Targeting β-Cell Plasticity: A Promising Approach for Diabetes Treatment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this review article, the authors reviewed and discussed the mechanisms behind β-cell dedifferentiation and trans-differentiation and provided insights into mitigating these processes by identifying new therapeutic targets, medications, and treatments.

Comments:

This is an interesting review article. The reviewer has some concerns as follows:

1.     This manuscript for reviewing the role of β-cell plasticity in diabetes treatment is interesting and novel. The references are generally appropriate and adequate to related and previous work. However, the introduction can be strengthened on the previously related literature, such as Honzawa and Fujimoto, 2021 (The Plasticity of Pancreatic β-Cells. Metabolites. 2021 Apr 2;11(4):218. doi: 10.3390/metabo11040218).

2.     In page 32, "see Box 1", but where is the box 1? In lines 63-91, there are only text descriptions here without box. Please check this issue.

3.     In Table 1, please confirm the completeness of the thick black line throughout the table.

4.     Regarding the format of literature citations within the manuscript, please revise it according to the Journal’s format guideline.

 

5.    Overall, this manuscript is well written, but some revisions are recommended.

 

Author Response

Dear Reviewer,

I hope this message finds you well.

I am pleased to submit the revised version of our manuscript titled “Targeting β-cell Plasticity: A Promising Approach for Diabetes Treatment” for your consideration. We have carefully addressed all the comments and suggestions provided by you and the other reviewers.

Below, you will find detailed responses along with corresponding revisions/corrections highlighted or tracked changes in the resubmitted files.

We believe these revisions have significantly strengthened the manuscript, and we are grateful for the constructive feedback that has guided these improvements.

Thank you for your time and effort in reviewing our work. We look forward to your feedback.

Best regards,

Esmaeel

 

In this review article, the authors reviewed and discussed the mechanisms behind β-cell dedifferentiation and trans-differentiation and provided insights into mitigating these processes by identifying new therapeutic targets, medications, and treatments. 

Comments:

This is an interesting review article. The reviewer has some concerns as follows:

1.This manuscript for reviewing the role of β-cell plasticity in diabetes treatment is interesting and novel. The references are generally appropriate and adequate to related and previous work. However, the introduction can be strengthened on the previously related literature, such as Honzawa and Fujimoto, 2021 (The Plasticity of Pancreatic β-Cells. Metabolites. 2021 Apr 2;11(4):218. doi: 10.3390/metabo11040218). 

Answer: The following part was added to introduction

“The concept of β-cell dedifferentiation was introduced by Gershengorn et al. in 2004 after they observed that β-cells in human pancreatic islets vanished and were replaced by fibroblast-like precursor cells. They also noted that these cells could redifferentiate into α and β-cells under certain growth conditions. This concept was further confirmed by the findings of Dor et al. (2007), Accili et al. (2012), Wang et al. (2014), and Cinti et al. (2016) ¹. β-cell dedifferentiation is characterized by the appearance of progenitor-like biomarkers such as Ngn3, Oct4, Aldh1a3, Nanog, L-Myc, and Sox9, and the disappearance or downregulation of β-cell-enriched biomarkers, including MafA, insulin, Nkx6.1, GLUT2 and Pdx-1. This shift results in β-cells losing their functionality, which can ultimately lead to the incidence of diabetes ^1,2. Similar to rodents, β-cell dedifferentiation occurs in human pancreatic islets. However, unlike in rodents, Ngn3 is not upregulated in human pancreatic islets during dedifferentiation. Aldh1a3, on the other hand, is upregulated in both rodents and humans ³. Transdifferentiation of β-cells into other pancreatic cell types such as a and d cells are another form of β-cell plasticity that occurs in both rodents and humans, much like dedifferentiation ². Transdifferentiation of β-cells into α-cells is typically marked by the loss of β-cell differentiation markers and the acquisition of α-cell biomarkers, such as glucagon and the Arx transcription factor ⁴. Transdifferentiation of pancreatic endocrine cells, including α and δ cells, into β-cells is observed, alongside reports of pancreatic exocrine cells, like acinar cells, also transdifferentiating into β-cells. This suggests a novel approach for treating diabetes ⁵.

Dedifferentiation and transdifferentiation of β-cells are observed across all diabetes types. In T1D, inflammatory reactions are pivotal in triggering these processes, inducing ER stress and modifying gene expression and protein synthesis in β-cells ⁶. T2D, metabolic stresses linked with obesity, insulin resistance, and high levels of glucose and lipids similarly contribute to β-cell dedifferentiation and transdifferentiation ⁷. "

 

2.In page 32, "see Box 1", but where is the box 1? In lines 63-91, there are only text descriptions here without box. Please check this issue.

Answer: The box was highlighted for easier identification.

3.In Table 1, please confirm the completeness of the thick black line throughout the table.

Answer: The table has been updated and the necessary revisions have been implemented.

5.Overall, this manuscript is well written, but some revisions are recommended.

Answer: Some revisions have been done on the paper.

 

 

 

1            Honzawa, N. & Fujimoto, K. The plasticity of pancreatic β-cells. Metabolites 11, 218 (2021).

2            Son, J. & Accili, D. Reversing pancreatic β-cell dedifferentiation in the treatment of type 2 diabetes. Experimental & Molecular Medicine 55, 1652-1658 (2023).

3            Cinti, F. et al. Evidence of β-cell dedifferentiation in human type 2 diabetes. The Journal of Clinical Endocrinology & Metabolism 101, 1044-1054 (2016).

4            Gao, T. et al. Pdx1 maintains β cell identity and function by repressing an α cell program. Cell metabolism 19, 259-271 (2014).

5            Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. nature 455, 627-632 (2008).

6            Eizirik, D. L. et al. The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines. PLoS genetics 8, e1002552 (2012).

7            Efrat, S. Beta-cell dedifferentiation in type 2 diabetes: concise review. Stem Cells 37, 1267-1272 (2019).

 

Reviewer 2 Report

Comments and Suggestions for Authors

The review manuscript by Gojani et al titled: "Targeting β-cell Plasticity: A Promising Approach for Diabetes Treatment" is an interesting review that discusses various molecular mechanisms whereby beta-cell responses could be modulated in manners that are beneficial to Diabetes treatment. The work is well organized and covers an interesting topic with clinical significance. The reviewer would like to raise some points for authors to consider. 

1. It would be interesting to discuss beta cell plasticity in the context of immunity especially given the relationship between immunity and T1DM.

2. ATF4 is a transcription factor that not only responds to ER stress but also ISR. This is something that needs to be pointed out since nutritional stress in the form of essential amino acid reaction may be a way in which caloric restriction may be mimicked and research in the field also involving mTOR has suggested that approach.

3. Body composition especially muscle mass and amino acid intake seems to extend an interplay with beta cells and insulin resistance. It is interesting to briefly discuss this viewpoint a potential manuscript that may help to that end is the following: 

Maykish, A.; Sikalidis, A.K. Utilization of Hydroxyl-Methyl Butyrate, Leucine, Glutamine and Arginine Supplementation in Nutritional Management of Sarcopenia—Implications and Clinical Considerations for Type 2 Diabetes Mellitus Risk Modulation. J. Pers. Med. 2020, 10, 19. https://doi.org/10.3390/jpm10010019

4. Consider discussing the diet as a whole in relation to beta-cell plasticity as well as physical activity. For example physical activity especially regular aerobic even more so when it produces weight loss tends to improve insulin resistance in part due to increased mitochondrial mass. These concepts would be helpful to be discussed because they provide also some avenues of thought towards recommendations and potentially management/clinical practices. 

Author Response

Dear Reviewer,

I hope this message finds you well.

I am pleased to submit the revised version of our manuscript titled “Targeting β-cell Plasticity: A Promising Approach for Diabetes Treatment” for your consideration. We have carefully addressed all the comments and suggestions provided by you and the other reviewers.

Below, you will find detailed responses along with corresponding revisions/corrections highlighted or tracked changes in the resubmitted files.

We believe these revisions have significantly strengthened the manuscript, and we are grateful for the constructive feedback that has guided these improvements.

Thank you for your time and effort in reviewing our work. We look forward to your feedback.

Best regards,

Esmaeel

 

 

The review manuscript by Gojani et al titled: "Targeting β-cell Plasticity: A Promising Approach for Diabetes Treatment" is an interesting review that discusses various molecular mechanisms whereby beta-cell responses could be modulated in manners that are beneficial to Diabetes treatment. The work is well organized and covers an interesting topic with clinical significance. The reviewer would like to raise some points for authors to consider. 

1.It would be interesting to discuss beta cell plasticity in the context of immunity especially given the relationship between immunity and T1DM.

Answer:  “The following part was added to the paper” The ablation of b-cells, which is associated with T1D, induces the transdifferentiation of a-cells into b-cells ¹. this indicates that cell plasticity could be used as a potential treatment for T1D. it is worth mentioning that in the context of T1D the immunity system does not attack to a-cells ². Insulitis, inflammation of b-cells, is most associated with T1D. under this condition b-cells are usually exposed to verity of cytokines, including IFNg, IL-1b, TNFa and IL-17, all of which are able to trigger ER stress-relating to b-cells reverse dedifferentiation ³. There are some evidence indicating that the direct interaction of autoimmune T cells with β cells may be responsible but not enough for changes in β cell identity in T1D patients ⁴. However, the islet microenvironment in T1D has been reported to generate CD4 T cells that target proinsulin, suggesting Proinflammatory mediators from the islets may cause β cell dedifferentiation in individuals with T1D ⁵. Moreover, it has been found that exposing β cells to double-stranded RNA, mimicking a viral infection that could trigger T1D, reduces the expression of β cells enriched genes like MAFA and INS while increasing progenitor markers. This effect is driven by NF-kB within the beta cells and interferon alpha released by neighboring cells ^6,7. The ablation of b-cells, which is associated with T1D, induces the transdifferentiation of a-cells into b-cells ¹. this indicates that cell plasticity could be used as a potential treatment for T1D. it is worth mentioning that in the context of T1D the immunity system does not attack to a-cells ².”

2. ATF4 is a transcription factor that not only responds to ER stress but also ISR. This is something that needs to be pointed out since nutritional stress in the form of essential amino acid reaction may be a way in which caloric restriction may be mimicked and research in the field also involving mTOR has suggested that approach.

Answer: The following part was added to the paper

“ATF4 is also a key player in integrated stress response (ISR). ISR is a complex cellular signaling pathway in eukaryotic cells that manages and mitigates various stress stimuli. Essential for maintaining cellular homeostasis, the ISR can be triggered by intrinsic and extrinsic factors such as hypoxia, amino acid deprivation, glucose deprivation, viral infections, and oxidative stress ⁸. The ISR is driven by the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four kinases: PERK, GCN2, PKR, or HRI. This phosphorylation reduces global protein synthesis, conserving resources and alleviating the load on the protein-folding machinery. At the same time, it promotes the translation of specific genes, such as the transcription factor ATF4, which, in turn, aids in cellular adaptation to stress by upregulating genes involved in both protein folding and amino acid metabolism ⁹. The activation of ISR in b-cells is found to regulate oxidative stress, a key player in b-cell dedifferentiation. this supports with the observations indicating that the phosphorylation of eIF2α has protective impact on b-cells against oxidative stress caused by insulin resistance, through a ATF4-dependent mechanism ¹⁰. Amino acid deficiency, another inducer of the ISR, stimulates the binding of uncharged tRNA to GCN2, leading to the autophosphorylation of GCN2 and subsequent phosphorylation of eIF2α ¹¹. This phosphorylation of eIF2α is associated with an increase in ATF4 levels, which upregulates genes involved in the synthesis and metabolism of amino acids ¹². he upregulation of ATF4 also increases the expression of sesterin2, thereby inhibiting mTORC1. It is noteworthy that chronic activation of mTORC1 during the insulin resistance phase negatively impacts β-cell mass and proliferation¹³.

In summary, the ATF4 upregulation whether through ER stress or through ISR has great impact on b-cell fate.“

3. Body composition especially muscle mass and amino acid intake seems to extend an interplay with beta cells and insulin resistance. It is interesting to briefly discuss this viewpoint a potential manuscript that may help to that end is the following: 

Maykish, A.; Sikalidis, A.K. Utilization of Hydroxyl-Methyl Butyrate, Leucine, Glutamine and Arginine Supplementation in Nutritional Management of Sarcopenia—Implications and Clinical Considerations for Type 2 Diabetes Mellitus Risk Modulation. J. Pers. Med. 2020, 10, 19. https://doi.org/10.3390/jpm10010019

Answer: The following part was added to the paper

“ Muscle mass reduction can significantly impact glucose metabolism and increase the risk of diabetes. Since approximately 80% of glucose clearance occurs in muscle tissue under normal glycemic conditions, any decrease in muscle mass or quality can adversely affect blood glucose regulation, leading to hyperglycemia, insulin resistance, metabolic stress, and impaired β-cell function and maintenance. Consequently, a correlation has been observed between sarcopenia (age-related loss of muscle mass and function) and the incidence T2D. To address this issue, dietary supplements containing specific amino acids such as HMB (β-hydroxy β-methylbutyrate), leucine, glutamine, and arginine have been proposed as potential interventions. These supplements may help prevent sarcopenia and, consequently, reduce the risk of developing insulin resistance and T2D. By maintaining muscle mass and function, these amino acids could support better glucose regulation and metabolic health, particularly in aging populations or those at risk for muscle loss ¹⁴. “

3.Consider discussing the diet as a whole in relation to beta-cell plasticity as well as physical activity. For example physical activity especially regular aerobic even more so when it produces weight loss tends to improve insulin resistance in part due to increased mitochondrial mass. These concepts would be helpful to be discussed because they provide also some avenues of thought towards recommendations and potentially management/clinical practices.

Answer: The following part was added to the paper:

“Physical activity has been demonstrated to enhance pancreatic β-cell function and may play a crucial role in preventing or reversing β-cell dedifferentiation in the context of T2D. Exercise promotes β-cell function in T2D patients by increasing β-cell mass, improving glucose sensing, enhancing insulin secretion, and augmenting insulin content ^15,16. The protective effects of exercise on β-cells are mediated through multiple mechanisms: 1. Reducing Metabolic Stress: Physical activity facilitates increased glucose uptake in peripheral tissues, thereby lowering blood glucose levels and reducing the metabolic load on β-cells¹⁷. 2. Alleviating ER and Oxidative Stress: Exercise may attenuate ER stress and oxidative stress in β-cells, which are critical contributors to β-cell dysfunction. 3. Modulating Inflammation: Physical activity has the potential to modulate the immune environment of the pancreas, thereby reducing inflammatory processes that contribute to β-cell damage ¹⁶. IL-6, produced by muscle cells during contraction, is associated with elevated levels of IL-1RA, IL-10, and GLP-1, which regulate abdominal fat lipolysis and promote β-cell protection, growth, and mass ^18,19.  Additionally, certain myokines like angiopoietin and osteoprotegerin, specifically produced by triceps, exhibit anti-inflammatory effects ²⁰. Exercise can also increase levels of adiponectin, an adipokine synthesized by adipose tissue with anti-inflammatory properties ²¹. Adiponectin has been shown to induce a shift of M1 (proinflammatory) macrophages to the M2 (anti-inflammatory) phenotype by reducing proinflammatory cytokines, including TNFα ²².  Notably, inflammatory responses by macrophages resident in the pancreas play a critical role in the fate of β-cells ²³.

Besides IL-6, exercise also positively influences brain-derived neurotrophic factor (BDNF) ²⁴, and C-X3-C motif chemokine ligand 1 (CX3CL1) ²⁵. Similar to GLP-1, these factors have beneficial effects on insulin secretion, glucose-stimulated insulin secretion (GSIS), and β-cell function ^{24 25}.

Irisin, an exerkine released by muscle tissue ^26,27, osteocalcin, produced by bone marrow ^28,29, and FGF21 from hepatic tissues ³⁰, are three exerkines elevated during exercise known to enhance β-cell proliferation, function, and maintenance.

β-cell Dedifferentiation and Exercise: Although direct evidence linking exercise to the prevention or reversal of β-cell dedifferentiation is limited, various studies suggest that exercise may support the maintenance of β-cell identity and function. In animal models, voluntary running has been shown to prevent β-cell failure in susceptible islets³¹. Apelin, an exerkine that is involved in various physiological processes and produced and secreted by various tissues, including adipose tissue, heart, and skeletal muscle, has been shown to reverse b-cell dedifferentiation ³². Exercise is also linked to the activation of the GABAergic system ³³. There is evidence suggesting that GABA and activation of the GABAergic system might trigger the conversion of α-cells into β-cells, presenting a potential treatment avenue for Type 1 diabetes (T1D) ^34,35.

Combined Effects of Diet and Exercise: The synergistic effects of dietary intervention and physical exercise on β-cell health are notable. Both strategies can alleviate metabolic stress on β-cells, and their combination, particularly through diet-induced weight loss, may enhance the benefits of each intervention. This dual approach has the potential to mitigate the adverse effects of T2D on both β-cells identity ¹⁶.”

 

 

1            Thorel, F. et al. Conversion of adult pancreatic α-cells to β-cells after extreme β-cell loss. Nature 464, 1149-1154 (2010).

2            Brissova, M. et al. α cell function and gene expression are compromised in type 1 diabetes. Cell reports 22, 2667-2676 (2018).

3            Eizirik, D. L. et al. The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines. PLoS genetics 8, e1002552 (2012).

4            Md Moin, A. S. et al. Increased hormone-negative endocrine cells in the pancreas in type 1 diabetes. The Journal of Clinical Endocrinology & Metabolism 101, 3487-3496 (2016).

5            Michels, A. W. et al. Islet-derived CD4 T cells targeting proinsulin in human autoimmune diabetes. Diabetes 66, 722-734 (2017).

6            Diedisheim, M. et al. Modeling human pancreatic beta cell dedifferentiation. Molecular metabolism 10, 74-86 (2018).

7            Oshima, M. et al. Virus-like infection induces human β cell dedifferentiation. JCI insight 3 (2018).

8            Kulkarni, A., Muralidharan, C., May, S. C., Tersey, S. A. & Mirmira, R. G. Inside the β cell: molecular stress response pathways in diabetes pathogenesis. Endocrinology 164, bqac184 (2023).

9            Pakos‐Zebrucka, K. et al. The integrated stress response. EMBO reports 17, 1374-1395 (2016).

10          Koromilas, A. E. M (en) TORship lessons on life and death by the integrated stress response. Biochimica Et Biophysica Acta (BBA)-General Subjects 1863, 644-649 (2019).

11          Zaborske, J. M. et al. Genome-wide Analysis of tRNA Charging and Activation of the eIF2 Kinase Gcn2p*♦. Journal of Biological Chemistry 284, 25254-25267 (2009).

12          Kilberg, M. S., Shan, J. & Su, N. ATF4-dependent transcription mediates signaling of amino acid limitation. Trends in Endocrinology & Metabolism 20, 436-443 (2009).

13          Kanno, A. et al. GCN2 regulates pancreatic β cell mass by sensing intracellular amino acid levels. JCI insight 5 (2020).

14          Maykish, A. & Sikalidis, A. K. Utilization of hydroxyl-methyl butyrate, leucine, glutamine and arginine supplementation in nutritional management of sarcopenia—implications and clinical considerations for type 2 diabetes mellitus risk modulation. Journal of Personalized Medicine 10, 19 (2020).

15          Lyngbaek, M. P. et al. The effects of different doses of exercise on pancreatic β-cell function in patients with newly diagnosed type 2 diabetes: study protocol for and rationale behind the “DOSE-EX” multi-arm parallel-group randomised clinical trial. Trials 22, 244 (2021).

16          Lv, C. et al. β-cell dynamics in type 2 diabetes and in dietary and exercise interventions. Journal of Molecular Cell Biology 14, mjac046 (2022).

17          Chow, L. S. et al. Exerkines in health, resilience and disease. Nature Reviews Endocrinology 18, 273-289 (2022).

18          Wedell-Neergaard, A.-S. et al. Exercise-induced changes in visceral adipose tissue mass are regulated by IL-6 signaling: a randomized controlled trial. Cell metabolism 29, 844-855. e843 (2019).

19          Lehrskov, L. L. et al. Interleukin-6 delays gastric emptying in humans with direct effects on glycemic control. Cell metabolism 27, 1201-1211. e1203 (2018).

20          Rutti, S. et al. Angiogenin and Osteoprotegerin are type II muscle specific myokines protecting pancreatic beta-cells against proinflammatory cytokines. Scientific reports 8, 10072 (2018).

21          Becic, T., Studenik, C. & Hoffmann, G. Exercise increases adiponectin and reduces leptin levels in prediabetic and diabetic individuals: systematic review and meta-analysis of randomized controlled trials. Medical sciences 6, 97 (2018).

22          Ohashi, K. et al. Adiponectin promotes macrophage polarization toward an anti-inflammatory phenotype. Journal of Biological Chemistry 285, 6153-6160 (2010).

23          Ghasemi-Gojani, E., Kovalchuk, I. & Kovalchuk, O. Cannabinoids and terpenes for diabetes mellitus and its complications: from mechanisms to new therapies. Trends in Endocrinology & Metabolism (2022).

24          Fulgenzi, G. et al. Novel metabolic role for BDNF in pancreatic β-cell insulin secretion. Nature communications 11, 1950 (2020).

25          Strömberg, A. et al. CX3CL1—a macrophage chemoattractant induced by a single bout of exercise in human skeletal muscle. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 310, R297-R304 (2016).

26          Liu, S. et al. Effects and underlying mechanisms of irisin on the proliferation and apoptosis of pancreatic β cells. PloS one 12, e0175498 (2017).

27          Natalicchio, A. et al. The myokine irisin is released in response to saturated fatty acids and promotes pancreatic β-cell survival and insulin secretion. Diabetes 66, 2849-2856 (2017).

28          Wei, J., Hanna, T., Suda, N., Karsenty, G. & Ducy, P. Osteocalcin promotes β-cell proliferation during development and adulthood through Gprc6a. Diabetes 63, 1021-1031 (2014).

29          Kover, K. et al. Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions. Biochemical and biophysical research communications 462, 21-26 (2015).

30          Cui, X. et al. Pancreatic alpha cell glucagon–liver FGF21 axis regulates beta cell regeneration in a mouse model of type 2 diabetes. Diabetologia 66, 535-550 (2023).

31          Curran, M. et al. The benefits of physical exercise for the health of the pancreatic β‐cell: A review of the evidence. Experimental Physiology 105, 579-589 (2020).

32          Tanday, N., Irwin, N., Moffett, R. C., Flatt, P. R. & O'Harte, F. P. Beneficial actions of a long‐acting apelin analogue in diabetes are related to positive effects on islet cell turnover and transdifferentiation. Diabetes, Obesity and Metabolism 22, 2468-2478 (2020).

33          Li, H.-q. & Spitzer, N. C. Exercise enhances motor skill learning by neurotransmitter switching in the adult midbrain. Nature communications 11, 2195 (2020).

34          Ben-Othman, N. et al. Long-term GABA administration induces alpha cell-mediated beta-like cell neogenesis. Cell 168, 73-85. e11 (2017).

35          Li, J. et al. Artemisinins target GABAA receptor signaling and impair α cell identity. Cell 168, 86-100. e115 (2017).

 

Reviewer 3 Report

Comments and Suggestions for Authors

The malfunction of β-cells is characterized by dedifferentiation and transdifferentiation primarily. The present paper seeks to analyze the complex processes that drive β-cell dedifferentiation. Please address the following issues.

1. The process of β-cell dedifferentiation and transdifferentiation has to be explained clearly.

2. Figure 1 requires the inclusion of the data source. Correction of mTOR is required at line 58 of the legends.

3. The present study does not provide any information about the harm caused to β-cells by chemicals. For what reason?

4. In line 1043, diosmin is suggested as a possible inhibitor of DYRK1A, although references are needed to substantiate this claim.

5. To summarize, studies have shown that blocking the glucagon receptor, either on its own or in conjunction with GLP-1 agonism and DYRK1A inhibition, has been successful in repairing destroyed β-cells in diabetes animals. However, other references are needed to corroborate these findings.

6. The present report may be strengthened by its limitations.

Author Response

 

Dear Reviewer,

I hope this message finds you well.

I am pleased to submit the revised version of our manuscript titled “Targeting β-cell Plasticity: A Promising Approach for Diabetes Treatment” for your consideration. We have carefully addressed all the comments and suggestions provided by you and the other reviewers.

Below, you will find detailed responses along with corresponding revisions/corrections highlighted or tracked changes in the resubmitted files.

We believe these revisions have significantly strengthened the manuscript, and we are grateful for the constructive feedback that has guided these improvements.

Thank you for your time and effort in reviewing our work. We look forward to your feedback.

Best regards,

Esmaeel

 

1.The process of β-cell dedifferentiation and transdifferentiation has to be explained clearly.

Answer: A section regarding β-cell dedifferentiation and transdifferentiation history, along with a brief overview of the processes of β-cell dedifferentiation and transdifferentiation, has been added to the introduction.

2. Figure 1 requires the inclusion of the data source. Correction of mTOR is required at line 58 of the legends.

Answer: Figure 1 has been created based on all the information regarding the mechanism of β-cell dedifferentiation and transdifferentiation included in the paper. An explanation caption has been added to the caption of figure.

3. The present study does not provide any information about the harm caused to β-cells by chemicals. For what reason?

Answer: The following part was added to the paper:

“Compounds like ethanol, STZ, glucocorticoids, and environmental pollutants impact different aspects of diabetes, including the dedifferentiation and transdifferentiation of β-cells.

Alcohol addiction is a pervasive global issue, responsible for 5.9% of all deaths worldwide. Regular alcohol consumption heightens the risk of metabolic diseases, including T2DM, compared to non-drinkers. Chronic alcohol consumption results in reduced insulin levels, β-cell apoptosis, and decreased β-cell mass, along with upregulated FGF21 and its receptor mRNA levels, indicating FGF21 resistance. Short-term ethanol exposure does not immediately impair β-cell function, but prolonged exposure progresses to significant dysfunction. Chronic binge drinking is linked to reductions in insulin, the insulin-to-glucagon ratio, GSIS response and NKX2.2 and PDX1 levels, highlighting alcohol's impact on β-cell dedifferentiation ¹. Furthermore, Additionally, there is a negative correlation between alcohol consumption and blood adiponectin levels ², which dimmish insulin expression and secretion ³ and also β-cell survival ⁴.

STZ is a naturally occurring compound used to induce diabetes in experimental models by selectively destroying insulin-producing β-cells in the pancreas through DNA alkylation and oxidative stress. It targets β-cells specifically due to their high expression of the GLUT2 glucose transporter, which facilitates STZ uptake and leads to cell death. However, some β-cells with lower GLUT2 expression survive the initial damage but experience long-term functional and phenotypic changes, remaining in a low-GLUT2 state  It has been found that some interventions such as islet transplantation and glucose normalization can partially restore β-cell function and GLUT2 expression^5,6.  STZ treatment potentially promotes β-cell regeneration and transdifferentiation in pancreatic islets. This includes the emergence of vimentin+/MafB+ cells, which can differentiate into insulin-producing β-like cells, and the possibility of α-cells transdifferentiating into β-cells under specific conditions ^7,8.

Glucocorticoids, such as dexamethasone and hydrocortisone, can induce β-cell dedifferentiation through various mechanisms, including triggering ER stress, changes in gene expression⁹, impaired β-cell glucose metabolism and reduced sensitivity to glucose stimulation, leading to altered insulin secretion patterns^10,11and decreased GSIS⁴⁶. Excess glucocorticoid exposure during fetal development can permanently alter β-cell mass and function, resulting in long-term consequences for glucose homeostasis ¹².

Bisphenols (BPs) and phthalates are classes of industrial chemicals widely used in the production of plastics and other consumer products. These chemicals have been linked to higher diabetes rates, particularly affecting β-cells. Exposure to these chemicals disrupts electrical activity, mitochondrial function, gene expression and β-cell function, leading to altered GSIS and impaired β -cell proliferation ¹³. Studies show mixed results, with some indicating heightened insulin secretion and content, while others suggest diminished GSIS or insulin levels. These substances can also impact β-cell survival, with higher doses or prolonged exposures often leading to increased cell death ¹⁴. BPs induce endoplasmic reticulum stress, mitochondrial dysfunction, and oxidative stress in β-cells, contributing to cellular damage and apoptosis ^15,16. Effects on β-cell proliferation vary, with some studies showing increased proliferation at lower doses¹⁷ and others observing reduced growth at higher concentrations ¹⁸. BPA acts as an endocrine disruptor, altering gene expression in β-cells during development and producing long-term metabolic disturbances. Early-life exposure to BPA increases β-cell mass and proliferation through estrogen receptor β (ERβ), resulting in hyperinsulinemia that precedes insulin resistance ^19,20.

Phthalates affect insulin secretion in β-cells variably depending on the duration and concentration of exposure. Acute high-concentration exposure increases basal insulin secretion (BIS) without affecting GSIS. In contrast, prolonged exposure typically inhibits both BIS and GSIS, indicating impaired insulin synthesis and β-cell function, often coupled with reduced β-cell survival and increased cellular stress ¹³.”

4. In line 1043, diosmin is suggested as a possible inhibitor of DYRK1A, although references are needed to substantiate this claim.

Answer: The reference was added to the text:

“Molecular docking analysis revealed diosmin, a flavonoid compound found in various plants such as Zanthoxylum chalybeum Engl., as a potential inhibitor of DYRK1A. In vitro experiments showed that the extract promoted cell viability and proliferation, particularly under conditions of palmitate exposure. However, in vivo results indicated only mild β-cell regenerative potential, suggesting further investigation is needed to understand the molecular interactions underlying these effects, particularly regarding diosmin's role as a DYRK1A inhibitor ²¹.”

5.To summarize, studies have shown that blocking the glucagon receptor, either on its own or in conjunction with GLP-1 agonism and DYRK1A inhibition, has been successful in repairing destroyed β-cells in diabetes animals. However, other references are needed to corroborate these findings.

Answer: The following part was added to the paper:

“The DREAM complex plays a pivotal role in maintaining quiescence in human β cells. Comprising transcriptionally repressive proteins, this complex forms in response to DYRK1A kinase activity, enforcing cellular quiescence. When DYRK1A activity is inhibited, DREAM subunits transition into the pro-proliferative MMB complex. Small molecule DYRK1A inhibitors stimulate replication in human β cells by shifting the DREAM complex from its repressive state to the proliferative MMB configuration ²².

Small-scale suppression of DYRK1A holds promise for pharmaceutical intervention in β-cell regeneration for diabetes, addressing a significant therapeutic gap. However, due to DYRK1A's involvement in critical signaling pathways, it is essential to maintain its levels similar to those found in healthy individuals. In silico tests have identified several plant-based compounds as potential ligands for β-cell regeneration. These compounds include 3-[6-(3-methyl-but-2-enyl)-1H-indolyl]-6-(3-methyl-but-2-enyl)-1H-indole, Lanceolatin B, Lysicamine, Pratorinine, Pratorimine, Lanceolatin A, Lanuginosine, Hippacine, (-)-Semiglabrin, Aegyptinone B, 3'-Prenylnaringenin, and 8-C-p-hydroxybenzylluteolin. Computational analyses suggest these compounds have good intestinal absorption and favorable drug-like properties, making them promising candidates for new medications, pending further in vivo validation ⁵.

Various synthetic small molecules have shown potential in promoting β-cell regeneration by inhibiting DYRK1A activity. AC27, a compound with superior selectivity compared to harmine, not only inhibits DYRK1A but also reduces its cellular levels, resulting in increased β-cell proliferation and enhanced insulin and C-peptide secretion in response to glucose. These effects were stable over a 12-day period and persisted after the inhibitor was withdrawn. AC27's benefits were validated in insulinoma cell lines, iPSC-derived β-cell organoids, and isolated mouse pancreatic islets, with its effects further enhanced by TGF-β inhibition. ²³.

Gua et al. conducted a chemical screen in zebrafish to identify small molecules that increase the number of incretin-expressing cells. One of the identified compounds, AZ Dyrk1B 33, was found to be a potent inhibitor of DYRKs and effectively boosted incretin+ cell populations in zebrafish. Moreover, it demonstrated improved glucose regulation in mouse models. The research suggests that AZ Dyrk1B 33 likely influences enteroendocrine cell differentiation through NFAT signaling pathways. Unlike DYRK1A inhibitors known to enhance β-cell proliferation, AZ Dyrk1B 33 did not affect β-cell mass in this study, highlighting its specific role in enteroendocrine cells regulated by DYRK1B ²⁴. 

While DYRK1A inhibitors such as AC27 hold potential for enhancing β-cell proliferation and function by targeting β-cell cycle pathways, significant obstacles persist. These include achieving β-cell specificity, refining evaluation techniques, and overcoming the low proliferation rates of human β-cells. To address these issues, structural modifications are needed to improve targeting and minimize off-target effects. Furthermore, integrating DYRK1A inhibitors with other anti-diabetic medications could enhance their therapeutic efficacy ²⁴.

Common DYRK1A inhibitors can induce β-cell proliferation but with low rates and limited specificity. It has been shown that combining any GLP1R agonist with any DYRK1A inhibitor results in a significant synergistic increase in human β-cell replication (5 to 6%), leading to an actual increase in human β-cell count. This combination did not cause β-cell dedifferentiation and was effective for both normal human β-cells and those derived from individuals with T2D ²⁸⁰.

GLP1 induces insulin-glucagon-positive cells in rat pancreatic islets, potentially indicating a to b cell transdifferentiation. Molecular analyses suggest GLP1 promotes b-cell function through the PI3K/AKT/FOXO1 pathway, enhancing PDX1 and MAFA expression while reducing MAFB, suggesting a mechanism for alpha cell inhibition and beta cell promotion by GLP1 ²⁵.

Treatment using REMD 2.59, a competitive human GCGR antagonist, lowered blood glucose levels while elevating plasma levels of glucagon and somatostatin. Notably, administration of a GCGR monoclonal antibody substantially increased α-cell and δ-cell populations in both normal and T1D mice. This increase in δ-cell mass was linked to enhanced proliferation and neogenesis originating from pancreatic ducts. Remarkably, the observed δ-to-β-cell trans-differentiation in T1D mice post-treatment suggests a promising avenue for restoring β-cell numbers ²⁶.

Additionally, it has been found that REMD 2.59 induces α-cell regression to progenitors, facilitating β-cell neogenesis, and upregulates regeneration-related genes in mouse islets. This highlights GCGR mAb's potential for β-cell regeneration in T2D, supporting α-to-β cell conversion and progenitor-driven β-cell formation ²⁷.

GCGR mAb treatment in PANIC-ATTAC mice, which triggers caspase-8–mediated apoptosis specifically within the pancreatic β-cell, stimulates α-cell to β-cell conversion, leading to sustained improvements in glycemic control and effectively curing diabetes in this model. While α-cell hyperplasia may pose challenges in T2D, in T1D, it offers a potential source for generating new insulin-producing cells ²⁸.

10-4 Other pathways involved in β-cell proliferation

In addition to the aforementioned methods to induce β-cell proliferation and regeneration, there are other approaches, such as inhibiting TBK1/IKKε and salt-inducible kinases (SIKs).

Xu et al found that TBK1/IKKε inhibitors (TBK1/IKKε-Is) as potent enhancers of β-cell regeneration in a zebrafish model of type 1 diabetes, validated across mammalian systems including human and rat β-cells, and STZ-induced diabetic mice. The mechanism involves TBK1/IKKε inhibition promoting β-cell proliferation via the cAMP-PKA-mTOR signaling axis through PDE3, highlighting a novel role for TBK1/IKKε in β-cell mass modulation. Notably, TBK1/IKKε-Is like amlexanox and PIAA selectively enhance β-cell proliferation without inducing proliferation in other cell types, showing promise for targeted therapy ²⁹.

An in vivo study by Charbord et al. identified HG-9-91-01(HG) as a β cell mitogen that enhances β cell proliferation in zebrafish, mouse, and human β cells by inhibiting salt-inducible kinases (SIKs), specifically SIK1–SIK3, members of the AMPK-related protein kinase family. In contrast to harmine, which targets DYRK1–DYRK4 and monoamine oxidase A, HG stimulates β cell proliferation through UPR activation via ATF6–IRE1 pathways. Despite showing specificity for SIK1, HG's pharmacokinetic limitations currently preclude its consideration as a drug candidate ³⁰.”

6.The present report may be strengthened by its limitations.

Answer: The following part was added to the paper:

“12. Limitations:

 

1. Lack of Review on Experimental Models: In this study, we did not review the experimental models and the methods used to model β-cell dedifferentiation and transdifferentiation. A comprehensive examination of the various approaches to model β-cell dedifferentiation and transdifferentiation in both in vitro and in vivo settings would be highly beneficial.
2. Limited Scope on Cell Plasticity: This review primarily focused on β-cell plasticity and did not cover the plasticity of other cell types. It would be valuable to briefly explore the plasticity of other cells such as hepatocytes, hepatic stellate cells, epidermal stem cells, melanocytes, astrocytes, intestinal stem cells, cardiac fibroblasts, club cells, podocytes, and others. Investigating the plasticity of these diverse cell types could offer insights into the broader applications and implications of cellular plasticity.
3. Context of Cell Plasticity in Other Diseases: While this review centered on β-cell plasticity, it would have been beneficial to study cell plasticity in the context of other diseases such as liver diseases, neurodegenerative diseases, and cancer. Understanding how cellular plasticity manifests and contributes to the pathology of these diseases could inform the development of new therapeutic strategies and enhance our knowledge of disease progression and treatment.”

 

 

 

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made a reasonable effort to address the reviewer's points. I believe the manuscript has improved and I recommend acceptance for publication.

Comments on the Quality of English Language

English language is fine overall.