NAD+ Precursors Repair Mitochondrial Function in Diabetes and Prevent Experimental Diabetic Neuropathy
, Mohammad Salimian 1, Muhammed Ikbal Arvas 1, Ahmad F. Hedayat 1, Yanni Kevas 1, Anand Kadakia 1 and James W. Russell 1,2,3,*Round 1
Reviewer 1 Report
ijms-1664997
Chandrasekaran et al
NAD+ Precursors Repair Mitochondrial Function in Diabetes 2
and Prevent Experimental Diabetic Neuropathy 3
This paper is well written and appears scientifically sound. The topic is of interest, although as NAD+ supplements are available OTC a study in humans would seem more relevant.
Some minor comments:
1) Line 383: Please state what proportion of the STZ-treated animals fulfilled the inclusion criteria and what dictated the choice of the latter.
2) Line 384: Chronic NMN injections – please state route of injection. Was vehicle also injected? As this is detailed later in section 4.2 it would be best to delete this statement from this section.
3) In general, the potential stress-habituation response induced by chronic ip. injections on the outcome of the study should be discussed.
Author Response
We would like to thank the reviewers for reviewing the manuscript and for their helpful suggestions. We have answered each question in turn.
Reviewer # 1
ijms-1664997
Chandrasekaran et al
NAD+ Precursors Repair Mitochondrial Function in Diabetes and Prevent Experimental Diabetic Neuropathy
This paper is well written and appears scientifically sound. The topic is of interest, although as NAD+ supplements are available OTC a study in humans would seem more relevant.
Some minor comments:
Line 383: Please state what proportion of the STZ-treated animals fulfilled the inclusion criteria and what dictated the choice of the latter. This statement is added in line 411.
Response: Animals which had a weight loss of more than 20% from the baseline were euthanized and were not included in the study. Sixty percent of the STZ-treated animals fullfilled the inclusion criteria.
Line 384: Chronic NMN injections – please state route of injection. Was vehicle also injected? As this is detailed later in section 4.2 it would be best to delete this statement from this section.
Response: One-week after confirmation of diabetes, NMN was administered. Nicotinamide Mononucleotide (NMN) was dissolved in saline and injected intraperitoneally (IP) into diabetic rats at a dose of 50 mg/kg or 100 mg/kg on alternate days (Monday. Wednesday and Friday) for 3 months. The control rats were injected IP with saline (vehicle) on alternate days.
In general, the potential stress-habituation response induced by chronic ip. injections on the outcome of the study should be discussed.
Response: This is an excellent point. Overall, the protocol used would have minimized stress and increased habituation. Previously, chronic IP administration of NMN at a dose of 500 mg/kg/daily for 10 days was found to safe and effective [17]. In this study, NMN or saline was injected on alternate days for 3 months (36 injections total) and we did not observe any visible adverse effects in the mouse. The stress-habituation response should have been similar between experimental and control animals. Control animals received an equal number of identical injections (but with vehicle) as the experimental animals. We have discussed this in line 391.
Reviewer 2 Report
The authors tested if the administration of NAD+ precursors, nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), will prevent DPN in models of Type 1 and Type 2 diabetes. While this is an interesting topic to study, there are things the authors need to clarify or avoid:
Introduction
1) No need for a subsection
2) Line 36: Could add lifestyle changes such as exercise
3) Line 51: The link between NDA+ and fatty acid oxidation in the context of DPN should be highlighted as these two are indicated separately in the section
4) Line 64-65: Is confusing especially the last part
5) The entire document requires some editorial input. For example, Line 67 to 69 should read '..NR, by increasing NAD+ levels in DRG neurons, can improve...'.
Results:
1) SD should be spelled out at first mention (Line 73)
2) Line 75: Would like to see a comparison of the change in body weight from baseline to the end of the study between the groups.
3) Line 166-9: Could be reworded, with 'triglycerides and NEFA levels' mentioned together as such.
4) IP-GTT should be defined at first mention
5) Line 171: Since the study is about T1D and T2DM-induced DPN, it might be necessary to show HOMA-IR to establish whether these animals were insulin resistant (IR) or not, especially on the HFD-fed animals.
6) Line 184: The CD group had significantly lower SMNCV as compared with control mice in the STZ treated groups. See TSNCV as well. Were these mice the same age?
7) Line 186-7: is there a reference range diagnosing DPN with these parameters without looking at the differences between the groups? This is important since TSNCV is considerably low on the HFD control animals as compared with the STZ control.
8) Line 190: How long were the animals kept in the study. Please clarify the timeline for the HFD-fed mice.
9) Line 192: Important to discuss whether the HFD-induced PN is a consequence of diabete or just hyperlipidaemia, and to do that, authors need to show whether T2DM was established in the model
10) Line 215-16: Misplaced. Move it to the discussion
11) Line 237-40: Make sure this statement means what you intended to say.
Discussion:
1) Line 261: the role of NMN on Mt respiratory capacity wasnt studied.
2) Line 266-8: Add a reference
3) Line 276: Why was NMN not the one studied instead of NR?
4) Line 278: The discussion is heavy on gene networks, but the authors did not do any mRNA expression assays. This should be clarified and authors should avoid overreaching (see line 286-89)
5) Line 299-301: add a reference. Check and add references to other statements that are based on evidence generated from previous studies.
6) Line 317: Avoid mentioning 'show' twice in one statement. Check others as well.
Materials and Methods:
1) Editorial input such as in line 380 ('could have' should be replaced with 'had')
2) Line 384: When did the treatments start?
3) Section 4.2: It should be 'diabetes induction with HFD'. Also, the 2 sections should specify the type of diabete being induced.
4) Section 4.3 is misplaced
5) Line 425: Why was dose translation not performed to ensure that the treatments take into account the surface area?
6) Line 426: These tests are not indicated on the rat section.
7) Line 427: How was IP-GTT performed?
8) Line 432: Why was NR not tested in the STZ group?
9) Line 439: Use animals and not mouse as both rats and mice were used.
Author Response
Manuscript ID: ijms-1664997
Chandrasekaran et al
NAD+ Precursors Repair Mitochondrial Function in Diabetes and Prevent Experimental Diabetic Neuropathy
Reviewer # 2
The authors tested if the administration of NAD+ precursors, nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), will prevent DPN in models of Type 1 and Type 2 diabetes. While this is an interesting topic to study, there are things the authors need to clarify or avoid:
Introduction
No need for a subsection
Response: Deleted
Line 36: Could add lifestyle changes such as exercise
Response: There is emerging evidence that lifestyle interventions are effective in individuals with established diabetic neuropathy. In addition, there is evidence that effects on biochemical pathways that improve muscle function affect other organ systems including peripheral nerve reviewed in [2]. Zilliox, L.A.; Russell, J.W. Physical activity and dietary interventions in diabetic neuropathy: a systematic review. Clin Auton Res 2019, 29, 443-455, doi:10.1007/s10286-019-00607-x.
Line 51: The link between NDA+ and fatty acid oxidation in the context of DPN should be highlighted as these two are indicated separately in the section
Response: Added lines 67-69. “We propose that NMN and NR by increasing NAD+ levels in DRG neurons can furnish reducing equivalents (NADH) to the mitochondrial electron transport chain to generate ATP and with increased NAD+ sirtuins can deacetylate proteins including mitochondrial priteins to regulate fatty acid oxidation and thereby prevent diabetic neuropathy”.
Line 64-65: Is confusing especially the last part
Response: We modified the sentence “Although nicotinamide riboside (NR) may oppose diabetes and diabetic neuropathy in T2D [19], it is unknown if nicotinamide mononucleotide (NMN) can prevent experimental diabetic neuropathy” to clarify this: “Nicotinamide riboside (NR) may improve diabetes and diabetic neuropathy in T2D [19]. However, it is unknown if nicotinamide mononucleotide (NMN) can prevent experimental diabetic neuropathy and the NR-regulated mechanisms that prevent neuropathy.”
The entire document requires some editorial input. For example, Line 67 to 69 should read '..NR, by increasing NAD+ levels in DRG neurons, can improve...'.
Response: Revised
Results:
SD should be spelled out at first mention (Line 73)
Response: SD spelled out as Sprague Dawley
Line 75: Would like to see a comparison of the change in body weight from baseline to the end of the study between the groups.
Response: Added line 78: “Comparison of the changes in body weight from baseline to end of the study showed that administration of STZ irrespective of NMN treatment caused a decrease in body weight”.
Line 166-9: Could be reworded, with 'triglycerides and NEFA levels' mentioned together as such.
Response: reworded to “triglyceride (TG) and NEFA levels” were decreased
IP-GTT should be defined at first mention
Response: IP-GTT defined as “Intraperitoneal glucose tolerance test”
Line 171: Since the study is about T1D and T2DM-induced DPN, it might be necessary to show HOMA-IR to establish whether these animals were insulin resistant (IR) or not, especially on the HFD-fed animals.
Response: HOMA-IR index vales were calculated. “The calculated Homeostatic Model Assessment Insulin Resistance (HOMA-IR) index were CD = 46 ± 5, CD+NR = 42 ± 5, HFD = 106 ± 9, HFD+NR (150) = 99 ± 9 and HFD+NR (300) = 95 ± 9.6, suggesting that the HFD mice were insulin resistant (IR) with or without NR treatment. These results suggest that NR treatment does not influence IR”. Results were added to Table 3.
Line 184: The CD group had significantly lower SMNCV as compared with control mice in the STZ treated groups. See TSNCV as well. Were these mice the same age?
Response: STZ and HFD mice were of different age. STZ mice were 5 months-old whereas HFD-fed mice were 3 months-old.
Line 186-7: is there a reference range diagnosing DPN with these parameters without looking at the differences between the groups? This is important since TSNCV is considerably low on the HFD control animals as compared with the STZ control.
Response: As mentioned above, the STZ mice were older at the start of that experimental study compared with the HFD-fed mice. As with humans, there is a normative curve for the nerve conduction studies .
Line 190: How long were the animals kept in the study. Please clarify the timeline for the HFD-fed mice.
Response: The following clarification has been added: One-week after HFD-diet feeding, NMN was administered. Nicotinamide Mononucleotide (NMN) was dissolved in saline and injected IP into diabetic rats at a dose of 50 mg/kg or 100 mg/kg on alternate days (Monday, Wednesday, and Friday) for 3 months. The control animals were injected IP with saline on alternate days. Chronic IP administration of NMN at a dose of 500 mg/kg/daily for 10 days was found to safe and effective [17]. The total length of the HFD study was 30 weeks.
Line 192: Important to discuss whether the HFD-induced PN is a consequence of diabetes or just hyperlipidemia, and to do that, authors need to show whether T2DM was established in the model
Response: According to human blood glucose parameters, the HFD mice would have met criteria for diabetes because the glucose was >200 mg/dl, the HBA1C was > 6.5%, and the intraperitoneal GTT was consistent with diabetes. The mechanism leading to peripheral neuropathy are complex but likely hyperglycemia, hyperlipidemia, and other factors are implicated. We have discussed this in line 337 of the revised manuscript.
10) Line 215-16: Misplaced. Move it to the discussion
Response: Moved to discussion, line 289
11) Line 237-40: Make sure this statement means what you intended to say.
Response: Revised the statement: “Maximal oxygen consumption rate induced by the uncoupler FCCP in cultured neurons from CD mice (513 ± 22 pmol O2/min) was significantly higher compared to DRG neurons from HFD mice (335 ± 15 pmol O2/min)”.
Discussion:
Line 261: the role of NMN on Mt respiratory capacity wasn’t studied.
Response: We studied the effect of NMN administration on mitochondrial respiratory function in hippocampal brain tissue but not in DRG neurons. That’s why it was not included. The results on isolated mitochondria from hippocampal tissue was published (Nicotinamide Mononucleotide Administration Prevents Experimental Diabetes-Induced Cognitive Impairment and Loss of Hippocampal Neurons, Krish Chandrasekaran, Joungil Choi, Muhammed Ikbal Arvas, Mohammad Salimian, Sujal Singh, Su Xu, Rao P Gullapalli, Tibor Kristian and James William Russell, Int. J. Mol. Sci. 2020, 21, 3756). The results showed that administration of NMN significantly increased both ADP-stimulated (State 3) and uncoupled respiration. Spare respiratory capacity was calculated after subtracting the basal respiration from uncoupled respiration and was also significantly higher in NMN-treated hippocampal mitochondria (p < 0.05 in Non-Diabetic vs. Diabetic and p < 0.01 Diabetic + NMN vs. Diabetic).
Line 266-8: Add a reference
Response: References added Line 278
Line 276: Why was NMN not the one studied instead of NR?
Response: We studied the effect of NMN administration on mitochondrial respiratory function in hippocampal brain tissue but not in DRG neurons. That’s why it was not included. Discussed in Response 1 under Discussion.
Line 278: The discussion is heavy on gene networks, but the authors did not do any mRNA expression assays. This should be clarified, and authors should avoid overreaching (see line 286-89)
Response: We have deleted the overreaching statement. Line 301.
5) Line 299-301: add a reference. Check and add references to other statements that are based on evidence generated from previous studies.
Response: Relevant references added, and one reference deleted.
6) Line 317: Avoid mentioning 'show' twice in one statement. Check others as well.
Response: Corrected, Line 331.
Materials and Methods:
Editorial input such as in line 380 ('could have' should be replaced with 'had')
Response: replaced with had, Line 393
Line 384: When did the treatments start?
Response: Three-weeks-old male C57BL6 mice were fed with HFD. One week after HFD feeding, NMN was administered.
Section 4.2: It should be 'diabetes induction with HFD'. Also, the 2 sections should specify the type of diabetes being induced.
Response: Replaced the section with “T2D induction with HFD”.
Section 4.3 is misplaced
Response: Changed to Section 4.7
Line 425: Why was dose translation not performed to ensure that the treatments take into account the surface area?
Response: Considering the surface area of rat and mice, the dose of 100 mg/kg in rats would correspond to 50 mg/kg in mice. The dose of 100 mg/kg in mice would represent a higher dose of NMN.
Line 426: These tests are not indicated on the rat section.
Response: Included
Line 427: How was IP-GTT performed?
Response: Reference to our previous publication in the revised manuscript line 535, included. “Animal was fasted for 6 hours prior to the glucose tolerance test. The animal had free access to drinking water during the fasting period. A sterile 20 % D-glucose stock solution in water was prepared. Prior to performing a glucose tolerance test, body weight and a baseline glucose level were recorded for each animal. A small drop of tail blood was placed on the glucometer test strip and the baseline blood glucose value was recorded (in mg/dL). The animal was injected i.p. with a dose of 2 mg glucose/gram body weight. Additional blood samples are obtained at 15, 30, 60, and 120 minutes to measure post-challenge glucose levels. Statistical data for area under the curve for the glucose tolerance test was calculated”.
Line 432: Why was NR not tested in the STZ group?
Response: The effect of NR on STZ mice was not tested as a prevention protocol but was tested to see if it would reverse after STZ-induced DPN and will be presented separately.
Line 439: Use animals and not mouse as both rats and mice were used.
Response: changed to animals
Round 2
Reviewer 2 Report
None
