Is it possible to isolate insulin from homogenate

Here, we developed a simple nonselective inductive culture system rather than spontaneous differentiation of EC cells. It has been previously shown that the expansion of nestin-positive cells is not required for the activation of pancreatic differentiation [37]. Overall, our observations demonstrated that the differentiation of EC cells generated islet-like clusters, without selection of nestin-positive cells.

A similar finding was documented by Blyszczuk et al. In accord with the earlier works, we demonstrated that MPE could induce pancreatic markers, proinsulin, insulin and insulin receptor beta. These factors produced by pancreatic islet cells during development and tissue regeneration.

Both intracellular and secreted insulin enhanced when the glucose concentration in the medium was increased.

It should be emphasized that immunoreactivity for both proinsulin and insulin in the current study indicated the presence of endogenously produced insulin. The observation is in line with previous findings of Fujikawa et al.

Moreover, intracellular insulin level after exposure to 25 mM glucose was lower in comparison to secreted insulin Figure 4 B. Therefore, it can be concluded that the hormone produced by the differentiated IPCs cells rather than taken up from the culture medium.

PDX-1 is a crucial regulator of pancreatic development [44] and its expression is very important in the in vitro differentiation of ES cells along pancreatic cell lineages [45]. These findings are in accordance with previous studies from our laboratory on IPCs differentiation of P19 cells by pancreas conditioned medium submitted. These transcription factors are markers for pluripotency and self-renewal in embryonic stem cells [46].

Functionally, Nanog blocks differentiation; thus, negative regulation of Nanog is required to promote differentiation during embryonic development [49]. Compared with P19 cells, the relatively reduced stem cell marker expression in IPCs may indicate their higher differentiation properties.

Thus, it is possible that when EC cells were cultured as EBs, MPE could increase the level of specific pancreatic genes, while decreasing the expression of stem cell markers.

In this study, we used mutual rank MR instead of Pearson correlation coefficient since difference in gene expression correlation commonly follows a logarithmic manner rather than arithmetic average; consequently, geometric average MR index is more accurate as discussed by Obayashi and Kinoshita [26].

The fact that EP has high correlation with master transcription factors such as Sp2 transcription factor Table S2 which has binding sites on the promoter regions of many genes reinforces this statement. To best of our knowledge, this is the first report demonstrating the differentiation of P19 EC cells into IPCs in a simple and accessible way. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool to study pancreatic development and function.

This method may provide a new way to substitute the cytokines required in IPCs induction of stem cells. All together, bioinformatics approaches employed in this study helps in discovery of new elements of insulin-producing cell mechanism and gene discovery based on network construction. Highly co-expressed genes with PDX1 transcription factor in different microarray experiments. Highly co-expressed genes with EP transcription factor in different microarray experiments.

Highly co-expressed genes with CREB transcription factor in different microarray experiments. Relationships and references constructing WAC regulatory network Figure 8.

The authors would like to express deep thanks to Dr. The work carried out in the institute was supported by grant from Shahrekord University. We would like to thank Dr. Conceived and designed the experiments: FE. Wrote the paper: FE. Edited the manuscript: EE. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Introduction Diabetes mellitus is one of the most common chronic diseases which directly affects millions of people [1].

Download: PPT. Statistical analysis All experiments were repeated at least three times, and statistical significances were measured using Student's t-test, when there were two groups to be compared and one-way analysis of variance ANOVA and Duncan's Test, when there were more than two groups. Results Characterization of P19 EC cells on the basis of cell morphology and expression of pluripotent markers The experiments reported here were carried out with the P19 line of murine EC stem cells.

Figure 1. Representative micrographs showing morphology of exponentially growing P19 cells at low A and high B magnification. Figure 2.

Table 2. Figure 3. Figure 4. Quantitative analysis of gene expressions Real-time qPCR analysis was used to examine the expression of pancreatic specific genes in differentiated cells. Figure 5. Quantitative analysis of genes involved in differentiation of pancreatic cells derived from P19 EC cells. Figure 6. Co-expression based network of transcription factors involved in generation of insulin producing cells. Figure 7. Discussion In the present study, we developed for the first time, a simple, accessible nonselective way to generate functional IPCs from P19 EC cell line within a short period of time 7—15 days.

Conclusion To best of our knowledge, this is the first report demonstrating the differentiation of P19 EC cells into IPCs in a simple and accessible way. Supporting Information.

Figure S1. Table S1. Table S2. Table S3. Table S4. Table S5. Table S6. Table S7. Table S8. Acknowledgments The authors would like to express deep thanks to Dr.

Author Contributions Conceived and designed the experiments: FE. References 1. Pandey S stem cell transplantation: A future for diabetic paitient. View Article Google Scholar 2. Proceedings of the National Academy of Sciences — View Article Google Scholar 3.

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A hypoglycemic polypeptide was extracted from M. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE and its amino acid composition, it was concluded that it was a plant insulin The objective of the present study was to characterize an insulin-like protein in B.

Leaves of B. After thoroughly washing, leaf blades were freeze-dried and then reduced to a fine powder for further analysis. The antibodies used were a guinea pig polyclonal anti-human insulin antibody acquired from Peninsula Laboratories, Inc. Leaves were thoroughly washed, cut into pieces 0. For immunocytochemical localization, sections of approximately 60 nm were cut from the resin blocks and collected on Formvar films. The sections were then incubated with a guinea pig anti-human insulin antibody in the above buffer for 2 h.

After washing, sections were incubated with a guinea pig anti-IgG antibody conjugated with colloidal gold 10 nm at dilution for 2 h and washed again. The sections were examined and photographed with an Omega Zeiss transmission electron microscope operating at 80 KV. Controls were run using only the guinea pig anti-IgG antibody conjugated with colloidal gold.

The crystalline inclusions in leaves of B. After fixation with 2. Chloroplasts were isolated by the procedure described by Marcos and Flores 14 , with some modifications. Five grams of B. The supernatant was then centrifuged at g for 10 min at the same temperature. The pellet containing chloroplasts was resuspended in 2 mL of grinding buffer and the quality of the preparation was analyzed by phase contrast light microscopy.

After electrophoresis ten gels the band, with a mass similar to that of bovine insulin, was sliced horizontally into 0. This procedure was repeated five times. Next, the gel material was collected into a 2-mL Eppendorf tube and 1 mL of water was added.

The mixture was vortexed for 30 s and left at room temperature for 5 min. The gel material was pelleted by centrifugation at 12, g for 1 min and the supernatant was collected. The column was previously equilibrated with 0. Fractions 2 mL were collected and absorbance at , and nm was measured.

Fractions were analyzed for the presence of insulin-like antigens by dot blot data not shown and quantified by ELISA see below using a bovine insulin antibody. Absorbance was recorded at nm and the fractions were analyzed for the presence of insulin with bovine insulin antibody by a modified ELISA.

Bovine and human insulins were subjected to the same procedure as controls. The solutions were then discarded and the wells were washed for 1 h with 0. The wells were washed for 1 h with 0. The plates were then washed three times with TPBS as described above. The amount of insulin present was determined from standard curves obtained from the bovine insulin response.

One hundred microliters of a saline 0. Five days after the alloxan inoculation, high glucose levels in the blood of the animals were verified by the orthotoluidine method Five groups of mice of 5 animals each were used in the experiment. All samples were dissolved in 0. After 1 h, blood was collected from the ophthalmic venous plexus and the serum collected after centrifugation. Microscopic analysis of leaf tissue sections showed that anti-insulin antibodies labeled the chloroplasts of the parenchyma cells Figure 1 , panels A,B,C.

We also observed labeled crystals present in the cell vacuole Figure 2 , panels A,B. Scanning electron microscopy of these crystals Figure 2 , panel D and analysis by energy-dispersive X-ray microanalysis Figure 3 , panels A and B showed that they consisted mostly of calcium, probably in the form of calcium oxalate Additional experiments were carried out using isolated chloroplasts.

Figure 4 shows the electrophoretic profile of proteins from the B. A protein band from the leaf extract with a molecular mass similar to that of bovine insulin was recognized by a human insulin antibody lane 2' and was accompanied by leaf pigments lane 3.

To investigate the nature of the pigment associated with this band, an absorption spectrum of the protein extracted from the gels was run and absorption peaks data not shown at protein , and nm characteristic of chlorophyll were observed.

A solution of the insulin-like protein extracted from the gels was filtered through a G Sephadex column Figure 5 , panel A. The eluted fractions 2 mL were monitored at , , and nm and tested by dot blot analysis Figure 5 , panel B for the presence of insulin-like molecules. G-2 fraction Figure 5 , which was free of pigments and peroxidase activity, was then submitted to reverse-phase chromatography through a C column. A positive reaction by ELISA using both human and bovine anti-insulin antibodies data not shown was observed for F7 fraction Figure 6 , panel A , that corresponds to a component of both human and bovine insulin, as seen in Figure 6 , panel B.

The effect of the insulin-like protein on serum glucose levels of diabetic mice was determined in experiments in which alloxan-treated mice were injected intravenously with the preparation. The leaf extract and the insulin-like protein isolated from leaves of B. The hypoglycemic activity of the insulin-like protein isolated from leaves was similar to that of commercial swine insulin used as control.

Figure 1. Electron micrographs of Bauhinia variegata leaf blades using transmission electron microscopy. Figure 2. Transmission electron micrographs of Bauhinia variegata leaf blades, showing a crystal arrows present in the parenchyma cell.

Figure 3. Figure 4. Lanes 1 and 1 ', 5. Figure 5. Fractions 2 mL were collected and assayed by dot blot B with an antibody to human insulin. Absorbance was measured at , , and nm. In B , samples are as follows: 1 G-1 fraction, 2 control for the G-1 fraction, 3 G-2 fraction; 4 control for the G-2 fraction. Figure 6. Panel A , Fraction G-2; panel B , bovine and human insulin.

F7 means the seventh fraction. Absorbance read at nm. Figure 7. Comparative effects of swine insulin 1 IU , the extract from the leaves of Bauhinia variegate 0. The leaves of plants of many Bauhinia species Leguminoseae are used in antidiabetic treatments by many populations of the world These plants, which are collectively known as cow's foot and "pata-de-vaca" in Portuguese because of the shape of their leaves, are very common in Brazil where investigations have been done in the search for the active principle The presence of insulin-like molecules was recently demonstrated in the leaves of B.

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