Hypoxia causes pancreatic β-cell dysfunction by activating a transcriptional repressor BHLHE40

  1. Tomonori Tsuyama
  2. Yoshifumi Sato
  3. Tatsuya Yoshizawa
  4. Takaaki Matsuoka
  5. Kazuya Yamagata

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Abstract

Hypoxia can occur in pancreatic β-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on β-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human β-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or in the β-cells of ob/ob mice reversed the insulin secretion. Mechanistically, BHLHE40 represses expression of Mafa , which encodes the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic β-cells was recovered by MAFA expression. Collectively, this work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in β-cells and its implication in the β-cell dysfunction in type 2 diabetes.

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    Reply to the reviewers

    1. General Statements [optional]

    Thank you for the peer review of our manuscript entitled “Hypoxia causes pancreatic β____-cell dysfunction by activating a transcriptional repressor BHLHE40” (RC-2022-01560). We greatly appreciate the reviewers’ constructive suggestions and your invitation to revise the manuscript. Below, we address the comments point-by-point and provide details of the changes we are planning to or have implemented. We believe that the revision plan will meet with the approval of the editor and reviewers. We also would be happy to respond to any further questions and comments that you may have.

    2. Description of the planned revisions

    Response to comments of reviewer 1

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The authors examine the role of hypoxia-induced transcriptional repression in mediating loss of β-cell function in type2 diabetes. Transcriptional profiling of mouse and human islets exposed to low oxygen conditions revealed downregulation of β-cell identity and oxidative phosphorylation genes, and upregulation of genes associated with hypoxia. Identification of genes commonly upregulated in Min6 cells, mouse and human islets under hypoxic conditions, revealed induction of two transcriptional repressors BHLHE40 and ATF3. The authors further show that Bhlhe40 deficiency rendered β-cells resistant to hypoxic stress and restored glucose- and KCl-stimulated insulin secretion. This rescue in β-cell function was at least, in part, due to restoration of ATP generation and exocytosis of insulin granules. Furthermore, transcriptional profiling of Min6 cells overexpressing Bhlhe40 indicated down-regulation of key β-cell genes including Mafa. The authors elegantly show that BHLHE40 blocks PDX1 binding to Mafa transcription start site by binding to two E-box sites within the Mafa promoter/enhancer region. Lastly, Cre-mediated β-cell-specific deletion of Bhlhe40 in ob/ob mice restored expression of Mafa and exocytotic genes, accompanied by improvements in ATP generation and insulin secretion.

    __Major comments

    1. The authors conclude that BHLHE40 regulates insulin secretion at two key steps: ATP generation and exocytosis. However, insulin secretory profiles with glucose and KCl seem to be similar with genetic manipulations of Bhlhe40 both in vivo and ex vivo. As the authors indicate in line 176, this suggests a more prominent role of BHLHE40 in regulating exocytotic events downstream of Ca2+ influx. Further experiments are therefore necessary to adequately address the effects on ATP generation. Given the observation that PGC1____α, a regulator of mitochondrial biogenesis is suppressed by BHLHE40, mitochondrial assessments would be crucial. Additionally, the effect on mitochondrial mass in Fig 3K seem to be marginal and need to be confirmed using additional measurements listed below. __

    We appreciate your constructive suggestion. According to your suggestions, we will explore the role of BHLHE40 in mitochondrial function in more detail.

    a. In fig 3F, the authors show no change in KCl stimulated Ca2+ influx. Glucose stimulated Ca2+ influx needs to be examined to confirm regulation of ATP generation.

    We thank the reviewer for pointing this out. We performed the experiment according to the suggestion. Please see section 3.

    b. OXPHOS subunits, TOM20 levels by western blotting

    We thank the reviewer for pointing this out. We will perform Western blotting to check the protein levels of OXPHOS subunits and TOM20 in control (Ctrl) and Bhlhe40 knockdown (KD) MIN6 cells cultured under 20% or 5% O­­­2.

    c. mtDNA content, transcript levels by qRT-PCR

    We will perform qRT-PCR to check the mtDNA content in Ctrl and Bhlhe40 KD MIN6 cells cultured under 20% or 5% O­­­2.

    __d. Functional assessments: Changes in mitochondrial membrane potential or oxygen consumption __

    We will evaluate mitochondrial membrane potential by MitoTracker Red staining in Ctrl and Bhlhe40 KD MIN6 cells cultured under 20% or 5% O­­­2.

    __2. Data presented in Figure 4 and 5 indicates transcriptional repression of Mafa by BHLHE40 as a mechanism of beta-cell dysfunction under hypoxic conditions. However, additional experiments are necessary to confirm that repression of PDX1-Mafa binding specifically is responsible for defects in GSIS - __

    a. Fig 5G shows inhibition of PDX1-binding to Mafa with overexpression of Bhlhe40. This needs to be confirmed under hypoxic conditions.

    We thank the reviewer for pointing this out. Hypoxia for 16 to 24 hours decreases the expression levels of* Pdx1* in mouse islets and MIN6 cells (Figure 1A and Sato Y., PLoS One 2014). In that condition, it is difficult to assess whether the reduction in PDX1 binding to Mafa enhancer is attributed to inhibition of PDX1 binding or PDX1 downregulation. Therefore, we will aim to determine the hypoxia exposure time during which Mafa expression is downregulated but Pdx1 expression is not affected. If we fail to identify the time, we plan to generate Pdx1-overexpressing MIN6 cell lines and evaluate PDX1 binding to Mafa enhancer in hypoxic conditions.

    Sato Y. et al. Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes. PLoS One. 2014;9(12):e114868.

    b. Fig 4H and 4I show restoration of insulin secretion normalized to total protein with AAV-Mafa. This needs to be supplemented with insulin content as MAFA has been implicated in regulating insulin gene expression (PMID: 25500951).

    We thank the reviewer for pointing this out and agree with the comment on our original manuscript. We will evaluate insulin content by insulin ELISA assay in samples from AAV-Ctrl and AAV-Mafa-overexpressing MIN6 cells cultured under 20% or 5% O2. If the insulin content is affected by *Mafa *overexpression, insulin secretion will be adjusted by the intracellular insulin content.

    c. qRT-PCR of exocytosis genes and ATP generation with hypoxia and AAV-Mafa.

    We thank the reviewer for pointing this out. We will evaluate expression of exocytosis genes and ATP generation in AAV-Ctrl and AAV-Mafa-overexpressing MIN6 cells cultured under 20% or 5% O2.

    __d. Would mutation of A and C E-box sites restore PDX1 binding to Mafa TF region under hypoxia? __

    To address this question, we plan to introduce mutations in A and C sites of the* Mafa* gene in MIN6 cells by using CRISPR-Cas9 technology and then to examine PDX1 binding to the Mafa gene by ChIP assay under hypoxic conditions.

    3. β-dedifferentiation has been proposed to be involved in loss of insulin secretion in T2D (PMID: 22980982, 16123366). One can speculate that transcriptional repression of Mafa by BHLHE40 is a component of a larger dedifferentiation phenomenon occurring under hypoxia, as other ____β-cell genes were decreased with hypoxia (Fig 1A) and Bhlhe40-OE in Fig 4A. Identifying differences in dedifferentiation and ____β-cell disallowed genes with Bhlhe40 overexpression (RNA seq, qRT-PCR) would therefore potentially reveal a dedifferentiation mechanism.

    We thank the reviewer for pointing this out. Please see section 3.

    4. The authors identify Atf3 as another transcriptional repressor enriched under hypoxia although to a lesser degree than Bhlhe40. The role of ATF3 in hypoxia-induced apoptosis and adaptive UPR has been previously suggested (PMID: 20519332, 20349223). Additionally, hypoxia represses adaptive UPR in models of T2D and drives ____β-cell apoptosis (PMID: 27039902). The authors discuss the role of ATF3 under hypoxia in the discussion (lines 319-324) and addressing these research gaps regarding ATF3 function would be insightful.

    We are grateful for the reviewer’s comment. We will generate Atf3 knockdown MIN6 cell lines and examine the effect of ATF3 on hypoxia-induced apoptosis by PI/AnnexinV staining. If ATF3 is involved in hypoxia-induced apoptosis, we will also measure the mRNA expression levels involved in adaptive UPR.

    __Minor comments

    1. In Fig 2E, increasing replicates would confirm no induction of Bhlhe40 with Thapsigargin. __

    Thank you for pointing out this issue. We will perform additional experiments to confirm the effect of thapsigargin on Bhlhe40 expression.

    2. In Fig 2B, BHLHE40 bands need to be quantified to show time-dependent increase in protein levels.

    Thank you for pointing out this issue. Please see section 3.

    3. In Fig 3C, insulin content needs to be shown with Bhlhe40-OE as in Fig 3B with hypoxia.

    Thank you for pointing out this issue. Please see section 3.

    4. In Fid 4E-F, band intensities need to quantified by densitometry to determine degree of downregulation of MAFA.

    We performed three independent experiments for Figure 4E. Please see section 3. We plan to perform additional experiments to determine the expression levels of MAFA (Figure 4F).

    5. In Fig4H and 6G, insulin content needs to be shown as stated above.

    We thank the reviewer for pointing this out. We will perform additional experiments to check the insulin content in these settings.

    6. In Supplemental Figure 3C, apoptosis induced by hypoxia was assessed by PI staining that detects late apoptosis. No significant changes were observed with Bhlhe40-KD, but additional cell death assessments can be used to confirm that B40 does not affect ____β-cell death.

    We thank the reviewer for mentioning this issue. To address this concern, we plan to investigate the effects of Bhlhe40 KD on the number of Annexin V-positive cells (early apoptosis) and cleaved (activated) caspase 3 expression in Ctrl and Bhlhe40 KD MIN6 cells under hypoxic conditions.

    7. It would be interesting to see the rates of diabetes incidence in Bhlhe40KO: ob/ob mice and if Bhlhe40 deficiency protects against or delays development of diabetes.

    Thank you for mentioning this issue. Please see section 3.

    8. Knockdown efficiency shown in Supplementary figure 3A needs to be estimated by quantifying band intensities.

    We plan to perform additional experiments to quantify the band intensities.

    9. Line 43 should say "...reversed defects in insulin secretion."

    We apologize for our incorrect explanation in the original manuscript. We have corrected this error in the text. Please see section 3.

    Reviewer #1 (Significance (Required)):

    The data presented provides novel mechanistic insights into the role of hypoxia in β-cell dysfunction. Studies in multiple models of type 2 diabetes (T2D) have shown the loss of signature β-cell genes including Ins1, Pdx1, Mafa, Slc2a2 as a result of excess nutrient stimulation and hypoxia; the precise causal mechanisms, however, still remain to be determined (PMID: 22980982, 28270834). A previous paper from the same group demonstrated downregulation of β-cell signature genes with hypoxia by a HIF1α independent mechanism (PMID: 25503986). Data presented in this report extend those observations and reveal a previously unappreciated role for transcriptional repressor BHLHE40 in the downregulation of a key β-cell gene Mafa. As the authors have identified additional transcriptional repressors including ATF3 and differentially expressed genes in both human and rodent β-cells, this paper would be of great value in understanding the effects of hypoxia. Moreover, studies in mouse models of T2D extend the association of BHLHE40 to clinical β-cell dysfunction and diabetes.

    My areas of interest are pancreatic β-cell and mitochondrial physiology. GSE analysis and repression of PGC1α by BHLHE40, as appropriately discussed by the authors, point towards impaired mitochondrial function and ATP generation. Additional experiments would greatly support the role of BHLHE40 in mitochondrial dysfunction under hypoxia.

    We thank the reviewer for his/her valuable comments.

    Response to comments of reviewer 2

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): This study examines the role of BHEH40 in beta-cell function and its role in mediating the changes with 'hypoxia'. Most of the studies use 5% oxygen which is probably close to normal oxygen tension for islets, although it is not great for islet survival once the islets are removed from their normal vasculature. The human islet studies use 2% oxygen which would be actual hypoxia.

    __Major comments: Why were different oxygen concentrations used for mouse and human islets? What were the effects of 5% oxygen in human islets? Why was 5% oxygen chosen? 5% is close to normal oxygen tension that islets are exposed to in vivo, whereas 2% is not physiological. __

    We apologize for the lack of explanation in the original manuscript. Please see section 3.

    If there was only 1 human donor, are the 2 and 3 RNA-seq technical replicates? If so, they do not show high replicability. Please discuss.

    The reviewer is correct. We analyzed human islets from one donor for RNA-seq (Figure 1A). It would be preferable to obtain additional data derived from different human donor samples. Therefore, we plan to analyze human islets from another donor to show the replicability of increased expression of Bhlhe40 under hypoxic conditions.

    Are the sequencing results from individual mice? Were the same mouse's islets used for normal and 5% oxygen or are they all different animals?

    We apologize for the lack of explanation in the original manuscript. Results of RNA-seq were obtained from different animals. Please see section 3.

    The whole gels with appropriate size markers need to be shown for all Westerns - they are not able to be appropriately reviewed in their current formats.

    We apologize for the mistake. Please see section 3.

    The histology in Figure 1K does not appear to match with the Western blot results in 1B, 1C which show a smaller but still clear band in the control 20% conditions, and in figures 1I and J in ob/ob and db/db controls. Lower power views showing most of the pancreas with a zoom-in shot of an example islet would be more appropriate. The immunofluorescence should be repeated to also include insulin so that beta-cells can be identified.

    We thank the reviewer for mentioning this issue. We will repeat the immunohistochemical analysis according to the reviewer’s comments. We also plan to show the data on insulin staining.

    What was the ____β-cell deletion efficiency of the knockdown mouse?

    We apologize for the lack of explanation in the original manuscript. Please see section 3.

    In the setting of hypoxia, would it be 'clinically' beneficial to have increased insulin secretion and thus metabolic demand? Please discuss.

    We thank the reviewer for mentioning this important issue. Our results show that BHLHE40 controls at least two steps of insulin secretion: exocytosis and ATP generation. A relatively smaller reduction of Ppargc1a might suggest a more prominent role of BHLHE40 in regulating exocytosis rather than ATP generation (oxygen consumption step). Chronic hyperglycemia induces b-cell damage and impairs insulin secretion, a process known as glucotoxicity (Weir GC, Diabetes 2004, 2020). We believe that inactivation of BHLHE40 may help to reduce glucotoxicity by increasing insulin secretion. However, we would like to discuss this topic in more detail once we have investigated the roles of BHLHE40 in ATP generation (as suggested by reviewer 1).

    Weir GC. et al. Five stages of evolving beta-cell dysfunction during progression to diabetes. Diabetes. 2004;53 Suppl 3:S16-21.

    Weir GC. Glucolipotoxicity, β-Cells, and Diabetes: The Emperor Has No Clothes. Diabetes. 2020;69(3):273-278.

    __- Are the key conclusions convincing? Hard to assess the data in some cases - see above.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Yes, some of the conclusions are too strongly worded. An example is "However, hyperactivation of HIF in ____β-cells impairs insulin secretion by switching glucose metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis (14-16)," this is too broad a statement. Hyperactivation of HIF in ____β-cells BY VHL DELETION impairs insulin secretion by that mechanism. Other ways of increasing HIF in ____β-cells do not all have this effect. So, the following part "suggesting that activation of HIF underlies ____β-cell dysfunction and glucose intolerance in hypoxia." Is not warranted. It would be fair to say "suggesting that unregulated over-activation of HIF may cause ____β-cell dysfunction.__

    We apologize for our incorrect explanation in the original manuscript. Please see section 3.

    The paper is not off to a good start when the author spell Abstract as Abstruct - it suggests a spell-check was not performed. For the last sentence of the abstract, 'and its implication' - what implication?

    We apologize for the typo and the poor description in the original manuscript. Please see section 3.

    __Line 64 High glucose conditions generate RELATIVE, not absolute hypoxia in beta-cells. This statement should also be referenced. __

    We apologize for our inaccurate explanation in the original manuscript. Please see section 3.

    - Would additional experiments be essential to support the claims of the paper? See above.

    - Are the data and the methods presented in such a way that they can be reproduced? Not enough detail for methods, but what is presented looks OK.

    - Are the experiments adequately replicated and statistical analysis adequate? Unclear, see above.

    - Specific experimental issues that are easily addressable.

    - Are prior studies referenced appropriately? No, only the body of work on VHL, not HIFs.

    - Are the text and figures clear and accurate? See above comments.

    - Do you have suggestions that would help the authors improve the presentation of their data and conclusions? See comments about gels etc above.

    We thank the reviewer for his/her valuable comments. Please see section 3 regarding to references.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    __The authors conclude that BHLHE40 regulates insulin secretion at two key steps: ATP generation and exocytosis. However, insulin secretory profiles with glucose and KCl seem to be similar with genetic manipulations of Bhlhe40 both in vivo and ex vivo. As the authors indicate in line 176, this suggests a more prominent role of BHLHE40 in regulating exocytotic events downstream of Ca2+ influx. Further experiments are therefore necessary to adequately address the effects on ATP generation. Given the observation that PGC1____α, a regulator of mitochondrial biogenesis is suppressed by BHLHE40, mitochondrial assessments would be crucial. Additionally, the effect on mitochondrial mass in Fig 3K seem to be marginal and need to be confirmed using additional measurements listed below. __

    a. In fig 3F, the authors show no change in KCl stimulated Ca2+ influx. Glucose stimulated Ca2+ influx needs to be examined to confirm regulation of ATP generation.

    We thank the reviewer for pointing this out. In accordance with the reviewer’s comments, we examined the glucose-stimulated Ca2+ influx and found that the influx stimulated by 22mM glucose in Bhlhe40-overexpressing (OE) MIN6 cells was significantly smaller than that in control MIN6 cells (Figure 3, J and K). We have added this information to the manuscript, as follows: “In addition, glucose-stimulated [Ca2+]i levels were significantly attenuated by Bhlhe40 overexpression (Figure 3, J and K). These results indicate that BHLHE40 suppresses glucose-stimulated ATP generation and the increase of [Ca2+]i levels in MIN6 cells” (lines 197 to 200).

    3. β-dedifferentiation has been proposed to be involved in loss of insulin secretion in T2D (PMID: 22980982, 16123366). One can speculate that transcriptional repression of Mafa by BHLHE40 is a component of a larger dedifferentiation phenomenon occurring under hypoxia, as other ____β-cell genes were decreased with hypoxia (Fig 1A) and Bhlhe40-OE in Fig 4A. Identifying differences in dedifferentiation and ____β-cell disallowed genes with Bhlhe40 overexpression (RNA seq, qRT-PCR) would therefore potentially reveal a dedifferentiation mechanism.

    We thank the reviewer for pointing this out. To check whether genes involved in dedifferentiation and the expression of b-cell disallowed genes are controlled by BHLHE40, we examined the expression of these genes in Bhlhe40 OE MIN6 cells and found that it was not increased by BHLHE40. However, because the findings were detected under limited experimental conditions, at this point we cannot conclude that BHLHE40 does not cause dedifferentiation of b-cells and induction of b-cell disallowed genes.

    __Minor comments

    1. In Fig 2B, BHLHE40 bands need to be quantified to show time-dependent increase in protein levels.__

    Thank you for pointing out this issue. We performed three independent experiments and showed statistically significant upregulation of BHLHE40 (Figure 2B).

    3. In Fig 3C, insulin content needs to be shown with Bhlhe40-OE as in Fig 3B with hypoxia.

    Thank you for pointing out this issue.* Bhlhe40* OE did not affect the insulin content in MIN6 cells (Supplemental Figure 3F).

    4. In Fid 4E-F, band intensities need to quantified by densitometry to determine degree of downregulation of MAFA.

    We performed three independent experiments for Figure 4E. Bhlhe40 OE led to a 67.4% decrease in the expression of MAFA in MIN6 cells (Figure 4E).

    7. It would be interesting to see the rates of diabetes incidence in Bhlhe40KO: ob/ob mice and if Bhlhe40 deficiency protects against or delays development of diabetes.

    Thank you for pointing out this issue. We found that nonfasting blood glucose concentrations were similar in Ctrl:ob/ob (239.9 ± 19.6 mg/dl; n = 9) and bB40KO:ob/ob mice (215.2 ± 17.1 mg/dl; n = 6) at 12 weeks of age (Supplemental Figure 5F). We have added this information to the revised manuscript, as follows: “In these mice, BHLHE40 deficiency in b-cells had no effect on obesity (Figure 6B), insulin sensitivity (Supplemental Figure 5E), or nonfasting glucose concentrations (Supplemental Figure 5F)” (lines 282 to 285).

    9. Line 43 should say "...reversed defects in insulin secretion."

    We apologize for our incorrect explanation in the original manuscript and have corrected it accordingly.

    Response to comments of reviewer 2

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): This study examines the role of BHEH40 in beta-cell function and its role in mediating the changes with 'hypoxia'. Most of the studies use 5% oxygen which is probably close to normal oxygen tension for islets, although it is not great for islet survival once the islets are removed from their normal vasculature. The human islet studies use 2% oxygen which would be actual hypoxia.

    __Major comments: Why were different oxygen concentrations used for mouse and human islets? What were the effects of 5% oxygen in human islets? Why was 5% oxygen chosen? 5% is close to normal oxygen tension that islets are exposed to in vivo, whereas 2% is not physiological. __

    We apologize for the lack of explanation in the original manuscript. We previously reported that hypoxic responses occur at 5% to 7% oxygen tension in MIN6 cells and mouse islets and that 3% hypoxia for 24 hours markedly increases MIN6 cell death (Sato Y., J Biol Chem 2011, Sato Y., PLoS One 2014). We also examined whether hypoxic responses occur at the same oxygen tension in human islets. Interestingly, in human islets, exposure to 2% but not 5% oxygen tension induced the upregulation of the SLC2A1 gene without apparent cell death. Another group also reported a hypoxic response in human islets in 2% oxygen (Puri S., Genes Dev. 2013). We have no adequate explanation as to why hypoxic responses occur at different oxygen tensions in mouse and human islets, but because of these findings, we used 5% oxygen in MIN6 cells and mouse islets and 2% oxygen in human islets. We have added this information to the text (lines 98 to 104) and Supplemental Figure 1A.

    Sato, Y. et al. Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro. J Biol Chem. 2011;286(14):12524-32.

    Sato, Y. et al. Moderate hypoxia induces β-cell dysfunction with HIF-1-independent gene expression changes. PLoS One. 2014;9(12):e114868.

    Puri S. VHL-mediated disruption of Sox9 activity compromises β-cell identity and results in diabetes mellitus. Genes Dev. 2013;27(23):2563-2575.

    Are the sequencing results from individual mice? Were the same mouse's islets used for normal and 5% oxygen or are they all different animals?

    We apologize for the lack of explanation in the original manuscript. Each sample was derived from different mice. We have clarified this point by describing “n = 3” as “n = 3 mice/group” and have added this information to the legends of Figure 1 and Supplemental Figure 1.

    The whole gels with appropriate size markers need to be shown for all Westerns - they are not able to be appropriately reviewed in their current formats.

    We apologize for the inappropriate presentation of the data. We have included whole gel images with molecular weight markers as supplemental material.

    What was the ____β-cell deletion efficiency of the knockdown mouse?

    We apologize for not including this information. Although we showed the expression levels of Bhlhe40 in the islets of bB40KO mice (original Supplemental Figure 5B), we did not explain the data in the text. The deletion efficiency in the islets was 74.1%. We have added this information to the revised text (lines 274 to 275).

    - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Yes, some of the conclusions are too strongly worded. An example is "However, hyperactivation of HIF in ____β-cells impairs insulin secretion by switching glucose metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis (14-16)," this is too broad a statement. Hyperactivation of HIF in ____β-cells BY VHL DELETION impairs insulin secretion by that mechanism. Other ways of increasing HIF in ____β-cells do not all have this effect. So, the following part "suggesting that activation of HIF underlies ____β-cell dysfunction and glucose intolerance in hypoxia." Is not warranted. It would be fair to say "suggesting that unregulated over-activation of HIF may cause ____β-cell dysfunction.

    We apologize for our incorrect explanation in the original manuscript. We have corrected this accordingly, as follows: “However, hyperactivation of HIF in b-cells by von Hippel-Lindau (VHL) deletion impairs insulin secretion by switching glucose metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis (15-17), suggesting that unregulated overactivation of HIF may cause b-cell dysfunction (12, 14)” (lines 73 to 77).

    The paper is not off to a good start when the author spell Abstract as Abstruct - it suggests a spell-check was not performed. For the last sentence of the abstract, 'and its implication' - what implication?

    We apologize for the typo and the poor description in the original manuscript. The spell check was performed by an editing company, and we did not notice the error. We have changed the last sentence of the Abstract, as follows: “Collectively, this work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in b-cells that negatively regulates insulin secretion by suppressing MAFA expression” (lines 47 to 49).

    __Line 64 High glucose conditions generate RELATIVE, not absolute hypoxia in beta-cells. This statement should also be referenced. __

    We apologize for our inaccurate explanation in the original manuscript. We have corrected this accordingly, as follows: “high glucose conditions generate relative hypoxia in b-cells because these cells consume large amounts of oxygen (Sato Y., J Biol Chem 2011; Bensellam M., PLoS One 2012; Bensellam M., J Endocrinol 2018; Ilegems E, Sci Transl Med 2022)” (lines 64 to 65).

    Sato, Y. et al. Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro. J Biol Chem. 2011;286(14):12524-32.

    Bensellam, M. et al. Glucose-induced O₂ consumption activates hypoxia inducible factors 1 and 2 in rat insulin-secreting pancreatic beta-cells. PLoS One 2012;7(1):e29807.

    Bensellam M. et al. Mechanisms of β-cell dedifferentiation in diabetes: recent findings and future research directions. J Endocrinol. 2018;236(2):R109-R143

    Ilegems, E. et al. HIF-1α inhibitor PX-478 preserves pancreatic β cell function in diabetes.* Sci Transl Med*. 2022;14(638):eaba9112.

    - Are prior studies referenced appropriately? No, only the body of work on VHL, not HIFs.

    We apologize for our inadequate references about the involvement of HIFs in hypoxia-induced β-cell dysfunction. We have included the following references in the text (line 77):

    Catrina, S.B. et al. Hypoxia and hypoxia-inducible factors in diabetes and its complications. Diabetologia. 2021;64(4):709-716

    Gulton JE. Hypoxia-inducible factors and diabetes. *J Clin Invest. *2020; 130(10):5063-5073

    4. Description of analyses that authors prefer not to carry out

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This study examines the role of BHEH40 in beta-cell function and its role in mediating the changes with 'hypoxia'. Most of the studies use 5% oxygen which is probably close to normal oxygen tension for islets, although it is not great for islet survival once the islets are removed from their normal vasculature. The human islet studies use 2% oxygen which would be actual hypoxia.

    Significance

    Major comments:

    Why were different oxygen concentrations used for mouse and human islets? What were the effects of 5% oxygen in human islets? Why was 5% oxygen chosen? 5% is close to normal oxygen tension that islets are exposed to in vivo, whereas 2% is not physiological.

    If there was only 1 human donor, are the 2 and 3 RNA-seq technical replicates? If so, they do not show high replicability. Please discuss.

    Are the sequencing results from individual mice? Were the same mouse's islets used for normal and 5% oxygen or are they all different animals?

    The whole gels with appropriate size markers need to be shown for all Westerns - they are not able to be appropriately reviewed in their current formats.

    The histology in Figure 1K does not appear to match with the Western blot results in 1B, 1C which show a smaller but still clear band in the control 20% conditions, and in figures 1I and J in ob/ob and db/db controls. Lower power views showing most of the pancreas with a zoom-in shot of an example islet would be more appropriate. The immunofluorescence should be repeated to also include insulin so that beta-cells can be identified.

    What was the β-cell deletion efficiency of the knockdown mouse?

    In the setting of hypoxia, would it be 'clinically' beneficial to have increased insulin secretion and thus metabolic demand? Please discuss.

    • Are the key conclusions convincing? Hard to assess the data in some cases - see above.
    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Yes, some of the conclusions are too strongly worded. An example is "However, hyperactivation of HIF in β-cells impairs insulin secretion by switching glucose metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis (14-16)," this is too broad a statement. Hyperactivation of HIF in β-cells BY VHL DELETION impairs insulin secretion by that mechanism. Other ways of increasing HIF in β-cells do not all have this effect. So, the following part "suggesting that activation of HIF underlies β-cell dysfunction and glucose intolerance in hypoxia." Is not warranted. It would be fair to say "suggesting that unregulated over-activation of HIF may cause β-cell dysfunction. The paper is not off to a good start when the author spell Abstract as Abstruct - it suggests a spell-check was not performed. For the last sentence of the abstract, 'and its implication' - what implication? Line 64 High glucose conditions generate RELATIVE, not absolute hypoxia in beta-cells. This statement should also be referenced.
    • Would additional experiments be essential to support the claims of the paper? See above.
    • Are the data and the methods presented in such a way that they can be reproduced? Not enough detail for methods, but what is presented looks OK.
    • Are the experiments adequately replicated and statistical analysis adequate? Unclear, see above.
    • Specific experimental issues that are easily addressable.
    • Are prior studies referenced appropriately? No, only the body of work on VHL, not HIFs.
    • Are the text and figures clear and accurate? See above comments.
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? See comments about gels etc above.
  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The authors examine the role of hypoxia-induced transcriptional repression in mediating loss of β-cell function in type2 diabetes. Transcriptional profiling of mouse and human islets exposed to low oxygen conditions revealed downregulation of β-cell identity and oxidative phosphorylation genes, and upregulation of genes associated with hypoxia. Identification of genes commonly upregulated in Min6 cells, mouse and human islets under hypoxic conditions, revealed induction of two transcriptional repressors BHLHE40 and ATF3. The authors further show that Bhlhe40 deficiency rendered β-cells resistant to hypoxic stress and restored glucose- and KCl-stimulated insulin secretion. This rescue in β-cell function was at least, in part, due to restoration of ATP generation and exocytosis of insulin granules. Furthermore, transcriptional profiling of Min6 cells overexpressing Bhlhe40 indicated down-regulation of key β-cell genes including Mafa. The authors elegantly show that BHLHE40 blocks PDX1 binding to Mafa transcription start site by binding to two E-box sites within the Mafa promoter/enhancer region. Lastly, Cre-mediated β-cell-specific deletion of Bhlhe40 in ob/ob mice restored expression of Mafa and exocytotic genes, accompanied by improvements in ATP generation and insulin secretion.

    Major comments

    1. The authors conclude that BHLHE40 regulates insulin secretion at two key steps: ATP generation and exocytosis. However, insulin secretory profiles with glucose and KCl seem to be similar with genetic manipulations of Bhlhe40 both in vivo and ex vivo. As the authors indicate in line 176, this suggests a more prominent role of BHLHE40 in regulating exocytotic events downstream of Ca2+ influx. Further experiments are therefore necessary to adequately address the effects on ATP generation. Given the observation that PGC1α, a regulator of mitochondrial biogenesis is suppressed by BHLHE40, mitochondrial assessments would be crucial. Additionally, the effect on mitochondrial mass in Fig 3K seem to be marginal and need to be confirmed using additional measurements listed below.
      • a. In fig 3F, the authors show no change in KCl stimulated Ca2+ influx. Glucose stimulated Ca2+ influx needs to be examined to confirm regulation of ATP generation.
      • b. OXPHOS subunits, TOM20 levels by western blotting
      • c. mtDNA content, transcript levels by qRT-PCR
      • d. Functional assessments: Changes in mitochondrial membrane potential or oxygen consumption
    2. Data presented in Figure 4 and 5 indicates transcriptional repression of Mafa by BHLHE40 as a mechanism of beta-cell dysfunction under hypoxic conditions. However, additional experiments are necessary to confirm that repression of PDX1-Mafa binding specifically is responsible for defects in GSIS -
      • a. Fig 5G shows inhibition of PDX1-binding to Mafa with overexpression of Bhlhe40. This needs to be confirmed under hypoxic conditions.
      • b. Fig 4H and 4I show restoration of insulin secretion normalized to total protein with AAV-Mafa. This needs to be supplemented with insulin content as MAFA has been implicated in regulating insulin gene expression (PMID: 25500951).
      • c. qRT-PCR of exocytosis genes and ATP generation with hypoxia and AAV-Mafa.
      • d. Would mutation of A and C E-box sites restore PDX1 binding to Mafa TF region under hypoxia?
    3. β-dedifferentiation has been proposed to be involved in loss of insulin secretion in T2D (PMID: 22980982, 16123366). One can speculate that transcriptional repression of Mafa by BHLHE40 is a component of a larger dedifferentiation phenomenon occurring under hypoxia, as other β-cell genes were decreased with hypoxia (Fig 1A) and Bhlhe40-OE in Fig 4A. Identifying differences in dedifferentiation and β-cell disallowed genes with Bhlhe40 overexpression (RNA seq, qRT-PCR) would therefore potentially reveal a dedifferentiation mechanism.
    4. The authors identify Atf3 as another transcriptional repressor enriched under hypoxia although to a lesser degree than Bhlhe40. The role of ATF3 in hypoxia-induced apoptosis and adaptive UPR has been previously suggested (PMID: 20519332, 20349223). Additionally, hypoxia represses adaptive UPR in models of T2D and drives β-cell apoptosis (PMID: 27039902). The authors discuss the role of ATF3 under hypoxia in the discussion (lines 319-324) and addressing these research gaps regarding ATF3 function would be insightful.

    Minor comments

    1. In Fig 2E, increasing replicates would confirm no induction of Bhlhe40 with Thapsigargin.
    2. In Fig 2B, BHLHE40 bands need to be quantified to show time-dependent increase in protein levels.
    3. In Fig 3C, insulin content needs to be shown with Bhlhe40-OE as in Fig 3B with hypoxia.
    4. In Fid 4E-F, band intensities need to quantified by densitometry to determine degree of downregulation of MAFA.
    5. In Fig4H and 6G, insulin content needs to be shown as stated above.
    6. In Supplemental Figure 3C, apoptosis induced by hypoxia was assessed by PI staining that detects late apoptosis. No significant changes were observed with Bhlhe40-KD, but additional cell death assessments can be used to confirm that B40 does not affect β-cell death.
    7. It would be interesting to see the rates of diabetes incidence in Bhlhe40KO: ob/ob mice and if Bhlhe40 deficiency protects against or delays development of diabetes.
    8. Knockdown efficiency shown in Supplementary figure 3A needs to be estimated by quantifying band intensities.
    9. Line 43 should say "...reversed defects in insulin secretion."

    Significance

    The data presented provides novel mechanistic insights into the role of hypoxia in β-cell dysfunction. Studies in multiple models of type 2 diabetes (T2D) have shown the loss of signature β-cell genes including Ins1, Pdx1, Mafa, Slc2a2 as a result of excess nutrient stimulation and hypoxia; the precise causal mechanisms, however, still remain to be determined (PMID: 22980982, 28270834). A previous paper from the same group demonstrated downregulation of β-cell signature genes with hypoxia by a HIF1α independent mechanism (PMID: 25503986). Data presented in this report extend those observations and reveal a previously unappreciated role for transcriptional repressor BHLHE40 in the downregulation of a key β-cell gene Mafa. As the authors have identified additional transcriptional repressors including ATF3 and differentially expressed genes in both human and rodent β-cells, this paper would be of great value in understanding the effects of hypoxia. Moreover, studies in mouse models of T2D extend the association of BHLHE40 to clinical β-cell dysfunction and diabetes. My areas of interest are pancreatic β-cell and mitochondrial physiology. GSE analysis and repression of PGC1α by BHLHE40, as appropriately discussed by the authors, point towards impaired mitochondrial function and ATP generation. Additional experiments would greatly support the role of BHLHE40 in mitochondrial dysfunction under hypoxia (as discussed under comments).

  4. Version published to 10.1101/2022.06.28.498031v1 on bioRxiv