Hyperaminoacidemia induces pancreatic α cell proliferation via synergism between mTORC1 and CaSR-Gq signaling pathways

doi:10.21203/rs.3.rs-798563/v1

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Hyperaminoacidemia induces pancreatic α cell proliferation via synergism between mTORC1 and CaSR-Gq signaling pathways

https://doi.org/10.21203/rs.3.rs-798563/v1

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published 16 Jan, 2023

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Glucagon has emerged as the main regulator of extracellular amino acid homeostasis. Insufficient glucagon signaling results in hyperaminoacidemia, which drives adaptive proliferation of glucagon-producing α cells. Aside from mammalian target of rapamycin complex 1 (mTORC1), the role of other amino acid sensors in the α cell proliferation has not been described. Here, using gcgr-deficient zebrafish and cultured mouse islets, we show that α cell proliferation requires the calcium sensing receptor (CaSR) and downstream extracellular signalregulated protein kinase (ERK1/2). Inactivation of casr dampened α cell proliferation, which can be rescued by re-expression of CaSR or activation of the downstream Gq, but not Gi, signaling in α cells. CaSR was also unexpectedly necessary for mTORC1 activation in α cells. Furthermore, co-activation of Gq and mTORC1 induced α cell proliferation independent of hyperaminoacidemia. These results reveal another amino acid sensitive mediator, and identify major pathways necessary and sufficient for hyperaminoacidemia-induced α cell proliferation.

Endocrinology & Metabolism

General Cell Biology & Physiology

hyperaminoacidemia

glucagon

mTORC1

CaSR

Amino acids are one of the main building blocks and fuel sources of life. As such, plasma amino acid levels are tightly regulated¹ and remain constant during protein restriction and short-term fasting². Glucagon has emerged as a key hormone regulating plasma amino acid homeostasis during the past a few years^3,4. Glucagon stimulates amino acid catabolism by activating ureagenesis and gluconeogenesis in the liver⁵. Impaired glucagon signaling leads to increased plasma amino acid levels (hyperaminoacidemia), which in turn leads to compensatory proliferation of the glucagon-producing α cells and hyperglucagonemia^6–8. Liver specific inactivation of Gcgr⁹ or its downstream Gnas1¹⁰ also leads to α cell hyperplasia in mice. Persistent loss of glucagon signaling eventually results in glucagonoma, tumors of a cells^11,12. These recent studies thus reveal an α cell-liver axis in plasma amino acid homeostasis. Glucagon receptor antagonism (GRA) has been shown to be very effective in lowering blood glucose in preclinical diabetes models. This axis is a roadblock for clinical use of GRA therapy for diabetes¹³. However, how hyperaminoacidemia induces α cell proliferation is not fully understood.

A few mediators have been implicated in hyperaminoacidemia-induced α cell proliferation. Unsurprisingly, the amino acid sensor mTORC1 is necessary for the hyperaminoacidemia-induced α cell proliferation^14–16. The preferential response of α cells to hyperaminoacidemia is due in part to an mTORC1-dependent induction of a glutamine transporter Slc38a5 as its ablation dampens the proliferation^14,15. In the Slc38a5-/- mice and in humans where SLC38A5 expression is absent in α cells, however, hyperaminoacidemia still causes α cell proliferation^14,15. Furthermore, although genetic activation of mTORC1 alone in mouse α cells induces α cell proliferation in adult mice, no change is detected in new born¹⁷. As activated mTORC1 enhances a cell secretion^7,18, leading to hyperglucagonemia and downregulation of hepatic GCGR signaling, the increased a cell proliferation may be secondary to hepatic glucagon resistance. Therefore, other cell intrinsic signaling pathways may also be necessary for the compensatory α cell proliferation.

CaSR is a class C G- protein-coupled receptor (GPCR) that senses L-amino acids and Ca²⁺, among other endogenous ligands ^19,20. It was initially identified because its loss of function and gain of function mutations caused familial hypocalciuric, hypercalcaemia and autosomal dominant hypocalcaemia, respectively²¹. In addition to calcitropic tissues (for example, parathyroid glands, kidneys and breast), CaSR is also expressed in noncalcitropic tissues including enteroendocrine and pancreatic endocrine cells¹⁹. In the pancreatic islet, CaSR is highly expressed in the α and β cells^22,23 and regulates pancreatic endocrine cell secretion^23,24. CaSR couples to multiple heterotrimeric G-protein subtypes (Gq/11, Gi/o, and G12/13) to activate intracellular signal transduction cascades^25,26. These cascades lead to increased cytosolic calcium concentrations and activation of the mitogen-activated protein kinase (MAPK) pathway, which ultimately results in phosphorylation and activation of ERK1/2 ^25,27. Of note, CaSR has also been shown to regulate cell proliferation^28,29. Its mutation or aberrant activation is associated with various types of cancer and altered pancreatic islet mass^24,30,31. However, whether CaSR contributes to the hyperaminoacidemia-induced pancreatic α cell proliferation remains unclear.

In the present study, using zebrafish and cultured mouse islets as models, we set out to determine the molecular mechanism of hyperaminoacidemia-induced pancreatic α cell proliferation (Fig. 1a). We deciphered that the CaSR-Gq-ERK1/2 pathway is another mediator of hyperaminoacidemia-induced pancreatic α cell proliferation. We show that CaSR activated MEK1/2 also contributes to mTORC1 activation. Importantly, co-activation of CaSR-Gq and mTORC1 in α cells causes proliferation in the gcgr-intact background. These results identify the major pathways that are necessary and sufficient for hyperaminoacidemia-induced α cell proliferation, and illustrate a previously unidentified physiological role of CaSR.

Activation of mTORC1 is insufficient for α cell proliferation

We previously demonstrated that the α cell-liver axis is conserved in zebrafish. In zebrafish lacking both gcgra and gcgrb (gcgr^DKO), there are more α cells due to increased proliferation³². In addition, the hyperplasia is rapamycin-sensitive and depends on slc38a5b¹⁴. To fully characterize the phenotypic conservation, we determined the free amino acid levels in whole larvae and in adult serum. Compared to the control groups, the free amino acid level was markedly elevated in larvae and in adult serum of gcgr^DKO zebrafish (Supplementary Fig 1a, b). We also assessed mTORC1 activity in α cells using serine 240/244 phosphorylation of S6 ribosomal protein (pS6) as a readout. The number of pS6 positive α cells and pS6 signal intensity were both increased in the α cells of gcgr^DKO zebrafish (Supplementary Fig 1c, d and e). These data confirm that loss of glucagon signaling causes hyperaminoacidemia and activates mTORC1 in α cells^14-16^,³³.

Whether mTORC1 activation is sufficient for α cell proliferation is unknown. To answer this question, we established two transgenic lines, Tg(gcga:MTOR^L1460P, cryaa:tagRFP) and Tg(gcga:Rheb^S16H, cryaa:YFP) (Tgα^CA-MTOR and Tgα^CA-Rheb hence forward), that express a constitutively active mTOR^L1460P (CA-MTOR) or Rheb^S16H (CA-Rheb) only in α cells. Lens fluorescent markers were used to facilitate genotyping. As expected, the pS6 signal was overtly increased in both transgenic lines (Supplementary Fig 1f), indicating functional transgene expression. We used EdU incorporation to label the dividing and newly divided α cells³². However, there was no significant increase in either total α cell number or EdU-positive α cells in either of the transgenic lines (Fig 1b, c, e, and f). Nonetheless, α cells in both transgenic lines exhibited hypertrophy compared to the control (Fig 1d). These results indicate that mTORC1 activation leads to α cell hypertrophy but not hyperplasia. However, adult Tgα^CA-MTOR fish did have significantly increased α cell area compared to WT control (supplementary Fig 1g, h), likely a secondary effect of hyperglucagonemia and subsequent glucagon resistance. Since high concentrations of amino acid are sufficient to induce α cell proliferation in primary islets^14-16, we hypothesize that other amino acid-sensitive pathway(s) may also be required for hyperaminoacidemia-induced α cell proliferation.

Hyperaminoacidemia-induced α cell proliferation requires CaSR cell-autonomously

Amino acids are capable of stimulating certain GPCRs which subsequently, can induce cell proliferation. We assessed 3 amino acid-sensitive GPCRs, gprc6a, tas1r3 and casr, all of which have previously been implicated in the regulation of endocrine cell function, to determine if these GPCRs impact α cell proliferation^34-37. Using efficient CRISPR mutagenesis³⁸, we knocked down gprc6a, tas1r3 and casr in gcgr^DKO zebrafish individually. The results revealed only the casr knockdown significantly reduced α cell number down to that of the control group (Fig 2a), suggesting that CaSR is involved in α cell proliferation. Hence, we established 2 independent casr mutant lines for cross validation (Fig 2b). Loss of casr function did not affect gross appearance or body length at 5 dpf (Supplementary Fig 2a, b), the stage most of the analyses were performed at in this study. As expected, the mutants have hypercalcemia at 7 dpf (Supplementary Fig 2c) and impaired calcium deposition in the notochord at 10 dpf (Supplementary Fig 2d, e). In 2-month-old adult zebrafish, casr mutants displayed skeletal dysplasia (Supplementary Fig 2f, g) and marked growth retardation (Supplementary Fig 2h, i). The mutant lines did not survive past 3 months of age. These results are reminiscent of casr-deficient mice³⁹^,⁴⁰, supporting that the mutations are loss of function.

To delineate the role of CaSR in α cell proliferation, we first evaluated α cell proliferation levels in casr mutants. Compared to wild-type and heterozygotes (casr+/-), there were significant decreases in the total α cell number and EdU-labelled α cell number in casr-/- zebrafish (Supplementary Fig 3a, b, c and d). These results indicate that CaSR plays a role in α cell proliferation in normal aminoacidemia. To test whether CaSR is required for hyperaminoacidemia-induced α cell proliferation, we crossed the casr mutant into gcgr^DKO background to generate the triple mutants (casr-/-;gcgr^DKO). The α cell number in 5 dpf casr-/-;gcgr^DKO fish was significantly reduced compared to that of gcgr^DKO, and was similar to that of wild-type controls (Supplementary Fig 3e, f). These results indicate that CaSR is essential for hyperaminoacidemia-induced α cell proliferation.

It is unknown whether the α cell function of CaSR is cell autonomous. Therefore, we re-expressed the human CASR gene (CASR)⁴¹ specifically into α cells using Tg(gcga:CASR,cryaa:tagRFP) (Tgα^CaSR) in casr-/-;gcgr^DKO zebrafish. We observed that both total α cell number and the number of EdU-labelled α cells in Tgα^CaSR;casr-/-;gcgr^DKO zebrafish were restored to the levels of the gcgr^DKO zebrafish (Fig 2c-f). To determine whether CaSR’s role was conserved in mammals, we used Calhex 231⁴² to inhibit CaSR in primary mouse islets cultured in high amino acid medium (HAA)¹⁴. We observed that Calhex 231 significantly suppressed HAA-induced α cell proliferation (Fig 2g). These results suggest that CaSR functions cell-autonomously in hyperaminoacidemia-induced α cell proliferation and this function is conserved in mammals.

Gq mediates hyperaminoacidemia-induced α cell proliferation

We used designer receptors exclusively activated by designer drugs (DREADDs) to determine the CaSR downstream signaling pathways involved in hyperaminoacidemia-induced α cell proliferation⁴³. CaSR can couple to Gq and Gi to activate intracellular signals²⁵^,²⁶. To determine whether Gq or Gi mediates α cell proliferation, we established 2 transgenic lines, Tg(gcga:hM3Dq, cryaa:tagRFP) and Tg(gcga:hM4Di, cryaa:tagRFP) (Tgα^Gq and Tgα^Gi). These engineered receptors can be activated by CNO (Clozapine N-oxide) to initiate Gq and Gi signaling, respectively. We bred these transgenes into the casr-/-;gcgr^DKO and gcgr^DKO background and treated the progenies with 20 μM CNO for 48 h from 3 to 5 dpf (Fig 3a). CNO treatment restored α cell number and proliferation in the Tgα^Gq;casr-/-;gcgr^DKO group to that of the gcgr^DKO group, but did not increase α cell number and proliferation further in the Tgα^Gq;gcgr^DKO group (Fig 3b, c, d and e). However, CNO did not increase either α cell number or α cell proliferation in the Tgα^Gi;casr-/-;gcgr^DKO group (Supplementary Figure 4a, b, c, and d). Overall, these results suggest that activation of Gq, not Gi, signaling leads to α cell proliferation and CaSR may signal through Gq to induce α cell proliferation.

Synergism of CaSR and mTORC1 pathways is sufficient to induce α cell proliferation

To determine whether activation of mTORC1 and Gq pathways are sufficient to cause α cell proliferation independent of hyperaminoacidemia, we crossed Tgα^Gq to Tgα^CA-Rheb. We treated the progeny with 1 µM or 20 µM CNO for 48 hours from 3 to 5 dpf to activate the Gq pathway in α cells. Activation of Gq pathway with 1 µM CNO was insufficient to cause α cell proliferation in Tgα^Gq fish, but significantly increased α cell number in Tgα^Gq;Tgα^CA-Rheb (Fig 4a). These results suggest that Gq and mTORC1 pathways act synergistically to induce α cell proliferation. Interestingly, 20 µM CNO was sufficient to increase α cell number and proliferation in gcgr-intact fish to the same level of the gcgr^DKO group (Fig 4b, c, d and e). This degree of activation is likely supraphysiological as the ligand concentration is over 1000-fold of its EC50. This indicates that Gq hyperactivation is sufficient for α cell proliferation.

CaSR-Gq induces α cell proliferation via the ERK1/2 Pathway

GPCR coupling of Gq activates multiple intracellular transduction cascades²⁵. Pertinent to cell proliferation is the MAPK pathway that leads to phosphorylation and consequent nuclear translocation of ERK1/2⁴⁴. Hence, we next explored whether ERK1/2 is activated in α cells by hyperaminoacidemia. We found that the pERK1/2 positive α cells and nuclear pERK1/2 signal intensity were markedly increased in the gcgr^DKO group compared to that of the control group, but not in the casr-/-;gcgr^DKO group (Fig 5a, b and c). These results indicate that hyperaminoacidemia-induced activation of CaSR activates ERK1/2. To determine whether CaSR activates ERK1/2 through Gq, we assayed the effect of Gq activation on pERK1/2 levels in α cells by using DREADD.

We found that CNO treatment significantly increased the pERK1/2 positive α cell percentage and nuclear pERK1/2 signal intensity in Tgα^Gq;casr-/-;gcgr^DKO fish to the same levels of the gcgr^DKO fish (Fig 5d, e and f). Interestingly, CNO even increased the pERK1/2 positive α cell percentage further in the Tgα^Gq;gcgr^DKO group (Fig 5d, e). These results indicate that Gq activation leads to ERK1/2 phosphorylation in α cells. Therefore, hyperaminoacidemia activates the CaSR-Gq-ERK1/2 pathway in α cells.

It is unknown whether ERK1/2 activation is essential in hyperaminoacidemia-induced α cell proliferation. To answer this question, we utilized the MEK1/2 inhibitors U0126 and PD0325901⁴⁵^,⁴⁶ to suppress ERK1/2 phosphorylation in control, gcgr^DKO and casr-/-;gcgr^DKO zebrafish for 48h (Fig 6a). ERK1/2 is the only known physiological substrate of MEK1/2⁴⁷. As expected, both of U0126 and PD0325901 decreased the nuclear pERK1/2 levels in α cells of gcgr^DKO islets (Supplementary Fig 5a). Both inhibitors also reduced the α cell number and proliferation in gcgr^DKO fish to that of control and casr-/-;gcgr^DKO groups (Fig 6b, c, d and e and Supplementary Fig 5b, c, d, e). Notably, U0126 even reduced α cell number and proliferation in the control fish (Fig 6b, c, d and e).

It is unknown whether MEK1/2 is also necessary for amino acids induced a cell in mammalian α cells. We therefore examined the effect of U0126 on HAA-stimulated α cell proliferation in cultured mouse islets¹⁴. We found that U0126 significantly decreased the percentage of Ki67 positive α cells in the islets cultured in HAA medium (Fig 6f, g). These results indicate ERK1/2 activation is essential for hyperaminoacidemia-induced α cell proliferation. Taken together, these findings suggest that CaSR-Gq mediates hyperaminoacidemia-induced α cell proliferation via MEK1/2-ERK1/2.

Hyperaminoacidemia-induced mTORC1 activation in α cells requires CaSR and MEK1/2

Previous studies demonstrated an essential role of mTORC1 in hyperaminoacidemia- induced α cell proliferation^14-16^,³³. Since α cell proliferation also depended on CaSR, we tested whether mTORC1 activation is intact in casr-/-;gcgr^DKO fish. Unexpectedly, pS6 levels decreased significantly in casr-/-;gcgr^DKO fish when compared to gcgr^DKO fish (Fig 7a, b, c), suggesting that CaSR is required for mTORC1 activation. To establish if mTORC1 activation requires the downstream MEK1/2, we treated gcgr^DKO fish with U0126. U0126 decreased the percentage of pS6 positive α cells and the intensity of α cell pS6 signal in gcgr^DKO fish to the same levels of wild-type fish (Fig 7d, e and f). These results suggest hyperaminoacidemia-induced mTORC1 activation in α cells requires CaSR and MEK1/2.

Gq hyperactivation-induced α cell proliferation requires mTORC1 activity

As Gq hyperactivation caused α cell hyperplasia in gcgr-intact animals, we examined whether it also required mTORC1. Indeed, rapamycin abolished α cell hyperplasia induced by Gq hyperactivation (Fig 8a, b). Consequently, Gq hyperactivation also increased pS6 levels in Tgα^Gq;casr-/-;gcgr^DKO fish (Fig 8c, d and e), and further amplified the percentage of pS6 positive α cells in Tgα^Gq;gcgr^DKO fish compared to untreated controls (Fig 8c and d). Furthermore, the Gq hyperactivation even increased pS6 levels in gcgr-intact zebrafish, although the pS6 positive α cell number was still less than gcgr^DKO group (Fig 8f, g and h). Therefore, Gq hyperactivation simultaneously activates ERK1/2 and mTORC1, and both are necessary for α cell proliferation.

Together, these data revealed activation of ERK1/2 and mTORC1 is necessary and sufficient for inducing α cell proliferation. The study also uncovered a previously unidentified role of the CaSR.

Glucagon is a key hormone which regulates plasma amino acid homeostasis by stimulating amino acid catabolism in the liver^3-5. Impaired glucagon signaling leads hyperglucagonemia and hyperaminoacidemia, leading to compensatory α cell proliferation^7-9^,¹⁸^,³². Here, by combining gain-of-function and loss-of-function interventions, both in zebrafish and cultured mouse islets, we found that the CaSR-Gq-ERK1/2 pathway is also essential for mediating hyperaminoacidemia-induced α cell proliferation. We showed that synergy between CaSR/Gq and mTORC1 pathways is sufficient to cause α cell proliferation independent of hyperaminoacidemia. We also demonstrated that mTORC1 activation is CaSR-Gq-ERK/2 dependent during α cell proliferation. Therefore, we deciphered the major pathways that are necessary and sufficient for α cell proliferation and uncovered a previously unidentified physiological role for the CaSR (Fig 9).

We have illustrated activation of mTORC1 is insufficient to induce α cell proliferation in WT larvae. We observed that mTORC1 activated α cells are not hyperplastic but hypertrophic, similar to a recent study in which mTORC1 is activated by knocking out Tsc2 in mouse a cells¹⁷. In the mouse study, no α cell hyperplasia is observed in neonatal islets¹⁷, consistent with our results. However, adult Tgα^CA-MTOR zebrafish had significantly increased α cell area, similar to adult mice with α cell specific mTORC1 activation¹⁷. In mice, this is due to the secondary effect of liver glucagon resistance resulting from increased glucagon secretion and hyperglucagonemia and is thus not an α cell autonomous effect. The increased α cell area in adult Tgα^CA-MTOR fish is likely due to the same secondary effect (Fig S1g). We speculate that glucagon resistance has not been established in the liver at 5 dpf in zebrafish.

We discovered CaSR is cell autonomously required for hyperaminoacidemia-induced α cell proliferation. Since mTORC1 activation is insufficient to cause α cell proliferation cell autonomously, we speculated a role of other amino acid sensitive pathways and identified CaSR. Through loss of function and rescue studies, we demonstrated that CaSR is required cell-autonomously for hyperaiminoacidemia-induced α cell proliferation (Fig 2c, d, e and f). We further confirmed a necessary role of CaSR in high amino acid induced α cell proliferation in mouse primary islets. Although it has been shown that CaSR is highly expressed in pancreatic endocrine cells¹⁹^,²²^,²³, we established a role for CaSR in hyperaiminoacidemia-induced α cell proliferation for the first time. Previous studies also revealed a role for CaSR in other proliferating cell types in vitro³⁰^,⁴⁸, while our study showed a necessary role for CaSR in α cell proliferation in vivo.

Although CaSR is known to be sensitive to amino acids based on in vitro pharmacology⁴⁹^,⁵⁰, to our knowledge, the present study is the first to genetically demonstrate an amino acid sensitive function of CaSR in vivo. L-Phe and L-Trp are slightly more potent in activating CaSR in the presence of physiological Ca²⁺⁵¹, however, their concentrations in blood are lower than 100 µM, even under conditions of hyperaminoacidemia, much lower than the EC50 of 2.5 mM⁵¹. Therefore, activation of CaSR by hyperaminoacidemia in vivo results from the action of all amino acids, particularly Gln and Ala, the two major amino acids preferentially increased under conditions of glucagon signaling interruption¹⁴^,¹⁵. In normal conditions, blood amino acid levels are tightly maintained within a small range, and thus unlikely to play a role in regulating CaSR in most tissues. However, the amino acid concentration increases substantially in the GI tract after feeding. This surge likely activates CaSR in the enteroendocrine cells¹⁹. Indeed, several in vitro studies have demonstrated a role of CaSR in the secretion of gut hormones⁵⁰^,⁵²^,⁵³. Confirmation of these studies in vivo by genetic means will establish additional physiological roles of amino acid sensing by CaSR.

Our study suggests that CaSR acts through Gq signaling to induce α cell proliferation. CaSR regulates cell proliferation in many tissues^28-30. However, few reports have determined which G protein mediates CaSR action in cell proliferation. Using DREADDs, we tested the 2 major CaSR activated pathways and found that activation of Gq, but not Gi, induced α cell proliferation (Fig 3b, c, d and e; Supplementary Fig 4a, b, c and d). These results suggest that the Gq pathway may be responsible for CaSR mediated proliferation. However, we cannot rule out a role for the Gs pathway, which can be activated by CaSR in some cell types⁵⁴^,⁵⁵. We recognize that the CaSR-Gq connection is based on correlative evidence. Interestingly, Gq and Gs activation increases β cell mass and proliferation⁵⁶^,⁵⁷, while Gi activation inhibits β cell proliferation⁵⁸. The positive and negative effects on β cell proliferation correlate with the stimulation and inhibition of insulin secretion, respectively. This correlation does not hold true in α cells since several Gi-coupled GPCRs stimulate glucagon secretion⁵⁹. The reason why Gi activation fails to promote α cell proliferation is unknown and may be a subject for future investigation.

Downstream of Gq, CaSR is necessary for hyperaminoacidemia-mediated MEK1/2-ERK1/2 activation in α cell proliferation. MEK1/2 phosphorylation of ERK1/2 and subsequent nuclear localization is essential for cell proliferation and is implicated in many cancer types⁶⁰^,⁶¹. As expected, we found that increased nuclear pErk1/2 correlates with α cell hyperplasia (Fig 5) and inhibition of MEK1/2 significantly reduced α cell proliferation both in gcgr-deficient zebrafish and in mouse islets cultured in high amino acid medium (Fig 6; Supplementary Fig 5). Although it is unsurprising that MEK1/2 activity is required for hyperaminoacidemia-induced α cell proliferation, it is interesting that CaSR is necessary for their activation. We propose that CaSR is a component of the amino acid sensor in α cells that maintains amino acid homeostasis.

A surprising finding is that CaSR is necessary for hyperaminoacidemia-mediated mTORC1 activation. mTORC1 activation is known to be required for hyperaminoacidemia-induced α cell proliferation, and it is thought that increased intracellular amino acids caused by amino acid transporters are the primary driver of mTORC1 activation by hyperaminoacidemia^14-16^,³³. However, we found that CaSR, presumably activated by increased extracellular amino acids and acting through Gq-MEK1/2, is also required for mTORC1 activation. A previous study showed that the α1A adrenergic receptor promotes mTORC1 signaling via the Gq pathway⁶². This may result from ERK1/2, PI3K and TSC2 activation^63-65. The exact mechanism of CaSR mediated mTORC1 activation in α cells requires further studies.

We demonstrated that Gq and mTORC1 pathways act synergistically to induce α cell proliferation (Fig 4 a). Activation individually failed to cause α cell proliferation in gcgr-intact fish. However, a high concentration of CNO alone was also sufficient for α cell proliferation in gcgr-intact Tgα^Gq fish (Fig4 b, c, d and e). We do not think such degree of activation is achievable in physiological conditions. In the transgenic fish, the Gq-DREADD is under the strongest promoter in α cells and is likely expressed at much higher levels than endogenous GPCRs. The high agonist concentration would likely achieve supraphysiological levels of Gq activation that is sufficient to turn on both MEK1/2-ERK1/2 and mTORC1 signaling.

In summary, we revealed that activation of mTORC1 and CaSR-Gq-ERK1/2 is necessary and sufficient for α cell proliferation and identified a previously unknown physiological role of CaSR (Fig 9). Using zebrafish and cultured mouse islets, the present study demonstrated that the CaSR-Gq-ERK1/2 pathway is another mediator of hyperaminoacidemia-induced α cell proliferation and CaSR-Gq-MEK1/2 also contributes to mTOR activation. Importantly, co-activation of CaSR-Gq and mTORC1 in α cells causes proliferation independent of hyperaminoacidemia. Future studies should decipher the mechanism of the interplay between Gq-ERK1/2 and mTORC1 during hyperaminoacidemia-induced α cell proliferation. Understanding how hyperaminoacidemia induces α cell proliferation may provide new insights for glucagonoma treatment and for mitigating the undesired effects of the proposed GRA therapy for diabetes.

Zebrafish maintenance.

Zebrafish (Danio rerio) were raised at 28°C in an Aquatic-Habitats system and embryos were raised at 28.5°C in an incubator on a 14/10-hour light/dark cycle. All animals were staged by days postfertilization (dpf). In this study, Tg(gcga:EGFP)⁶⁶ was used to mark α cells for imaging and/or counting α cells. Transgenic and mutant strains used for this study are listed in Supplementary Table 1. The gcgra-/-;gcgrb-/- fish were described previously³². Mutations in casr were generated using CRISPR-Cas9 as described previously⁶⁷, by using recombinant Cas9 (PNA Bio). Two mutant lines (8 bp deletion and 11 bp insertion) in casr were characterized for an initial cross-validation before selecting the 8 bp deletion line for all the experiments. Transgenic lines expressing constitutively active mTOR^L1460P or Rheb^S16H and CaSR, hM3Dq or hM4Di in α cells were generated using the Tol2 System⁶⁸.

Free amino acids assay.

Free amino acids level was determined using the published method⁶⁹. Briefly, a pool of forty larvae or 10 μL serum of adult fish was homogenized in perchloric acid (Weight ratio: 1:50). The homogenate centrifuged at 15,000 g for 10 min. The supernatant was neutralized with 0.8 M KOH (using a volume of KOH 1/19th of the supernatant). After the addition of KOH, the samples were cooled on ice water for about 10 min and centrifuged at 15,000 g for 10 min. 10μL of supernatant were mixed with 3 mL of OPA-MET reagent. After 2–10 min the fluorescence above the OPA-MET reagent was read in cuvettes on a spectrofluorometer using an excitation wavelength of 340 nm and an emission wavelength of 440 nm. In order to calculate the concentration of the amino acids in the sample, 10 μL of the standard mixture of amino acids (prepared as described above and diluted 1 : 10, 1 : 100 and 1 : 1,000) were mixed with 3mL of OPA-MET reagent and the fluorescence was read as for the sample. Six 40-fish pools and 6 adult fish sera of each genotype were measured respectively.

Proliferation analysis in zebrafish.

Proliferation was analyzed using the Click-iT EdU Alexa Fluor 594 Imaging Kit (C10339; Invitrogen). To identify proliferating α cells, embryos were pericardially injected with 1-2 nl of 0.1 mM 5-ethynyl-2-deoxyuridine (EdU) at 4 dpf. After 24 h, they were euthanizied and fixed. EdU was detected according to modified manufacturer’s instructions. All images were collected using Zeiss LSM780 (Carl Zeiss, Jena, Germany) and analyzed by Imaris (Oxford Instruments).

Isolation and culture of mouse pancreatic islets.

All mice used in these studies were 10-18-week-old C57BL/6J males. Mouse pancreata were digested with collagenase P (Roche, Basel, Switzerland), and islets were isolated via density gradient centrifugation as previously described¹⁴. The islets were then handpicked to > 99% purity and cultured in media with high or normal amino acids, and with an inhibitor or vehicle for 3 days. The islet culture was based on a RPMI 1640 media formula accept that each amino acid concentration was individually formulated to mimic the concentrations found in the serum of either wildtype (Low AA) or Gcgr-/- (High AA) mice¹⁴ with 10% Fetal Bovine Serum (Atlanta Biologicals), 1% Penicillin/Streptomycin (GIBCO), and 5.6mM D-glucose. To inhibit CaSR, 10 µM Calhex 231 (Cayman) was used. To inhibit MEK1/2, 10 µM U0126 was used.

After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (GIBCO Life Technologies or HyClone GE Healthcare) for 5-10 min by gentle pipetting to obtain single or very small cell clusters. Dispersed islet cells were recovered by centrifugation in RPMI media containing 5.6 mM glucose, 10% FBS, and 1% Penicillin/Streptomycin. The resulting cell pellet was resuspended in 100 μl of media and centrifuged onto a glass slide using a cytospin (Thermo Scientific, Waltham, MA) centrifuge. Air-dried slides were stored at -80℃until use, thawed, and immediately fixed in 4% paraformaldehyde before immunofluorescence. Slides were mounted with Aqua-Poly/Mount (Warrington, PA).

Immunofluorescence and imaging.

Immunofluorescence on larval zebrafish islet-containing sections was performed as below: zebrafish larvae were fixed in 4% paraformaldehyde (PFA) at 5 dpf, equilibrated in 20% sucrose and embedded into O.C.T. for making cryosections. Islet-containing cryosections (12 μm) were selected for immunofluorescence. Immunofluorescence of cultured mouse islets was performed as described¹⁴.

Primary antibodies used were Anti-GFP (1:300, Thermo fisher scientific, A-11120, Mouse), Anti-pS6 (Ser240/244) (1:300, Cell signaling technology, 5364, Rabbit), Anti-pERK1/2 (Thr202/Tyr204) (1:150, Cell signaling technology, 4370, Rabbit), Anti-glucagon (1:200, Cell signaling technology, 2760, Rabbit) and Anti-Ki67 (1:150, Abcam ab15580, Rabbit). Followed by Alexa Fluor 488 or Alexa Fluor 568 conjugated secondary antibody for visualization (1:1,000, Thermo Fisher Scientific). Nuclei were stained with Hoechst 33342 (0.5 μg/ml, Thermo Fisher Scientific). The coverslips were mounted on slides with ProLong Diamond mounting medium (Thermo Fisher Scientific). Images were collected using a Zeiss LSM880 (Carl Zeiss, Jena, Germany) and analyzed by Imaris (Oxford Instruments).

Ca²⁺concentration assay, alizarin red S (ARS) staining and Mirco-CT.

Ca²⁺concentration was measured by ICP-MS. Briefly, 1.5 mL polypropylene microcentrifuge tubes (Fisher Scientific) were immersed and washed three times in 10% nitric acid (67–69%, Optima™; Fisher Chemical) overnight. After acid treatment, tubes were rinsed thoroughly with ultrapure water (Processed by Milli-Q™). Afterward, embryos (7 dpf) were washed 3 times with cold ultrapure water, and four 20-fish pools were placed in tubes for each group. For metal measurement, 200 μL nitric acid was added to digest samples at 85°C until dry. Samples were thensuspended in nitric acid and dried again. 200 μL hydrogen peroxide (30%, Optima™; Fisher Chemical) was added and evaporated at 85°C twice. Eventually, samples were diluted in 1.5 mL 2% nitric acid. Empty tubes, processed identically as tubes containing embryos, work as mock treatments. Ca²⁺ level was normalized to sulfur, and the standard curves were generated to measure the content of Ca²⁺ by Agilent 7700 series ICP-MS (Agilent Technologies).

ARS staining was used for larval zebrafish skeletal mineralization. Briefly, zebrafish were fixed in 4 % PFA at 10 dpf, then stained for 15 min with 0.01 % ARS (3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt, from Sigma-Aldrich, St. Louis, MO) dissolved in 70 % ethanol. After rinsing 3 times/5 min in PBS, the samples were imaged immediately under 510 nm excitation fluorescent with Zeiss LSM780 (Carl Zeiss, Jena, Germany) and analyzed by Imaris (Oxford Instruments).

3 zebrafish (60 dpf) were sacrificed for each group and kept in 75% alcohol at -20 °C until use. The sample was then placed in a sample holder in which a sponge was used to fix sample. Micro-CT scanning was run via μCT 50 (Scanco Medical, Switzerland). High-resolution scans (5 μm voxel resolution) were obtained using the following parameters: X-ray energy 45 kVp, current 88 μA, sampling rate 2048, 1000 proj/180°, integration time 300 ms, which were modified according to the previously published methods⁷⁰. Image processing was performed using custom software and the bone development of zebrafish remodeled by micro-CT-scan was described as total volume (TV), bone volume (BV), bone volume fraction (BV/TV) and tissue mineral density (TMD).

Chemical treatment in zebrafish experiment.

All drugs were made in 1,000X stock solution and stored in light-protected Eppendorf tubes at -20°C. CNO (20 mmol/L; Tocris), PD0325901 (10 mmol/L; Sigma-Aldrich), U0126 (10 mmol/L; LC laboratories) and Rapamycin (2.5 mmol/L; Cayman) were dissolved in DMSO at the indicated concentrations. Zebrafish were exposed to 20 μM or 1 μM CNO, 10 μM PD0325901 or U0126 and 2.5 μM Rapamycin for 2 days, respectively, starting at 3 dpf and injected with EdU at 4 dpf. Then larvae were fixed and stained at 5 dpf for α cell number and proliferation assay.

Statistics.

For group comparisons, either one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) post hoc test or two-tailed unpaired Student’s t test was used. A P value less than 0.05 was considered statistically significant. Values represent means ± SEM. Analyses were done using GraphPad Prism 8.

Study approval.

The animals were handled in compliance with guidelines approved by the Vanderbilt University Institutional Animal Care and Use Committee protocols (protocol no. M1700143-01).

Acknowledgements.

We thank members of the Chen, Powers and Dean groups for helpful discussions. The CASR-Tango plasmid with codon optimized human CaSR coding sequence was provided by Brian Roth via Addgene (plasmid # #66235). The coding sequence for constitutively active rat RHEB was provided by Mustafa Sahin via Addgene (plasmid # 32520). The coding sequence for constitutively active human MTOR was provided by David Sabatini via Addgene (plasmid # 69006). Human islets were from IIDP. Confocal imaging and image analysis were performed in part through the use of the Vanderbilt Cell Imaging Shared Resource. IP-MS was done at the Vanderbilt Mass Spectrometry Research Center (MSRC). Micro-CT was performed at the VUMC Institute of Imaging Science (VUIIS) Center for Small Animal Imaging (CSAI). This study is supported by DK117147 (WC and ACP) and DK117969 (EDD). YG is supported by China Scholarship Council (#201904910575). BAC is supported by the METP training grant (T32 DK 7563-32). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Author Contributions.

Conceptualization: WC. Funding acquisition: WC, ACP, EDD; Investigation: YG, DZ, ZT, KCC, WAS, KS, BAC. Methodology: BY, LYin, LYang, RSP. Supervision: WC, ACP, EDD. Visualization: YG, DZ, KCC, WAS. Writing – original draft: YG, WC; Writing – review & editing: KCC, EDD, BAC, ACP.

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Hyperaminoacidemia induces pancreatic α cell proliferation via synergism between mTORC1 and CaSR-Gq signaling pathways

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