Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ signalling to control glucose-dependent insulin secretion
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Evaluation Summary:
This paper will be of interest not only to the field of insulin release and beta cell biology but also of general interest to the community interested in peptide hormone secretion. It shows that the preservation of the tissue context, in particular the local interaction with integrins at the capillary interface, is important in preserving cell function when using cultured cell or organ isolates in vitro.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
A developing understanding suggests that spatial compartmentalisation in pancreatic β cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell subcellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact β-cell structure, and enhances glucose-dependent Ca 2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase (FAK), in regulating β cells. Integrins and FAK are exclusively activated at the β-cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose-dependent Ca 2+ responses and insulin secretion. We conclude that FAK orchestrates the final steps of glucose-dependent insulin secretion within the restricted domain where β-cell contact the islet capillaries.
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Author Response:
Reviewer #1 (Public Review):
In this detailed study the authors show that in isolated islets the polarity of the secretory apparatus is largely lost while it is preserved in slices where the capillary network remains intact. The authors then go on to show that the integrin/FAK pathway appears to be responsible for inducing and maintaining polarity, which involves concentration of active zone proteins and calcium channels at the contact sites and a higher sensitivity and potency of insulin secretion to glucose stimulation.
Generally, the data appear to be of high quality, being carried out with state-of-the-art technology, and the manuscript is lavishly illustrated. Since as a neuroscientist I am not sufficiently familiar with the field of the cell biology of insulin release it is difficult for me to judge whether …
Author Response:
Reviewer #1 (Public Review):
In this detailed study the authors show that in isolated islets the polarity of the secretory apparatus is largely lost while it is preserved in slices where the capillary network remains intact. The authors then go on to show that the integrin/FAK pathway appears to be responsible for inducing and maintaining polarity, which involves concentration of active zone proteins and calcium channels at the contact sites and a higher sensitivity and potency of insulin secretion to glucose stimulation.
Generally, the data appear to be of high quality, being carried out with state-of-the-art technology, and the manuscript is lavishly illustrated. Since as a neuroscientist I am not sufficiently familiar with the field of the cell biology of insulin release it is difficult for me to judge whether there is sufficient advance in knowledge. A higher degree of organization of release sites including a role of active zone proteins was previously demonstrated from other endocrine organs involving the release of large dense-core vesicles such as chromaffin cells. Thus, the differences between the highly organized and rapidly responding exocytotic sites in neurons and the slower reacting release sites of peptide/protein containing granules are not fundamental but rather gradual, despite the principal cell biological differences between the biogenesis and recycling pathways of the secretory organelles.
In summary, the work adds new aspects to the understanding of the regulation of exocytosis in pancreatic beta cells. Aside from corrections of figure descriptions and experimental details, my only major comment relates to the data shown in Fig. 4. It appears that the difference in the time-to-peak between the two preparation is mainly caused by a (rather variable?) delay between glucose addition and the onset of the rise since the rate of increase is apparently not different between the preparations. Is this due a delay in depolarization, i.e. a delay in the closure of the ATP-K channels? This should be clarified. Also, the authors should show a comparative histogram of the delay times (between glucose addition and the inflection point at the onset of the rise).
The delay observed is due to a slower response in islets vs slices, which given the potentiating effects we show of the KATP channel drugs (diazoxide and now glibenclamide) is likely explained by a delay in KATP closure. However, since we are measuring the Ca2+ response we cannot directly prove this. We feel this is adequately discussed with reference to glucose-dependent triggering (where the KATP channel is a key component). In direct response to the referee’s comment about variability, we have re-expressed the data to show frequency histogram comparisons of the delay to peak (new Fig 4J).
Reviewer #2 (Public Review):
- The authors present an investigation of subcellular distribution and dynamics of known presynaptic proteins in a relatively new approach, pancreatic slices, mastered by a limited number of laboratories, and which is currently the best method to largely preserve capillary networks. They demonstrate the advantage of this method by detailed cellular and subcellular optical analysis comparing isolated islets, islets in pancreatic slices, isolated islet cells and isolated islet cells on ECM (laminin) covered surfaces. This work provides good proof that preservation of capillary networks and corresponding distribution of proteins (laminin, liprin, integrin beta1 etc) is required for insulin secretion at the apical surface of islet cells. Moreover, in these pancreatic slices they observe a restriction of exocytotic sites at the vascular surfaces. The role of the extracellular matrix is also well investigated here by experiments on dispersed or single beta cells attached either to a glass-BSA interface or to a glass-laminin interface. However, the authors have already previously published in 2014 a restricted polarized insulin secretion in cultured islets as well as the preservation of localized liprin and laminin distribution (as well as RIM2 and piccolo; DOI 10.1007/s00125-014-3252-6). It is not clear why these data cannot be reproduced now again in isolated islets (see Fig. 1 and 2) .
We thank the referee for their comments. To clarify the specific issue around our past work. All our live sub-cellular resolution experiments have previously been performed with isolated islets – we have not, until recently been able to reliably get the slice to work. In contrast, our work with immunofluorescence of active zone proteins has been performed with fixed slices (including DOI 10.1007/s00125-014-3252-6, Low et al 2014).
- The authors try to gain insight which mechanisms control this specific spatial restriction and they provide evidence that Focal Adhesion kinase activity is implicated in glucose-induced calcium fluxes and insulin secretion by the use of a small molecule antagonist and the use of a purified monoclonal antibody. They conclude that FAK is a master regulator of glucose induced insulin secretion that controls positioning of presynaptic scaffold proteins and the functioning of calcium channels. Although FAK may be a regulator, the claim that FAK controls functioning of calcium channels can certainly not be made. Ratio measurements of cellular calcium levels do not suffice for that (patch or sharp would be required). Moreover, the fact that KCl-induced insulin secretion (which bypasses nutrient metabolism and leads directly to opening of voltage-dependent calcium channels) is not altered by the FAK antagonist strongly argues against a role of FAK in calcium channel regulation. Indeed, the presented data suggest that FAK may intervene far more upstream from exocytosis such as in nutrient metabolism or granule mobility/maturation.
Our data clearly shows that integrin/FAK activation is part of the glucose dependent control of Ca2+ and insulin secretion. It is not relevant to this conclusion how we measure Ca2+ responses – they are obviously affected by all manipulations of integrin/FAK. We note that the referee is specifically correct in saying that we do not have evidence that Ca2+ channel function is a direct target of integrins/FAK and we have reworded the text to make this clear.
Further, our work does not define where in the glucose pathway integrin/FAK are acting. The referee is correct in saying the KCl data suggests it is upstream of the final stages of Ca2+ channel and exocytosis. Consistent with this we see effects of integrin/FAK manipulation on ELKS and liprin positioning (Figs 7 and 8) and, given the published data showing that ELKS enhances Ca2+ channel current (Ohara-Imaizumi et al 2019) we think it is plausible integrin/FAK intersect with this pathway to regulate Ca2+ channel activity. With reference to the high K responses, KCl rapidly depolarises the cells to recruit Ca2+ channels, in contrast glucose slowly depolarises cells. This difference will affect Ca2+ channel behaviour and altered CaV1.2 function, such as lowered voltage threshold might specifically only be apparent in the glucose responses.
- The authors present data that islets in pancreatic slices are considerably more sensitive to glucose, inducing a response already at basal glucose levels (2.8 mM). In the same vein the authors observe a considerably shortened delay between stimulus and response (this delay is general due to nutrient metabolism and initial filling of intracellular calcium stores). The authors take these phenomena as evidence for a superior and more physiological quality of their islet slices as compared to conventional purified islets.
However, contrary to their interpretation, these observations considerably questions whether the slice preparation used here in this work has physiological qualities. Indeed, the authors observe considerable activity of islet beta-cells already far below the set-point of around 6 or 7 mM in rodents, very well characterized through a number of studies in-vivo, in-vitro and even in-situ (10.1113/jphysiol.1995.sp020804), and their preparations reach almost full activity around the set-point. This is also surprising as such a hypersensitivity has not been reported by several other groups using the same preparation, i.e. pancreatic slices (10.1152/ajpendo.00043.2021; 10.1371/journal.pone.0054638; 10.3389/fphys.2019.00869; 10.1371/journal.pcbi.1009002; 10.1038/nprot.2014.195) even using patch clamp (10.3390/s151127393). >Moreover, even human islets, known for a lower set-point, are inactive in slices at 3 mM (10.1038/s41467-020-17040-8) in line with the physiological requirement to avoid insulin secretion in low glucose states as to avoid life-threatening hypoglycaemia. The same applies for the shortened delay between application of a stimulus (glucose) and start of the response, which has also not been observed by other groups in pancreatic slices (refs see above).
We are cognisant that our data challenges the dogma and talked around this point in the discussion. Evidence that our findings might be correct include the responses seen by Henquin to glucose concentrations below 6 mM (Gembal et al 1992) and the long-standing evidence of heterogeneous responses in isolated cells that show responses to very low glucose concentrations (Van Schravendijk et al 1992). As such, our data is not as unusual as it might initially appear. Furthermore, as discussed in detail below the findings from others using the slice preparation is not directly or easily compared to our work.
In general, such an increased glucose sensitivity is observed in prediabetic states or experiments mimicking such a condition. To the best of my recollection such an apparently increased sensitivity can also be observed in brain slices due to leakage. Unfortunately, no independent measures of islet quality in slices are provided.
We have previously characterised increased insulin secretion in “prediabetes” in mice and demonstrated a clear effect on the mechanisms of granule fusion such as an increase in compound exocytosis (Do et al 2016). We do not think this is relevant to this slice preparation where normal mice were used for both the slice and the islet experiments and our data in slices and islets both show normal granule fusion and not compound exocytosis.
Within the same vein the comparison between slices and islets (Fig 5) is not in favour of a more physiological aspect of slices and the different cell morphology and small number of observations shed more doubt, especially in view of the well known normal beta-cell heterogeneity (which may explain differences and may have been missed here due to a small sample size).
We acknowledge that beta cell heterogeneity is a potential confounding factor. However, our sample sizes are not small, in each islet or slice we record Ca2+ responses from ~10 cells (see Fig 3) and have repeated preparations from each mouse with the total dataset from >3 mice. It is true that the sample size for Ca2+ waves is small for the isolated islets, but this is because these are such rare events which is explained by the fragmented capillaries and compromised cell structure (eg Fig 1) in isolated islets.
In a larger context this glucose supersensitivity may also shed doubts on the proposed important role of FAK as its role may be far less preponderant in preparations corresponding to physiological criteria.
We agree that the relative importance of FAK might be different in different in vitro models. But it is clear that FAK plays an important role in vivo and the data from FAK KO mice show both defective glucose homeostasis and lower insulin secretion (Cai et al 2012) directly demonstrating physiological relevance.
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Evaluation Summary:
This paper will be of interest not only to the field of insulin release and beta cell biology but also of general interest to the community interested in peptide hormone secretion. It shows that the preservation of the tissue context, in particular the local interaction with integrins at the capillary interface, is important in preserving cell function when using cultured cell or organ isolates in vitro.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
In this detailed study the authors show that in isolated islets the polarity of the secretory apparatus is largely lost while it is preserved in slices where the capillary network remains intact. The authors then go on to show that the integrin/FAK pathway appears to be responsible for inducing and maintaining polarity, which involves concentration of active zone proteins and calcium channels at the contact sites and a higher sensitivity and potency of insulin secretion to glucose stimulation.
Generally, the data appear to be of high quality, being carried out with state-of-the-art technology, and the manuscript is lavishly illustrated. Since as a neuroscientist I am not sufficiently familiar with the field of the cell biology of insulin release it is difficult for me to judge whether there is sufficient …
Reviewer #1 (Public Review):
In this detailed study the authors show that in isolated islets the polarity of the secretory apparatus is largely lost while it is preserved in slices where the capillary network remains intact. The authors then go on to show that the integrin/FAK pathway appears to be responsible for inducing and maintaining polarity, which involves concentration of active zone proteins and calcium channels at the contact sites and a higher sensitivity and potency of insulin secretion to glucose stimulation.
Generally, the data appear to be of high quality, being carried out with state-of-the-art technology, and the manuscript is lavishly illustrated. Since as a neuroscientist I am not sufficiently familiar with the field of the cell biology of insulin release it is difficult for me to judge whether there is sufficient advance in knowledge. A higher degree of organization of release sites including a role of active zone proteins was previously demonstrated from other endocrine organs involving the release of large dense-core vesicles such as chromaffin cells. Thus, the differences between the highly organized and rapidly responding exocytotic sites in neurons and the slower reacting release sites of peptide/protein containing granules are not fundamental but rather gradual, despite the principal cell biological differences between the biogenesis and recycling pathways of the secretory organelles.
In summary, the work adds new aspects to the understanding of the regulation of exocytosis in pancreatic beta cells. Aside from corrections of figure descriptions and experimental details, my only major comment relates to the data shown in Fig. 4. It appears that the difference in the time-to-peak between the two preparation is mainly caused by a (rather variable?) delay between glucose addition and the onset of the rise since the rate of increase is apparently not different between the preparations. Is this due a delay in depolarization, i.e. a delay in the closure of the ATP-K channels? This should be clarified. Also, the authors should show a comparative histogram of the delay times (between glucose addition and the inflection point at the onset of the rise).
-
Reviewer #2 (Public Review):
1. The authors present an investigation of subcellular distribution and dynamics of known presynaptic proteins in a relatively new approach, pancreatic slices, mastered by a limited number of laboratories, and which is currently the best method to largely preserve capillary networks. They demonstrate the advantage of this method by detailed cellular and subcellular optical analysis comparing isolated islets, islets in pancreatic slices, isolated islet cells and isolated islet cells on ECM (laminin) covered surfaces. This work provides good proof that preservation of capillary networks and corresponding distribution of proteins (laminin, liprin, integrin beta1 etc) is required for insulin secretion at the apical surface of islet cells. Moreover, in these pancreatic slices they observe a restriction of …
Reviewer #2 (Public Review):
1. The authors present an investigation of subcellular distribution and dynamics of known presynaptic proteins in a relatively new approach, pancreatic slices, mastered by a limited number of laboratories, and which is currently the best method to largely preserve capillary networks. They demonstrate the advantage of this method by detailed cellular and subcellular optical analysis comparing isolated islets, islets in pancreatic slices, isolated islet cells and isolated islet cells on ECM (laminin) covered surfaces. This work provides good proof that preservation of capillary networks and corresponding distribution of proteins (laminin, liprin, integrin beta1 etc) is required for insulin secretion at the apical surface of islet cells. Moreover, in these pancreatic slices they observe a restriction of exocytotic sites at the vascular surfaces. The role of the extracellular matrix is also well investigated here by experiments on dispersed or single beta cells attached either to a glass-BSA interface or to a glass-laminin interface.
However, the authors have already previously published in 2014 a restricted polarized insulin secretion in cultured islets as well as the preservation of localized liprin and laminin distribution (as well as RIM2 and piccolo; DOI 10.1007/s00125-014-3252-6). It is not clear why these data cannot be reproduced now again in isolated islets (see Fig. 1 and 2) .2. The authors try to gain insight which mechanisms control this specific spatial restriction and they provide evidence that Focal Adhesion kinase activity is implicated in glucose-induced calcium fluxes and insulin secretion by the use of a small molecule antagonist and the use of a purified monoclonal antibody. They conclude that FAK is a master regulator of glucose induced insulin secretion that controls positioning of presynaptic scaffold proteins and the functioning of calcium channels.
Although FAK may be a regulator, the claim that FAK controls functioning of calcium channels can certainly not be made. Ratio measurements of cellular calcium levels do not suffice for that (patch or sharp would be required). Moreover, the fact that KCl-induced insulin secretion (which bypasses nutrient metabolism and leads directly to opening of voltage-dependent calcium channels) is not altered by the FAK antagonist strongly argues against a role of FAK in calcium channel regulation. Indeed, the presented data suggest that FAK may intervene far more upstream from exocytosis such as in nutrient metabolism or granule mobility/maturation.3. The authors present data that islets in pancreatic slices are considerably more sensitive to glucose, inducing a response already at basal glucose levels (2.8 mM). In the same vein the authors observe a considerably shortened delay between stimulus and response (this delay is general due to nutrient metabolism and initial filling of intracellular calcium stores). The authors take these phenomena as evidence for a superior and more physiological quality of their islet slices as compared to conventional purified islets.
However, contrary to their interpretation, these observations considerably questions whether the slice preparation used here in this work has physiological qualities. Indeed, the authors observe considerable activity of islet beta-cells already far below the set-point of around 6 or 7 mM in rodents, very well characterized through a number of studies in-vivo, in-vitro and even in-situ (10.1113/jphysiol.1995.sp020804), and their preparations reach almost full activity around the set-point. This is also surprising as such a hypersensitivity has not been reported by several other groups using the same preparation, i.e. pancreatic slices (10.1152/ajpendo.00043.2021; 10.1371/journal.pone.0054638; 10.3389/fphys.2019.00869; 10.1371/journal.pcbi.1009002; 10.1038/nprot.2014.195) even using patch clamp (10.3390/s151127393). Moreover, even human islets, known for a lower set-point, are inactive in slices at 3 mM (10.1038/s41467-020-17040-8) in line with the physiological requirement to avoid insulin secretion in low glucose states as to avoid life-threatening hypoglycaemia. The same applies for the shortened delay between application of a stimulus (glucose) and start of the response, which has also not been observed by other groups in pancreatic slices (refs see above).
In general, such an increased glucose sensitivity is observed in prediabetic states or experiments mimicking such a condition. To the best of my recollection such an apparently increased sensitivity can also be observed in brain slices due to leakage. Unfortunately, no independent measures of islet quality in slices are provided.
Within the same vein the comparison between slices and islets (Fig 5) is not in favour of a more physiological aspect of slices and the different cell morphology and small number of observations shed more doubt, especially in view of the well known normal beta-cell heterogeneity (which may explain differences and may have been missed here due to a small sample size).In a larger context this glucose supersensitivity may also shed doubts on the proposed important role of FAK as its role may be far less preponderant in preparations corresponding to physiological criteria.
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Reviewer #3 (Public Review):
The authors have - using 2-photon imaging of exocytosis - compared insulin secretion in isolated islets maintained in tissue culture with that in islets of acutely prepared slices of the pancreas.
They demonstrate that i) secretion is highly polarized and direct towards the capillaries in the latter preparation, is ii) that the concentration dependence of insulin secretion is shifted towards lower concentrations; and iii) that the difference involves presynaptic scaffold proteins and focal adhesion kinase (FAK).
The manuscript is well written with a clear narrative and the data are logically presented.
The experimental data are of high quality (typifying the work of this team), the results are unexpected but of great significance. The findings bridge the gap between in vivo and in vitro studies.
Insulin …
Reviewer #3 (Public Review):
The authors have - using 2-photon imaging of exocytosis - compared insulin secretion in isolated islets maintained in tissue culture with that in islets of acutely prepared slices of the pancreas.
They demonstrate that i) secretion is highly polarized and direct towards the capillaries in the latter preparation, is ii) that the concentration dependence of insulin secretion is shifted towards lower concentrations; and iii) that the difference involves presynaptic scaffold proteins and focal adhesion kinase (FAK).
The manuscript is well written with a clear narrative and the data are logically presented.
The experimental data are of high quality (typifying the work of this team), the results are unexpected but of great significance. The findings bridge the gap between in vivo and in vitro studies.
Insulin secretion becomes defective in type 2 diabetes (T2D; the commonest form of diabetes afflicting close to 400 million individuals worldwide) and if these data can be extended to human pancreases they might be of highly relevant to the understanding of the aetiology of T2
In general, I am enthusiastic about the paper. Possibly, the mechanisms underlying the differences between isolated islets and slices could be explored in somewhat greater detail and there a actually a few loose ends but they may be possible to resolved by textual changes and may not require additional work.
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