Screening of amino acids as a safe energy source for isolated rat pancreatic acini

Abstract

Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acinar cells. Adequate nutritional support is important for acute pancreatitis treatment. However, some amino acids, such as arginine and lysine, are toxic for pancreatic acinar cells in high concentrations. The study aimed to select the candidate amino acids as the best non-toxic energy sources for supplemental therapy of acute pancreatitis.
Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1–20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell necrosis and apoptosis were evaluated with fluorescent microscopy and DNA gel electrophoresis.
Among the tested amino acids, glutamate, glutamine, alanine, lysine and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, while arginine, histidine and asparagine were not. Lysine, arginine and glutamine (20 mM) caused complete necrosis of acinar cells after 24 h of incubation. Glutamine also caused early (2–4 h) cell swelling and blebbing. Aspartate, asparagine and glutamate only moderately increased the number of necrotic cells, while alanine and histidine were not toxic. No significant apoptosis developed after incubation with amino acids.
In conclusion, we propose alanine and glutamate as safe candidate amino acid supplements for patients with acute pancreatitis.

Full text

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Screening of amino acids as a safe energy source
for isolated rat pancreatic acini
Anastasiia M. Zub (  anastasiya.yakubovska@lnu.edu.ua )
Ivan Franko National University of Lviv
Bohdan O. Manko
Ivan Franko National University of Lviv
Volodymyr V. Manko
Ivan Franko National University of Lviv
Research Article
Keywords: amino acids, necrosis, pancreatic acinar cells, mitochondrial respiration
Posted Date: July 13th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3153597/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

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Abstract
Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acinar
cells. Adequate nutritional support is important for acute pancreatitis treatment. However, some amino
acids, such as arginine and lysine, are toxic for pancreatic acinar cells in high concentrations. The study
aimed to select the candidate amino acids as the best non-toxic energy sources for supplemental therapy
of acute pancreatitis.
Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1–20 mM) on uncoupled
respiration of isolated acini were studied with a Clark electrode. Cell necrosis and apoptosis were
evaluated with uorescent microscopy and DNA gel electrophoresis.
Among the tested amino acids, glutamate, glutamine, alanine, lysine and aspartate were able to stimulate
the uncoupled respiration rate of isolated pancreatic acini, while arginine, histidine and asparagine were
not. Lysine, arginine and glutamine (20 mM) caused complete necrosis of acinar cells after 24 h of
incubation. Glutamine also caused early (2–4 h) cell swelling and blebbing. Aspartate, asparagine and
glutamate only moderately increased the number of necrotic cells, while alanine and histidine were not
toxic. No signicant apoptosis developed after incubation with amino acids.
In conclusion, we propose alanine and glutamate as safe candidate amino acid supplements for patients
with acute pancreatitis.
Introduction
Pancreatic acinar cells rely on highly active amino acid transport (Rooman et al. 2013) to continuously
produce and secrete digestive enzymes (Dijk et al. 2019). Beyond protein synthesis, amino acids play a
vital and presently less recognized role in the metabolism and survival of these cells. Some amino acids
are primary energy and metabolite sources for pancreatic acinar cells. Glutamine and glutamate were
shown to substantially increase maximal uncoupled respiration in isolated rat pancreatic acini (Manko et
al. 2019). It was also reported that murine pancreatic slices prefer alanine and leucine to glucose as
substrates for energy metabolism (Danielsson and Sehlin 1974).
Amino acid metabolism is very important in acute pancreatitis pathogenesis. On one hand, serum amino
acid composition is drastically changed in patients with acute pancreatitis (Sandstrom et al. 2008). On
the other hand, the disruption of amino acid transport or metabolism leads to high incidence of acute
pancreatitis. Asparaginase treatment is well-known to cause acute pancreatitis (Oparaji et al. 2017),
possibly due to the important role of asparagine synthesis in pancreatic acinar cells (Tsai et al. 2020).
Acute pancreatitis is also associated with several rare genetic diseases related to amino acid metabolism
or transport, such as type II citrullinemia, citrin aspartate/glutamate carrier deciency (Komatsu et al.
2008), lysinuric protein intolerance, a deciency of SLC7A7 – a component of cationic amino acid
transporter (Mauhin et al., 2017, Parto et al., 1994) and propionyl CoA carboxylase deciency, which

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disrupts metabolism of amino acids valine, isoleucine, methionine, and threonine (Pena and Burton
2012).
Nutritional support in acute pancreatitis plays an important role in preventing malnutrition, correcting a
negative nitrogen balance, and reducing incidence of infectious complications and mortality rate (Wu et
al. 2018; Yang 2021). The inuence of high-energy diet on acute pancreatitis recovery is actively studied
(Juhász et al. 2022; Márta et al. 2017). Amino acid supplements seem to be a viable strategy to supply
the necessary energy and metabolites to pancreatic acinar cells and ameliorate acute pancreatitis. There
is evidence that early intravenous alanyl-glutamine dipeptide administration is associated with better
clinical outcomes in severe acute pancreatitis patients (Xue et al. 2008). Lysine (10 mg/kg bw) treatment
inhibited inammation and enhance antioxidant activity in mice with acute pancreatitis (Al-Malki 2015).
However, the benets from amino acids administration might be outweighed by potential harmful effects,
as particular amino acids were shown to be toxic to the pancreas at high doses. In isolated murine
pancreatic acinar cells, arginine, ornithine and histidine (20 mM) cause cell necrosis within 6–10 h of
action (Zhang et al. 2019). Furthermore, intraperitoneal injection of arginine (4 g/kg), lysine (2 g/kg)
ornithine (3 g/kg) or histidine (8 g/kg) causes necrotizing pancreatitis in rodents (Biczó et al. 2011;
Rakonczay et al. 2008; Zhang et al. 2019). L-arginine overdose might also cause of acute pancreatitis as
suggested by two clinical cases (Binet et al. 2018; Saka et al. 2004). Mechanisms of toxic amino acid
effects in pancreatic acinar cells are not fully understood, but mitochondrial damage appears to be a
central event in amino acid induced pancreatitis (Biczo et al. 2018; Biczó et al. 2011; Kui et al. 2014).
Thus, the aim of present study was to select the candidate amino acids (among alanine, glutamine,
glutamate, asparagine, aspartate, lysine, arginine and histidine) as the best non-toxic energy sources for
supplemental therapy of acute pancreatitis. We have performed
in vitro
cell toxicity screening and
investigation of acinar cell respiration. In our recent studies (Manko et al. 2019; Mazur et al. 2019), we
have shown that that the ability of cells to transport and oxidize substrates such as Krebs cycle
intermediates or amino acids can be studied by creating an articial demand for reducing equivalents in
mitochondria with protonophore, such as FCCP. At high concentrations, FCCP causes a collapse of
mitochondrial membrane potential and a decrease of the uncoupled respiration rate, which can be
delayed by additional substrate supply (Manko et al. 2019). In the present study, we employ this method
to evaluate the capacity of pancreatic cell mitochondria to oxidize the amino acids.
Materials and methods
Animal experiments
All experiments were carried out in accordance with the "European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientic Purposes" (Council of Europe № 123,
Strasbourg 1985). All animal procedures were approved by the Committee for the Care and Use of
Animals of the Ivan Franko National University of Lviv under protocol number 34-04-2023. Experiments

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were carried out on male Wistar rats (250–300 g). Animals were kept under the standard conditions of
vivarium at a constant temperature with a 12:12-hour light-dark cycle and on a basic diet. Animals were
starved for 12 hours before the experiment. Animals were sacriced by decapitation, and pancreatic acini
were immediately isolated.
Materials
Reagents used in experiments were purchased from Sigma-Aldrich (St. Louis, Mo): sodium chloride,
potassium chloride, D-glucose, N-2-hydroxyethylpiperazineN′-2-ethanesulfonic acid (HEPES), soybean
trypsin inhibitor, bovine serum albumin (BSA), sodium pyruvate, L-glutamine, L-glutamic acid, L-alanine, L-
lysine, L-arginine, L-asparagine, L-aspartic acid, L-histidine, FCCP, collagenase type IV, proteinase K,
Hoechst 33358, ethidium bromide, ethylenediaminetetraacetic acid (EDTA), agarose, nonyl
phenoxypolyethoxylethanol (NP-40), Triton X-100, tris(hydroxymethyl)aminomethane (Tris). Calcium
chloride dehydrate was purchased from Merck Chemicals (Burlington, Mass). All other reagents were of
the purest available grade.
Pancreatic acini isolation
A suspension of isolated pancreatic acini was obtained with collagenase (type IV, 0.2 mg/mL) as
previously reported (Manko et al. 2013). Pancreatic tissue and isolated acini were incubated in a medium,
containing the following: NaCl, 140.0 mM; KCl, 4.7 mM; CaCl2, 1.3mM; MgCl2, 1.0 mM; HEPES, 10.0 mM;
glutamine, 2.0 mM; sodium pyruvate, 2.0 mM; glucose, 10.0 mM; BSA, 2.5 mg/mL; soybean trypsin
inhibitor, 0.1 mg/mL and minimum essential medium (MEM) amino acid supplement; pH was brought to
7.4 with NaOH. Basic extracellular solution for cell respiration and viability studies was similar in
composition, but did not contain amino acid supplement, glutamine, sodium pyruvate or glucose. Cell
counting and initial viability assessment were performed with trypan blue staining using a
hemocytometer.
Cell respiration studies
Cell respiration was studied by measuring the rate of oxygen consumption with the Clark oxygen
electrode (Biological Oxygen Monitor YSI 5300; Yellow Springs Instruments, Yellow Springs, Ohio) in the
closed 1.6-mL glass respiration chamber at 37°C. Before respiration rate measurement, the suspension of
isolated pancreatic acinar cells was preincubated for 15 minutes at 37°C in basic extracellular solution 1)
without oxidation substrates or 2) with amino acid, 3) only with glucose, and 4) amino acid and glucose.
Protonophore FCCP (0.5–2 µM) was added directly into the respiratory chamber to reach the maximal
uncoupled respiration rate as described earlier (Manko et al. 2019). The respiration rate was rst
normalized by cell number and scaled relative to the mean basal respiration rate in the control.
DNA extraction and gel electrophoresis
DNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction. Both lower DNA
phase and intermediate phase containing unseparated protein-DNA complexes were used to ensure total
DNA extraction. Afterwards, proteinase K (nal concentration, 2.5 µg/mL) in SDS-Sodium Chloride-Tris-

Page 5/16
EDTA buffer was added to DNA samples and incubated at 50°C overnight. After the addition of 0.5
volume of 10 M ammonium acetate, DNA was precipitated with 2.5 volume of ethanol, dissolved in gel
loading buffer, and separated by electrophoresis in 1% agarose gels in Tris-Borate-EDTA buffer containing
ethidium bromide (at 70 V).
Fluorescent studies of pancreatic acinar cells viability and
morphology
Fluorescent imaging was performed using an IX73 microscope and DP-74 camera (Olympus, Tokyo,
Japan). Images were processed using ImageJ 1.53e software (NIH, Bethesda, Md). Isolated pancreatic
acini were incubated either in a basic extracellular solution containing glucose (10 mM) or in Dulbecco-
modied Eagle’s medium with glucose (10 mM) but without glutamine and pyruvate. The studied amino
acids were added at 20 mM. Pancreatic acinar cell necrosis was studied with ethidium bromide and
Hoechst 33258 staining as described earlier (Manko et al. 2021). Cell blebbing counting was performed
only for peripheral cells in acini in one focal plane. To study cell swelling, two perpendicular cell
diameters were measured using CellSens Dimension software. Cell area was calculated with the
equation:
where A and B are a major and minor axes length.
Statistical analysis
At least 5 separate preparations of isolated cells obtained from different animals were used in each
experimental series (“n” always represents number of animals). Results are presented as means ±
standard error of the mean (SEM). Statistical analysis was performed using OriginPro 2018 software
(OriginLab, Northampton, Mass) software. Signicance of difference between the groups was determined
with a 2-way repeated-measures analysis of variance (ANOVA) followed by Holm-Bonferroni corrected
post hoc t-tests. Student’s t test was performed when comparing the experiments with two groups only.
Results
The effects of amino acids on uncoupled respiration of
pancreatic acini
In our previous work, we have shown that extracellular oxidative substrates are able to signicantly
enhance the maximal uncoupled respiration rate and stability of uncoupled respiration in pancreatic acini
(Manko et al. 2019). A similar experimental approach was used in this study. Acini were incubated in a
basic extracellular solution with amino acids (0.1–20 mM) for 15 min to allow sucient accumulation in
cells. Afterwards, basal respiration was recorded followed by titration with FCCP (0.5-2µM) (Fig.1). We
performed two experimental series –without and with glucose (10 mM) in solution.
S
=
π
× (
A
×
B
/4),

Page 6/16
In all experiments, amino acids did not signicantly affect the basal respiration rate of isolated
pancreatic acini (Table1). Among the studied amino acids glutamate, glutamine, alanine, lysine and
aspartate had signicant dose-dependent effects on uncoupled respiration (Table1, Fig.1,
Supplementary Fig.1–7). In general, the effects of amino acids were more pronounced when higher
(inhibitory) FCCP concentrations were used. Glutamate was different from other amino acids as it
signicantly stimulated respiration by up to 200%. This effect was signicant for a wide range of this
amino acid (1–20 µM) and uncoupler concentrations (0.5-2 µM) (Table1, Supplementary Fig.1). The
effect of 0.1 mM of glutamate was not studied. Glutamine (1–20 mM) enhanced respiration by 30–50%
only upon 2 µM FCCP (Table1, Supplementary Fig.2). In contrast to glutamate and glutamine, higher
concentrations (5 or 20 mM) of alanine, lysine and aspartate were necessary to cause signicant
stimulation of uncoupled respiration (Table1, Fig.1, Supplementary Fig.3, 4). We did not nd sucient
evidence that asparagine, arginine or histidine are used as an energy source by the uncoupled
mitochondria of pancreatic acinar cells despite sporadic small statistically signicant effects on oxygen
consumption (Table1, Supplementary Fig.5–7).
The availability of glucose as the main energy source decreased the contribution of lysine, alanine and
aspartate to the uncoupled respiration rate of pancreatic acini (Table1, Fig.1b, Supplementary Fig.3b,
4b). This was not the case for glutamine and glutamate (Table1, Supplementary Fig.1b, 2b).
The effects of amino acids on morphology and viability of
pancreatic acini
Immediately after isolation, the viability of pancreatic acini was in the range of 93–97%; cells did not
display signicant morphological issues such as blebbing. The effects of amino acids (20 mM, 2 h) on
pancreatic acinar cells viability and morphology were rst studied in a basic extracellular solution
containing sole glucose as an energy source. In control, about 18% of cell nuclei were stained with
ethidium bromide indicating their necrosis (Fig.2a, b). In this experiment, arginine and lysine both caused
a signicant increase in cell necrosis, while the presence of glutamine or alanine signicantly reduced the
number of dead cells compared to the control (Fig.2b). No increase in cell blebbing was detected
(Fig.2c). Interestingly, glutamine (but not any other amino acid) caused an almost two-fold increase in
the average cell area (Fig.2d).
The results of this experiment proved that the lack of amino acid source in extracellular solution is
detrimental to pancreatic acinar cells. Therefore, we have tested the toxicity of studied amino acids (20
mM) in Dulbecco-modied Eagles medium (DMEM) containing a range of different amino acids and
vitamins.
After 2 h of incubation in DMEM, the necrosis level of pancreatic acinar cells in control was about 11% –
lower than in the experiment with basic extracellular solution use (Supplementary. Figure9a). Moreover,
the studied amino acids did not affect the viability of cells. Neither did they cause cell death upon 4 h of
incubation (Supplementary. Figure8, 9b). We have also estimated the apoptosis by the presence of cells

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with condensed or fragmented nuclei (Supplementary. Figure9e). The apoptosis levels as assessed with
nuclei condensation or fragmentation were very low (< 1%) and did not change with amino acid
supplementation (Supplementary. Figure9c, d). Progressive cell blebbing was seen when glutamine was
present (Fig.3a, b). Notably, cells with blebs usually did not display signs of apoptosis. A signicant
increase in cell blebbing was detected after 2, but not 4 h, of incubation with aspartate (Fig.3a, b).
Glutamine, again, induced cell swelling after 2 and 4 h of incubation (Fig.3c, d).
After 24 h of incubation, in control the majority (75%) of cells were still viable (Fig.3f). Among all tested
amino acids, only alanine and histidine did not decrease the viability of pancreatic acini (Fig.3f,
Supplementary. Figure8). Glutamate, asparagine and aspartate displayed only moderate toxicity
(Fig.3f). The observed similar effects of aspartate and asparagine indicate that the increase in aspartate
concentration due to asparaginase treatment is most likely not the cause of acute pancreatitis.
Surprisingly, glutamine caused complete loss of cell viability (Fig.3f). We have also conrmed high
in
vitro
toxicity of arginine and lysine, but not of histidine (Fig.3f), as was previously shown on murine
isolated acinar cells (Zhang et al. 2019). It is worth noting that histidine toxicity
in vitro
was shown when
cells were incubated under suboptimal conditions of amino acid and vitamin deprivation. Besides, we
have studied rat acini instead of murine isolated acinar cells which might have different metabolism.
We have also performed DNA electrophoresis of pancreatic acinar cells after 24 h of incubation with
amino acids in DMEM (Fig.3e). We have detected substantial DNA fragmentation which was most
pronounced in case of lysine, arginine and glutamine action and the least in control (glucose), alanine
and histidine groups (Fig.3e). The light DNA fragments did not form discrete bands proving cells died by
necrosis and not apoptosis.
Discussion
Pancreatic mitochondria from mice and rats with experimental acute pancreatitis display a decreased
ability to generate the membrane potential (Biczo et al. 2018). Therefore, it is important to nd oxidation
substrates contributing to restoration of mitochondrial oxidative capacity of and ATP production increase
for acinar cell protection. In our previous study, we have shown that glutamine in moderate concentration
(2 mM) prevents mitochondrial membrane depolarization and pancreatic acini necrosis caused by a
combination of ethanol and cholecystokinin (Manko et al., 2021).
In present study, the uncoupled respiration experiment was designed to demonstrate the capacity or
inability of mitochondria in pancreatic acinar cells to oxidize the tested amino acids in conditions of
extreme demand for reducing equivalents. Such conditions may partly mimic the pathologic state of
acinar cells during acute pancreatitis, with both decreased mitochondrial membrane potential and ATP
generation. If amino acid in saturating concentration does not affect oxygen consumption under such
conditions, the conclusion is that mitochondria are largely not able to oxidize this amino acid. In the
opposite case, a positive effect of amino acid on uncoupled respiration proves the principal capacity of
mitochondria to oxidize it under the experimental conditions, in the presence or absence of glucose as an

Page 8/16
alternative energy source. This does not prove, however, that mitochondria of pancreatic acinar cells
prefer to oxidize the tested amino acids at physiological conditions.
In light of this, the results of the experiments show that mitochondria of rat pancreatic acinar cells cannot
oxidize arginine, asparagine and histidine. This is in accordance with the lack of expression of key
enzymes in the pancreas. Pancreas express the rst enzyme of histidine catabolism pathway – histidine
ammonia-lyase (Kumar et al. 2023), but not urocanase / urocanate hydratase 1
(https://www.proteinatlas.org/ENSG00000159650-UROC1) or histidine decarboxylase
(https://www.proteinatlas.org/ENSG00000140287-HDC). Asparaginase is also not expressed in the
pancreas (https://www.proteinatlas.org/ENSG00000166183-ASPG). Several arginine metabolism
pathways are not functional in healthy pancreas (e.g., enzymes arginine decarboxylase, arginase, and NO
synthase), except for creatine synthesis pathway with proven expression of arginine:glycine
amidinotransferase and guanidinoacetate N-methyltransferase (Emerald et al. 2022; da Silva et al. 2014).
However, this pathway does not directly lead to mitochondrial oxidation.
Among the amino acids that are oxidized in the acinar cells of the pancreas, lysine is obviously the least
preferred candidate for supplementation in acute pancreatitis patients, as its positive effects on
uncoupled mitochondrial respiration were observed only at high concentrations (5–20 mM), while
incubation with 20 mM lysine caused a rapid loss of cell viability. Although there is evidence of a positive
effect of low-concentration lysine on the course of pancreatitis in mice (Al-Malki 2015), we suggest that
long-term intake of lysine supplements may lead to increased damage to the pancreas. Glutamate, on the
other hand, showed moderate toxicity at high concentrations while being able to enhance uncoupled
respiration at much lower concentrations. Aspartate proved to be a weakly oxidized amino acid, and only
in the absence of an alternative energy source (glucose). Alanine seems to be the best option for
pancreatitis therapy, as it is both non-toxic for pancreatic acinar cells and is readily utilized as an energy
source, conrming the previous study (Danielsson and Sehlin 1974).
The most unexpected result of this study is the long-term toxicity of glutamine in pancreatic acinar cells.
It was previously shown that
in vivo
glutamine enhanced acinar cell apoptosis in rats with severe acute
pancreatitis (Xu et al. 2006). Evidently, the mechanism of cell death is different from lysine or arginine-
induced necrosis. Glutamine induced a signicant cell swelling and blebbing followed by necrosis.
Notably, glutamate, a direct metabolite of glutamine, did not cause such detrimental effects in pancreatic
acinar cells. Glutamine is known to spontaneously decompose in solutions producing ammonia (Ozturk
and Palsson 1990). Glutamine is also implicated in astrocyte swelling caused by ammonia (Albrecht et
al. 2010), but the mechanisms need further studies. We assume that ammonia accumulation is the main
cause of swelling and necrosis of pancreatic acinar cells induced by glutamine.
In a randomized controlled clinical trial, enteral administration of glutamine in patients with severe acute
pancreatitis did not meet the primary endpoints (Arutla et al. 2019). Due to the instability of glutamine in
solution, supplementation of alanyl-glutamine dipeptide was also studied in patients with acute
pancreatitis (Xue et al. 2008). We assume that the positive effect of this treatment was mainly caused by

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alanine. Considering our data, alanine supplementation seems to be a safer option than glutamine.
Alanine can be catabolized to pyruvate by alanine transaminase (McCommis et al. 2015). Pyruvate
in
vitro
enhances the oxidative and ATP-productive capacity of mitochondria and protects pancreatic acinar
cells from toxic substances (Manko et al., 2021; Peng et al., 2018).
In conclusion, this study showed that alanine and glutamine might be safe and effective amino acid
supplements that support high oxidative capacity of pancreatic acinar cell mitochondria. Further studies
are required to elucidate the effect of the amino acid on pancreatic acinar cell injury and pancreatitis
in
vivo
.
Declarations
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this
manuscript.
Competing interests
The authors have no competing interests to declare that are relevant to the content of this article.
Author contributions
The authors conrm contribution to the paper as follows: study conception and design: Bohdan O.
Manko, Volodymyr V. Manko, Anastasiia M. Zub; data collection: Anastasiia M. Zub; analysis and
interpretation of results: Anastasiia M. Zub, Bohdan O. Manko; draft manuscript preparation: Anastasiia
M. Zub, Bohdan O. Manko, Volodymyr V. Manko. All authors reviewed the results and approved the nal
version of the manuscript.
Ethical approval
All experiments were carried out in accordance with the "European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientic Purposes" (Council of Europe № 123,
Strasbourg 1985). All animal procedures were approved by the Committee for the Care and Use of
Animals of the Ivan Franko National University of Lviv under the protocol number34-04-2023.
Consent to participate: Not applicable.
Consent for publication: Not applicable.
Availability of data and material: Not applicable.
Data availability
Supporting data will be available from the corresponding author upon reasonable request.

Page 10/16
References
1. Albrecht J, Zielińska M, Norenberg MD. Glutamine as a mediator of ammonia neurotoxicity: A critical
appraisal. Biochem Pharmacol. 2010;80(9):1303–8. https://doi.org/10.1016/j.bcp.2010.07.024
2. Al-Malki AL. Suppression of acute pancreatitis by L-lysine in mice. BMC Complement Altern Med.
2015;15 15:193. https://doi.org/10.1186/s12906-015-0729-x
3. Arutla M, Raghunath M, Deepika G, Jakkampudi A, Murthy HVV., Rao GV., et al. Ecacy of enteral
glutamine supplementation in patients with severe and predicted severe acute pancreatitis – A
randomized controlled trial. Indian J Gastroenterol. 2019;38(4):338–47.
https://doi.org/10.1007/s12664-019-00962-7
4. Biczó G, Hegyi P, Dósa S, Shalbuyeva N, Berczi S, Sinervirta R, et al. The crucial role of early
mitochondrial injury in L-lysine-induced acute pancreatitis. Antioxid Redox Signal. 2011;15(10):
2669–81. https://doi.org/10.1089/ars.2011.4065
5. Biczo G, Vegh ET, Shalbueva N, Mareninova OA, Elperin J, Lotshaw E, et al. Mitochondrial
dysfunction, through impaired autophagy, leads to endoplasmic reticulum stress, deregulated lipid
metabolism, and pancreatitis in animal models. Gastroenterology. 2018;154(3):689–703.
https://doi.org/10.1053/j.gastro.2017.10.012
. Binet Q, Dufour I, Agneessens E, Debongnie J-C, Aouattah T, Covas A, et al. The second case of a
young man with l-arginine-induced acute pancreatitis. Clin J Gastroenterol. 2018;11(5):424–27.
https://doi.org/10.1007/s12328-018-0862-4
7. Danielsson A, Sehlin J. Transport and oxidation of amino acids and glucose in the isolated exocrine
mouse pancreas: effects of insulin and pancreozymin. Acta Physiol Scand. 1974;91(4):557–65.
https://doi.org/10.1111/j.1748-1716.1974.tb05710.x
. Dijk DPJ, Horstman AMH, Smeets JSJ, Dulk M, Grabsch HI, Dejong CHC, et al. Tumour-specic and
organ‐specic protein synthesis rates in patients with pancreatic cancer. J Cachexia Sarcopenia
Muscle. 2019;10(3):549–56. https://doi.org/10.1002/jcsm.12419
9. Emerald BS, Mohsin S, D’Souza C, John A, El-Hasasna H, Ojha S, et al. Diabetes mellitus alters the
immuno-expression of neuronal nitric oxide synthase in the rat pancreas. Int J Mol Sci.
2022;23(9):4974. https://doi.org/10.3390/ijms23094974
10. Juhász MF, Vereczkei Z, Ocskay K, Szakó L, Farkas N, Szakács Z, et al. The effect of dietary fat
content on the recurrence of pancreatitis (EFFORT): Protocol of a multicenter randomized controlled
trial. Pancreatology. 2022;22(1):51–7. https://doi.org/10.1016/j.pan.2021.10.002
11. Komatsu M, Yazaki M, Tanaka N, Sano K, Hashimoto E, Takei Y, et al. Citrin deciency as a cause of
chronic liver disorder mimicking non-alcoholic fatty liver disease. J Hepatol. 2008;49(5):810–20.
https://doi.org/10.1016/j.jhep.2008.05.016
12. Kui B, Balla Z, Végh ET, Pallagi P, Venglovecz V, Iványi B, et al. Recent advances in the investigation of
pancreatic inammation induced by large doses of basic amino acids in rodents. Lab Invest.
2014;94(2):138–49. https://doi.org/10.1038/labinvest.2013.143

Page 11/16
13. Kumar N, Rachagani S, Natarajan G, Crook A, Gopal T, Rajamanickam V, et al. Histidine enhances the
anticancer effect of gemcitabine against pancreatic cancer via disruption of amino acid
homeostasis and oxidant–antioxidant balance. Cancers (Basel). 2023;15(9):2593.
https://doi.org/10.3390/cancers15092593
14. Manko BO, Bilonoha OO, Manko VV. Adaptive respiratory response of rat pancreatic acinar cells to
mitochondrial membrane depolarization. Ukr Biochem J. 2019;91(3):34–45.
https://doi.org/10.15407/ubj91.03.034
15. Manko BO, Bilonoha OO, Voloshyn DM, Zub AM, Ivasechko II, Manko VV. Pyruvate and glutamine
dene the effects of cholecystokinin and ethanol on mitochondrial oxidation, necrosis, and
morphology of rat pancreatic acini. Pancreas. 2021;50(7):972–81.
https://doi.org/10.1097/MPA.0000000000001864
1. Manko BO, Klevets MYu, Manko VV. An implication of novel methodology to study pancreatic acinar
mitochondria under
in situ
conditions. Cell Biochem Funct. 2013;31(2):115–21.
https://doi.org/10.1002/cbf.2864
17. Márta K, Szabó AN, Pécsi D, Varjú P, Bajor J, Gódi S, et al. High versus low energy administration in
the early phase of acute pancreatitis (GOULASH trial): protocol of a multicentre randomised double-
blind clinical trial. BMJ Open. 2017;7(9):e015874. https://doi.org/10.1136/bmjopen-2017-015874
1. Mauhin W, Habarou F, Gobin S, Servais A, Brassier A, Grisel C, et al. Update on lysinuric protein
intolerance, a multi-faceted disease retrospective cohort analysis from birth to adulthood. Orphanet J
Rare Dis. 2017;12(1):3. https://doi.org/10.1186/s13023-016-0550-8
19. Mazur HM, Merlavsky VM, Manko BO, Manko VV. Dependence of the mitochondrial adaptive capacity
of hepatocytes on the oxidative substrates availability. Ukr Biochem J. 2019;91(6):5–14.
https://doi.org/10.15407/ubj91.06.005
20. McCommis KS, Chen Z, Fu X, McDonald WG, Colca JR, Kletzien RF, et al. Loss of mitochondrial
pyruvate carrier 2 in the liver leads to defects in gluconeogenesis and compensation via pyruvate-
alanine cycling. Cell Metab. 2015;22(4): 682–94. https://doi.org/10.1016/j.cmet.2015.07.028
21. Oparaji J-A, Rose F, Okafor D, Howard A, Turner RL, Orabi AI, et al. Risk factors for asparaginase-
associated pancreatitis. J Clin Gastroenterol. 2017;51(10):907–13.
https://doi.org/10.1097/MCG.0000000000000827
22. Ozturk SS, Palsson BO. Chemical decomposition of glutamine in cell culture media: effect of media
type, pH, and serum concentration. Biotechnol Prog. 1990;6(2):121–8.
https://doi.org/10.1021/bp00002a005
23. Parto K, Kallajoki M, Aho H, Simell O. Pulmonary alveolar proteinosis and glomerulonephritis in
lysinuric protein intolerance: Case reports and autopsy ndings of four pediatric patients. Hum
Pathol. 1994;25(4):400–7. https://doi.org/10.1016/0046-8177(94)90150-3
24. Pena L, Burton BK. Survey of health status and complications among propionic acidemia patients.
Am J Med Genet A. 2012;158A(7):1641–6. https://doi.org/10.1002/ajmg.a.35387

Page 12/16
25. Peng S, Gerasimenko JV., Tsugorka TM, Gryshchenko O, Samarasinghe S, Petersen OH, et al.
Galactose protects against cell damage in mouse models of acute pancreatitis. J Clin Invest.
2018;128(9):3769–78. https://doi.org/10.1172/JCI94714
2. Rakonczay Z, Hegyi P, Dósa S, Iványi B, Jármay K, Biczó G, et al. A new severe acute necrotizing
pancreatitis model induced by l-ornithine in rats. Crit Care Med. 2008;36(7):2117–27.
https://doi.org/10.1097/CCM.0b013e31817d7f5c
27. Rooman I, Lutz C, Pinho AV., Huggel K, Reding T, Lahoutte T, et al. Amino acid transporters expression
in acinar cells is changed during acute pancreatitis. Pancreatology. 2013;13(5):475–85.
https://doi.org/10.1016/j.pan.2013.06.006
2. Saka M, Tüzün A, Ateş Y, Bağci S, Karaeren N, Dağalp K. Acute pancreatitis possibly due to arginine
use: a case report. Turk J Gastroenterol. 2004;15(1):56–8.
29. Sandstrom P, Trulsson L, Gasslander T, Sundqvist T, von Dobeln U, Svanvik J. Serum amino acid
prole in patients with acute pancreatitis. Amino Acids. 2008;35(1):225–31.
https://doi.org/10.1007/s00726-007-0557-5
30. da Silva RP, Clow K, Brosnan JT, Brosnan ME. Synthesis of guanidinoacetate and creatine from
amino acids by rat pancreas. Br J Nutr. 2014;111(4):571–7.
https://doi.org/10.1017/S0007114513003012
31. Tsai CY, Kilberg MS, Husain SZ. The role of asparagine synthetase on nutrient metabolism in
pancreatic disease. Pancreatology. 2020;20(6):1029–34. https://doi.org/10.1016/j.pan.2020.08.002
32. Wu P, Li L, Sun W. Ecacy comparisons of enteral nutrition and parenteral nutrition in patients with
severe acute pancreatitis: a meta-analysis from randomized controlled trials. Biosci Rep. 2018;38(6).
https://doi.org/10.1042/BSR20181515
33. Xu XF, Lou WH, Wang DS, Jin DY, Ni XL, Wu ZH. Inuence of glutamine on pancreatic blood ow and
apoptosis of pancreatic acinar in rats with severe acute pancreatitis. Chin J Dig Dis. 2006;7(2):121–
6. https://doi.org/10.1111/j.1443-9573.2006.00250.x
34. Xue P, Deng LH, Xia Q, Zhang ZD, Hu W-M, Yang XN, et al. Impact of alanyl-glutamine dipeptide on
severe acute pancreatitis in early stage. World J Gastroenterol. 2008;14(3):474–8.
https://doi.org/10.3748/wjg.14.474
35. Yang AL. Nutrition and acute pancreatitis. J Clin Med. 2021;10(4):836.
https://doi.org/10.3390/jcm10040836
3. Zhang X, Jin T, Shi N, Yao L, Yang X, Han C, et al. Mechanisms of pancreatic injury induced by basic
amino acids differ between L-arginine, L-ornithine, and L-histidine. Front Physiol. 2019;9:1922.
https://doi.org/10.3389/fphys.2018.01922
Tables
Table 1󰁅Effects (% from control) of amino acids on uncoupled respiration rate. Red – decreased compared to
control, green – increased compared to control, and white – close to control levels. Bold font – a statistically
significant difference compared to control respiration with P < 0.05 according to two-way ANOVA and post-hoc
Holm-Bonferroni test; n = 5-9

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󰁅 Amino acid (mM) Amino acid (mM) + glucose (10 mM)
󰁅 0.1󰁅 1󰁅 5󰁅 20󰁅 0.1󰁅 1󰁅 5󰁅 20󰁅
󰁅
󰁅 Glutamate Glutamate󰁅+󰁅
glucose
FCCP,
µM 0 - 17.98󰁅 14.20󰁅 -2.08󰁅 - 1.41󰁅 -3.09󰁅 -3.79󰁅
0.5 - 20.72󰁅*21.01󰁅* 41.65󰁅*󰁅 - 18.73󰁅 19.92󰁅 31.66󰁅
1 - 23.31󰁅 58.65󰁅*󰁅 107.36󰁅*󰁅- 30.94󰁅*34.15󰁅*󰁅66.82󰁅*
1.5 - 34.37󰁅*91.40󰁅* 168.49󰁅*󰁅- 36.56󰁅*45.49󰁅* 95.83󰁅*󰁅
2 - 36.25󰁅 114.38󰁅*192.74󰁅*󰁅- 62.58󰁅*87.04󰁅* 130.70󰁅*
󰁅 󰁅 Alanine Alanine󰁅+󰁅
glucose
FCCP,
µM 04.36 1.15 -3.94 -13.58 -2.99 -12.06 -4.34 7.64
0.5 16.50󰁅*-3.11 0.07 -3.73 6.96 5.60 -12.24 -3.28
17.56 10.28 12.45 -3.55 -4.91 -7.93 -15.50 -3.86
1.5 15.96 19.25 41.19󰁅* 11.91 -2.93 6.76 -5.22 11.57
2 30.77 49.55 95.84󰁅* 77.25󰁅* 17.84 34.75 38.87 75.04󰁅*
󰁅
󰁅 Glutamine Glutamine󰁅+󰁅
glucose
FCCP,
µM 07.30󰁅 3.34 11.23 -5.97 -23.07 -10.07 -12.15 -12.95
0.5 -3.57󰁅 -4.60 -1.48 -16.28 -16.40 -5.18 -11.34 -3.11
1-1.61󰁅 -0.87 -1.17 -14.92 -11.35 -8.79 -13.12 -9.56
1.5 1.62󰁅 18.09 11.42 -5.92 -7.63 6.32 1.75 7.45
2 20.42󰁅 44.60󰁅*47.25󰁅* 23.87 1.75 41.32󰁅*32.46󰁅* 31.09󰁅*
󰁅
󰁅 Lysine Lysine󰁅+󰁅
glucose
FCCP,
µM 04.07 -3.40 0.62 0.06 8.10 4.27 -1.48 2.06
0.5 1.50 -4.54 -7.91 14.39 4.55 -5.76 -7.84 1.42
1-7.63 -9.34 -1.31 11.85 -0.29 -14.00 -3.96 0.29
1.5 17.42 4.08 51.39󰁅* 42.59󰁅* -1.08 -11.66 17.67 9.40
28.43 3.13 15.59 52.33󰁅* 13.65 8.85 43.27󰁅* 40.21
󰁅 󰁅
Aspartate Aspartate󰁅+󰁅
glucose
FCCP,
µM 0-3.15 6.81 0.52 -7.17 -16.11 -2.23 -4.66 -23.33
0.5 1.39 1.30 6.82 -2.33 -12.02 -3.33 -4.40 -23.58
19.75 17.27 22.66 7.39 -10.43 2.01 5.77 -7.55
1.5 6.84 19.02 35.73󰁅* 24.47 -20.59 -9.48 0.38 3.42
28.43 16.28 42.99󰁅* 26.94 -27.99󰁅*-15.51 1.50 10.72
󰁅 󰁅
Asparagine Alanine󰁅+󰁅
glucose
FCCP,
µM 06.57 2.99 -7.26 -12.22 -11.00 -3.65 -15.37 -20.57
0.5 4.52 2.93 10.05 16.21 -10.75 -5.17 -14.05 -19.71
17.97 17.62 23.34 26.56 -8.04 -6.93 -6.43 -10.58
1.5 10.16 20.97 24.90 19.00 -8.09 -4.11 5.41 -7.76
2 13.39 27.38 23.51 -1.21 -12.37 -6.16 8.58 -9.68
󰁅 󰁅
Arginine Arginine󰁅+󰁅
glucose
FCCP,
µM 0-3.13 4.78 5.61 8.08 10.72 17.51 20.47 10.52
0.5 -2.53 3.91 18.36 20.14 3.73 5.24 7.68 2.83
1-6.46 8.37 22.34󰁅* 2.78 5.32 1.90 8.64 -4.99
1.5 3.94 23.33 18.69 -7.33 -14.27 -2.59 8.63 -19.99
2-6.60 18.63 2.56 -4.88 -8.56 -12.34 13.59 -18.04
󰁅 󰁅
Histidine Histidine󰁅+󰁅
glucose
FCCP,
µM 05.33 16.01 7.32 9.27 -2.28 -17.82 2.51 -8.24
0.5 3.71 27.13 11.98 11.36 -0.17 -18.75 -0.04 0.97
1 13.69 23.13 14.98 23.30 -0.25 -17.72 6.94 4.29
1.5 14.76 31.80 21.35 16.06 5.15 -13.03 13.63 3.80
27.51 23.97 24.12 4.56 26.45 -10.62 36.89󰁅* 2.25

Page 14/16
󰁅* - P󰁅<󰁅0.05 vs. control
Figures
Figure 1
The effect of alanine on the basal ([FCCP] = 0) and uncoupled ([FCCP] = 0.5–2 µM) respiration rate of rat
pancreatic acini in the basic extracellular solution without (a) or with (b) glucose: [glucose] = 10 mM,
[alanine] compared to control respiration with P < 0.05 according to two-way ANOVA and post-hoc Holm-
Bonferroni test; n = 6

Page 15/16
Figure 2
Viability and morphology of pancreatic acini exposed to a high concentration of amino acids during 2 h
incubation in basic extracellular solution with glucose: light (b/w) and uorescent (Hoechst 33258
staining of all nuclei, ethidium bromide staining of necrotic cell nuclei) microscopy of pancreatic acini (a);
cell necrosis rates (b); the number of cells with blebs (c); the effect of glutamine on average cell area (d);
[glucose] = 10 mM, [amino acid] = 20 mM; * – a statistically signicant difference compared to control
respiration with P < 0.05 according to two-way ANOVA and post-hoc Holm-Bonferroni test; n = 6-7

Page 16/16
Figure 3
Viability and morphology of pancreatic acinar cells under the inuence of amino acids in a high
concentration during incubation in DMEM: number of cells with blebs at 2 h (a) and 4 h (b); the cell area
at 2 h (c) and 4 h (d); electrophoresis in agarose gel during long-term incubation (24 h) (e); necrosis at 24
h (f); [glucose] = 10 mM, [amino acid] = 20 mM, glucose was always present; * – a statistically signicant
difference compared to control respiration with P < 0.05 according to two-way ANOVA and post-hoc
Holm-Bonferroni test; n = 8
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