UGGT1 retains proinsulin in the endoplasmic reticulum in an arginine dependent manner

https://doi.org/10.1016/j.bbrc.2020.04.158Get rights and content

Highlights

  • UGGT1 binds to proinsulin in the absence of arginine.

  • Arginine competes with proinsulin and binds to UGGT1 in the ER.

  • Released proinsulin moves to Golgi apparatus and secretory vesicles to secrete.

Abstract

We sought to clarify a pathway by which L- and dD-arginine simulate insulin secretion in mice and cell lines and obtained the following novel two findings. (1) Using affinity magnetic nanobeads technology, we identified that proinsulin is retained in the endoplasmic reticulum (ER) through UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) when arginine availability is limited. (2) L- and d-arginine release proinsulin from UGGT1 through competition with proinsulin and promote exit of proinsulin from the ER to Golgi apparatus. The ability of arginine to release proinsulin from UGGT1 closely correlates with arginine-induced insulin secretion in several models of β cells indicating that UGGT1-proinsulin interaction regulates arginine-induced insulin secretion.

Introduction

Insulin secreted from pancreatic β cells plays a central role in glucose homeostasis and the impairment in insulin secretion leads to diabetes [[1], [2], [3]]. Glucose is a principal stimulator of insulin secretion and glucose metabolism in pancreatic β cells is coupled with insulin secretion [4]. Glucose is taken up by β cells through glucose transporter 1 and 2 (GLUT1 and 2, solute carrier family 1 and 2; SLC2A1 and 2) is converted to glucose-6-phosphate by glucokinase (GCK) to initiate glucose metabolism that ultimately closes KATP channel and induces Ca2+ influx by opening the voltage-gated Ca2+ channels leading to exocytosis of insulin granules [4].

The semi-essential amino acid arginine that is mainly supplied from food augments insulin secretion when there is a permissive concentration of glucose [1,2,5,6], and isomer d-arginine also stimulates insulin secretion [5]. When arginine reaches the pancreatic β cells, arginine is transported into β cells by cationic amino acid transporter 1–2 (CAT1-2, SLCA1-2)[[7], [8], [9]]. It is proposed that uptake of arginine by the electrogenic transporter CAT1-2 generates a depolarizing current initiating Ca2+ influx and insulin secretion in the presence of a permissive concentration of glucose [4,6]. As arginine induced insulin secretion is considered to be independent from metabolism through the TCA cycle and oxidative phosphorylation, it has been widely used to evaluate metabolism independent secretory function of β cells in vivo and in vitro [6,10,11]. However, there appears to be additional pathways by which arginine promotes insulin secretion. Interestingly, both L- and D-isomers of arginine are capable of inducing insulin secretion [5]. As CAT1-2 are stereospecific and only mediate uptake of l-amino acid, a mechanism by which d-arginine stimulates insulin secretion is unanswered.

Other known membrane receptors for arginine include GPRC6A (G-protein-coupled receptors, GPCR [12,13]) and amino acid taste receptor (TAS1R5-24[14]). In addition to l-amino acids, ligands of GPRC6A include osteocalcin, steroids, and androgen, some of which are not strong insulin secretagogues making GPRC6A a less likely mediator of stimulation of insulin secretion. Moreover, d-amino acids do not serve as ligands of GPRC6A [5,13]. As for amino acid taste receptors, the expression of TAS1R5-24 is low in pancreas leaving its contribution to insulin secretion questionable. Thus, arginine transporters and membrane receptors are unlikely to mediate arginine’s action to promote insulin secretion.

Alternatively, arginine may stimulate insulin secretion through intracellular targets. Arginine is known to bind to several proteins which serve as potential intracellular targets including nitric oxide synthase (NOS), arginase, Cellular arginine sensor for mTORC1 (CASTOR1), phosphofructokinase 1 and 2 (PFK1 and 2), RuvB-like 1, and RuvB-like 2 [[15], [16], [17], [18]]. However, we previously reported that an arginine analogue, arginine methyl ester (AME), stimulates insulin secretion to similar extent as arginine, despite AME inhibition of NOS activity, indicating that arginine-induced insulin secretion cannot be explained by arginine-NOS-NO signals [15]. In addition, a non-selective inhibitor of NOS, NG-Nitro-l-arginine methyl ester (l-NAME), also stimulates insulin secretion as much as L- and d-arginine do [15]. These data indicate that NOS is a less likely target of arginine to increase insulin secretion. Arginine is also reported to activate mTORC1 through CASTOR1 in eukaryotes [17,18], but its expression in human and mouse β cells appears to be low [19]. Thus, the pathway by which L- and d-arginine stimulate insulin secretion remains unclear.

While Ca2+ influx is primarily responsible for initiating exocytosis of insulin granules, continuous production of insulin granules is critically important for insulin secretion as well [4]. Insulin undergoes highly regulated processing after translation of its precursor protein preproinsulin to form mature insulin granules [20,21]. After translation, preproinsulin is immediately translocated to the endoplasmic reticulum (ER) where a signal peptide is removed to produce proinsulin. At the ER, proinsulin undergoes disulfide bond formations and then moves to the Golgi network where proinsulin is cleaved into mature insulin in the secretory granules ready for secretion [1,2]. Animal models of insulin gene mutations (Akita and Munich mice) and humans with insulin gene mutations caused a dominant negative form of diabetes (maturity-onset diabetes of the young type 10, MODY10) and revealed that a proper folding of insulin in the ER is critically important for transit of proinsulin out of ER [22]. However, it is unknown whether there is an additional factor that regulates retention and release of proinsulin at the ER in β cells. Interestingly, we found that arginine depletion increases the retention of proinsulin and arginine acutely promotes exits of proinsulin from the ER in insulin secreting cells [23]. Here, we identified UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a protein known as gatekeeper of N-glycoprotein quality control (NGPQC) [24], serves as a molecular scaffold to retain proinsulin in the ER when arginine availability is low. Arginine disrupts the binding of proinsulin to UGGT1 and promotes transport of proinsulin out of the ER. UGGT1 is known to selectively re-glucosylate unfolded or mutated N-glycoproteins, thus providing quality control for protein transport out of the ER. The current study has revealed a new role UGGT1 plays as a regulator of arginine dependent transport of proinsulin out of the ER.

Section snippets

Antibodies and cell culture

The following antibodies were purchased: β-actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), glyceraldehyde 3-phosphate dehydrogenase (ab9485, Abcam, Cambridge, UK), insulin (L6B10, Cell Signaling Technology, Danvers, MA, USA, and sc-9168, Santa Cruz Biotechnology), UGGT1 (14170-1-AP, Proteintech, Rosemont, IL, USA; sc-374565, Santa Cruz Biotechnology; and ab13520-50, Abcam), KDEL (ER staining, SPA-827, Stressgen, Victoria, BC, Canada), gp96 (2104S, Cell Signaling Technology),

Arginine releases proinsulin from the endoplasmic reticulum (ER)

To analyze intracellular distribution of insulin, mCherry-tagged insulin expression vector was established and transfected to HEK293 FT cells with higher efficiency [15]. At first, mCherry-tagged insulin secretion is induced by arginine-administration after arginine-depletion for 30 min (Fig. 1a). In these time period, time lapse microscopy was taken HEK293 FT cells transfected with insulin-mCherry and signal-GFP-KDEL vectors (Fig. 1b and Movie S1). After arginine-depletion for 30 min (time 0),

Conclusion

In summary, we combined chemical, cell-based, and a transgenic mouse model to demonstrate that arginine sensitive interaction between proinsulin and UGGT1 contributes to the release of insulin in response to both L- and d-arginine.

In mice, we observed that arginine stimulates the mobilization of proinsulin from the ER after fasting. Combining biochemical and NIT-1 cell models, we demonstrated that proinsulin binds to UGGT1 when arginine availability is low in the ER, leading to the retention of

Discussion

It has been known that the isomer d-arginine stimulates insulin secretion like l-arginine. However, the stimulation of insulin secretion by d-arginine occurs without Ca2+ influx and the opening of voltage-gated channels [11,12] leaving a mechanism responsible for the stimulation of insulin secretion by d-arginine unanswered. In the present study, we have shown that both L- and d-arginine stimulate insulin secretion by promoting release of proinsulin bound to UGGT1 (Fig. 1, Fig. 4g, and

Funding

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT 18659493), the Japan Science and Technology Agency (A-STEP-AS2312036G and FY2013–SICP) and NCGG (28–25) to TI, the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 17H06112, and MEXT-Supported Program for the Strategic Research Foundation at Private Universities S1411011 to H.H. Y.H is supported by Grant Number 18H02779. Yu I is financially supported by

Author contribution

JC, MH, YM, and SS performed the experiments, TI designed the experiments, analyzed the data, and wrote the manuscript. YoI, YuI and HH interpreted the data and wrote the manuscript.

Author information

Readers are welcome to comment on the online version of the paper. Correspondence should be addressed to TI ([email protected]).

Data and materials availability

Requests for data and materials should be addressed to TI ([email protected]).

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgments

We are grateful to our department members in NCGG for their helpful discussions and Dr. N. Maekawa, M Umeda and Y Tsugawa for technical assistance, and Pr. T Satoh in Nagoya City University for protein structure analysis, and Pr. T. Miyata and Dr. Y. Hattori in Nagoya University for time-lapse photography.

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