Referee 1's review:

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication, the manuscript has been revised in light of these comments and to address other editorial requirements.
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PAPER COMMENTS and REVIEW:
The paper entitled "Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1" attempts to demonstrate a negative relationship between GLP1 and NOS expression/activity. The authors show that islets of GK rats have elevated NO generation that is associated with abnormal iNOS and ncNOS expression/activity in beta-cells and alpha-cells. They also suggest an associated between elevated NO and impaired glucose-stimulated insulin release, elevated glucagon secretion and impaired glucose-induced glucagon suppression. The authors also show that GLP1 can suppress iNOS and ncNOS expression and activity and can partially restore hormone secretion. The ability of GLP1 to suppress iNOS expression was shown to be reversed by the PKA inhibitor H89. Injection of glucose plus GLP-1 in GK rats showed that GLP-1 amplified the insulin response in vivo. They also suggest that increased NO production within diabetic islets may be a significant player in the pathogenesis of type 2 diabetes and this elevated NO may be counteracted by GLP-1 through PKA-dependent mechanism. Overall this is a nice paper but needs more work before it is ready for publication.

MAJOR COMMENTS:
1. This paper is an extension of the author's previous work looking at the role of GLP1 regulated NOS activity. Their previous work showed that GLP1 could inhibit glucose-stimulated NOS activity and now extend this to look at the role of GLP1 in regulating NOS activity in the diabetic GK rat. The authors fail to confirm the effects of GLP1 on NOS activity they found in the in vitro studies in the in vivo studies. They could easily treat with GLP1 in vivo during the ivGTT and then isolate islets and measure NOS by western and NOS activity. I admit that the effects on NOS activity in vivo may be only small since most of the effects of GLP1 should be on enhanced glucose-stimulated insulin release however I believe this is a critical point to this paper.
2. The studies of the involvement of the proteosome system in the degradation NOS are weak. No concentration of MG132 was given and no other inhibitors were investigated. Either more data needs to be generated here to definitively rule out this system or this data should all be removed. If the proteosome system is not degrading iNOS and ncNOS then what is? Reference 20 is only a minor paper looking at the regulation of NOS by the proteosome system. The authors need to find a better reference e.g. Musial A, Eissa NT. Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway. J Biol Chem. 2001 29;276(26):24268-73. In addition, the authors will also need to provide a better discussion of the role of this system and its role in the regulation of NOS is required.
3. A NOS inhibitor (e.g. NG-amino-L-arginine (NAA)) could be used to verify that NOS is involved in the effects on insulin and glucagon release in vitro and in vivo in the GK rat.

MINOR COMMENTS:
1. The term ex vivo used throughout the paper suggests that the author's techniques are unique from other islet studies. It is more common to use "in vitro" instead for primary culture of islets (whether the islets are used right after isolation or after culture).
2. Figure 1A and Figure 3: please provide the control Wistar data for comparison.
3. Although Wistar rats are commonly used as a control for studies using GK rats, the use of this control is still being debated since there are strain to strain and even colony to colony differences in the glucose responsiveness for rats and mice. This needs to be discussed.
4. The method of measuring the differences between ncNOS and iNOS needs more detail. The protocol describes how you measure iNOS but not ncNOS. Is ncNOS back calculated from the total NOS activity? It was also not clear how many islets you used in these studies (250 islets?).
5. Are the effects of GLP1 dose-dependent?
6. I am not sure if the term ncNOS is still used but as far as I know it is more commonly called nNOS.
7. Since the authors had an antibody to ncNOS, why didn't they do IHC for ncNOS.
8. Since the decrease in ncNOS and iNOS is a critical component to this paper, the western blot and/or IHC data needs to be quantified.
9. There is multiple published version of how to prepare KRB. Please define the ingredients of your KRB.
10. An alternative hypothesis for these studies is that the increased NADPH utilization by iNOS may decrease NADPH availability to act as a signaling molecule for glucose-regulated insulin release.