Main Research Findings

1) Results of this study reported that IGF-1 DES led to excitatory transmission in the CA1 region of the rat hippocampus and enhancement of fEPSP slopes in a dose-dependent manner.

2) The research team of Elis et. Al found in KID and KIR mice models, IGF-1 is crucial for the development of normal body, organ, and bone size.

3) Results of the study found that subcutaneous injection of IGF-1 DES at the onset of diabetes can act as a protective measure against predegenerative biochemical abnormalities.

 

Selected Data

1) The research team utilized male Sprauge-Dawley rats as test subjects for this experiment. Once the subjects reached 20-40 days old they were anesthetized with halothane in order for researchers to obtain slices of certain brain regions. Coronal hippocampal slices were prepared with a vibrating tissue slicer and maintained at room temperature in a solution of oxygenated artificial cerebrospinal fluid (ACSF). The ACSF contained 124mM NaCl, 3.3 mM KCl, 2.4 mM MgCl2, 2.5 mM CaCl2, 1.2 mM KH2PO4, 10 mM D-glucose, and 25.9 mM NaHCO3. The slices were saturated with the ACSF twice per minute during the observation period [2].

Researchers used NMDA receptor antagonists (APV), GABA receptor antagonists (BIC), and AMPA/KA receptor antagonists (DNQX) in order to pharmacologically isolate and observe evoked currents. IGF-1 DES was prepared as a stock solution in 0.1 N acetic acid. Various compounds were used in order to examine the signaling components of IGF-1, including, tyrosine kinase inhibitors, PI3K inhibitors and LY 294002 inhibitors. Like IGF-1 DES these inhibitors were made into a stock solution, however, instead of acetic acid dimethyl sulfoxide (DMSO) was used. All drugs were administered via ACSF [2].

In order to collect data electrodes were prepared and filled with a recording solution containing 120 mM K-gluconate, 10 mM KCl2, 5 N-(2,6-dimethyl-phenylcarbamoylmethyl)-triethylammonium bromide, 1mM EGTA, 0.1 mM CaCl2, 2 Mg-ATP, 0.2 tris-GTP, and 10 mM free acid (HEPES). Neuron voltages were clamped at -70 mV in order to record AMPA-mediated excitatory postsynaptic currents (EPSCs) while in the presence of antagonists, APV and BIC. Neuron voltages were clamped and -30 mV in order to record NMDA-mediated EPSCs while in the presence of DNQX and BIC. The synaptic currents were sent every 20 seconds by way of a 0.2 ms long electrical stimulation of the adjacent tissue to the electrode [2].

Field excitatory postsynaptic potentials (fEPSPs) were obtained under the same conditions and using the same recording equipment as the patch-clamp recordings. The only change made was the adjustment of the recording electrodes which were now placed in the apical dendritic field. The IGF-1 DES-induced concentration-response curves of EPSCs and fEPSPs were analyzed by one-way ANOVA and Newman-Keuls test for comparisons. Dunnett’s post hoc test and one-way ANOVA tests were used to compare inhibitory effects on IGF-1 DES-mediated changes in fEPSP slopes [2].

2) The study conducted by Elis et. Al aimed to find how bioavailable derivatives of IGF-1 affects somatic and skeletal growth as well as body composition and tissue integrity. The research team created two mutated mouse models with knock-in RE-IGF1 and IGF-1 DES. The first mutant substituted an E amino acid for an R at position three and was named knock-in R (KIR). While the second mutant is missing the first three amino acids of IGF-1 (IGF-1 DES) and was named knock-in D (KID). The two mutated mouse models represent an unbound form of IGF-1 with impaired post-translational control and increased bioavailability; they are then used to characterize the effects of KIR and KID in this scenario [3].

Serum levels of IGF-1 in KID and KIR mice were observed by using a radioimmunoassay (RIA) kit. The same RIA kit was used to measure recombinant RE-IGF1 and IGF-1 DES. The bioactivity of free IGF1 in serum was observed using a cell-based IGF-1 kinase receptor activation (KIRA) bioassay. This is used to determine the ability of the serum to stimulate IGF1R activity in vitro [3].

3) 12-week-old male Sprague-Dawley rats were assigned to different treatment groups and used for animal experimentation in accordance with NIH guidelines. The animals were split into two experimental groups and a control group, each group had 5 rats. All solutions were administered to the rats through Acrodisk filters. The experimental groups were intraperitoneally administered a 50 mg/kg dose of STZ in order to induce diabetes. The control group was labeled non-diabetic (ND) while the first experimental group was labeled at STZ-veh indicating the animal was diabetic and received vehicle treatments. The second experimental group was labeled STZ-des, the diabetic rats received an active dose of IGF-1 DES. Each treatment was administered through a subcutaneous osmotic minipump over the course of two weeks [4].

After two weeks of treatment the rats were euthanized and the eyes were preserved with 4% paraformaldehyde in phosphate buffered saline (PBS). The preserved eyes were set in paraffin and cut into slices measuring approximately 4 micrometers in length. Additionally, tail blood was collected in order to measure glucose assay 24 hours after STZ or vehicle treatment and again at 2 weeks after treatment. The retinal tissue slices were rehydrated through the use of xylene and various graded alcohol concentrations. The samples were then properly prepared by rinsing in PBS and incubation with the primary antibodies. The slices were stained in order to identify the number of immunoreactive cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL) throughout three randomly chosen segments [4].

 

Discussion

1) IGF-1 and Growth Hormone (GH) play an essential role in promoting growth and development in the body. Additionally, researchers Ramsey et. Al examined how IGF-1 DES can potentially affect the cognitive function of aged rats by treating the excitatory synapses in the CA1 region of the hippocampus. 15 minutes of IGF-1 DES application resulted in a concentration-dependent increase in the CA1 fEPSP slope by 39 􏰊+/- 6% [2].

Potentiation induced by IGF-1 DES was initiated within 5 minutes of application when the treatment was administered at concentrations of 40 ng/ml. The synaptic response to treatment with IGF-1 DES remained significantly increased in comparison to baseline levels. Researchers observed a dramatic difference in potentiation magnitude when subjects were given 5 ng/ml versus 20 or 40 ng/ml. The results reported that the potentiation magnitude plateaus at 20 ng/ml [2].

Figure 1: % changes in baseline fEPSP slopes depending on administered dose of IGF-1 DES.

The cell patch-clamp recordings in the CA1 pyramidal layer were used to determine which receptors were responsible for IGF-1 DES-induced improvement of fEPSP slopes. By applying a 40 mg/ml dose of IGF-1 DES for 15 minutes, there was a significant 34 +/- 7% increase in the amplitude of AMPA EPSCs. This is compared to NMDA EPSCs that experienced no significant changes in amplitude when IGF-1 DES was administered [2].

Figure 2: AMPA and NMDA receptor levels following 40 ng/ml dose of IGF-1 DES.

Hippocampal slices were treated with various inhibitors in order to observe changes in CA1 fEPSPs and EPSCs. The slices were treated with a 220 micrometer dose of the􏰁 tyrosine kinase inhibitor, genistein, a 1 micrometer dose of wortmannin, or a 10 micrometer dose of LY 294002. Application of 40 ng/ml of IGF-1 DES was continued throughout treatment with the different inhibitors.

In the presence of genistein there was no IGF-1 DES-mediated potentiation of the fEPSP slopes. The fEPSP slope actually showed significant decreases following application of IGF-1 DES in combination with genistein. It is important to note that potentiation of the fEPSP slope was inhibited by genistein and IGF-1 DES to the same extent as genistein administered alone. Genistein + IGF-1 DES led to a 38 +/- 6% inhibition in fEPSP slopes, while genistein alone led to a 40 +/- 5% inhibition. This allowed researchers to conclude that the inhibition of genistein was an independent effect elicited on excitatory transmission. Both wortmannin and LY 294001 led to a reduction in fEPSP slope potentiation at rates of 14 +/- 2% and 7 +/- 5%, respectively. Applying these PI3K inhibitors without IGF-1 DES did not lead to a significant reduction in potentiation [2].

Figure 3: Changes in fEPSP slope from baseline in response to different inhibitory compounds.

The results of the study caused the research team to hypothesize that elevation in IGF-1 can lead to improved cognitive performance in aged animals. While further research has to be conducted to solidify the link between cognition and IGF-1, IGF-1 has been proven to act at AMPARs to initiate changes in synaptic structure and protein expression. After a period of chronic administration of IGF-1 DES, these changes can lead to long-term benefits in learning and memory [2].

2) The RIA test using polyclonal antibody recorded reduction in serum IGF-1 levels at 4, 8, and 16 weeks of age. The recombinant R3-IGF1 and IGF-1 DES were measured with the same RIA kit; results reports that in concentrations <150 ng/ml, the precision of the kit was approximately 70% for IGF-1 DES and only 6% for R3-IGF1. The KIRA bioassay results contradicted the findings of the RIA kit. When comparing KID, KIR, and a control the KIRA bioassay found that bioactive IGF1 levels all reached a similar magnitude without any significant differences between the three groups. Since the data obtained from KID mice showed levels of bioactivity similar to the control group, the research team hypothesized that the difference in recorded results stemmed from the RIA kit only recording fragments of IGF-1 DES that were bound to IGF-1 binding proteins with a low affinity [3].

Figure 4: Serum IGF-1 levels in different experimental groups

In addition to changes in serum levels, both KID and KIR mice saw significant increases in body weight from 4 to 16 weeks in age. At 4, 8, and 16 weeks of age KID and KIR mice showed increased body length. NMR testing revealed that at 16 weeks of age, KIR and KID mice experienced a significant increase in lean body mass in both male and female subjects. This was in comparison to the control group where both males and females showed increased body adiposity while body adiposity and weight of gonadal fat pads decreased in KIR and KID mice [3].

Figure 5: Changes in the body weight and body length of both male and female KID, KIR, and control mice.

The research team attempted to understand how IGF-1 affects cortical and trabecular bone structures. The femurs were dissected from 16-week-old female mice and observed using microcomputed tomography. The data reported that IGF-1 significantly increased total cross-sectional area (tt.Ar.), cortical bone area ( Ct.Ar.), and cortical thickness (Ct.Th) in female KIR mice. The cortical changes were less dramatic in KID mice; there were only significant increases in Ct.Th and Ct.Ar. There was no large difference in tissue mineral density (TMD) between the control group and KID mice and the control group and KIR mice. However, there was a significant reduction of TMD in KIR mice when compared to KID mice [3].

Changes in morphology and mechanical properties were measured by a four-point bending test performed on the femora obtained from 16-week-old KID and KIR mice. Both of these groups exhibited increased stiffness in comparison to the control, however, the difference was not statistically significant. The four-point bending test also determined that maximum load was much higher in KIR mice in comparison to both the control group and the KID mice.

Any variations to the trabecular structure was assessed at the distal femur. Results showed that the bone volume/total volume fraction (BV/TV) increased in both KID and KIR mice. The researchers reported that the increase in BV/Tv was due to a significant increase in trabecular number (Tb.N) in KIR and KID mice. This was accompanied by a notable decrease in trabecular spacing (Tb.Sp.) in KIR and KID mice. However, there were no major differences in TMD or trabecular thickness (Tb.Th.) in either experimental group. It is important to note that the micro-CT data identified elevated levels of the bone formation marker, osteocalcin, in KIR mice. There were no observed changes in osteocalcin levels in KID mice or the control group [3].

3) Results of this study found that there was a low level of IGF-1 receptor immunoreactivity in the GCL, INL, and BRB in the retina of ND rats. Immunoreactivity was increased in the retina of STZ-vehicle rats, however, in STZ-des rats immunoreactivity was measured almost as low as in ND rats. Type 1 IGF receptor-immunoreactive cells were counted in the GCL and INL of the rats in all three groups in order to determine if the changes in immunoreactivity were significant. In comparison to the ND rats, immunoreactivity was significantly enhanced in the GCL and INL in the STZ-veh rats. It was confirmed that when rats with induced diabetes were treated with IGF-1 DES, immunoreactivity lowered to levels similar to the control group. There was also a significant reduction of immunoreactivity in STZ-des rats versus STZ-veh rats [4].

Figure 6: Changes in immunoreactive cell numbers in the two different experimental groups and control group

The research team of Kummer et. Al also tested the potential of IGF-1 DES to prevent increased vascular endothelial growth factor (VEGF) immunoreactivity. The retinal tissue of ND rats showed a baseline level of VEGF immunoreactivity that seemed to be associated with retinal endothelial cells. However, the retinal pigmented epithelial cells (RPEs) in STZ-veh rats exhibited signs of increased VEGF immunoreactivity. The researchers were able to conclude that the increase in VEGF immunoreactivity seen in RPEs was efficiently prevented by treating the diabetic rats with IGF-1 DES. It is important to note that, when stained, VEGF immunoreactivity was occasionally seen in the cells of the STZ-des and ND rats and this occurrence is not unusual [4].

Figure 7: (A) VEGF immunoreactivity in the retina of ND rats versus (C) VEGF immunoreactivity in CGL, INL, and BRB in STZ-des rats.

This study also examined changes in the basal level immunoreactivity of phospho-AKT, the apoptotic-stress response protein, in the GCL and INL of ND rats. The GCL and INL of STZ-veh rats exhibited significantly increased immunoreactivity. This is in comparison to STZ-des rats; these subjects experienced a reduction in phospho-Akt immunoreactivity. In order to determine if the differences in immunoreactivity were significant, the number of immunoreactive cells in the GCL and INL in rats from all three groups were recorded. STZ-veh rats showed increased levels of immunoreactivity in the GCL and INL regions. In the rats treated with IGF-1 DES, phospho-Akt immunoreactivity was reduced in the GCL and INL regions to a level comparable to the control group. When comparing STZ- des and STZ-veh, immunoreactivity levels were far lower in the STZ-des rats.

Figure 8: Changes in phospho-Akt immunoreactive cell count between the control group and two experimental treatment groups.

It is important to note that the efficacy of IGF-1 DES treatment was not dependent on metabolic control in the animals. Initial results of the study reported that IGF-1 DES treatment was not effective at treating hyperglycemia or promoting weight loss, except in excessively high doses. The low doses used in this study did not elicit any changes in hyperglycemic levels or rate of weight loss. Any recorded measurements were deemed not statistically significant.

Neural degeneration in the retinas was initially detected in the diabetic rats at 4 weeks. 2 weeks following induction of diabetes the researchers checked for any sign of predegenerative changes. Their observations as well as the previous results regarding weight loss and hyperglycemia, allowed the researchers to conclude that IGF-1 DES was able to protect against predegenerative retinal abnormalities regardless of metabolic control. Additionally, it was determined that there was a possibility the observed retinal abnormalities were not caused by acute hyperglycemia but rather the loss of IGF-1 activity in cases of diabetes [4].

 

Conclusion

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Ballard FJ, Wallace JC, Francis GL, Read LC, Tomas FM. Des(1-3)IGF-I: a truncated form of insulin-like growth factor-I. Int J Biochem Cell Biol. 1996 Oct;28(10):1085-7. doi: 10.1016/1357-2725(96)00056-8. PMID: 8930132.

[2] Ramsey, Melinda M., et al. “Functional Characterization of Des-IGF-1 Action at Excitatory Synapses in the CA1 Region of Rat Hippocampus.” Journal of Neurophysiology, vol. 94, 2005, pp. 247-254, https://journals.physiology.org/doi/pdf/10.1152/jn.00768.2004.

[3] Bailes J, Soloviev M. Insulin-Like Growth Factor-1 (IGF-1) and Its Monitoring in Medical Diagnostic and in Sports. Biomolecules. 2021 Feb 4;11(2):217. doi: 10.3390/biom11020217. PMID: 33557137; PMCID: PMC7913862.

[4] Kummer A, Pulford BE, Ishii DN, Seigel GM. Des(1-3)IGF-1 treatment normalizes type 1 IGF receptor and phospho-Akt (Thr 308) immunoreactivity in predegenerative retina of diabetic rats. Int J Exp Diabesity Res. 2003 Jan-Mar;4(1):45-57. doi: 10.1080/15438600303729. PMID: 12745670; PMCID: PMC2480499.