently display hypoglycemic activity in mice. It was further revealed that inhibitor against trypsin (TI) of MC directly docked into IR and activated the kinase activity of IR in a dose-dependent manner. In conclusion, the findings suggested that MCSE regulated glucose metabolism mainly via the insulin signaling pathway. Moreover, TI was newly identified as a novel IR-binding protein of MC that triggered the insulin signaling pathway via binding to IR

Desipramine increases HO-1 expression through Nrf2 activation in Mes23.5 dopaminergic neurons.

<p>(A) Cells were treated with desipramine (20 µM) for indicated time periods (60 or 120 min) and nuclear extracts were collected, and the binding activity of Nrf2 to Nrf2-DNA binding element was examined by EMSA analysis. The DNA binding activity of Nrf2 is significantly different between desipramine treatment group and control group (one-way ANOVA followed by Bonferroni’s post hoc test). Cells were pretreated with PD98059 or SP600125 with desipramine (20 µM), and nuclear extracts were examined by EMSA analysis. Lane 1 was loaded without nuclear extracts (probe only). Results are expressed as the means ± S.E.M. from three independent experiments. *, <i>p</i><0.05 as compared with the vehicle control group. #, p<0.05 as compared with the desipramine treatment group. (B) Cells were transfected with Control siRNA (100 nM) or Nrf2 siRNA (50 and 100 nM) for 24 h followed by stimulation with desipramine (20 µM) for another 24 h, and the protein levels of Nrf2 and HO-1 were determined by Western blot. The HO-1 expression is significantly different between Nrf2 siRNA group and control siRNA group (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, <i>p</i><0.05 as compared with the control group. #, p<0.05 as compared with the desipramine treatment alone group.</p

Role of HO-1 in neuroprotective effect in Mes23.5 dopaminergic neurons.

<p>Cells were treated with Copp IX (1 µM) (A and C) or hemin (B and D) for 8 h followed by treatment with 6-OHDA (50 µM) for another 16 h. Cell viability was determined by MTT assay and SRB assay. Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with vehicle group. #, <i>p</i><0.05 as compared with desipramine-treated group.</p

Desipramine and fluoxetine increase HO-1 expression in Mes23.5 dopaminergic neurons.

<p>Cells were incubated with various concentrations (5, 10 or 20 µM) of desipramine (A) or fluoxetine (C) for 24 h or with desipramine (20 µM; B) or fluoxetine (20 µM; D) for the indicated time periods (4, 8 and 24 h). Whole cell lysates were extracted and subjected to Western blot for detection of HO-1 expression. The HO-1 expression is significantly different between control group and desipramine or fluoxetine treatment groups (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, <i>p</i><0.05 as compared with the vehicle control group. Cells were incubated with various concentrations (5, 10 or 20 µM) of desipramine (E) or fluoxetine (G) for 8 h or with desipramine (20 µM; F) or fluoxetine (20 µM; H) for indicated time periods (4, 8 and 24 h). The quantitative data are shown in below. HO-1 mRNA expression was determined by RT-PCR. Results are the representatives of three independent experiments.</p

ERK and JNK signaling pathways are involved in desipramine- and fluoxetine-increased HO-1 expression in Mes23.5 dopaminergic neurons.

<p>Cells were pretreated with various of MAP kinase inhibitors SB203580 (10 µM), PD98059 (20 µM) or SP600125 (10 µM) for 30 min followed by stimulation with desipramine (20 µM; A) or fluoxetine (20 µM; D) for another 24 h. Whole cell lysates were subjected to Western blot for detection of HO-1 expression. Cells were incubated with desipramine or fluoxetine for the indicated time periods, and cell lysates were subjected to immunoblots with antibodies against phospho-JNK (B and C) and phospho-ERK (D and E). Results are the representatives of three or four independent experiments.</p

Desipramine induces Nrf2 translocation from cytoplasm to nucleus in Mes23.5 dopaminergic neurons.

<p>Cells were incubated with desipramine (20 µM) for indicated time periods (4 or 8 h), and Nrf2 expression levels in whole cell lysates (A) and nuclear extracts (B) were determined by immunoblotting with Nrf2-specific antibody. PCNA was used as the loading control for nuclear fraction. The quantitative data are shown in below. The Nrf2 expression is significantly different between desipramine treatment group and control group in nuclear extract (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with the vehicle control group. Cells were treated with or without desipramine (20 µM) for 2 h, and the levels of Nrf2 were determined by immunoflourescence (C). Note that the Nrf2 translocates from cytoplasm to nucleus in response to desipramine stimulation. Results are the representatives of three independent experiments.</p

Neuroprotection of desipramine on rotenone- and 6-OHDA-induced neurotoxicity.

<p>Mes23.5 dopaminergic neurons were pretreated with various concentrations of desipramine for 8 h and followed by treatment with rotenone (3 µM) for another 16 h. The cell viability was determined by MTT assay and SRB assay (A and C, respectively). The neuroprotective effects are significantly different between rotenone alone group and rotenone treated with desipramine group in both MTT and SRB assays (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with the vehicle group. #, <i>p</i><0.05 as compared with the desipramine-treated group. Cells were pretreated with various concentrations of desipramine for 8 h and followed by treatment with 6-OHDA (50 µM) for another 16 h. The cell viability was determined by MTT assay and SRB assay (B and D, respectively). The neuroprotective effect is significantly different between 6-OHDA alone group and 6-OHDA treated with desipramine group in SRB assay (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with the vehicle group. #, <i>p</i><0.05 as compared with the desipramine-treated group. Whole cell lysates were subjected to Western blot for detection of tyrosine hydroxylase (TH). Results are the representatives of at least three independent experiments.</p

Involvement of HO-1 in desipramine-mediated neuroprotective effect in Mes23.5 dopaminergic neurons.

<p>Cells were treated with ZnPP IX (0.1 or 0.3 µM) for 30 min and follow by treatment with desipramine for 8 h, and then treated with rotenone (A and C) or 6-OHDA (B and D) for another 16 h. Results are expressed as the means ± S.E.M. from four independent experiments. *, <i>p</i><0.05 as compared with vehicle group.</p

Histopathological examination of liver.

<p>For all the experimental groups, liver sections were prepared from the liver retrieved at 72 hpi, stained with H/E, and imaged under microscopic observation with magnification of 400×. Representative liver sections of PBS-control naïve mice (A), PBS-control diabetic mice (B), and the <i>K. pneumoniae</i>-infected naïve mice (C) are shown. Infiltrates of neutrophils and lymphocytes are indicated with arrows. Several characteristics revealed on the liver section from the <i>K. pneumoniae</i>-infected diabetic mice, including liquefactive necrosis with degeneration of liver parenchyma and inflammatory cells (D), accumulation of <i>K. pneumoniae</i> (E and F), ballooning degeneration of hepatocytes (G), and the formation of Councilman body (H) are shown with an indication of arrows. A large hepatic abscess was noted in the right liver lobe of a <i>K. pneumoniae</i>-infected diabetic mouse (I). Scale bar represents a distance of 20 μm. (J) Hepatic injury graded by the Knodell necroinflammatory scoring system. Livers were retrieved from <i>K. pneumoniae</i>-infected naïve mice (NI; n=4) and diabetic mice (DI; n=8). Statistical analysis by the Mann-Whitney U test (one-tailed) showed no significant difference between NI and DI groups.</p

Production of cytokines and chemokines in hepatic responses to <i>K. pneumoniae</i> infection.

<p>Liver lysates were prepared from PBS-control naïve mice (n=3), PBS-control diabetic mice (n=3), and the <i>K. pneumoniae</i>-inoculated naive and diabetic mice which had developed an extraintestinal infection at 72 hpi (the sample size in naïve and diabetic groups is 4 and 8, respectively). Protein levels of IL-2 (A), IL-6 (B), IL-10 (C), IL-17A (D), and IL-17F (E), IFN-γ (F), MIP-1α (G), MIP1β (H), MIP-2 (I), and IL-1β (J) were determined by ELISA and normalized with total protein amounts. Data are expressed as the mean ± SEM. An asterisk (*) represents a significant increase in the <i>K</i>. <i>pneumoniae</i>-infected naïve or diabetic group (slash bar) in comparison with the corresponding control group (empty bar) by the Mann-Whitney U test (one-tailed; <i>P</i> < 0.05).</p