Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

Reviewer #1 (Public review):

Summary:

The authors demonstrate impairments induced by a high cholesterol diet on GLP-1R dependent glucoregulation in vivo as well as an improvement after reduction in cholesterol synthesis with simvastatin in pancreatic islets. They also map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations and screened for key residues selected from these sites and performed detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviour, clustering, trafficking and signalling in pancreatic beta cells and primary islets, and describe an improved insulin secretion profile for the V229A mutant receptor.

These are extensive and very impressive studies indeed. I am impressed with the tireless effort exerted to understand the details of molecular mechanisms involved in the effects of cholesterol for GLP-1 activation of its receptor. In general the study is convincing, the manuscript well written and the data well presented. Some of the changes are small and insignificant which makes one wonder how important the observations are. For instance in figure 2 E (which is difficult to interpret anyway because the data are presented in percent, conveniently hiding the absolute results) does not show a significant result of the cyclodextrin except for insignificant increases in basal secretion. That is not identical to impairment of GLP-1 receptor signaling!

To me the most important experiment of them all is the simvastatin experiment, but the results rest on very few numbers and there is a large variation. Apparently, in a previous study using more extensive reduction in cholesterol the opposite response was detected casting doubt on the significance of the current observation. I agree with the authors that the use of cyclodextrin may have been associated with other changes in plasma membrane structure than cholesterol depletion at the GLP-1 receptor. The entire discussion regarding he importance of cholesterol would benefit tremendously from studies of GLP-1 induced insulin secretion in people with different cholesterol levels before and after treatment with cholesterol-lowering agents. I suspect that such a study would not reveal major differences.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors provided a proof of concept that they can identify and mutate a cholesterol-binding site of a high-interest class B receptor, the GLP-1R, and functionally characterize the impact of this mutation on receptor behavior in the membrane and downstream signaling with the intent that similar methods can be useful to optimize small molecules that as ligands or allosteric modulators of GLP-1R can improve the therapeutic tools targeting this signaling system.

Strengths:

The majority of results on receptor behavior are elucidated in INS-1 cells expressing the wt or mutant GLP-1R, with one experiment translating the findings to primary mouse beta-cells. I think this paper lays a very strong foundation to characterize this mutation and does a good job discussing how complex cholesterol-receptor interactions can be (ie lower cholesterol binding to V229A GLP-1R, yet increased segregation to lipid rafts). Table 1 and Figure 9 are very beneficial to summarize the findings. The lower interaction with cholesterol and lower membrane diffusion in V229A GLP-1R resembles the reduced diffusion of wt GLP-1R with simv-induced cholesterol reductions, although by presumably decreasing the cholesterol available to interact with wt GLP-1R. This could be interesting to see if lowering cholesterol alters other behaviors of wt GLP-1R that look similar to V229A GLP-1R. I further wonder if the authors expect that increased cholesterol content of islets (with loading of MβCD saturated with cholesterol or high-cholesterol diets) would elevate baseline GLP-1R membrane diffusion, and if a more broad relationship can be drawn between GLP-1R membrane movement and downstream signaling.

Weaknesses:

I think there are no obvious weaknesses in this manuscript and overall, I believe the authors achieved their aims and have demonstrated the importance of cholesterol interactions on GLP-1R functioning in beta-cells. I think this paper will be of interest to many physiologists who may not be familiar with many of the techniques used in this paper and the authors largely do a good job explaining the goals of using each method in the results section. The intent of some methods, for example the Laurdan probe studies, are better expanded in the discussion. I found it unclear what exactly was being measured to assess 'receptor activity' in Fig 7E and F.

Certainly many follow-up experiments are possible from these initial findings and of primary interest is how this mutation affects insulin homeostasis in vivo under different physiological conditions. One of the biggest pathologies in insulin homeostasis in obesity/t2d is an elevation of baseline insulin release (as modeled in Fig 1E) that renders the fold-change in glucose stimulated insulin levels lower and physiologically less effective. No difference in primary mouse islet baseline insulin secretion was seen here but I wonder if this mutation would ameliorate diet-induced baseline hyperinsulinemia.

I would have liked to see the actual islet cholesterol content after 5wks high-cholesterol diet measured to correlate increased cholesterol load with diminished glucose-stimulated inulin. While not necessary for this paper, a comparison of islet cholesterol content after this cholesterol diet vs the more typical 60% HFD used in obesity research would be beneficial for GLP-1 physiology research broadly to take these findings into consideration with model choice.

Another area to further investigate is does this mutation alter ex4 interaction/affinity/time of binding to GLP-1 or are all of the described findings due to changes in behavior and function of the receptor?

Lastly, I wonder if V229A would have the same impact in a different cell type, especially in neurons? How similar are the cholesterol profiles of beta-cells and neurons? How this mutation (and future developed small molecules) may affect satiation, gut motility, and especially nausea, are of high translational interest. The comparison is drawn in the discussion between this mutation and ex4-phe1 to have biased agonism towards Gs over beta-arrestin signaling. Ex4-phe1 lowered pica behavior (a proxy for nausea) in the authors previously co-authored paper on ex4-phe1 (PMID 29686402) and I think drawing a parallel for this mutation or modification of cholesterol binding to potentially mitigate nausea is worth highlighting.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors demonstrate impairments induced by a high cholesterol diet on GLP-1R dependent glucoregulation in vivo as well as an improvement after reduction in cholesterol synthesis with simvastatin in pancreatic islets. They also map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations and screened for key residues selected from these sites and performed detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviour, clustering, trafficking and signalling in pancreatic beta cells and primary islets, and describe an improved insulin secretion profile for the V229A mutant receptor.

These are extensive and very impressive studies indeed. I am impressed with the tireless effort exerted to understand the details of molecular mechanisms involved in the effects of cholesterol for GLP-1 activation of its receptor. In general the study is convincing, the manuscript well written and the data well presented.

Some of the changes are small and insignificant which makes one wonder how important the observations are. For instance in figure 2 E (which is difficult to interpret anyway because the data are presented in percent, conveniently hiding the absolute results) does not show a significant result of the cyclodextrin except for insignificant increases in basal secretion. That is not identical to impairment of GLP-1 receptor signaling!

We assume that the reviewer refers to Fig. 1E, where we show the percentage of insulin secretion in response to 11 mM glucose +/- exendin-4 stimulation in mouse islets pretreated with vehicle or MβCD loaded with 20 mM cholesterol. While we concur with the reviewer that the effect in this case is triggered by increased basal insulin secretion at 11 mM glucose, exendin-4 can no longer compensate for this increase by proportionally amplifying insulin responses in cholesterol-loaded islets, leading to a significantly decreased exendin-4-induced insulin secretion fold increase under these circumstances, as shown in Fig. 1F. We interpret these results as a defect in the GLP-1R capacity to amplify insulin secretion beyond the basal level to the same extent as in vehicle conditions. An alternative explanation is that there is a maximum level of insulin secretion in our cells, and 11 mM glucose + exendin-4 stimulation gets close to that value. With the increasing effect of cholesterol-loaded MβCD on basal secretion at 11 mM glucose, exendin-4 stimulation appears as working less well. A simple experiment to rule out this possibility would be to test insulin secretion following KCl stimulation under these conditions to determine if maximal stimulation has been reached or not. We will perform this control experiment in the revised manuscript to clarify this point. We will also include absolute insulin results as well as percentages of secretion to improve the completeness of the report.

To me the most important experiment of them all is the simvastatin experiment, but the results rest on very few numbers and there is a large variation. Apparently, in a previous study using more extensive reduction in cholesterol the opposite response was detected casting doubt on the significance of the current observation. I agree with the authors that the use of cyclodextrin may have been associated with other changes in plasma membrane structure than cholesterol depletion at the GLP-1 receptor.

We agree with the reviewer that the insulin secretion results in vehicle versus LPDS/simvastatin treated mouse islets (Fig. 1H, I) are relatively variable and we therefore plan to perform further biological repeats of this experiment for the paper revision to consolidate our current findings.

The entire discussion regarding the importance of cholesterol would benefit tremendously from studies of GLP-1 induced insulin secretion in people with different cholesterol levels before and after treatment with cholesterol-lowering agents. I suspect that such a study would not reveal major differences.

We agree with the reviewer that such study would be highly relevant. While this falls outside the scope of the present paper, we encourage other researchers with access to clinical data on GLP-1RA responses in individuals taking cholesterol lowering agents to share their results with the scientific community. We will highlight this point in the paper discussion to emphasise the importance of more research in this area.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors provided a proof of concept that they can identify and mutate a cholesterol-binding site of a high-interest class B receptor, the GLP-1R, and functionally characterize the impact of this mutation on receptor behavior in the membrane and downstream signaling with the intent that similar methods can be useful to optimize small molecules that as ligands or allosteric modulators of GLP-1R can improve the therapeutic tools targeting this signaling system.

Strengths:

The majority of results on receptor behavior are elucidated in INS-1 cells expressing the wt or mutant GLP-1R, with one experiment translating the findings to primary mouse beta-cells. I think this paper lays a very strong foundation to characterize this mutation and does a good job discussing how complex cholesterol-receptor interactions can be (ie lower cholesterol binding to V229A GLP-1R, yet increased segregation to lipid rafts). Table 1 and Figure 9 are very beneficial to summarize the findings. The lower interaction with cholesterol and lower membrane diffusion in V229A GLP-1R resembles the reduced diffusion of wt GLP-1R with simv-induced cholesterol reductions, although by presumably decreasing the cholesterol available to interact with wt GLP-1R. This could be interesting to see if lowering cholesterol alters other behaviors of wt GLP-1R that look similar to V229A GLP-1R. I further wonder if the authors expect that increased cholesterol content of islets (with loading of MβCD saturated with cholesterol or high-cholesterol diets) would elevate baseline GLP-1R membrane diffusion, and if a more broad relationship can be drawn between GLP-1R membrane movement and downstream signaling.

Membrane diffusion experiments are difficult to perform in intact islets as our method requires cell monolayers for RICS analysis. We do however agree that it would be interesting to perform further RICS analysis in INS-1 832/3 SNAP/FLAG-hGLP-1R cells pretreated with vehicle or MβCD loaded with 20 mM cholesterol, and we will therefore add this experiment to the paper revisions.

Weaknesses:

I think there are no obvious weaknesses in this manuscript and overall, I believe the authors achieved their aims and have demonstrated the importance of cholesterol interactions on GLP-1R functioning in beta-cells. I think this paper will be of interest to many physiologists who may not be familiar with many of the techniques used in this paper and the authors largely do a good job explaining the goals of using each method in the results section.

The intent of some methods, for example the Laurdan probe studies, are better expanded in the discussion.

To clarify the intent of the Laurdan experiments early in the manuscript, we will add the following text to the methods section in the paper revisions: “Laurdan, 6-dodecanoyl-2-dimethylaminonaphthalene (product D250) was purchased from ThermoFisher. Laurdan (40 μM) was excited using a 405 nm solid state laser and SNAP/FLAG-hGLP-1R labelled with SNAP-Surface Alexa Fluor 647 with a pulsed (80 MHz) super-continuum white light laser at 647 nm. Laurdan emission was recorded in the ranges of 420–460 nm (IB) and 470–510 nm (IR), and the general polarisation (GP) formula (GP = IB-IR/IB+IR) used to retrieve the relative lateral packing order of lipids at the plasma membrane. Values of GP vary from 1 to −1, where higher numbers reflect lower fluidity or higher lateral lipid order, whereas lower numbers indicate increasing fluidity.”

I found it unclear what exactly was being measured to assess 'receptor activity' in Fig 7E and F.

Figs. 7E and F refer to bystander complementation assays measuring the recruitment of nanobody 37 (Nb37)-SmBiT, which binds to active Gas, to either the plasma membrane (labelled with KRAS CAAX motif-LgBiT), or to endosomes (labelled with Endofin FYVE domain-LgBiT) in response to GLP-1R stimulation with exendin-4. This assay therefore measures GLP-1R activation specifically at each of these two subcellular locations. We will add a schematic of this assay to the methods section in the paper revisions to clarify the aim of these experiments.

Certainly many follow-up experiments are possible from these initial findings and of primary interest is how this mutation affects insulin homeostasis in vivo under different physiological conditions. One of the biggest pathologies in insulin homeostasis in obesity/t2d is an elevation of baseline insulin release (as modeled in Fig 1E) that renders the fold-change in glucose stimulated insulin levels lower and physiologically less effective. No difference in primary mouse islet baseline insulin secretion was seen here but I wonder if this mutation would ameliorate diet-induced baseline hyperinsulinemia.

We concur with the reviewer that it would be interesting to determine the effects of the GLP-1R V229A mutation on insulin secretion responses under diet-induced metabolic stress conditions. While performing in vivo experiments on glucoregulation in mice harbouring the V229A mutation falls outside the scope of the present study, in the paper revisions we will include ex vivo insulin secretion experiments in islets from GLP-1R KO mice transduced with adenoviruses expressing SNAP/FLAG-hGLP-1R WT or V229A and subsequently treated with vehicle versus MβCD loaded with 20 mM cholesterol to replicate the conditions of Fig. 1E.

I would have liked to see the actual islet cholesterol content after 5wks high-cholesterol diet measured to correlate increased cholesterol load with diminished glucose-stimulated inulin. While not necessary for this paper, a comparison of islet cholesterol content after this cholesterol diet vs the more typical 60% HFD used in obesity research would be beneficial for GLP-1 physiology research broadly to take these findings into consideration with model choice.

We will include these data and compare islet cholesterol levels after the high cholesterol diet with those of HFD-fed mouse islets in the paper revisions.

Another area to further investigate is does this mutation alter ex4 interaction/affinity/time of binding to GLP-1 or are all of the described findings due to changes in behavior and function of the receptor?

To answer this question, we will perform exendin-4 binding affinity experiments in INS-1 832/3 SNAP/FLAG-hGLP-1R WT versus V229A cells for the paper revisions.

Lastly, I wonder if V229A would have the same impact in a different cell type, especially in neurons? How similar are the cholesterol profiles of beta-cells and neurons? How this mutation (and future developed small molecules) may affect satiation, gut motility, and especially nausea, are of high translational interest. The comparison is drawn in the discussion between this mutation and ex4-phe1 to have biased agonism towards Gs over beta-arrestin signaling. Ex4-phe1 lowered pica behavior (a proxy for nausea) in the authors previously co-authored paper on ex4-phe1 (PMID 29686402) and I think drawing a parallel for this mutation or modification of cholesterol binding to potentially mitigate nausea is worth highlighting.

While experiments in neurons are outside the scope of the present study, we will add this worthy point to the discussion and hypothesise on possible effects of the V229A mutation on central GLP-1R effects in the revised manuscript.