TFP5, a Peptide Derived From Cdk5 Activator p35, Protects Pancreatic β-Cells From Glucose Toxicity

doi:10.21203/rs.3.rs-1218842/v1

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TFP5, a Peptide Derived From Cdk5 Activator p35, Protects Pancreatic β-Cells From Glucose Toxicity

https://doi.org/10.21203/rs.3.rs-1218842/v1

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Type 2 diabetes mellitus (T2DM) is among the most important public health challenges. The pathological hyperactivity of multifunctional cyclin-dependent kinase 5 (Cdk5) contributes to the pathogenesis of T2DM. P5, a peptide derived from the Cdk5 activator p35, shows potential as a Cdk5 activity inhibitor. However, its inhibitory effect and functional regulation of Cdk5 activity need to be confirmed. In this study, we conjugated P5 with a Fluorescein Isothiocyanate (FITC) tag at the N-terminus and a TAT protein transduction domain containing an 11 amino acid peptide at the C-terminus to synthesize TFP5, which we used to inhibit Cdk5 activity. We then evaluated the efficiency of TFP5 in treating T2DM. We demonstrated that TFP5 effectively penetrated pancreatic β-cells, inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected MIN6 cells from apoptosis in pancreatic tissues of db/db mice (Type II diabetes mice). Furthermore, we found that TFP5 reduced inflammation in pancreatic islets by reducing the expression of inflammatory cytokines (including TGF-β, TNF-α, and IL-1β). These data indicate that the TFP5 peptide is a promising candidate for T2DM treatment.

TFP5

cdk5 activity

p25 activity

insulin

inflammation factor

apoptosis

We conjugated P5 with a Fluorescein Isothiocyanate (FITC) tag at the N-terminus and a TAT protein transduction domain containing an 11 amino acid peptide at the C-terminus to form TFP5, which inhibits Cdk5 activity, to explore the efficiency of TFP5 as a type 2 diabetes mellitus (T2DM) intervention. TFP5 effectively penetrated pancreatic β-cells, inhibited the hyperactivity of Cdk5, enhanced insulin secretion, and protected MIN6 cells from apoptosis in pancreatic tissues of db/db mice. TFP5 also reduced inflammation in pancreatic islets by reducing the expression of inflammatory cytokines (including TGF-β, TNF-α, and IL-1β).

Type 2 diabetes mellitus (T2DM), with incidence increasing at an alarming rate, is associated with high morbidity and mortality and has become a global public health problem¹. However, no effective strategies for hindering the progression of T2DM have been established. In the progression of T2DM, the deficient insulin secretion by pancreatic β-cells and resistance to insulin in peripheral target tissues is a critical pathological feature, and it is correlated with aberrant activation and dysregulation of multiple proteins.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that is activated by specific coactivators, namely, p35 and the p39 active complex². Recent studies have demonstrated that Cdk5 is a key instigator that exhibits critical hyperactivity under diabetic conditions and is involved in mitochondrial dysregulation in podocytes in diabetic nephropathy³and ER stress (Endoplasmic reticulum stress) in chronic hyperglycemia⁴. Long term hyperglycemia stimulates the formation of advanced glycation end products (AGEs) in the body. AGEs and its receptors combine to produce oxidative reaction and produce a variety of oxidative stress products (ROS), which will destroy the islets β Cell mitochondrial structure, induce apoptosis, activate nuclear transcription factor κB signaling pathway, cause cellular inflammatory response⁵. Besides, oxidative stress interferes with the phosphorylation of insulin receptor and insulin receptor substrate, affects the activation of phosphatidylinositol 3-kinase and induces insulin resistance⁶. And in our recent studies, we have done some research about the relevance of oxidative stress level and diabetes.

In addition, researchers have shown that aberrant Cdk5 activation can cause pancreatic β-cell failure, inhibit differentiation and neogenesis7. More importantly, it has recently been reported that Cdk5/p35 is an attractive candidate for disease therapy^8,9. Therefore, we hypothesized that Cdk5/p35 is a novel therapeutic target for T2DM treatment.

In our previous studies, we demonstrated that the activity of p35, which is cleaved by the calpain protease into p25 and p10 fragments, was significantly increased under pathological high-glucose conditions and played a pivotal role in Cdk5 hyperactivation and pancreatic β-cell dysfunction1, ^,11.Additionally, we found that the P5 fragment of p35 (a peptide of 24 amino acid residues) shows inhibitory potential against Cdk5/p25 pathological hyperactivity¹². Then, we found that TFP5 can inhibit Cdk5/p25 activity effectively without affecting the endogenous Cdk5/p35 or other Cdks^13,14. The efficiency of TFP5 treatment has been verified in Alzheimer’s disease, Parkinson's disease, and amyotrophic lateral sclerosis mouse models^{15 − 17}. And in our previous research, we found that TFP5 could inhibits the hyperactivity of Cdk5 induced by high glucose, protects pancreatic cells from apoptosis, and recovers the insulin secretion.

In this study, we focused on determining whether TFP5 can impact insulin secretion and pancreatic β-cell survival. We incubated high-glucose-stressed MIN6 cells with the TFP5 peptide and found that pathological Cdk5 hyperactivity was inhibited and that the secretion of insulin was reestablished. Similarly, purified pancreatic islet cells obtained from db/db mice exposed to TFP5 also showed reestablished insulin secretion. The reduction in inflammation and apoptosis rate was the potential mechanism. Together, these results indicated that TFP5 may be a therapeutic candidate for T2MD because it reduces pathological Cdk5 hyperactivation during T2MD progression.

Animals and treatment groups

All animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communication Disorders (NINDS/NIDCD) (protocol is ASP: 1231-11) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (protocol K058-KDB-10) in Bethesda, Maryland. The db/db mice and C57BL/6J mice (8-weeks-old males) were obtained from Jackson Laboratory (Bar Harbor, ME, USA).

Twelve db/db mice and 12 C57BL/6J mice were used in this study. The C57BL/6J and db/db mice (Type II diabetes mice) were randomly assigned to 2 groups with equal size, and 6 mice in each group received TFP5 (C57BL/6J + TFP5, n=6), and 6 other mice received a scrambled peptide (C57BL/6J + SCB, n=6); similarly, 6 of the db/db mice received TFP5 (db/db + TFP5, n=6) and 6 mice received the scrambled peptide (db/db + SCB, n=6). All treatment was administered by i.p. injection, and the dose and time of injection was determined on the basis of previous research¹⁹. All mice were housed with a 12-h light/dark cycle and had ad libitum access to standard chow diet and water¹⁸.The timeline and dosage details are shown in Supplementary Figure S1.

Antibodies and reagents

Anti-Cdk5 (C-8) (1:1000), anti-p35 (C-19) (1:1000), anti-Bax (1:1000), anti-Bcl-2 (1:1000), and anti-GFP (1:500) polyclonal antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas). Anti-cleaved Caspase-3 (Asp¹⁷⁵) (1:1000) and anti-tubulin antibodies (Sigma, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA). Anti-TGF-β1, anti-TNF-α, and anti-IL-1β antibodies (1:500-1000) were obtained from Abcam (Cambridge, MA). Secondary horseradish peroxidase-conjugated antibodies (1:2000) were obtained from Amersham Biosciences (Piscataway, NJ). Secondary fluorescence-conjugated Oregon Green and Texas Red antibodies (Molecular Probes, Eugene OR) were used after dilution at 1:400. Collagenase XI was purchased from Sigma. A rat/mouse insulin ELISA kit was obtained from Millipore (Billerica, MA).

Design and synthesis of TFP5

TFP5 was designed and synthesized as described previously¹⁰^,¹¹. Both TFP5 and the scrambled peptide (SCB) were synthesized by Peptide 2.0 (Chantilly, VA, USA). The TFP5 sequence was FITCGGGKEAFWDRCLSVINLMSSKMLQINAYARAARRAARR, and the SCB sequence was FITCGGGGGGFWDRCLSGKGKMSSKGGGINAYARAARRAARR.

Generation of recombinant adenoviruses

We used an adenoviral vector (pAdTrack-CMV) packaging system to construct the p5 forward primer, TTTGCGGCCGCCATGGCATCAATGCAGAAGCTGATCTCAGAGGAGGACCTGATGAAGGAGGCCTTTTGGGACCG, and the reverse primer, TTTGATATCTTAGGCATTTATCTGCAGCATCTTT. The generation of the recombinant adenovirus was carried out as described previously¹⁰.

Cell culture and treatment

MIN6 cells (Mouse islet beta cells) were cultured in DMEM with 4.5 mM glucose, 1 mM sodium pyruvate and 10% fetal bovine serum (FBS) with 100U/ml penicillin G and 100 μg/ml streptomycin¹⁰. MIN6 cells from passages 12-25 were used in experiments. Cells were seeded in 6-well plates with 0.3× 10⁵ cells/cm² one day before use. The cells were infected with adenovirus-p5 or adenovirus-p35 separately for 24 h-48 h and then exposed to TFP5 or SCB (500 nM) for 24 h. Then, the cells were starved overnight with glucose-free medium, treated with different concentrations of glucose (low glucose, 5 mM, and high glucose, 25 mM) and incubated at 37°C for 24 h. The cells were fixed for immunohistochemistry analysis or lysed for immunoprecipitation, kinase activity, and Western blot analyses.

Mouse pancreatic islet isolation and culture

Mouse pancreatic islet isolation was carried out using the method described by Li et al²⁰. Each pancreas was perfused with a 3-ml injection of collagenase XI solution (1000 U/ml in 1× HBSS) via the common bile duct. Then, the pancreas was removed, and 2 ml of collagenase XI solution was used for digestion at 37°C for 20 min with 2-3 brief shakes of the tube by hand. Digestion was stopped when putting the tube on ice, then added 25 ml of washing solution (1 mM CaCl₂ into 1× HBSS) and centrifugated at 290 g for 30 s at 4°C. After washing twice, the resuspended solution was filtered through a 7-µm nylon cell strainer, and the isolated islets were picked up using a manually operated pipette. To allow cell recover, the hand-picked islets (purity >95%) were cultured overnight in DMEM containing 10% FBS, 100 U/mL penicillin, and streptomycin (HyClone) in a humidified atmosphere of 5% CO₂ and 95% air at 37°C and then cultured in RPMI 1640 culture medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 mM L-glutamine, 11 mM glucose, 10% (vol/vol) fetal calf serum, and 1% penicillin/streptomycin.

Insulin secretion test

β-cells were treated with TFP5 and SCB for 24 h and washed twice with basal Krebs-Ringer’s solution bicarbonate HEPES buffer (5 mM glucose, 124 mM NaCl, 5.6 mM KCl, 2.5 mM CaCl2, 20 mM HEPES at pH 7.4, and 0.5% BSA). The cells were then incubated with glucose-free KRBH for overnight starvation: After the medium was discarded, the cells were incubated in either basal KRBH or KRBH containing high glucose (5 mM and 25 mM) for another 2 h. The supernatant was then collected to measure the secreted insulin. Insulin released into the supernatant and insulin remaining in cells was measured with LINCO ELISA kits, and insulin secretion is expressed as the concentration of insulin per million cells secreted every 2h.

Western blot analysis

Western blot analysis was performed as described previously10. Briefly, cells were harvested by scraping and then lysed in ice-cold lysis buffer. The protein content was determined using a BCA protein assay (Pierce, Rockford, Illinois). An equal amount of total protein (20 mg of protein/lane) was resolved using 4-20%, 15%, or 8% SDS-polyacrylamide gel electrophoresis and blotted onto a PVDF membrane. The membrane was incubated in blocking buffer for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. The membrane was then washed and incubated with goat anti-mouse or goat anti-rabbit IgG (H+L)-HRP conjugated secondary antibodies (Amersham Biosciences, 1: 2500) for 2 h at room temperature. Western blots were analyzed using an enhanced chemiluminescence kit (Pierce) following the manufacturer’s instructions.

In vitro Cdk5 kinase assays

Kinase assays were performed as previously described²¹. Cdk5 was immunoprecipitated overnight from the supernatants of lysed cells using the polyclonal C-8 antibody at 4°C, and immunoglobulin isolation was performed with protein A-Sepharose beads for 2h at 4°C. Kinase assays were performed in the same lysis buffer used for immunoprecipitation supplemented with 1 mM DTT, 0.1 mM ATP and 0.185 MBq [g-32P] ATP with 20 mg of histone H1 as the substrate. The phosphorylation assay was performed at a final volume of 50 ml and incubation at 300°C for 60 min. The reaction was stopped by the addition of 10% SDS sample buffer and then heated at 95°C for 5 min. Samples were separated by SDS-PAGE, the gels were stained with Coomassie blue, destained, dried, and exposed via autoradiography.

Immunocytochemistry

MIN6 cells were treated with TFP5, SCB or adenovirus-p5 on glass coverslips. After 24 h, the cells were starved overnight with glucose-free medium, treated with different concentrations of glucose (5 mM or 25 mM) and incubated at 37°C for 24 h. The cells were then washed twice with PBS, fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, and permeabilized with buffer (25 mM Tris, pH 7.4; 150 mM NaCl; and 0.1% Triton X-100) for 15 min. The coverslips were incubated overnight at 40°C with primary antibodies. All antibodies were diluted in PBS with 1% Triton-X-100. After three washes with PBS, the coverslips were incubated with fluorescein goat anti-mouse IgG or Texas Red goat anti-rabbit IgG for 1 h at room temperature, followed by three washes with PBS. Cell nuclei were counterstained with Hoechst 33342 (Sigma), and then, the cells were mounted in aqueous medium (Biomeda). Fluorescent images were obtained with a Zeiss LSM-510 laser-scanning confocal microscope, and images were managed with Adobe Photoshop.

Statistical analysis

All data analyses presented are based on three replicates of experimental outcomes. A two-tailed Student’s test was used to compare two groups, with statistical significance determined as p≤0.05. The data are presented as the standard means ± SEM. Histology and immunofluorescence images were generated using Photoshop and quantified using Image J software.

TFP5, derived from p35, effectively inhibited pathological Cdk5 hyperactivity

As demonstrated in our previous studies, the small truncated peptide from p35 (residues 154-279), also known as Cdk5 inhibitory peptide (CIP), is a highly effective and specific inhibitor of Cdk5/p25 activity¹². To produce a smaller, more permeable, and easier-to-detect inhibitory peptide of p35/p25 for potential therapeutic use, we conjugated P5 with a FITC tag at the N-terminus and a TAT protein transduction domain containing an 11 amino acid peptide at the C-terminus of P5 to synthetize TFP5 (Fig 1A-B). As predicted, TFP5 was an effective inhibitory peptide of pathological Cdk5 hyperactivity after cell penetration and was identified in cells together with co-transfected p35 via the GFP-tagged antibody (Fig 1C, a and b). By performing an immunofluorescence assay, we observed the expression of TFP5 and SCB in both the MIN6 cells when treated with TFP5 or SCB (Fig 1D, a and b), and the C57BL/6J mouse pancreas when injected with TFP5 or SCB (Fig 1D, c and d). After performing in vitro kinase assays, we found that the 24-residue peptide GST-p5 (Lys254–Ala277) was a more effective inhibitor than CIP (Fig 1E). We then compared the inhibitory effect of viral-infected p5 with TFP5 through the in vitro kinase assays, and the results indicated that TFP5 inhibited Cdk5 activity as efficiently as infection with p5 (Fig 1E). Clearly, the modified peptide successfully penetrated MIN6 cells and the pancreas, as shown in Fig 1D.

TFP5 effectively inhibited pathological Cdk5 overactivation in vitro and in vivo

First, the effect of TFP5 on pathological Cdk5 overactivation was investigated. We found that the high glucose group showed higher p25 expression than the low glucose group in vitro (Fig 2A, lanes 1 and 2). The statistical analysis was shown in Fig 2B. TFP5 treatment significantly decreased the expression of p25 under high glucose conditions (Fig 2A, lanes 2 and 3). Consistent with the p25 expression profiles, Cdk5/p25 activity was significantly increased under high glucose conditions (Fig 2E, lane 2) compared to that under low glucose conditions (Fig 2E, lane 1), while TFP5- and p5-treated cells exhibited significantly reduced Cdk5 activity (Fig 2E, lanes 3 and 4). In addition, we isolated the islets from mice in each group, and p25 expression was observed only in the db/db mouse islets (Fig 2C-D). The Cdk5 kinase assays showed that exposure to TFP5 significantly reduced Cdk5 pathological hyperactivity in the db/db mice (Fig 2F, lane 3 compared with SCB-treated lane 2). Although the protein level of Cdk5 in C57BL/6J mice was similar to that in db/db mice, the activity of Cdk5 was lower in nonpathological C57BL/6J mice, and TFP5 exposure did not affect Cdk5 activity in the C57BL/6J mice, which indicated that only pathological overactivation of Cdk5 effectively inhibited by TFP5 in vivo.

TFP5 reestablished insulin secretion from pancreatic β-cells

Then, we explored the therapeutic efficiency of TFP5. Interestingly, we found that the reduced insulin secretion from the MIN6 cells treated with high glucose (Fig 3A, lane 2 compared to the control) was restored when cells were treated with TFP5 or P5 (Fig 3A, lanes 3 and 4 compared to lane 2). Furthermore, the effect of TFP5 on insulin secretion was detected in vivo. We found that insulin secretion was reestablished in TFP5-treated db/db mice (Fig 3B and C, lane 3 compared to lane 2). These data demonstrate that TFP5 can effectively reestablish insulin secretion from pancreatic β-cells.

TFP5 might protect pancreatic β-cells from high glucose-induced apoptosis

Next, in order to investigate the effect of TFP5 exposure on the apoptosis of pancreatic β-cells, we performed western blot analysis and immunohistochemistry assay to detect the expression of a specific apoptosis marker, caspase 3. MIN6 cells were treated with TFP5 peptide under high-glucose conditions. MIN6 cells underwent apoptosis when exposed to high glucose levels. However, apoptosis can be attenuated when cells were co-infected with CIP¹². Therefore, we hypothesized that TFP5 might protect pancreatic β-cells against high glucose-induced apoptosis. To determine whether TFP5 treatment had the same protective role as CIP, MIN6 cells were treated with TFP5 or SCB. After 24 h, the cells were starved overnight in glucose-free medium and then treated with 5 mM or 25 mM glucose for 24 h. Results showed that TFP5 exposure decreased the expression level of cleaved caspase 3, and the total caspase 3 level remained the same in cells treated with high glucose (Fig 4A and B, lane 4 compared to lane 3). In addition, these results were confirmed in the db/db mouse model. We found that elevated cleaved caspase 3 expression induced by high glucose exposure was decreased significantly upon TFP5 peptide exposure (Fig 4C and D, panel 3 compared to panel 2). These results indicate that TFP5 might protect pancreatic β-cells from high glucose-induced apoptosis.

TFP5 protected pancreatic β-cells by reducing inflammation

To further explore the potential protection mechanisms induced by TFP5 exposure in pancreatic β-cells and db/db mice. As before, MIN6 cells were treated with TFP5 or SCB. After 24 h, the cells were starved overnight in glucose-free medium and then treated with 5 mM or 25 mM glucose for 24 h. Given that inflammation is one of the main pathologic features of T2DM, we used western blot analysis to detect the effects of TFP5 on inflammatory factors. Results showed that TGF-β, TNF-α, and IL-1β expression levels were significantly increased in cells treated with high glucose, but was decreased significantly upon TFP5 peptide exposure (Fig 5A). The increased expression of cytokines in the pancreatic β-cells was significantly reduced upon TFP5 treatment(Fig 5B-D). These results indicate that TFP5 might protect pancreatic β-cells from high glucose-induced inflammation. In addition, these results were confirmed in the db/db mouse model. Injection paradigm was as described above in db/db mice. Purified pancreatic islet tissue was processed for Western blotting to detect the effects of TFP5 on inflammatory factors. We found that TGF-β, TNF-α, and IL-1β expression levels were significantly increased in the pancreatic islets of db/db+SCB mice but were significantly decreased in those of db/db +TFP5-treated mice (Fig 5E). The increased expression of cytokines in the db/db mice was significantly reduced by 30%-50% upon TFP5 treatment (Fig 5F-H).

The inhibition of inflammation by TFP5 in pancreatic islets

To confirm the function of TFP5 in inhibiting inflammation, immunofluorescence staining was performed to detect the expressions of inflammatory cytokines in pancreatic tissue (Fig 6A). It showed that the expressions of TGF-β, TNF-α, and IL-1β significantly increased in the db/db + SCB mice, while the expression of inflammatory cytokines was decreased by 50% in the pancreatic tissues of db/db mice treated with TFP5 (Fig 6B-D).

T2DM accounts for more than 90% of adult diabetes cases and severely affects health and life worldwide. Insulin resistance is the major cause of chronic hyperglycemia in diabetic patients. Currently, therapeutic interventions targeting T2DM are largely limited to control of hyperglycemia. Therefore, exploring promising therapies for T2DM is extremely urgent.

Recent findings strongly suggest that Cdk5-mediated phosphorylation, such as PPARgamma and sirt1 phosphorylation, may be involved in the pathogenesis of insulin resistance in diabetes²³. Here, we β-cells from glucose toxicity and act as a therapeutic candidate for T2DM therapy.

T2DM and Alzheimer's diseases are degenerative diseases, and their incidence continues to increase globally²³. Although insights into the pathogenesis of these diseases are limited, similarities in the pathological changes of brain neurons and pancreatic β-cells have been reported^24–26. Previous studies have indicated that neuronal dysfunction in AD patients (Alzheimer’s disease patients) is partly linked to hyperactivity of multifunctional Cdk5 kinase. It has been proposed that under neurotoxic conditions, p35, the normal regulator of Cdk5, is cleaved into p25 and a p10 fragment by calpain (a calcium-dependent protease)^27,28. Cdk5 complexed with p25 is stabilized, hyperactivated, and involved in the hyperphosphorylation of tau, causing inflammation and amyloidosis, and eventually leading to neuronal death^16,29. Thus, it is reasonable to speculate that regulating Ckd5/p35 is a promising pathway for T2DM therapy.

In previous studies, Cdk5 and p35 have been identified in pancreatic islets and β-cells and have been shown to play important roles in insulin secretion^30–32. We hypothesized that the model of activation of Cdk5/p25 in neurons is the same in stressed pancreatic β-cells and is critical for reduced insulin secretion and decreased apoptosis rates. Our previous studies have shown that long-term exposure of MIN6 cells to a high concentration of glucose can trigger p25 expression, which causes Cdk5 hyperactivity, induces apoptosis, and inhibits insulin secretion^11,12. These results indicated that aberrant Cdk5 activity is involved in the pathogenesis of T2DM. Our data also demonstrated that the Cdk5 inhibitory peptide CIP can specifically inhibit Cdk5/p25 activity, protect β-cells from apoptosis, and restore insulin secretion in MIN6 cells without affecting the activity of endogenous p35 or other kinase⁸. These data suggest that inhibition of Cdk5 pathological hyperactivity might be an efficacious approach to T2DM therapy. Although several potent chemical inhibitors of Cdk5 have been identified and studied^10,33,34, most of these inhibitors compete for ATP binding. Therefore, these compounds lack specificity and induce serious toxic side effects, since all kinases are equally dependent on ATP binding. As a therapeutic agent, Although CIP specifically inhibits Cdk5/p25 activity, it is not a small molecule by weight, that cannot penetrate cells, thus cannot cause higher side effects either. P5, a much smaller peptide derived from CIP, was previously identified as a more effective Cdk5/p25 inhibitor than CIP³⁴. However, it requires a carrier for transfection into cells. To overcome the low transfection rates and carcinogenicity of viral vector transfection, we used TFP5 in this study. TFP5 is a modified P5 that readily penetrates cells and shows an inhibitory effect equal to that of virally infected p5 on Cdk5/p25-induced pathology of stressed MIN6 cells in vitro and db/db pancreas tissues in vivo following i.p. treatment. In our previous studies, we confirmed that the application of TFP5 can reduce the blood glucose and weight of the db/db mice³⁵.

Furthermore, the potential mechanism of the effect of TFP5 on T2DM treatment was also explored. Interestingly, we found that inflammation, which is a critical pathogenic fatctor of pancreatic islet dysfunction, was significantly inhibited in T2DM. Compelling evidence shows that the apoptosis of pancreatic cells is associated with inflammation and that targeting inflammation is a potential approach to the treatment of T2DM³⁶. Thus, inhibition of inflammation-related apoptosis of pancreatic cells is a potential mechanism of TFP5 treatment.

However, there are some limitations to this study. The results reflect only short-term TFP5 exposure. Therefore, long-term exposure to TFP5 is required to assess its effects in vivo, including the effect on blood biochemical indicators, such as the levels of insulin and glycosylated hemoglobin, and should therefore be a goal of further study. The molecular mechanism of the effects of TPF5 on inflammation should also be investigated in future studies.

In this study, we explored the inhibitory function of TFP5 on the aberrant activation of Cdk5/p25 and the regulation of insulin secretion in vivo and in vitro. We demonstrated that TFP5 can protect MIN6 cells from high glucose-induced apoptosis, reduce inflammatory stress in vitro and attenuate diabetes in db/db mice by reducing the hyperactivation of Cdk5 in vivo. These data suggest that future studies of TFP5 can provide information on the mechanisms of drug action as a means of gaining further insight into the etiology of diabetes.

Acknowledgements

We thank Dr. Philip Grant for carefully reading and editing the manuscript. We also thank Dr. Varsha Shukla and Dr. Yong-Ning Huang for support with the animal experiments.

Authors’ contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; SYL, SLC contributed to conception and design, data acquisition, drafting the article, final approval of the version to be published; HYL, XZ, LB contributed to conception and design, data analysis and interpretation, drafting the article;JE, BL, XML, GQZ, XB drafted the article or critically revising it for important intellectual content; YLZ, HCP drafted the article or critically revising it for important intellectual content.

Funding

This work was supported by the following grant sponsors: National Natural Science Foundation of China [grant numbers 81460161, 81860136]; Natural Science Foundation of Ningxia Province [ grant numbers NZ14160, NZ17186]; The Key Research and Development Program of Ningxia Province Region projects [grant numbers 2018BFG0210] (Eastern and Western collaboration program), and an NIH intramural research grant (a collaboration with NINDS/NIH). These research supporters played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary materials.

Ethics approval and consent to participate

It is confrmed that the study was carried out in compliance with the ARRIVE guidelines. All animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communication Disorders (NINDS/NIDCD) (protocol is ASP: 1231-11) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (protocol K058-KDB-10) in Bethesda, Maryland. The db/db mice and C57BL/6J mice (8-weeks-old males) were obtained from Jackson Laboratory (Bar Harbor, ME, USA).

Consent for publication

Not applicable.

Competing interests

The authors declared no potential conflicts of interest.

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No competing interests reported.

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TFP5, a Peptide Derived From Cdk5 Activator p35, Protects Pancreatic β-Cells From Glucose Toxicity

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