Long-chain acylcarnitines decrease the phosphorylation of the insulin receptor at tyr1151 through a ptp1b-dependent mechanism

Article abstract of DOI:10.3390/ijms22126470

The accumulation of lipid intermediates may interfere with energy metabolism pathways and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B). Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium (IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmitoylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to insulin stimulation.

Full text of DOI:10.3390/ijms22126470

International Journal of
Molecular Sciences
Article
Long-Chain Acylcarnitines Decrease the Phosphorylation of the
Insulin Receptor at Tyr1151 through a PTP1B-Dependent
Mechanism
Karlis Vilks 1,2,* , Melita Videja 1,3 , Marina Makrecka-Kuka 1 , Martins Katkevics 4 , Eduards Sevostjanovs 5,
Aiga Grandane 6 , Maija Dambrova
1,3
and Edgars Liepinsh 1
1
Laboratory of Pharmaceutical Pharmacology, Latvian Institute of Organic Synthesis, Aizkraukles Str. 21,
LV-1006 Riga, Latvia; melita.videja@farm.osi.lv (M.V.); makrecka@farm.osi.lv (M.M.-K.);
maija.dambrova@farm.osi.lv (M.D.); ledgars@farm.osi.lv (E.L.)
Faculty of Biology, University of Latvia, Jelgavas Str. 1, LV-1004 Riga, Latvia
Faculty of Pharmacy, Riga Stradins University, Dzirciema Str. 16, LV-1007 Riga, Latvia
Biologically Active Compound Synthesis Laboratory, Latvian Institute of Organic Synthesis,
LV-1006 Riga, Latvia; martins@osi.lv
Laboratory of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, LV-1006 Riga, Latvia;
eduards@osi.lv
Organic Synthesis Group, Latvian Institute of Organic Synthesis, LV-1006 Riga, Latvia; aiga@osi.lv
Correspondence: karlis.vilks@farm.osi.lv
2
3
4
5
6
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: The accumulation of lipid intermediates may interfere with energy metabolism pathways
and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been
linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components
of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation
of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B).
Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is
not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium
Citation: Vilks, K.; Videja, M.;
Makrecka-Kuka, M.; Katkevics, M.;
Sevostjanovs, E.; Grandane, A.;
Dambrova, M.; Liepinsh, E.
Long-Chain Acylcarnitines Decrease
the Phosphorylation of the Insulin
Receptor at Tyr1151 through a
PTP1B-Dependent Mechanism. Int. J.
Mol. Sci. 2021, 22, 6470. https://
doi.org/10.3390/ijms22126470
(
IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR
phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect
the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmi-
toylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in
insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease
InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to
insulin stimulation.
Academic Editors: Adam Kr e˛ towski
and Lukasz Szczerbinski
Received: 3 May 2021
Accepted: 14 June 2021
Published: 16 June 2021
Keywords: palmitoylcarnitine; protein tyrosine phosphatase 1B; long-chain acylcarnitines; Akt;
insulin receptor; insulin resistance
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
. Introduction
Insulin signalling plays a central role in the regulation of glucose and lipid metabolism.
The activation of insulin signalling begins when insulin binds to the extracellular sub-
units of the insulin receptor (InsR), causing conformational changes that promote the
autophosphorylation of intracellular subunits, including the Tyr1151 phosphorylation
site [1]. This step of insulin signalling is regulated by tyrosine phosphatases; among these
tyrosine phosphatases, the most noteworthy is protein-tyrosine phosphatase 1B (PTP1B,
also referred to as PTPN1), which is considered the main regulator of InsR phosphoryla-
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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tion [
insulin signal transduction [
downstream kinases, insulin signalling reaches protein kinase B (Akt), a central member of
the insulin signalling pathway. Akt is activated when it binds to the cellular membrane [ ],
2]. Activated PTP1B catalyses the dephosphorylation of InsR, thus downregulating
3
]. After the sequential phosphorylation events mediated by
4,5
4
.0/).
Int. J. Mol. Sci. 2021, 22, 6470. https://doi.org/10.3390/ijms22126470
https://www.mdpi.com/journal/ijms

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exposing two main phosphorylation sites, namely, Tyr308 and Ser473. Akt phosphoryla-
tion is regulated by ceramide-activated protein phosphatase 2A (PP2A) [
multiple diacylglycerol (DAG)-dependent isoforms of protein kinase C (PKC) are able to
regulate Akt phosphorylation [ ]. Insulin-stimulated Akt phosphorylation at Ser473 ini-
tiates physiological processes associated with facilitating glucose uptake into the cell by
glucose transporter type 4 [ ] and limiting fatty acid metabolism by inhibiting carnitine
palmitoyltransferase 1 (CPT1)-mediated long-chain acylcarnitine (LCAC) synthesis [ 10].
6]. In addition,
7
8
9
,
Insulin resistance is associated with increased levels of lipid metabolism intermediates,
such as DAG, ceramides, and long-chain fatty acids and their acylcarnitines [11]. The role
of LCAC in the regulation of intracellular signalling pathways and the contribution of
LCAC to insulin resistance have received relatively little attention. To date, researchers
have determined that LCAC dictates energy metabolism patterns in mitochondria by
inhibiting pyruvate-lactate oxidation [12,13]. Increased levels of LCAC in mice induce
marked hyperinsulinaemia, insulin resistance, and glucose intolerance [14]. The role of
LCAC in the regulation of insulin signalling has been highlighted in studies showing that
LCAC induces a decrease in Akt phosphorylation in ex vivo [15
,16] and in vivo [14] models,
although the exact molecular mechanism has not yet been defined.
In this study, we selected the CHO INSR 1284 cell line (CHO), which continuously
overexpresses human InsR [17]. Because the expression of InsR in CHO cells is higher
than that in most cell cultures, this cell line is an appropriate system for studies of the
regulation of metabolism [17]. As shown in our previous study, a highly abundant LCAC,
palmitoylcarnitine, increases the concentration of circulating insulin in vivo [14]. We used
the insulin-secreting cell line RIN5F to further investigate the effects of LCAC on insulin
release [18]. Insulin-sensitizing bis-(maltolato)-oxovanadium (IV) (BMOV) and the non-
competitive PTP1B inhibitor 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione
(BVT948) were used as inhibitors of PTP1B [19
,20]. In addition, a zinc-containing compound
(zinc sulfate) was used as an inhibitor of PP2A [21], and sotrastaurin was used as an
inhibitor of both conventional and novel DAG-dependent PKC isoforms [22].
2. Results
2
.1. Palmitoylcarnitine Diminishes Insulin-Stimulated Phosphorylation of InsR and Akt
®
Stimulation of the CHO INSR 1284 cell line (ATCC CRL-3307
™
) with 10 nM insulin
significantly increased the phosphorylation of InsR at Tyr1151 by 25-fold (Figure 1A,B) and
the phosphorylation of Akt at Ser473 by approximately 50-fold (Figure 1A,C), indicating
subsequent amplification of the insulin signal. When cells were stimulated with 10 nM
insulin in the presence of 10
µM palmitoylcarnitine, the insulin-induced phosphorylation
of InsR at Tyr1151 was decreased by 35
insulin alone (Figure 1B). Compared to insulin alone, the addition of palmitoylcarnitine to
the cell culture media also resulted in a subsequent reduction in Akt Ser473 phosphorylation
±
24% (p < 0.05) compared to cells stimulated with
by 66
±
22% (p < 0.005) (Figure 1C). These findings suggest that palmitoylcarnitine affects
both InsR phosphorylation and Akt Ser473 phosphorylation. The baseline levels of InsR
Tyr1151 and Akt Ser473 phosphorylation in unstimulated CHO cells were very low, and the
addition of 10 µM palmitoylcarnitine to the CHO cell media did not cause measurable
changes in Akt phosphorylation (Figure 1A,C). Treatment with the CPT1 inhibitor etomoxir
did not affect insulin-stimulated InsR and Akt phosphorylation (Figure 1D–F), indicating
that endogenous acylcarnitine synthesis in CHO cells does not produce a sufficient amount
of LCACs to affect insulin signalling.

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Figure 1. Palmitoylcarnitine (PC) and etomoxir altered the phosphorylation of InsR and Akt in insulin-stimulated CHO
InsR 1284 cells. CHO cells were incubated in DMEM low glucose media with and without 10 nM insulin and 10
palmitoylcarnitine for 10 min. Representative bands from insulin-stimulated cells ( ). Semiquantitative data showing
the relative phosphorylation of InsR at Tyr1511 ( ) and Akt at Ser473 ( ). The band intensities were normalized by
dividing each value by the intensity of the total Akt or InsR band. The band intensities of the insulin control were set to 1.
p < 0.05 compared to the unstimulated control group and # p < 0.05 compared to the insulin-stimulated group (one-way
ANOVA, Tukey’s multiple comparison test). The results are presented as the means standard deviation. Sample size of
µM
A,D
B
,E
C,F
*
±
the groups in sections B and C: control n = 9, PC n = 4, insulin control n = 8, and insulin + PC n = 7. Sample size of groups in
section E: insulin n = 6, insulin + etomoxir n = 4. Sample size of groups in section F: insulin n = 6, insulin + etomoxir n = 5.
2
.2. Inhibition of PTP1B Alleviates the Palmitoylcarnitine-Induced Dephosphorylation of InsR
Tyr1151
We used the insulin sensitizer vanadium-containing compound BMOV, which is known
to increase InsR phosphorylation through PTP1B inhibition, to determine whether the effect
of palmitoylcarnitine on the dephosphorylation of InsR was related to PTP1B. In CHO cells
stimulated with 100
Figure 2A) were similar to those observed in insulin-stimulated cells (Figure 1A). We ob-
served 25-fold and 50-fold BMOV-stimulated increases in InsR Tyr1151 (Figure 2B) and
Akt Ser473 (Figure 2C) phosphorylation, respectively. The addition of 10 M palmitoyl-
µM BMOV, the levels of InsR Tyr1151 and Akt Ser473 phosphorylation
(
µ
carnitine to BMOV-stimulated cells did not induce any decrease in the phosphorylation
of InsR at Tyr1151 (Figure 2B). However, despite the preserved phosphorylation of InsR,
the level of Akt Ser473 phosphorylation in palmitoylcarnitine-treated cells was significantly
decreased by 69
±
15% (p < 0.005) (Figure 2C) compared to that in BMOV-stimulated
cells. Based on these results, palmitoylcarnitine influences InsR through the activation
of PTP1B, and palmitoylcarnitine reduces the level of Akt phosphorylation independent
of its effect on InsR. Since the palmitoylcarnitine-induced dephosphorylation of InsR at
Tyr1151 was abolished after the inhibition of PTP1B by BMOV, we determined whether
palmitoylcarnitine induced the dephosphorylation of InsR through the direct activation
of the tyrosine phosphatase PTP1B. We tested the effect of palmitoylcarnitine in a puri-
fied tyrosine phosphatase PTP1B enzyme activity assay. At a concentration of 100
BMOV decreased PTP1B activity by 62 4% (Figure 2D), but 10 M palmitoylcarnitine
did not cause any changes in PTP1B activity. Therefore, palmitoylcarnitine indirectly
µM,
±
µ

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activates PTP1B and stimulates the dephosphorylation of InsR at Tyr1151. We used the
non-competitive PTP1B inhibitor BVT948 together with insulin to confirm these results.
BVT948 completely blocked palmitoylcarnitine-induced dephosphorylation of InsR and
Akt (Figure 3A–C), supporting the hypothesis that PTP1B is a target of LCAC action.
Figure 2. Effects of bis-(maltolato)-oxovanadium (IV) (BMOV) on the phosphorylation of InsR and
Akt in CHO InsR 1284 cells and on the activity of purified protein tyrosine phosphatase 1B (PTP1B).
CHO cells were preincubated with 100
added 10 min before the cells were harvested for WB. Representative bands from BMOV-stimulated
cells are shown ( ). Semiquantitative data showing the relative phosphorylation of InsR at Tyr1511
) and Akt at Ser473 ( ). Activity of purified PTP1B in the presence of PC and BMOV ( ). The band
µM BMOV for 1 h, and 10 µM palmitoylcarnitine (PC) was
A
(B
C
D
intensities were normalized by dividing each value by the intensity of the total Akt or InsR band.
The band intensities of the BMOV control were set to 1. * p < 0.05 compared to the unstimulated
control group and # p < 0.05 compared to the BMOV-stimulated group (one-way ANOVA, Tukey’s
multiple comparison test). The results are presented as the means
of groups in sections B and C: control n = 9, BMOV control n = 8, and BMOV + PC n = 7. Sample size
of groups in section D: control n = 3, PC n = 3, and BMOV n = 3.
±
standard deviation. Sample size

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Figure 3. Effects of BVT948 on the palmitoylcarnitine (PC)-induced changes in InsR and Akt phos-
phorylation in insulin-stimulated CHO InsR 1284 cells. CHO cells were preincubated with 25
BVT948 for 1 h, and 10 M PC and 10 nM insulin were added 10 min before the cells were har-
vested for WB. Representative bands show InsR and Akt phosphorylation ( ). Semiquantitative
data showing the relative phosphorylation of InsR at Tyr1511 ( ) and Akt at Ser473 ( ). The band
µM
µ
A
B
C
intensities were normalized by dividing each value by the intensity of the total Akt or InsR band.
The band intensities of the insulin-BVT948-stimulated control were set to 1. * p < 0.05 compared to the
unstimulated control group (Kruskal-Wallis test, Dunn’s multiple comparison test). The results are
presented as the means
n = 12, and BVT948 + PC n = 12.
±
standard deviation. Sample size of groups: control n = 5, insulin + BVT948
Wortmannin did not affect insulin-stimulated phosphorylation of InsR at Tyr1151
(
Figure 4A,B) but completely prevented Akt Ser473 phosphorylation (Figure 4A,C).
Figure 4. Effects of wortmannin on the phosphorylation of InsR (
284 cells. The band intensities were normalized by dividing each value by the intensity of the total Akt or InsR band.
The band intensities of the insulin-stimulated control were set to 1. # p <0.05 compared to the insulin control group (one-way
A,B) and Akt (A,C) in insulin-stimulated CHO InsR
1
ANOVA, Tukey’s multiple comparison test). The results are presented as the means
groups: insulin n = 3, insulin + wortmannin n = 3.
±
standard deviation. Sample size of

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2.3. Palmitoylcarnitine Decreases Akt Phosphorylation Independently of PP2A and PKC Activity
We used sotrastaurin and zinc sulfate, which are inhibitors of PKCs and PP2A, to deter-
mine whether the effects of palmitoylcarnitine on Akt phosphorylation were mediated by
these enzymes. Stimulation of the cells with insulin in the presence of 100 M zinc sulfate
Figure 5A,B) and 100 nM sotrastaurin (Figure 5C,D) did not exert a significant effect on Akt
Ser473 phosphorylation compared to stimulation with insulin alone. When palmitoylcarni-
µ
(
2+
tine was added to the insulin- and Zn -stimulated cells, the level of Akt Ser473 phosphory-
lation was completely diminished (Figure 5A,B). This finding indicates that palmitoylcarni-
tine does not induce the dephosphorylation of Akt Ser473 through PP2A (Figure 5B). A sim-
ilar effect was observed when sotrastaurin was used. Since 100 nM sotrastaurin did not al-
leviate the effects of palmitoylcarnitine on insulin stimulation (Figure 5D), DAG-dependent
PKC isoforms are likely not involved in the palmitoylcarnitine-induced dephosphoryla-
tion of Akt. Taken together, palmitoylcarnitine induces Akt Ser473 dephosphorylation
independently of PP2A and PKC activity.
Figure 5. Effects of the PP2A inhibitor Zn2+ and the PKC inhibitor sotrastaurin on palmitoylcarnitine-induced Akt
phosphorylation in insulin-stimulated CHO InsR 1284 cells. CHO cells were preincubated in DMEM low glucose media
containing 100
in the presence or absence of 10
) or 100 nM sotrastaurin ( ). Semiquantitative data showing the relative phosphorylation of Akt at Ser473 (B and D).
µM zinc sulfate (A) or 100 nM sotrastaurin (
C) for 50 min and then stimulated with 5 nM insulin for 10 min
µM palmitoylcarnitine (PC). Representative bands from cells treated with zinc sulfate
(
A
C
The band intensities were normalized by dividing each value by the intensity of the total Akt band. The band intensities of
the insulin control were set to 1. Quantification of relative Akt phosphorylation and PC-induced effects in the presence
of zinc sulfate (
compared to the insulin/Zn -stimulated group (one-way ANOVA, Tukey’s multiple comparison test). The results are
presented as the means
standard deviation. Sample size of the groups in section B: insulin n = 6, Zn2+ + insulin n = 5, and
PC + Zn + insulin n = 6; section D: insulin n = 3, sotrastaurin + insulin n = 4 and PC + sotrastaurin + insulin n = 4.
B) and sotrastaurin (D) is shown. * p < 0.05 compared to the unstimulated control group and # p < 0.05
2+
±
2
+
2
.4. Palmitoylcarnitine Increases Insulin Release by Limiting Akt Ser473 Phosphorylation in
Insulin-Producing RIN5F Cells
We used the insulin-producing RIN5F cell line and measured the levels of Akt
Ser473 phosphorylation and insulin release in the presence of palmitoylcarnitine to eluci-
date the possible mechanisms underlying LCAC-induced hyperinsulinaemia. When RIN5F

Int. J. Mol. Sci. 2021, 22, 6470
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cells were incubated in cell media containing palmitoylcarnitine for 1 h, a dose-dependent
increase in insulin release was observed (Figure 6A). At a 1
carnitine did not affect insulin release, while palmitoylcarnitine applied at concentrations
of 5 M and 10 M stimulated an increase in the insulin concentration by 3.6- and 6.7-
µM concentration, palmitoyl-
µ
µ
fold, respectively, compared to the control (Figure 6A). Despite the approximately 7-fold
increase in the insulin concentration, we did not observe a statistically significant increase
in Akt Ser473 phosphorylation (Figure 6B,C), indicating that despite the high insulin con-
centration, palmitoylcarnitine hindered the ability of the cells to detect insulin through
the inhibition of Akt Ser473 phosphorylation (Figure 6B,C and Figure 7), and a higher
concentration of insulin is required to prevent insulin release.
Figure 6. Effects of palmitoylcarnitine (PC) on insulin-producing RIN5F cells. Insulin release in the presence of PC (A).
The insulin secretion in the media was compared by normalizing the insulin concentration to that of the control group
incubated without PC. The graph shows the relative insulin concentration compared to the control group treated without PC.
Insulin release was measured 1 h after the cell growth media was changed to DMEM high glucose media (11 mM glucose)
containing various concentrations of PC. Representative bands from RIN5F cells (
Akt Ser473 phosphorylation ( ). The phosphorylation of Akt at Ser473 in RIN5F cells was determined using WB after a 1 h
incubation with various concentrations of PC. The band intensities were normalized by dividing each value by the intensity
of the total Akt band. The band intensities of the control were set to 1. The results are presented as the means standard
deviation. * p < 0.05 compared to the control group (one-way ANOVA, Tukey’s multiple comparisons test). Sample size of
B). Semiquantitative data showing relative
C
±
the groups in section A: control n = 4, PC 1
section C: control n = 9, PC 10 µM n = 9.
µM n = 4, PC 5 µM n = 4, and PC 10 µM n = 4. Sample size of the groups in
3. Discussion
Here, we showed for the first time that palmitoylcarnitine affects the insulin signalling
pathway by inducing the dephosphorylation of InsR at Tyr1151 (Figure 7). The effects
of palmitoylcarnitine on the dephosphorylation of InsR at Tyr1151 were related to the
indirect activation of PTP1B. Additionally, palmitoylcarnitine significantly decreased the
insulin- and BMOV-stimulated phosphorylation of Akt at Ser473. Thus, the inhibitory
effect of palmitoylcarnitine on Akt activity is independent of the upstream effects on InsR
phosphorylation. The inhibition of PP2A and PKCs did not diminish the palmitoylcarnitine-
induced decrease in Akt Ser473 phosphorylation, indicating that PP2A and PKC are not
involved in the palmitoylcarnitine-induced dephosphorylation of Akt. Based on these re-
sults, palmitoylcarnitine affects insulin signalling pathways through different mechanisms
from ceramide and DAG. Finally, our results show that the molecular origins of insulin
resistance and hyperinsulinaemia, which are observed in insulin-resistant individuals,
are potentially explained by the effects of LCAC on the insulin signalling pathway in
insulin-secreting cells (Figure 8).

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BMOV
PTP1B
Ins
-
P
InsR
Tyr1151
P
Zn2+
PP2A
PKC
BVT948
PK
Sotrastauin
Akt
Ser473
P
Downstream
signals
Figure 7. Summary of the effects of palmitoylcarnitine (PC) on cells. PC inhibits insulin signalling
by decreasing the phosphorylation of InsR and Akt. PC does not decrease the phosphorylation of
InsR if it is stimulated by the PTB1B inhibitor bis-(maltolato)-oxovanadium (IV) (BMOV). However,
PC still markedly reduces the phosphorylation of the downstream target Akt in the presence of BMOV.
Abbreviations and symbols: blue—normal physiological response, red—PC-induced changes. InsR—
insulin receptor β, PTP1B—protein-tyrosine phosphatase 1B, Akt—protein kinase B, Ins—insulin,
“
-P”—dephosphorylation, and “+P”—phosphorylation.
The physiological importance of PTP1B in the insulin signalling pathway was previ-
ously established by the findings that the deletion of PTP1B results in insulin hypersen-
sitivity [23] and that overexpression of PTP1B causes insulin resistance, similar to that
observed in patients with type 2 diabetes [24]. A novel finding in this study is that palmi-
toylcarnitine decreases the phosphorylation of InsR at Tyr1151 through a PTP1B-dependent
mechanism. Moreover, palmitoylcarnitine does not decrease the phosphorylation of InsR
in cells that are treated with the PTB1B inhibitors BMOV and BVT948, suggesting that
indirect PTB1B activation is the main mechanism by which palmitoylcarnitine acts on
InsR. Because PTP1B is a crucial component of the insulin signalling pathway, the regula-
tion of PTP1B activity is a complex process that involves numerous stages and feedback
loops, and this process might be affected by palmitoylcarnitine. Akt plays a central role
in the insulin signalling pathway, and its activity is a measure of insulin sensitivity [25].
Akt Ser473 phosphorylation induces numerous insulin-dependent effects both in vitro and
in vivo. In this study, we observed a pronounced decrease in Akt Ser473 phosphorylation
after stimulation with insulin or BMOV. Since the inhibition of PTB1B fully restores the
phosphorylation of InsR, we conclude that palmitoylcarnitine decreases Akt phosphoryla-
tion independently of InsR. Importantly, the effects of LCAC on insulin signalling induce
changes in insulin-dependent glucose metabolism [14,15]. In addition to mediating the
insulin response in a cell, Akt serves as a positive regulator of InsR by phosphorylating
PTP1B at the Ser50 position, thus hindering its phosphatase activity [26] and indirectly
increasing InsR phosphorylation. By decreasing Akt activity, palmitoylcarnitine compro-
mises the Akt-dependent regulation of PTP1B activity (Figure 7), resulting in a higher InsR
dephosphorylation rate. Overall, the palmitoylcarnitine-induced decrease in InsR phos-
phorylation might at least partially depend on the palmitoylcarnitine-induced decrease
in Akt Ser473 phosphorylation. However, wortmannin treatment did not cause signifi-
cant changes in InsR Tyr1151 phosphorylation (Figure 4B), suggesting that the Akt-PTP1B
regulatory mechanism does not explain palmitoylcarnitine-induced effects on the CHO
cell line.

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We used BVT948, a non-competitive, irreversible inhibitor of the protein tyrosine phos-
phatases PTP1B (IC50 0.9
tyrosine-protein phosphatase nonreceptor type 11 (SHP-2) (IC50 0.09
common antigen-related protein tyrosine phosphatase (LAR) (IC50 1.5
µ
M), T-cell protein tyrosine phosphatase (TCPTP) (IC50 1.7
M), and leukocyte
M) to further clarify
µM),
µ
µ
the effects of palmitoylcarnitine [20]. BVT948 preserves InsR and Akt Ser473 phosphoryla-
tion in the presence of palmitoylcarnitine; therefore, all kinases in the insulin signalling
pathway are not inhibited by palmitoylcarnitine. Thus, the palmitoylcarnitine mechanism
of action is related to the activation of BVT948-targeted phosphatases. Furthermore, the ef-
fect of palmitoylcarnitine on Akt phosphorylation is not diminished by BMOV, suggesting
that palmitoylcarnitine-induced effects on Akt may be mediated by phosphatases that are
unaffected by BMOV but inhibited by BVT948 [20,27]. Overall, two phosphatases, LAR [28]
and TC-PTP [29], are most likely affected by palmitoylcarnitine, as both are described as
negative regulators of the insulin signalling pathway.
The physiological significance of PP2A and its involvement in insulin signalling have
been revealed by the findings that PP2A deletion leads to insulin hypersensitivity [30] but
PP2A overexpression induces insulin resistance [24]. PP2A has been described as a negative
regulator of Akt Tyr308 phosphorylation, but recent studies have also indicated its involve-
ment in the regulation of Akt Ser473 phosphorylation [31]. In this study, PP2A inhibition
by zinc did not alleviate the effects of palmitoylcarnitine on CHO cells, indicating that
palmitoylcarnitine does not decrease Akt phosphorylation through the same mechanism
2+
as ceramides. The insulin-sensitizing and mimetic effects of Zn are attributed not only
to the inhibition of PP2A [32] but also to the inhibition of multiple other phosphotyrosine
phosphatases [33], including phosphatase and tensin homologue (PTEN) [34], which are
2+
negative regulators of the Akt signalling pathway. Thus, since the Zn treatment did not
alter the effects of palmitoylcarnitine on Akt Ser473 phosphorylation, we conclude that
PP2A and PTEN are not involved in the effects of palmitoylcarnitine on insulin signalling
(
Figure 7).
The PKC family participates in the regulation of the insulin signalling pathway by
influencing Akt phosphorylation. Increased expression and activity of PKC isoforms
are associated with impaired insulin signalling in subjects with insulin resistance [22 35].
,
Due to the high similarity between the different PKC isoforms, we used the nonspecific
inhibitor sotrastaurin, which is a compound that inhibits most of the DAG-dependent PKC
isoforms, including α, βI, δ, ε, η, and θ [36]. Incubation of the cells with sotrastaurin did not
result in any changes in the insulin-stimulated phosphorylation of Akt at Ser473 and did
not alleviate the effects of palmitoylcarnitine. These results indicate that palmitoylcarnitine
affects the insulin signalling pathway through a mechanism that differs from PKC activation
by DAGs.
The mechanism underlying the autocrine control of insulin release from
been extensively studied for many years. First, Leibiger et al. noted that phosphoinositide
-kinase is required for the autocrine control of insulin synthesis in islet -cells [37]. Sub-
sequent studies elucidated that Akt controls the rate of insulin release, and alterations in
Akt signalling result in dysfunctional insulin release from -cells [38]. The accumulation of
LCAC increases insulin secretion from -cells in vitro 39] and the overall concentration of
circulating insulin in vivo 14]. In the present study, we observed lower Akt Ser473 phos-
β-cells has
3
β
β
β
[
[
phorylation in RIN5F cells treated with palmitoylcarnitine, despite the increased insulin
release and the corresponding increased concentration of insulin in the cell media. Thus,
palmitoylcarnitine-stimulated insulin release is most likely caused by palmitoylcarnitine-
induced suppression of Akt Ser473 phosphorylation (Figure 8).

Int. J. Mol. Sci. 2021, 22, 6470
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Ins
Ins
Ins
Ins Ins Ins
Ins
Ins
InsR
Ins
Ins
Ins
Akt
PC
Ser473
P
Figure 8. Effects of palmitoylcarnitine (PC) on insulin release. By decreasing Akt phosphoryla-
tion in the RIN5F cell line, PC interferes with the insulin-sensing feedback mechanism, thus pro-
moting excessive insulin release. Abbreviations and symbols: blue—normal physiological re-
sponse, red—PC-induced changes. InsR—insulin receptor
-P”—dephosphorylation, and “+P”—phosphorylation.
β, Akt—protein kinase B, Ins—insulin,
“
Our results further confirm recent findings showing that LCAC interacts with in-
sulin signalling [14 16 40 41]. In this study, we documented that the lower level of Akt
–
, ,
Ser473 phosphorylation is at least partially caused by LCAC-stimulated PTP1B-dependent
dephosphorylation of InsR. In a study by Pereyra et al. using a mouse model with muscle-
−
/−
specific CPT2 knockout (Sk
in an impairment in insulin and glucose tolerance in vivo
cose uptake nor the oxidation rate was assessed in the muscles of Sk
), an increased content of LCAC in muscle did not result
[
42]. Notably, neither glu-
−
/−
mice, which
would be important for in vivo studies where multiple tissues are involved in insulin-
dependent glucose uptake. Nevertheless, increased levels of LCAC significantly reduced
Akt Ser473 phosphorylation in liver and adipose tissue, as well as muscle from HFD-treated
mice [42]. These inhibitory effects of LCAC accumulation on Akt Ser473 phosphorylation
are consistent with our present study.
Since Randle et al. proposed the competition of lipid and glucose oxidative flux [43],
extensive research has been conducted to fully comprehend all aspects of lipid and glucose
metabolite crosstalk and their regulatory functions [44]. The role of these interactions
has become particularly relevant for understanding the mechanisms underlying insulin
resistance, which precedes the onset of type 2 diabetes. An elevated LCAC content is
associated with impaired insulin sensitivity in patients [15], and this association has
been further characterized in animal models of insulin resistance [45,46]. Our present
data highlight a role for LCAC in the regulation of the molecular mechanisms of insulin
signalling and explain the aspects of insulin resistance and hyperinsulinaemia, which are
associated with type 2 diabetes. Therefore, the results of this study suggest that reducing
LCAC levels might represent a novel treatment strategy for improving insulin sensitivity.
4. Materials and Methods
4
.1. CHO INSR 1284 Cell Culture
CHO INSR 1284 cells (ATCC® CRL-3307 ) were obtained from ATCC® (Lielpin,
™
Lomianki, Poland). The cells were grown in Ham’s F-12 medium with Glutamax (cat.
1765-035, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% v/v heat-
inactivated foetal bovine serum (FBS) (cat. F7524, Merck KGaA, Darmstadt, Germany) and
3
0
.34 mg/mL hygromycin B (cat sc-29067, Santa Cruz Biotechnology, Heidelberg, Germany)
◦
at 37 C in a HERAcell VIOS 160i CO incubator (Thermo Fisher Scientific) with 5% CO .
2
2
The cell cultures were maintained by subculturing the cells every 2–3 days. Before the
experiments, 1 mL of a cell suspension was plated in TC 24-well Cell+, F plates (cat,
5
8
3.3922.300, Starstedt, Nümbrecht, Germany) at a density of 10 cells/mL and incubated
overnight. Before the experiment, the cell media were changed to DMEM low glucose

Int. J. Mol. Sci. 2021, 22, 6470
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(cat. D5523, Merck KGaA) without FBS. The compounds or the respective vehicle controls
were added and incubated with the cells. After incubation, the cell media were removed
completely, and lysis buffer was added. All cell experiments were performed in serum-
and BSA-free media; however, our data indicate that the addition of BSA to cell media does
not change the palmitoylcarnitine-induced effect on insulin signalling (data not shown).
4
.2. RIN5F Insulin Release Assay
®
®
RIN-5F cells (ATCC CRL-2058
™
) were obtained from ATCC and grown in 100
µL
of RPMI 1640 medium (cat. 61870010, Thermo Fisher Scientific) supplemented with 10%
5
v/v heat-inactivated FBS at a density of 2
×
10 cells/mL. Twenty-four hours before the
assay, the media were changed to DMEM high glucose (cat. D1152, Merck KGaA) without
FBS. Immediately before the assay, the media were changed again to 100
high glucose containing various concentrations of palmitoylcarnitine (0–10
µ
L of DMEM
µM). After 1 h,
the cell media were collected and centrifuged at 380 g to safely remove any floating cells.
◦
The supernatant was frozen at
−
80 C until further analysis. Insulin concentrations were
measured using an insulin detection kit (Cat. No. EZRMI-13K, Merck KGaA). All cell
experiments were performed in serum- and BSA-free media.
4.3. Tests for Cell Culture Contamination
All the cell lines used in this study were routinely tested for mycoplasma contam-
ination using the MycoProbe Mycoplasma Detection Kit (CUL001B, R&D Systems, Inc.,
Minneapolis, MN, USA) according to the manufacturer’s protocol. Bacterial and fungal
contaminations were assessed by visually inspecting the cell media in each well using a
light microscope before performing the assay procedures.
4.4. The Protein Concentrations
Total protein concentration in the cell lysate was measured using the Lowry method
with bovine serum albumin (BSA) (cat. EQBAH66, Europa Bioproducts Ltd., Chelsworth,
UK) as the standard.
4.5. Preparation and Analysis of Protein Extracts Using Western Blotting
Protein extracts from CHO INSR 1284 cell cultures were prepared using a previously
described method [14] with some modifications. Briefly, the cells were homogenized in
urea buffer (4 mM urea (cat, U5378, Merck KGaA), 140 mM Tris base (cat, 648310, Merck
KGaA), 1% SDS (cat. L3771, Merck KGaA), 1 mM glycerol 2-phosphate (cat. G9891,
Merck KGaA), 1 mM NaF (cat. 60-013-87, Fisher Scientific Company, Pittsburgh, PA, USA),
1
0
µM leupeptin (cat 1167, Tocris Bioscience, Abingdon, UK), 1
µM pepstatin (cat 1190,
Tocris Bioscience), 1 M aprotinin (cat. A1153 Merck KGaA), and 100
µ
µM AEBSF (cat.
A8456, Merck KGaA)) for 20 s with an ultrasonic processor (Cole-Parmer, Vernon Hills,
◦
IL, USA) at a 20% amplitude. The samples were frozen at
The cell lysates were mixed with 6
glycerol (cat. G7886, Merck KGaA), 0.93% w/v DTT (cat. 3154 Tocris Bioscience), and 0.06%
w/v bromophenol blue (cat. 15613840, Fisher Scientific Company)), and 20 g of total
protein were loaded into each well of the gel. SDS-polyacrylamide gel electrophoresis
SDS-PAGE) gels consisting of a 2% stacking gel and an 8% resolving gel were cast in
Invitrogen Bolt Empty Mini Gel Cassettes (cat. NW2010 Thermo Fisher Scientific).
Electrophoresis was performed using a Thermo Scientific electrophoresis system (Thermo
Fisher Scientific). Then, the proteins were transferred to Invitrogen iBlot 2 Transfer
−
80 C or used immediately.
Laemli buffer (12% w/v SDS, 60 mM Tris pH 6.8, 47%
×
µ
(
™
™
™
™
Stack PVDF membranes (cat. IB24001, Thermo Fisher Scientific) using the iBlot 2 Dry
Blotting System (Thermo Fisher Scientific). After transfer, the polyvinylidene fluoride
(
(
PVDF) membranes were blocked with 5% BSA dissolved in TBS buffer (150 mM NaCl
S7653, Merck KGaA) and 50 mM Tris, pH 7.6) for 1 h at room temperature and then
◦
incubated overnight at 4 C with primary antibodies. After washes with TBS, the blots
were incubated for 1 h at room temperature with secondary HRP-linked anti-rabbit IgG

Int. J. Mol. Sci. 2021, 22, 6470
12 of 16
antibodies diluted 1:50,000 (cat. #7074, Lot: 26, Cell Signaling Technology, Danvers, MA) or
HRP-linked anti-mouse IgG antibodies diluted 1:50,000 (cat. #7076, Lot: 33, Cell Signaling
Technology) and then washed again with TBS. The blots were developed using Immobilon
Western Chemiluminescent HRP Substrate (cat. WBKLS0500, Merck KGaA) in an Azure
c400 Imaging System (Azure Biosystems, Dublin, CA, USA). The Western blot images
and band intensities were quantified using AzureSpor2.0 software (Azure Biosystems).
Phosphorylated proteins of interest were detected with the following antibodies: phospho-
Akt (Ser473) (D9E) XP® rabbit mAb diluted 1:2000 (cat. #4060, Lot: 23 Cell Signaling
Technology) and insulin receptor
β (Tyr1150/1151) (19H7) rabbit mAb diluted 1:1000 (cat.
#
3024, Lot: 15, Cell Signaling Technology). The obtained data were normalized using an
Akt (pan) (C67E7) rabbit mAb diluted 1:2000 (cat. #4691, Lot: 28, Cell Signaling Technology)
or purified mouse anti-actin Ab-5 C4 antibody diluted 1:4000 (cat. 612656, Lot: 7033721,
BD Bioscience, San Jose, CA, USA).
4.6. PTP1B Activity
The determination of PTP1B activity was performed by quantitatively measuring
a tyrosine phosphopeptide (RRLIEDAEpYAARG) (cat. 12-217, Merck KGaA) and RR-
src peptide (RRLIEDAEYAARG) (cat. BML-P308-0001, Enzo Life Sciences, Inc., Lausen,
Austria) in reaction media (100 mM Hepes pH 7.5 (Cat A0302497, ACROS Organics™—
Thermo Fisher Scientific), 20 mM EDTA (cat. 254045-500 g, Merck KGaA), 0.1% BSA,
and 0.015% Brij-35 (Cat. 8019621000, Merck KGaA)) using the UPLC/MS/MS method.
A Waters Acquity UPLC chromatograph (Waters UK, Elstree, UK) was coupled to a Waters
Xevo TQ-S tandem mass spectrometer (Waters UK). Chromatographic separation was
achieved on a Waters Acquity UPLC BEH C18 column (2.1
×
50 mm, 1.7 µm) (Waters
UK) with a mobile phase consisting of a 0.1% formic acid (Cat. 84865.260, Thermo Fisher
Scientific) aqueous solution (A) and acetonitrile (B) (Cat. 34851-2.5L-R, Merck KGaA).
The mobile phase gradient programme was as follows: 0 min—5% B; 2 min—65% B;
3
0
min—98% B; 4 min—98% B; 4.2 min—5% B; and 6 min—5% B. The flow rate was
.4 mL/min, and the column temperature was 40 C. The mass spectrometer was operated
◦
in positive ionization electrospray mode. Data acquisition was performed in MRM mode
with the MS/MS transitions m/z 533.7 > 392.1 (RRLIEDAEpYAARG) and m/z 507.30 > 392.1
RRLIEDAEYAARG). The concentrations of the pTyr peptide and RR-src were measured
against a four-point calibration curve. The calibration standards were prepared by spiking
blank reaction media with the pTyr peptide and RR-src in a concentration range of 2.5—
1
00
µM. The test samples were diluted 100-fold with a mixture of 0.1% formic acid in
acetonitrile/water (1:1, v/v) and used for UPLC/MS/MS analysis.
4.7. Synthesis of Palmitoylcarnitine
Palmitoyl-L-carnitine hydrochloride (PC) was synthesized from L-carnitine and palmi-
toyl chloride using a modified version of a protocol described by Nivet et al. [47]. Palmitoyl
chloride (0.59 mL, 1.95 mmol) was added dropwise to a solution of L-carnitine (286 mg,
◦
1
.77 mmol) in trifluoroacetic acid (3 mL). The solution was stirred at 70 C in the dark under
argon for 4 h and then at room temperature for 16 h (Figure 9). The resulting mixture was
cooled to room temperature, and diethyl ether (10 mL) was added. The white precipitate
was filtered and washed with diethyl ether. The crude material was recrystallized from
isopropanol (10 mL) to yield a white solid (475 mg, 61%).
Figure 9. Synthesis of palmitoyl-L-carnitine hydrochloride (PC).

Int. J. Mol. Sci. 2021, 22, 6470
13 of 16
NMR spectra were recorded at ambient temperature with a Varian 400 OXFORD
NMR spectrometer (400 MHz). LC-MS was performed on an Acquity UPLC system
(
(
Waters UK) connected to a Micromass Quatro microTM API tandem mass spectrometer
Micromass—Waters UK) operating in ESI (electrospray ionization) positive ion mode and
using an Acquity UPLC BEH HILIC column (1.7
µ
m, 2.1
×
100 mm) (Waters UK) with a
gradient of 80–50% acetonitrile/10 mM ammonium acetate (pH 4). Elemental analysis was
performed with a Carlo Erba EA 1108 instrument (CE Instruments Ltd., Hindley Green,
UK). The melting point was measured using an OptiMelt melting point apparatus (Stanford
◦
Research Systems, Sunnyvale, CA, USA) and is reported uncorrected. Mp 157–159 C.
IR (KBr, cm-1): 3488 (O-H), 1748 (C=O). 1H–NMR (400 MHz, DMSO–d6,
δ
): 12.72 (s, 1H),
.49–5.41 (m, 1H), 3.81 (dd, J = 14.2, 8.4 Hz, 1H), 3.65 (d, J = 14.2 Hz, 1H), 3.11 (s, 9H),
.74–2.63 (m, 2H), 2.38–2.24 (m, 2H), 1.58–1.46 (m, 2H), 1.32–1.17 (m, 24H), 0.89–0.81 (m,
H). 13C–NMR (100 MHz, DMSO–d6, ): 172.1, 170.5, 67.1, 64.9, 53.0, 37.1, 33.6, 31.3, 29.04,
5
2
3
2
3
δ
9.00, 28.9, 28.7, 28.4, 24.1, 22.1, 14.0. Anal. Calcd. for C23H46ClNO4: C, 63.35; H, 10.63; N,
.21. Found: C, 63.11; H, 10.79; N, 3.12. [α]D20 -14,4 (c = 1.00, MeOH).
4
.8. Other Materials Used
NovoRapid® insulin aspart 100 units/mL (Novo Nordisk, Bagsværd, Denmark),
sotrastaurin (Item No. 16726, Cayman Chemical, Ann Arbor, MI, USA) and (R)-(+)-etomoxir
sodium salt (Cat. No. 4539, Tocris Bioscience) were diluted before use in sterile filtered PBS
(
137 mM NaCl, 2.7 mM KCl (cat. P9541, Merck KGaA), 10 mM Na HPO (cat. 12695147,
2 4
ACROS Organics™), 1.8 mM KH PO (cat. 205920025, ACROS Organics™), pH 7.4).
2 4
BVT948 (Item No. 16615, Cayman Chemical) and wortmannin (Cat. No. 1232, Tocris
Bioscience) were dissolved in dimethyl sulfoxide (DMSO) (cat. D8418, Merck KGaA).
BMOV was synthesized using the method previously described by Caravan el al. [48]
4.9. Statistical Analysis
The results are reported as the means
±
standard deviations, and the statistical analysis
of the data was performed using GraphPad Prism computer software (GraphPad, Inc.,
San Diego, CA, USA). The data distribution was determined using the Shapiro—Wilk
normality test. The statistical significance of the experimental results was verified by
one-way ANOVA followed by Dunnett’s multiple comparison test for normally distributed
data. If the data were not normally distributed, the Kruskal–Wallis test followed by Dunn’s
multiple comparison test was used. The results were considered statistically significant if
the p-value was less than 0.05.
5. Conclusions
In conclusion, palmitoylcarnitine affects the insulin signalling pathway by activat-
ing PTP1B and inducing InsR Tyr1151 dephosphorylation, which consequently results in
diminished Akt Ser473 phosphorylation. Palmitoylcarnitine also affects the downstream
steps of the insulin signalling pathway and reduces Akt Ser473 phosphorylation indepen-
dent of its effect on InsR phosphorylation. Inhibition of PP2A and PKCs does not affect
the palmitoylcarnitine-induced dephosphorylation of Akt at Ser473, indicating different
mechanisms of action.
Author Contributions: Conceptualization, E.L., M.M.-K. and M.D.; methodology, K.V.; validation,
M.K., A.G. and E.S.; formal analysis, K.V., M.K., A.G. and E.S.; investigation, K.V. and M.V.; resources,
E.L., A.G. and M.D.; writing—original draft preparation, K.V.; writing—review and editing, M.M.-K.,
M.D. and E.L.; visualization, K.V.; supervision, M.D. and E.L.; project administration, E.L. and M.D.,
funding acquisition, K.V. and E.L. All authors have read and agreed to the published version of the
manuscript.
Funding: This study was supported by a student grant from the Latvian Institute of Organic Synthesis
IG-2017-03 and the Latvian Council of Science, project TRILYSOX, grant No. LZP-2018/1-0082.
Institutional Review Board Statement: Not applicable.

Int. J. Mol. Sci. 2021, 22, 6470
14 of 16
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated and analyzed during the current study are
available from the corresponding author on reasonable request.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,
or in the decision to publish the results.
Abbreviations
Akt
protein kinase B
BMOV
bis-(maltolato)-oxovanadium (IV)
BVT948 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione
®
CHO
CPT1
DAG
InsR
CHO INSR 1284 cell line (ATCC CRL-3307™)
carnitine palmitoyltransferase 1
diacylglycerol
insulin receptor
LCAC
PC
long-chain acylcarnitine
palmitoylcarnitine
PKC
protein kinase C
PP2A
PTEN
PTP1B
protein phosphatase 2A
phosphatase and tensin homolog
protein-tyrosine phosphatase 1B
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