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 Table of Contents  
Year : 2023  |  Volume : 40  |  Issue : 1  |  Page : 27-36

In Vitro investigation of insulin-like growth factor-i and mechano-growth factor on proliferation of neural stem cells in high glucose environment

1 Department of Genetics, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey
2 Department of Medical Genetics, Istanbul School of Medicine, Istanbul University, Istanbul, Turkey
3 Department of Neurosurgery, Haydarpaşa Numune Training and Research Hospital, Istanbul, Turkey

Date of Submission25-Jul-2022
Date of Decision26-Sep-2022
Date of Acceptance05-Oct-2022
Date of Web Publication29-Mar-2023

Correspondence Address:
Selcuk Sozer
MD, PhD, Department of Genetics, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/nsn.nsn_137_22

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Objective: High glucose levels cause metabolic and vascular complications by affecting the nervous system with an increased risk of stroke and seizures. There are still no effective treatment modalities for the high morbidity and mortality rates detected in such situations. Neural stem cells (NSCs) provide neurogenesis in the nervous system, but high glucose is detrimental to NSCs. This study investigates the intrinsic and extrinsic effects of the growth factors insulin-like growth factor-I (IGF-I) and mechano-growth factor (MGF) on NSCs when exposed to high glucose levels. Thus, the possibility of new treatment options for diabetes patients is explored. Materials and Methods: Rat NSCs grown in cell culture conditions were exposed to a control glucose concentration of 17.5 mM and high concentrations of 27.75, 41.75, and 83.75 mM for 24 h. The high glucose concentrations were designed to recapitulate the in vivo conditions of diabetes mellitus, diabetic ketoacidosis, and hyperglycemia hyperosmolar status. Then, 0.2 μg/ml IGF-I and MGF growth factors were separately added and their expressions in the NCSs investigated by real-time reverse transcription-polymerase chain reaction. The effects of exogenous IGF-I and MGF administration on NSC proliferation under high glucose conditions were measured by BrdU incorporation assay using flow cytometry analysis. Results: A significant increase was detected in the relative gene expression fold changes of IGF-I and MGF in the NSCs. The MGF relative fold change was greater than the IGF-I for each high glucose condition. NSCs exposed to 27.75 mM glucose revealed a 17-fold and 40-fold increase in the IGF-I and MGF gene expressions, respectively; the 41.75 mM glucose similarly revealed 68-and 161-fold increases and the 83.75 mM glucose 75-and 137-fold increases. Exogenous IGF-I administration increased its expression profile, while the administration of MGF lowered its expression. The NSC was in the growth (G0/G1) phase of the cell cycle during the 24 h culture time. The percentage of proliferated NSC decreased to 89% (17.5 mM), 85% (27.75 mM), 50.30% (41.75 mM), and 28.97% (83.75 mM). Surprisingly, the increase in both IGF-I and MGF saved the NSCs from cell death. Conclusion: Exogenous IGF-I and MGF administrations via high glucose environments increased NSC proliferation at the time of injury and protected the NSCs from cell death. The neuroprotective effect of MGF was greater than that of IGF-I. Thus, due to their neurogenesis potential, exogenous IGF-I and MGF could be applied in the treatment of diabetes patients to relieve neural damage.

Keywords: Growth factors, high glucose, insulin-like growth factor-I, mechano-growth factor, neural stem cells

How to cite this article:
Gurbuz TA, Güleç &, Toprak F, Toprak SF, Sozer S. In Vitro investigation of insulin-like growth factor-i and mechano-growth factor on proliferation of neural stem cells in high glucose environment. Neurol Sci Neurophysiol 2023;40:27-36

How to cite this URL:
Gurbuz TA, Güleç &, Toprak F, Toprak SF, Sozer S. In Vitro investigation of insulin-like growth factor-i and mechano-growth factor on proliferation of neural stem cells in high glucose environment. Neurol Sci Neurophysiol [serial online] 2023 [cited 2023 Jun 10];40:27-36. Available from: http://www.nsnjournal.org/text.asp?2023/40/1/27/372781

  Introduction Top

Glucose is an essential metabolic element that provides fuel to the brain and thus enables its physiological functioning. Glucose plays a critical role in both neural and nonneural cellular maintenance and in the generation of neurotransmitters in the central nervous system (CNS).[1] The brain is the body's most complex organ. Although it accounts for only ~2% of body weight in humans, it consumes ~20% of the body's glucose-derived energy.[2] Uncontrolled glucose levels cause alterations in cerebral energy homeostasis and metabolism by changing osmotic gradients,[3] hormonal regulations,[4] glucose utilization,[5] oxidative stress,[6] and the levels of ketone bodies.[7] High levels of glucose (hyperglycemia) result in metabolic and vascular disturbances affecting the CNS with increased risk of stroke, seizures, diabetic encephalopathy, and cognitive compromise.[8]

Glucose is also an essential element for neural stem cells (NSCs), which regenerate the CNS through their ability to differentiate into astrocytes, neurons, and oligodendrocytes.[9] The specialized microenvironment of NSCs, the NSC niche, plays an essential role in neurogenesis by simultaneously regenerating and repairing damaged brain tissue.[10]

It is well known that stem cells migrate to damaged areas.[11] The proliferation of cells specific to certain developmental stages, such as embryonic and postnatal NCS, has been proven to be dependent on glucose concentration in certain physiological and pathological conditions.[12],[13] For example, hyperglycemic conditions affect the proliferation and apoptosis of neural progenitor cells in the developing spinal tube[14] and significantly impair the proliferative potential of NCSs[15] while suppressing their differentiation capacity.[16] Contrarily, low glucose levels have also been shown to suppress the in vitro proliferation of NSCs while increasing their differentiation.[17]

The consequence of glucose level inconsistencies might affect many other cell types. Pertinently, it has been shown that diabetes impairs the function of hematopoietic,[18] skeletal muscle,[19] and osteoblast stem cells,[20] while high glucose has been shown to inhibit the in vitro proliferation, migration, and angiogenic ability of bone marrow-derived endothelial precursor cells[21] and alter the regenerative potential of mesenchymal cells.[22] Similarly, hyperglycemia is an independent risk factor in poor outcomes following stroke and causes the formation of free oxygen radicals, leading to increased cell death in the ischemic brain.[23],[24]

During CNS development, an essential role in neurogenesis is played by growth factors and their receptors, especially insulin-like growth factor-1 (IGF-1), mechano-growth factor (MGF), epidermal growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor.[25] IGF-1 is a neurotrophic factor responsible for repairing and developing neurons by promoting cell proliferation, differentiation, and survival, as demonstrated by numerous in vivo and in vitro studies.[26],[27],[28]

MGF is one of the alternative splice variants of IGF-1 and typically not expressed in healthy tissue. However, MGF has impact in ischemic, traumatic conditions, and degenerative pathologies including brain and bone tissues, and heart and skeletal muscles among others. It prompts stem cell activation as shown by Goldspink in quiescent muscle progenitor cells that induce satellite cell proliferation. MGF expression has been also detected in damaged tissue for various reasons.[29],[30] Unlike IGF-1, MGF is defined as a general tissue repair factor,[29] and studied by many researchers in the brain and heart particularly in the damage-resistant region after ischemia conditions.[31] MGF plays role on neural progenitor cell proliferation and migration, as well. The activation process is initiated and sustained by IGF-1 and processed to differentiation and maturation stages.[32],[33] Our recent in vitro study revealed that the combined application of IGF-I and MGF to rat NSCs has the highest proliferation rates that increase with culture time (to a maximum of 7 days tested).[28]

Recent studies have revealed that endogenous and transplanted NSCs can be activated by the external administration of growth factors and involve in neural regeneration.[10],[11],[34] However, the reported effects of hyperglycemia on NSCs and the role of IGF-I and MGF on NSC neurogenesis in both human and animal models recapitulating diabetes have been somewhat inconsistent. This study applied an in vitro model by exposing adult rat hippocampal NSCs to glucose concentrations ranging from average glucose through diabetes mellitus and diabetic ketoacidosis to hyperglycemia hyperosmolar status. In addition, IGF-1 and MGF were separately administered to the NSCs, and their effect on NSC proliferation and cell death was analyzed. Meanwhile, the changes in IGF-I and MGF gene expressions within these conditions were investigated. The results of this investigation could be useful since understanding the cellular biology of NSCs and the effect of growth factors in the high glucose environment may provide new opportunities for controlling the expansion of NSCs that can lead to the development of new therapy options for diabetic patients.

  Materials and Methods Top

Neural stem cell culture

An adult rat hippocampal NSC line (Sigma-Aldrich, Merck, Germany) was cultured with media containing 10 ml (×50) B-27 supplement (Invitrogen), 500 ml DMEM/F12 (Biochrom), 5 ml streptomycin (1%), 5 ml (200 mM) L-glutamine, and 20 ng/ml FGF-2 (PeproTech), henceforth, the “NSC medium.” Then, 150 mm culture plates were coated with 10 mg/ml poly-L-ornithine (PLO) (Sigma-Aldrich) and 6 μg/ml laminin (Sigma-Aldrich), as explained elsewhere.[28] The cells were incubated at 37°C in 5% CO2. When the NSCs reached a 70%–80% confluence in the plates, they were transferred to 24-well culture plates coated with PLO and laminin at a density of 6 × 105 cell/ml with 300 μl/well in the NSC medium.

Application of glucose and growth factors to neural stem cell

In order to investigate the effects of high glucose and growth factors on the survival and proliferation of NSCs, different concentrations of glucose were applied to the NSC medium in the 24-well culture plates. The NSC was incubated in vitro for 24 h under four different conditions of glucose concentration. Further to the 17.5 mM concentration already present in the NSC medium which was employed as a control representing the in vivo average glucose level concentrations of 27.75, 41.75, and 83.75 mM were attained by adding D-glucose (Sigma-Aldrich) to the NSC medium [Table 1]. These high glucose conditions were designed to represent diabetes mellitus, diabetic ketoacidosis, and hyperglycemia hyperosmolar status conditions, respectively.[35] To test the effect of growth factors, 0.2 μg/ml IGF-I (Sigma-Aldrich)[36] and 0.2 μg/ml MGF (Phoenix Peptide)[37] were added to culture wells. To compensate the osmotic change of cells caused by different glucose concentration, mannitol was also added accordingly to each culture condition [Table 1].
Table 1: Cell culture medium compositions for rat hippocampal-derived neural stem cells

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In total, six groups comprising (6 × 4) 24 samples were created. One group employed as the control group received just the four treatments of D-glucose. Further to this, one group received treatments of IGF-I and another of MGF and mannitol was also applied to the control and growth factor groups to eliminate the possible effect of osmolality on cell viability as an osmotic control. Thus, the three (control, IGF-I, and MGF) groups were supplemented by three mannitol groups. However, no statistically significant difference in osmotic pressure was measured by cell viability and cell death according to the sample analysis of the glucose and growth hormone (control, IGF-I and MGF) groups as compared to the respective mannitol groups. Therefore, the data for the three mannitol groups are not included in the figures provided.

Cell proliferation assay

An incorporated 5-bromo-2´-deoxyuridine (BrdU) assay was performed for proliferation, cell cycle, and cell death detection of NSCs with/without high glucose and IGF or MGF (and with/without mannitol). The BrdU Flow Kit (BD Pharmingen, US) was applied according to the manufacturer's instruction. Briefly, the NSCs were incubated in BrdU solution in the dark at 4°C for 2 h. After incubation, the cells were detached with 200 μl Accutase/well (Sigma-Aldrich) and incubated for 3–5 min at 37°C. Cell detachment was checked under a microscope. In order to inactivate the Accutase, 300 μl of the NSC medium was added to each well, and the cell suspension was transferred to the flow tubes. The BrdU staining protocol as per the manufacturer was applied to the cells, which were then analyzed with flow cytometry (BD FACSCalibur, US). The two separate sets of experiments were performed.

The incorporated BrdU was stained with a specific anti-BrdU fluorescent antibody. The staining was also coupled with 7-amino-actinomycin D (7-AAD), which binds to the total DNA. The DNA histogram of cells and each phase of the cell cycle gating strategies are explained in [Figure 3]a and elsewhere.[28] In brief, the cell cycle phases are shown thus: “G0/G1” represents the growth phase, “S” is for DNA synthesis with 2N, and “G2/M” is the DNA repairing and cell preparation phase for the coming mitosis. The cell cycle phases were identified in the DNA histograms and respective contour plots. The analysis revealed the percentage of proliferating cells by BrdU positive cells and the percentage of cells in each cell-cycle phase by 7-AAD fluorescence distribution. Apo gate in [Figure 3]a represent dead cells.
Figure 3: The effect of growth factors on NSC proliferation and cell death. (a) Representative gating strategy and analysis of the NSCs for the cell proliferation and cell cycle with flow cytometry. Each gate labelling shown as abbreviation for BrdU positive cells (BrdU+) and BrdU negative cells (BrdU-), synthase phase (S), growth phase (G0/G1), repairing and cell preparation phase (G2/M) and dead cells (Apo). (b) Proliferation analysis of the NSCs with growth factors with BrdU incorporation analyzed with flow cytometry. The BrdU positive cells (BrdU+) of NSCs after high glucose culture with growth were analyzed as the percentage of total cells. (c) 7-AAD analysis of the dead cells (Apo gate) *P < 0.05. NSC: Neural stem cell

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RNA isolation, cDNA synthesis, and real-time reverse transcription-polymerase chain reaction

Total RNA isolation was performed using a total RNA purification kit (Jena Bioscience, Germany). A SCRIPT cDNA Synthesis Kit (Jena Bioscience, Germany) was used for cDNA synthesis from RNA. Initially, 50 ng RNA was applied for the PCR with the relevant primers of the IGF-I and MGF genes. The forward primer for IGF-1 (GF-1Ea) was 5'-GCT TGC TCA CCT TTA CCA GC-3', and the reverse primer was 5'-AAG TGT ACT TCC TTC TGA GTC T-3' with 130 base pairs (bp) in fragment length. The forward primer for MGF was 5'-GCT TGC TCA CCT TTA CCA GC-3', and the reverse primer was 5'-AAG TGT ACT TCC TTT CCT TCT C-3', with a 130-bp length. Changes in each gene expression were detected with the SensiFAST SYBR No-ROX Kit (Bioline, Meridian Biosciences) by the real-time quantitative RT-PCR (Light cycler 480, Roche Diagnostics). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was applied as a housekeeping control in each run. The forward primer for GAPDH was 5'GGT GTG AAC GGA TTT GGC CGT AT-3', and the reverse primer was 5'CTC AGC ACC AGC GTC ACC CCA TT 3' with a 129 bp fragment length.

The gene expressional fold changes were examined for the NSC culture in varying glucose concentrations. These specific primers were applied for amplification, and the fluorescence emitted by dye above the baseline signal was detected using the software in real-time, recorded, and represented as the cycle threshold (CT). The arithmetic mean values of CTs, which were performed twice, were calculated for the statistical analysis. All samples were studied in duplicate. RNA samples directly isolated from a rat's hippocampal region were also applied as positive controls in reactions.

Statistical analysis

The 2-ΔΔCT method was performed to determine the relative fold change of each gene expression with the results of real-time RT-PCR. First, the Ct values of target mRNAs were normalized with the housekeeping control, GAPDH. Then, the fold changes of the target gene expressions were calculated respective to the ΔCT of the untreated controls applying the 2-ΔΔCT formula 2-(CTExample-CTReference: GAPDH)-(CTControl-CTReference).

The SPPS 25 (Statistics 25; IBM, Armonk, NY, USA) statistical package was used to evaluate the data. The variables applied were mean ± standard deviation and percentage and frequency values. The variables were evaluated after controlling for variance normality and homogeneity (Shapiro–Wilk and Levene tests). The Tukey HSD test was used to compare the one-way analysis of variance of three or more groups; in data analysis of the multiple comparison tests, statistically significant levels were taken as P < 0.05 and P < 0.01.

  Results Top

Glucose is an essential factor in the proliferation and differentiation of NSCs during neural stress. In order to test the effect of glucose environment on NSC activity, varying concentrations of glucose representing diabetes mellitus, diabetic ketoacidosis and hyperglycemia hyperosmolar status were generated and applied in vitro, as summarized in [Table 1] and [Figure 1]. The expressions of the IGF-I and MGF genes of NSCs were evaluated in the different glucose environments, and the relative effect of glucose on the gene expressions was analyzed. Further comparative analyses were performed for the roles of IGF-I and MGF by the external administration of growth factors in these environments. The relative gene expressional fold changes and NSC functional changes were evaluated.
Figure 1: Illustration of experimental design. The rat hippocampal-derived NSCs were cultured in vitro on flasks coated with pol-L-ornithine and laminin. After they reached 70% confluency, the NSCs were divided into 24-well cell culture plates for four different D-glucose concentrations of NSC media. As a representation of the in vivo average glucose level, 17.5 mM glucose was referred to as the control. In addition, 27.75, mM, and 83.75 mM concentrations were employed to represent diabetes mellitus, diabetic ketoacidosis, and hyperglycemia hyperosmolar status conditions, respectively. Mannitol was applied to control for the osmotic pressure of the glucose on cells. The growth factors IGF-I and MGF were administered at 0.2 μg/ml to the cultured NSC. Combining these (four) glucose concentrations (alone, as the control group) with the (two) growth factors (for the IGF-I and MGF groups) and then mannitol added to these three groups, a total of (6 × 4) 24 samples were investigated. The RNA isolations were performed for the gene expressional fold change analysis of IGF-I and MGF by RT-PCR analysis. The cell cycle and proliferation and cell death analysis were performed by BrdU incorporation assay using flow cytometry. IGF: Insulin-like growth factor, MGF: Mechano-growth factor, NSC: Neural stem cell, RT-PCR: Reverse transcription-polymerase chain reaction

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A high glucose environment increases the relative gene expressions of insulin-like growth factor-I and mechano-growth factor of neural stem cell

The IGF-I and MGF expressions of NSCs were determined by the real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis by exposing the cells to different glucose concentrations (17.5, 27.75, 41.75, and 83.75 mM) and growth factors (IGF-I and MGF) during 24 h culture period, with the 17.5 mM concentration referred to as a control and the glucose only (nongrowth factor) group of (four) samples regarded as the control group.

There was a significant fold change in the IGF-I and MGF expressions for NSCs exposed to glucose conditions as compared to the control group. NSCs exposed to 27.75 mM glucose revealed a 17-fold increase in IGF-I and a 40-fold increase in MGF expressions. A 68-fold increase for IGF-I and a 161-fold increase for MGF expressions were detected in NSCs exposed to 41.75 mM glucose. In NSCs exposed to 83.75 mM glucose, a 75-fold increase was detected for IGF-I and 137-fold for MGF. These significant increases demonstrate that IGF-I and MGF genes were expressed at the time of damage and that their expressions are concordant with the glucose concentrations [Figure 2]a and [Figure 2]b.
Figure 2: (a) The expression of IGF-I and in NSCs treated with growth factors. The study was performed with the addition of growth factors alone to the NSC culture and analysis of the expressional changes during 24 h period by real-time RT-PCR. The stacked proportion bar chart is sorted by increasing relative gene expression based on the glucose environment for individual treatments *P < 0.05. (b) The expression of MGF in NSCs treated with growth factors. The study was performed with the addition of growth factors alone to the NSC culture and analysis of the expressional changes during 24 h period by real-time RT-PCR. IGF: Insulin-like growth factor, MGF: Mechano-growth factor, NSC: Neural stem cell, RT-PCR: Reverse transcription-polymerase chain reaction

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Surprisingly, the administration of the growth factor of IGF-I and MGF to NCS culture dramatically changed the relative gene expressions in the NCS culture conditions. IGF-I expression was increased with the administration of IGF-I to the culture with the four glucose concentrations, by 60,180, 90, and 175 fold, respectively [Figure 2]a. MGF expression was suppressed by administration of MGF to the NCS culture but just at the higher (41.75 mM and 83.75 mM) glucose concentrations [Figure 2]b.

Summarizing, the NSC responds to the high glucose concentrations by increasing IGF-I and MGF gene expressions. External application of MGF in a high glucose environment decreases its relative gene expression, which is not the case for IGF-I.

Growth factors induce the proliferation of neural stem cells under high glucose concentrations

The NSC proliferation potential was tested by exposing the cells to different glucose concentrations and IGF-I and MGF growth factors during the 24 h culture period. The NSCs were labeled with BrdU and analyzed by flow cytometry [Figure 3]a. The high glucose concentrations (41.75 mM and 83.75 mM) were found to suppress cell proliferation (by 50.30% and 28.97%, respectively) [Figure 3]b.

Mannitol was applied as an osmotic control, but it was found that it normalizes the proliferative suppressor effect of the glucose at different concentrations on the cells. Therefore, there was no statistically significant difference compared to the control (P > 0.05); the external administration of IGF-1 and MGF also had no effect in this process (data not shown). Since the NSC proliferation decreased due to the increased glucose levels, the external administration of IGF-I and MGF to the NSC-exposed glucose concentrations was examined. The statistically significant increases were detected with the 83.75 mM glucose concentration, with IGF-I and MGF growth factors both positively affecting NSC proliferation (P < 0.05). The MGF application protected the cells and induced proliferation in all high glucose concentrations, and it induced proliferation better than IGF-I in the 41.75 mM glucose concentration [Figure 3]b.

Growth factors have a neuroprotective effect on high glucose

The response of NSCs with different glucose concentrations and with MGF and IGF-I was analyzed with the 7-AAD labeling [Figure 3]c. The analysis of cells exposed to high glucose concentrations revealed that the percentage of dead cells was higher in the 27.75 mM and 83.75 mM and lower in 41.75 mM glucose samples than in the control sample.

It was found that when IGF-I and MGF were added to the cultures, the percentages of dead cells were generally lower than in the controls. When the samples with and without growth factors were compared according to glucose concentration, the percentage of dead cells was found to be lower with IGF-I and MGF treatment in the 27.75-mM glucose concentration. The percentage of dead cells increased when the 41.75-mM glucose sample was treated with IGF-I but decreased when treated with MGF. The dead cell percentages were lower for the 83.75-mM glucose samples with both IGF-I and MGF [Figure 3]c. This result supports the idea that IGF and MGF have a neuroprotective effect in NSCs exposed to high glucose.

Growth factors are not mitogenic to neural stem cells under a high glucose environment

In order to determine the possible effect of growth factors in a high glucose environment on the activation and proliferation of NSCs, cell cycle analyses were performed with BrdU incorporation assay as previously shown elsewhere.[38] The samples treated with BrdU were analyzed with flow cytometry for each cell cycle phase, namely, the G0/G1, S, and G2/M phases [Figure 4]a. An overall analysis revealed a significant number of NSCs in G0/G1 phase compared to the S and G2/M phases (P < 0.01 at any time point), as shown in [Figure 4]a. Since NSCs are expected to be in a quiescent state, high glucose and growth factor administrations did not induce much difference in this regard in 24 h [Figure 4]b and ]Figure 4]d.
Figure 4: Cell cycle analysis of NSC with growth factors. (a) NSC cell cycle status after BrdU treatment and analysis by flow cytometry. (b) Growth phase (G0/G1), (c) Synthase (S) phase (d) Repairing and cell preparation (G2/M) phase. The green and blue boxes highlight the results of the externally administered IGF-I and MGF groups. IGF: Insulin-like growth factor, MGF: Mechano-growth factor, NSC: Neural stem cell

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The individual differences between the two growth factors for the NSC culture with four different glucose levels were not statistically significant. The IGF-I and MGF growth factors overall provided a minimal change in the S phase and tended to maintain the cells in the synthase phase during 1-day culture time [Figure 4]c. The growth factors induced mitosis in NSCs at the 27.75 mM glucose level.

  Discussion Top

Glucose is an essential factor for NSC regeneration following cerebral ischemia.[39] Conversely, it has been demonstrated by different groups that diabetes has a detrimental effect on NSCs. Thus, a finely tuned level of glucose is essential for NSC homeostasis. IGF-I acts as one of the leading growth factors during the development of NSCs[40],[41] and is induced in damaged regions of the brain after cerebral ischemia.[42] MGF, which is an alternative splicing variant of IGF-I, is not expressed in normal healthy tissues. Unlike IGF-I, it is a general tissue repair factor, however, and tissue damage induces its expression.[30]

Our study has demonstrated that the relative IGF-I and MGF gene expression fold changes dramatically increase when NSCs are exposed to a high glucose environment. Since these growth factors have a stress-related role this result is not surprising. However, comparing the relative fold change in gene expression between IGF-I and MGF revealed the latter to be higher than the former for all high glucose levels. In addition, external administration of MGF to NSCs under high glucose levels lowered the dead cell percentages more and had higher proliferation rates than did IGF-I for similar conditions. These findings support the view that MGF has a higher protection potential for NSCs at the time of high glucose damage than does IGF-I.

Li et al.(2009) investigated the effect of high glucose and growth factors on endothelial progenitor cells (EPCs).[43] They demonstrated that both IGF-I and MGF increased EPC proliferation under high glucose conditions. They also reported that MGF has a higher capacity to increase proliferation than does IGF-I. A study performed by Dłużniewska et al.(2004) also revealed that MGF is more protective for neurons than IGF-I.[33] The significant increase in MGF at the time of injury might be directly related to the increase in NSC survival. The results reported here provide further support for the neuroprotective effect of MGF in NSCs exposed to high glucose.

Our study also determined a decrease in proliferation of NSCs exposed to 41.75 mM and 83.75 mM glucose as compared to the control. There was no difference in the proliferation of NSCs exposed to 27.75 mM glucose. The results reported by Chen et al. performed on NSCs in high glucose environments further support these findings.[35] These results suggest the conclusion that significant changes in NSC proliferation do not result from high glucose but only to a certain level since the exceptionally high concentration of glucose (83.75 mM) representing diabetic ketoacidosis and hyperglycemia hyperosmolar status was found to suppress NSC proliferation. In addition, the administration of IGF and MGF provided a significant increase in NSC proliferation under 83.75 mM glucose concentrations, including the control.

According to the dead cell analysis of NSCs exposed to high glucose and the growth factors, the percentage of dead cells was higher in samples exposed to 27.75 mM and 83.75 mM glucose alone. The external administration of IGF-I and MGF protected the cells, as demonstrated by a lower dead cell percentage when compared to the untreated controls. This observation was detected as significant in NSCs with under 27.75 mM glucose for both growth factors. Furthermore, the results from cell cycle analysis revealed that the IGF-I and MGF growth factors overall tend to maintain the cells in the synthase phase.

Regarding to the limitations, the in vitro setup in these experiments was limited in time-wise analysis to 24 h. The changes observed with more prolonged exposures to high glucose levels might also be interesting since this might activate varying signaling pathways and related gene expressions. A more extended administration of growth factors to NSC in such high glucose conditions might also reveal enhanced protection.

  Conclusion Top

This study has found that NSC proliferation is reduced when exposed to increased glucose levels. At the same time, when IGF-I and MGF were administered externally to the NSCs exposed to high glucose, they positively affected NSC proliferation. It was found that there was IGF-I and MGF expression in NSCs exposed to high glucose and that MGF was more expressed than IGF-I. These findings reveal that IGF-I and especially MGF increase the proliferation of NSCs at the time of damage and provide a neuroprotective effect. It was found that exogenous IGF-I and MGF also increase NSC proliferation at the time of damage and that MGF is more neuroprotective than IGF-I.

It is known that stroke causes more severe damage in diabetic patients than in patients with normal blood glucose levels. The results reported here on the neuroprotective and proliferative effects of the external administration of MGF and IGF-I thus might indicate this approach as a treatment to alleviate the effects of neuronal damage due to strokes in diabetic patients.

Consent for publication


Author contributions

Concept: TAG, SS; Supervision: SS; Materials: TAG, SFT, SS; Data collection and/or processing: TAG, SFT, SS; Analysis and/or interpretation: ÇG, FT, SFT, SS; Literature search: TAG, FT, SFT; Writing: FT, SFT, SS; Critical reviews: SS., ÇG, FT.

Financial support and sponsorship

This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University (Project number: 58165).

Conflicts of interest

There are no conflicts of interest.

  References Top

Mergenthaler P, Lindauer U, Dienel GA, Meisel A. Sugar for the brain: The role of glucose in physiological and pathological brain function. Trends Neurosci 2013;36:587-97.  Back to cited text no. 1
Erbsloh F, Bernsmeier A, Hillesheim H. The glucose consumption of the brain & its dependence on the liver. Arch Psychiatr Nervenkr Z Gesamte Neurol Psychiatr 1958;196:611-26.  Back to cited text no. 2
Duarte JM. Metabolic alterations associated to brain dysfunction in diabetes. Aging Dis 2015;6:304-21.  Back to cited text no. 3
Lundqvist MH, Almby K, Abrahamsson N, Eriksson JW. Is the brain a key player in glucose regulation and development of type 2 diabetes? Front Physiol 2019;10:457.  Back to cited text no. 4
Nakrani MN, Wineland RH, Anjum F. Physiology, Glucose Metabolism. USA: StatPearls. Treasure Island (FL); 2021.  Back to cited text no. 5
Thiyagarajan R, Subramanian SK, Sampath N, Madanmohan Trakroo, Pal P, Bobby Z, et al. Association between cardiac autonomic function, oxidative stress and inflammatory response in impaired fasting glucose subjects: Cross-sectional study. PLoS One 2012;7:e41889.  Back to cited text no. 6
Kanikarla-Marie P, Jain SK. Hyperketonemia and ketosis increase the risk of complications in type 1 diabetes. Free Radic Biol Med 2016;95:268-77.  Back to cited text no. 7
Hardigan T, Ward R, Ergul A. Cerebrovascular complications of diabetes: Focus on cognitive dysfunction. Clin Sci (Lond) 2016;130:1807-22.  Back to cited text no. 8
Rietze RL, Reynolds BA. Neural stem cell isolation and characterization. Methods Enzymol 2006;419:3-23.  Back to cited text no. 9
Ottoboni L, Merlini A, Martino G. Neural stem cell plasticity: Advantages in therapy for the injured central nervous system. Front Cell Dev Biol 2017;5:52.  Back to cited text no. 10
Boese AC, Le QE, Pham D, Hamblin MH, Lee JP. Neural stem cell therapy for subacute and chronic ischemic stroke. Stem Cell Res Ther 2018;9:154.  Back to cited text no. 11
Saintonge J, Côté R. Brain development in relation to fetal weight and maternal glucose tolerance during normal gestation. Brain Dev 1987;9:26-32.  Back to cited text no. 12
Clodfelder-Miller B, De Sarno P, Zmijewska AA, Song L, Jope RS. Physiological and pathological changes in glucose regulate brain Akt and glycogen synthase kinase-3. J Biol Chem 2005;280:39723-31.  Back to cited text no. 13
Gao Q, Gao YM. Hyperglycemic condition disturbs the proliferation and cell death of neural progenitors in mouse embryonic spinal cord. Int J Dev Neurosci 2007;25:349-57.  Back to cited text no. 14
Coucha M, Abdelsaid M, Ward R, Abdul Y, Ergul A. Impact of metabolic diseases on cerebral circulation: Structural and functional consequences. Compr Physiol 2018;8:773-99.  Back to cited text no. 15
]Shahjalal HM, Shiraki N, Sakano D, Kikawa K, Ogaki S, Baba H, et al. Generation of insulin-producing β-like cells from human iPS cells in a defined and completely xeno-free culture system. J Mol Cell Biol 2014;6:394-408.  Back to cited text no. 16
Horie N, Moriya T, Mitome M, Kitagawa N, Nagata I, Shinohara K. Lowered glucose suppressed the proliferation and increased the differentiation of murine neural stem cells in vitro. FEBS Lett 2004;571:237-42.  Back to cited text no. 17
Ferraro F, Lymperi S, Méndez-Ferrer S, Saez B, Spencer JA, Yeap BY, et al. Diabetes impairs hematopoietic stem cell mobilization by altering niche function. Sci Transl Med 2011;3:104ra101.  Back to cited text no. 18
Fujimaki S, Kuwabara T. Diabetes-Induced dysfunction of mitochondria and stem cells in skeletal muscle and the nervous System. Int J Mol Sci 2017;18:E2147.  Back to cited text no. 19
Fadini GP, Ferraro F, Quaini F, Asahara T, Madeddu P. Concise review: Diabetes, the bone marrow niche, and impaired vascular regeneration. Stem Cells Transl Med 2014;3:949-57.  Back to cited text no. 20
Kolluru GK, Bir SC, Kevil CG. Endothelial dysfunction and diabetes: Effects on angiogenesis, vascular remodeling, and wound healing. Int J Vasc Med 2012;2012:918267.  Back to cited text no. 21
Cramer C, Freisinger E, Jones RK, Slakey DP, Dupin CL, Newsome ER, et al. Persistent high glucose concentrations alter the regenerative potential of mesenchymal stem cells. Stem Cells Dev 2010;19:1875-84.  Back to cited text no. 22
Weir CJ, Murray GD, Dyker AG, Lees KR. Is hyperglycaemia an independent predictor of poor outcome after acute stroke? Results of a long-term follow up study. BMJ 1997;314:1303-6.  Back to cited text no. 23
Dorsemans AC, Couret D, Hoarau A, Meilhac O, Lefebvre d'Hellencourt C, Diotel N. Diabetes, adult neurogenesis and brain remodeling: New insights from rodent and zebrafish models. Neurogenesis (Austin) 2017;4:e1281862.  Back to cited text no. 24
Toprak F, Toprak SF, Tokdemir SS. The role of growth factors in adult neurogenesis and neurodegenerative diseases. Experimed 2021;11:57-66.  Back to cited text no. 25
Ziegler AN, Levison SW, Wood TL. Insulin and IGF receptor signalling in neural-stem-cell homeostasis. Nat Rev Endocrinol 2015;11:161-70.  Back to cited text no. 26
Lunn JS, Sakowski SA, McGinley LM, Pacut C, Hazel TG, Johe K, et al. Autocrine production of IGF-I increases stem cell-mediated neuroprotection. Stem Cells 2015;33:1480-9.  Back to cited text no. 27
Tunç BS, Toprak F, Toprak SF, Sozer S. In vitro investigation of growth factors including MGF and IGF-1 in neural stem cell activation, proliferation, and migration. Brain Res 2021;1759:147366.  Back to cited text no. 28
Goldspink G. Impairment of IGF-I gene splicing and MGF expression associated with muscle wasting. Int J Biochem Cell Biol 2006;38:481-9.  Back to cited text no. 29
Matheny RW Jr., Nindl BC, Adamo ML. Minireview: Mechano-growth factor: A putative product of IGF-I gene expression involved in tissue repair and regeneration. Endocrinology 2010;151:865-75.  Back to cited text no. 30
Beresewicz M, Majewska M, Makarewicz D, Vayro S, Zabłocka B, Górecki DC. Changes in the expression of insulin-like growth factor 1 variants in the postnatal brain development and in neonatal hypoxia-ischaemia. Int J Dev Neurosci 2010;28:91-7.  Back to cited text no. 31
Tang JJ, Podratz JL, Lange M, Scrable HJ, Jang MH, Windebank AJ. Mechano growth factor, a splice variant of IGF-1, promotes neurogenesis in the aging mouse brain. Mol Brain 2017;10:23.  Back to cited text no. 32
Dluzniewska J, Sarnowska A, Beresewicz M, Johnson I, Srai SK, Ramesh B, et al. A strong neuroprotective effect of the autonomous C-terminal peptide of IGF-1 Ec (MGF) in brain ischemia. FASEB J 2005;19:1896-8.  Back to cited text no. 33
Zhang P, Li J, Liu Y, Chen X, Lu H, Kang Q, et al. Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri-infarct angiogenesis after focal cerebral ischemia. Neuropathology 2011;31:384-91.  Back to cited text no. 34
Chen J, Guo Y, Cheng W, Chen R, Liu T, Chen Z, et al. High glucose induces apoptosis and suppresses proliferation of adult rat neural stem cells following in vitro ischemia. BMC Neurosci 2013;14:24.  Back to cited text no. 35
Kleppisch T, Klinz FJ, Hescheler J. Insulin-like growth factor I modulates voltage-dependent Ca2+channels in neuronal cells. Brain Res 1992;591:283-8.  Back to cited text no. 36
Podratz JL, Tang JJ, Polzin MJ, Schmeichel AM, Nesbitt JJ, Windebank AJ, et al. Mechano growth factor interacts with nucleolin to protect against cisplatin-induced neurotoxicity. Exp Neurol 2020;331:113376. DOI: https://doi.org/10.1016/j.expneurol.2020.113376.  Back to cited text no. 37
Kim YH, Heo JS, Han HJ. High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways. J Cell Physiol 2006;209:94-102.  Back to cited text no. 38
Moreno M, Fernández V, Monllau JM, Borrell V, Lerin C, de la Iglesia N. Transcriptional profiling of hypoxic neural stem cells identifies calcineurin-NFATc4 signaling as a major regulator of neural stem cell biology. Stem Cell Reports 2015;5:157-65.  Back to cited text no. 39
Anlar B, Sullivan KA, Feldman EL. Insulin-like growth factor-I and central nervous system development. Horm Metab Res 1999;31:120-5.  Back to cited text no. 40
Ye P, Carson J, D'Ercole AJ. In vivo actions of insulin-like growth factor-I (IGF-I) on brain myelination: Studies of IGF-I and IGF binding protein-1 (IGFBP-1) transgenic mice. J Neurosci 1995;15:7344-56.  Back to cited text no. 41
Gluckman P, Klempt N, Guan J, Mallard C, Sirimanne E, Dragunow M, et al. A role for IGF-1 in the rescue of CNS neurons following hypoxic-ischemic injury. Biochem Biophys Res Commun 1992;182:593-9.  Back to cited text no. 42
Li W, Yang SY, Hu ZF, Winslet MC, Wang W, Seifalian AM. Growth factors enhance endothelial progenitor cell proliferation under high-glucose conditions. Med Sci Monit. 2009;15:BR357-63.  Back to cited text no. 43


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]


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