PID1 alters the antilipolytic action of insulin and increases lipolysis via inhibition of AKT/PKA pathway activation
PID1 alters the antilipolytic action of insulin and increases lipolysis via inhibition of AKT/ PKA pathway activation
Chunyan Yin 0 1 2
Wei hua Liu 0 1 2
Yuesheng Liu 0 1 2
Li Wang 0 1 2
Yanfeng XiaoID 0 1 2
0 The Second Affiliated Hospital of Xi'an Jiaotong University , Xi'an, Shan Xi , People's Republic of China
1 Science Foundation of China No. 81172689 to YFX and No. 81803262 to CY
2 Editor: Abdul Qadir Syed, Northwest University , UNITED STATES
Adipose tissue from HFD rats exhibited elevated PID1 expression, which showed a positive
correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that the
antilipolytic effect of insulin is mediated by AKT and that phosphorylated AKT results in the
promotion of PDE3B expression, the dephosphorylation of PKA and HSL and the suppression of
glycerol release. However, overexpression of PID1 and treatment with 1 ?M isoproterenol
and 100 nM insulin for 24 h resulted in an increased release of glycerol and a noticeable
inhibition of AKT phosphorylation, PDE3B expression and the phosphorylation of PKA/HSL in
3T3-L1 cells. In contrast, knockdown of PID1 and treatment with the above reagents
inhibited lipolysis and activated the phosphorylation of AKT, which resulted in the
dephosphorylation of PKA and HSL.
Our findings indicate that PID1 in adipose tissue increases lipolysis by altering the
antilipolytic action of insulin. This suggests that PID1 may represent a new therapeutic target to
ameliorate adipocyte lipolysis and hence improve insulin sensitivity.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Competing interests: The authors have declared
that no competing interests exist.
Obesity is an increasing global health problem that is usually accompanied by insulin
resistance (IR) and type 2 diabetes mellitus (T2DM). Elevated serum levels of free fatty acid (FFA)
are frequently observed in patients with T2DM. A wide body of evidence suggests that elevated
FFA levels are a consequence of inappropriate lipolysis, which is a major etiological factor for
IR and T2DM [
]. Thus, understanding the mechanism by which impaired insulin
suppresses fat cell lipolysis is critical for identifying the underlying defect in resistant adipose
tissues and ultimately for developing effective therapeutics.
In addition to regulating glucose metabolism, insulin plays a key role in promoting
lipogenesis and inhibiting lipolysis [
].The antilipolytic effect of insulin is believed to involve a
reduction in cyclic adenosine monophosphate (cAMP) levels and thus the activity of protein
kinase A (PKA). In this model, insulin signaling activates phosphodiesterase3b (PDE3b) via
the protein kinase B-mediated phosphorylation of Ser273 [
]. The activation of PDE3B
catalyzes the hydrolysis of cAMP, which reduces the cellular level of cAMP. The lowering of
cAMP further inhibits PKA activity and thereby results in a decrease in hormone-sensitive
lipase (HSL) and lipolysis ; however, recent results suggest that PDE3B activity in adipose
tissue is substantially reduced in obese patients [
]. Thus, decreased activity of the AKT/
PDE3B pathway may contribute to the diminished antilipolytic effect of insulin in obese
PID1 (also referred to as NYGGF4) is a novel gene that was initially isolated and
characterized in obese subjects. It is a 1527-bp cDNA containing 753 nucleotides of an ORF (open
reading frame) predicting 250 amino acids with a molecular mass of 28.27 kDa. Amino acid
sequence analysis revealed that PID1 has a phosphotyrosine-binding (PTB) domain, which
can bind to phosphorylated tyrosine residues, impair insulin signal transduction, and lead to
obesity-related IR [
]. The binding of insulin to its cell surface protein receptors causes
tyrosine phosphorylation, which results in the phosphorylation of insulin receptor substrates on
specific tyrosine residues and the activation and recruitment of PI3 kinase and its downstream
target, AKT. Given that PID1 is an important protein in AKT signaling [
] and a key player
in mediating the antilipolytic effect of insulin, we hypothesized that PID1 may influence the
AKT transduction pathway of insulin.
In this study, we examined the effects of PID1 on lipolysis in high-fat diet (HFD)-induced
obese rats and further investigated the potential molecular mechanisms that underlie these
effects in vitro using 3T3-L1 cells. We present evidence that PID1 alters the antilipolytic effect
of insulin by inhibiting the AKT/PKA pathway, which is activated by insulin and leads to
lipolysis in obese individuals.
Animal care and treatment schedule
Ninety-six male Sprague?Dawley (SD) rats (age: 3 weeks) were obtained from the Animal
Center of Xi?an Jiao tong University and individually housed in a humidity controlled room
with a 12 h light/dark cycle. All rats were fed a commercial diet for 1 week. Subsequently, the
animals were randomly allocated to one of two dietary groups at a ratio of 1:2, i.e., normal diet
[ND (n = 32), 12% kcal fat] and high-fat diet [HFD (n = 64), 60% kcal fat] groups. Eight rats
from the ND group and 16 rats from the HFD group were randomly selected, and their body
weights were measured at 8, 16, 20, and 24 weeks (w). The experimental protocols were
approved by the Animal Care and Protection Committee of Xi?an Jiao tong University.
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The rats were allowed access to food between 8 am and 10am on the day of execution. After
feeding, at 10.00 am, all rats were euthanized, and their peripheral and epididymal fat pads
were excised and weighed. Total body fat included peripheral and epididymal fat pads. Blood
samples were collected to measure the levels of insulin and glycerol by ELISA (Sigma) at 8, 16,
20 and 24 w. Enzymatic assay kits (Applygen) were used to determine serum glucose levels.
Samples of adipose tissue were collected to detect the mRNA expression of PID1 by RT-PCR.
Insulin binding to isolated fat cells
We studied insulin binding using fat cells isolated from rats in the HFD and ND groups at 24
w. Isolated fat cells from peripheral fat pads were prepared by Rodbell?s method and suspended
in buffer containing 35 mM Tris, 120 mM NaCl, 1.2 mM MgSO4, 2.5 mM KCl, 10 mM
glucose, 1 mM EDTA, and 1% bovine serum albumin at pH 7.6 and incubated with 125I-insulin
and unlabeled insulin in plastic flasks in a 24?C shaking water bath as previously described.
Optimal steady-state binding conditions were achieved at 24?C after 45 min of incubation.
The binding reaction was terminated as described by Gammeltoft and Gliemann[
removing 200-?L aliquots from the cell suspension and rapidly centrifuging the cells in plastic
microtubes in which 100 ?L dinonyl phthalate oil had been added. The supernatant was then
removed, and the cell-bound radioactivity was determined.
3T3-L1 cells were obtained from the American Tissue Culture Collection (ATCC) and
cultured in flasks (25 cm2) containing phenol red-free Dulbecco?s modified Eagle?s medium.
Differentiation was induced using protocols described elsewhere [
]. When >90% cells were
fully differentiated, 200-?L aliquots were placed into 5-mL polypropylene tubes, and 1 ?M
isoproterenol, a ?-adrenergic receptor agonist, and increasing concentrations of insulin (1?100
nmol/L) were added. Adipocytes were incubated for 1 h at 37?C in a shaking water bath (100
rpm), and the glycerol concentration in the cell medium was measured using free glycerol
reagent. To block the PKA pathway, the PKA inhibitor H-89 was added 12 h after exposure to
100 nM insulin and 1 ?M isoproterenol for 24h; subsequently, the culture medium and cells
were separated and stored.
Oil red O staining and quantification of lipid accumulation
Adipocytes were fixed for 40 min with 10% formalin, washed with PBS, stained for 2 h by
complete immersion in a working solution of Oil red O, and exhaustively rinsed with water. Excess
water was evaporated by placing the dishes at 37?C. The dye was extracted with 200 ?L of
isopropyl alcohol per well, and its absorbance was monitored spectrophotometrically at 510 nm.
Lipids were quantified using triolein (C18: 1, [cis]-9, Sigma) calibration curves as previously
Differentiated 3T3-L1 adipocytes cultured in 35-mm plates were deprived of serum for 12 h,
and the cells were treated with 1?M isoproterenol and different concentrations of insulin for
24h. Adipocytes were then washed with PBS and lysed in 0.1 mM hydrochloric acid. After
centrifuging at 2,000 ? g for 15 min at 4?C to remove insoluble materials, supernatants were
used to measure cAMP contents with a cAMP enzyme immunoassay kit (Amersham).
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An aliquot of the media (400 ?L) was collected, and glycerol release in cell culture medium
was determined using a colorimetric method (Sigma). The amount of glycerol was normalized
to protein concentration as an index of lipolysis.
3T3-L1 cells were cultured and differentiated on cover slips, fixed with 4% paraformaldehyde
for 20 min, permeabilized with 0.05% Triton X-100 in PBS (15 min), and blocked with 5%
BSA in PBST (1 h at room temperature). Staining with the PID1 antibody (1:1000; Santa Cruz,
CA, USA) was followed by incubation with AlexaFluor (488)-conjugated secondary antibodies
(Jackson), staining with 0.2 ?g/mL Nile Red (Sigma) for 5 min, and incubation with 0.1 ?g/mL
DAPI for 2 min.
Cells were lysed in ice-cold RIPA buffer. Total proteins or phosphorylated proteins were
extracted as described previously. Protein levels were quantified using the bicinchoninic acid
protein assay kit (Pierce, Rockford, IL, USA) in accordance with the manufacturer?s
instructions. After measuring protein concentration, the samples were mixed with Laemmli sample
buffer and subjected to polyacrylamide gel electrophoresis (PAGE) (10% acrylamide) and
Western blot analysis. After the electrotransfer of proteins onto a PVDF membrane
(Millipore), membranes were incubated overnight at 4?C with continual motion using specific
primary antibodies [AKT(ab108385, Abcam, USA, mouse, 1:500), p-AktSer473 (ab176657,
Abcam, USA, mouse, 1:500), PDE3B (ab42091, Abcam, USA, mouse, 1:1000), PKA (ab75996,
Abcam, USA, mouse, 1:500), p-PKAThr197 (ab75991, Abcam, USA, mouse, 1:1000), HSL
(#4107, CST, USA, mouse, 1:1000), p-HSLSer563(#4139, CST, USA, mouse, 1:1000), ATGL
(ab109251, Abcam, USA, mouse, 1:1000), and p-ATGLSer406 (ab135093, Abcam, USA, mouse,
1:1000)]. The detection of protein?antibody immune complexes was achieved using
horseradish peroxidase-conjugated secondary antibodies diluted 1:10000 in PBS with 0.05% Tween.
After the addition of the chemiluminescent substrate, films were exposed for 5 min. The bands
were detected using an enhanced chemiluminescence detection system (Amersham). ?-Actin
(Sigma) served as a control. Scanning densitometry was performed by acquisition into Adobe
Photoshop (Apple, Inc., Cupertino, CA), and analysis was performed using Quantity One
PID1 overexpression and silenced cell culture and treatment
PID1 is a 1527-bp cDNA encoding 250 amino acids with a molecular mass of 28.27 kDa. The
sequences of the two cDNA fragments (PID1siRNA, 50-AAGGTGAATAGACACATT-30;
andnegativecontrol, 50-GTTCTCCGAACGTGTCACG-3?) were subcloned into the
pGPU6/GFP/Neovector to generate an empty expression vector (pGPU6?NC?shRNA) or a
PID1-silenced vector (pGPU6- PID1-shRNA). The coding sequence of mouse PID1 was
subcloned into the HindIII and EcoRI sites of the pcDNA3.1Myc/His B vector using
oligonucleotides 5?-CCC AAG CTT ATG TTC AGC CTG CCC-3? and 5?-CGG GAA TTC CAG
CCA TCA TCG GA-3? to generate a plasmid expressing the PID1-6?His fusion protein. An
empty expression vector (pGPU6?NC?shRNA), a PID1-silenced vector (pGPU6?PID1?
shRNA), a pcDNA3.1Myc/HisB empty vector, or a PID1?pcDNA3.1Myc/His B expression
vector was stably transfected into 3T3-L1 preadipocytes using Lipofectamine 2000. The stably
transfected cells were grown in phenol red-free Dulbecco?s modified Eagle?s medium supplemented
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with 10% FBS (WISENT, CA) and 1% penicillin/streptomycin (P/S; WISENT, CA).
Preadipocytes were induced to differentiate into adipocytes using a method described previously. Fully
differentiated adipocytes were treated with 1 ?M isoproterenol and 100 nM insulin for 24 h.
Differentiated adipocytes were seeded at 1.5?105 cells in a 12-well plate and transfected with
75 nM AKT-siRNA or scrambled siRNA (Thermo Scientific). The targeting sequences were as
follows: AKT, GAGAGGACCUUCCAUGUAG and UGCCAUUCUACAACCAGGA. After 72 h,
cells were treated with 100 nM insulin and 1 ?M isoproterenol for 24 h and harvested. Equal
amounts of protein from different lysates were resolved by SDS-PAGE, and immunoblot
analyses were performed with the indicated antibodies.
The normality of the distribution of variables was assessed using the Kolmogorov?Smirnov
test. The results are expressed as the mean?standard error of the mean (SE). Between-group
differences were assessed using the t-test or one-way ANOVA with post hoc Bonferroni
correction as appropriate. Differences were considered significant at P<0.05.
Changes in body weight and adipose tissue weight
The body weight (BW) of rats in the HFD and ND groups was measured at 8, 16, 20 and 24
weeks. The initial mean BW in the 2 groups did not differ significantly. At week 8, the HFD
group had a significantly higher BW than the ND group, and the BWs remained higher
throughout the 24-week dietary period (Fig 1A). Similarly, after 8 weeks, the epididymal and
peripheral fat depot weights in the HFD group were heavier than those in the ND group (Fig
1B), which reflected increased body fat content induced by a high-fat diet.
Serum glucose, insulin, glycerol, triglyceride, cholesterol and FFA levels in
the HFD and ND groups
After 16 weeks on the diet, the triglyceride levels for the HFD groups were significantly
increased compared with the ND groups (Fig 1G). However, until 24 weeks, the cholesterol
levels in the HFD group were significantly higher than those in the ND group. At 20 weeks,
plasma glycerol and free fatty acid (FFA) levels in the HFD group were significantly higher
than those in the ND group (Fig 1D and 1F). Similarly, at 20 weeks, the insulin levels in the
HFD group were significantly higher than those in the ND group (Fig 1C). However, no
significant between-group difference was observed with respect to serum glucose level throughout
the 24-week dietary period (Fig 1E), suggesting a difference in the metabolic response to the
diet between the 2 groups. Moreover, a good correlation between insulin and glycerol levels
was observed at all time points (r = 0.57, P = 0.018). Since glycerol is an indicator of lipolysis,
we hypothesized that the antilipolytic effect of insulin was impaired in HFD rats.
Level of insulin binding to fat cells
We further measured the levels of insulin bound to receptors on fat cells in the 2 groups at 24
weeks. The results showed that adipocytes from the HFD rats bound significantly more insulin
at all insulin concentrations tested. At the lowest insulin concentration used (0.2 ng/mL),
insulin binding to adipocytes from HFD rats was 1.22?0.23% compared with 2.33?0.21% in
adipocytes from ND rats (Fig 1J).
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mRNA expression of PID1 in white adipose tissue
We also examined the mRNA expression of PID1 in the white adipose tissue (WAT) of HFD
and ND rats. At 20 weeks, the mRNA expression of PID1 in the HFD group was significantly
Fig 1. Body weight, total fat, insulin, glycerol, blood glucose, triglyceride, cholesterol and free fatty acid (FFA) levels in ninety-six Sprague-Dawley rats
fed either chow (normal diet, ND) or a high-fat diet(HFD) at 8, 16, 20, and 24 weeks (eight rats from the ND group and sixteen rats from the HFD group
were randomly selected at each time point). Insulin bound to the receptors of fat cells, and the expression of PID1 in white adipose tissue (WAT) was
determined. (a)Body weights of rats in the two groups. (b)Total fat (perirenal and epididymal fat pads) of rats in the two groups. (c)Plasma levels of insulin in
the two groups. (d) Plasma levels of glycerol in the two groups. (e) Plasma levels of glucose in the two groups. (f) Plasma levels of FFAs in the two groups. (g)
Plasma levels of triglycerides in the two groups. (h) Plasma levels of cholesterol in the two groups.(I)Relative mRNA expression of PID1 in WAT of the HFD
and ND groups; the control ratio was normalized to 1. (j)Insulin binding to perirenal adipocytes obtained from HFD and ND rats. Isolated fat cells were
incubated with mono-125I-(Tyr A14) insulin with or without various concentrations of unlabeled insulin. Specific insulin binding was determined. Data are
presented as the mean?SEM.
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higher than that in the ND group. At 24 weeks, PID1 mRNA expression in the HFD group was
further increased compared to that at 20 weeks and was significantly higher than that in the
ND group (Fig 1I). Furthermore, a positive correlation between PID1 mRNA expression and
glycerol levels was observed in the 2 groups (r = 0.38, P = 0.026), suggesting that PID1 may
play a role in lipolysis.
Effect of insulin exposure on isoproterenol-stimulated lipolysis in 3T3-L1
In contrast to the role of ?-adrenergic agonists in the activation of lipolysis, insulin is a key
inhibitor of lipolysis [
]. First, we separately tested the effects of isoproterenol and insulin on
lipolysis in differentiated 3T3-L1 cells. As expected, insulin significantly inhibited glycerol
release, while isoproterenol promoted glycerol release. In addition, PDE3B expression and
AKT phosphorylation were also increased upon exposure to insulin; however, the
phosphorylation of PKA and HSL was inhibited. On the other hand, the expression of the above
molecules was reversed upon exposure to isoproterenol (Fig 2A?2C).
Second, we examined the effect of different doses of insulin on lipolysis induced by
isoproterenol in 3T3-L1 cells. After 3T3-L1 cells were fully differentiated, the cells were treated with
different doses of insulin (1, 10, 100 nM) and 1 ?M isoproterenol for 24 h at 37?C;
subsequently, the glycerol concentration in the cell medium was measured. Consistent with the
results of previous studies [
], we found that insulin induced a concentration-dependent
decrease in glycerol release, with a significant reduction observed at 100 nM insulin (Fig 2F);
in addition, the intracellular lipids increased with increasing insulin dose. We used this
concentration in subsequent experiments (Fig 2D and 2E).
Insulin suppresses isoproterenol-stimulated lipolysis via phosphorylation
of the AKT signaling pathway
We assessed whether AKT was required for the inhibitory effect of insulin on the suppression
of isoproterenol-stimulated lipolysis and whether insulin inhibits isoproterenol-stimulated
lipolysis by affecting the level of cAMP and PDE3B expression. We found that insulin
increased AKT phosphorylation and PDE3B expression in 3T3-L1 cell lines in a
dose-dependent manner. In contrast, the level of cAMP was significantly reduced with increasing insulin
concentrations (Fig 2G?2K). Furthermore, differentiated adipocytes were transfected with
AKT siRNA and incubated in the presence of 100 nM insulin and 1 ?M isoproterenol for 24 h,
and we examined the effects of AKT knockdown on PDE3B expression, cAMP levels and
glycerol release in 3T3-L1 cells. AKT siRNA transfection led to >80% knockdown of the target
genes, decreased PDE3B expression and increased cAMP levels and glycerol release (Fig 3A?
3F). These results suggest that the antilipolytic effect of insulin on 3T3-L1 adipocytes is
mediated by AKT.
Insulin suppresses isoproterenol-stimulated lipolysis via the decreased
phosphorylation of PKA and HSL
The suppression of lipolysis by insulin involves the activation of PDE3B by AKT, which leads
to a decrease in PKA and the subsequent inactivation of HSL. Our results confirmed that the
antilipolytic effect of insulin is mediated by AKT; therefore, we further assessed whether
insulin inhibits isoproterenol-stimulated lipolysis by affecting the phosphorylation of PKA and
HSL. After the addition of different doses of insulin and 1 ?M isoproterenol for 24 h, we
analyzed the phosphorylation of HSL at its major PKA site and the phosphorylation of PKA. We
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Fig 2. Insulin inhibits isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes via the AKT/PKA/HSL pathway. We treated differentiated adipocytes with
100 nM insulin and 1 ?M isoproterenoland examined the effects on lipolysis(in triplicate). (a)Western blot analyses ofAKT, PDE3B, PKA, HSL and ATGL
protein levels in the insulin, isoproterenol and control groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL)
and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (b)The phosphorylated protein/total protein
ratios of AKT, PKA and HSL were calculated, and the control ratio was normalized to 1. (c)Western blot analyses of PDE3B protein levels in the insulin,
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isoproterenol and control groups. Differentiated 3T3-L1 adipocytes were treated with different doses of insulin (1, 10,100 nM) and 1 ?M isoproterenol for 24 h
(in triplicate). (d) Cellular triglycerides were stained with Oil red O. Bar, 50 ?m. (e) The amount of lipids was quantified by the Oil red O staining technique. (f)
The concentration of glycerol in the medium was detected in different groups(g, i)Western blot analyses of AKT, PDE3B, PKA, HSL and ATGL protein levels
in different groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL)
expression was normalized to their total protein level as a loading control. (h) The phosphorylated protein/total protein ratios were calculated, and the control
ratio was normalized to 1.(j)cAMP levels in different groups. (k) The expression of PDE3Bwas determined in different groups by Western blot.
observed a significant decrease in the phosphorylation levels of PKA and HSL upon exposure
to insulin (Fig 2G, 2I and 2H). On the other hand, AKT siRNA promoted the phosphorylation
of HSL and PKA after treatment with insulin and isoproterenol (Fig 3A?3C). Western blot
analysis showed that pretreatment with PKA inhibitors also caused a dose-dependent decrease
Fig 3. The effects of the depletion of AKT or PKA on the regulation of lipolysis by insulin. Differentiated adipocytes were transfected with AKT
siRNAortreated with the PKA inhibitor and incubated in the presence of 100 nM insulin and 1 ?M isoproterenol for 24 h (in triplicate). (a-b)Protein
expression of PDE3B, PKA, HSL and ATGL in 3T3-L1 adipocytes transfected with AKT siRNA. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA),
phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (c-d)Relative
protein expression of PDE3B and the phosphorylated protein/total protein ratios for PKA, HSL, and ATGL; the control ratio was normalized to 1. (e)
Determination of cAMP levels. (f) Glycerol released into the medium after transfection with an siRNA targeting AKT. (g)Western blot analyses of HSL and
phosphorylated HSL (p-HSL) protein expression in 3T3-L1 adipocytes treated with different doses of the PKA inhibitor. (h) The phosphorylated protein/total
protein ratio for HSL in 3T3-L1 adipocytes treated with different doses of the PKA inhibitor. (i) Glycerol released into the medium after treatment with
different doses of the PKA inhibitor. P<0.05; P<0.01.
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in HSL phosphorylation and the release of glycerol by 3T3-L1 cells (Fig 3G?3I). These data
confirm that insulin-induced AKT phosphorylation results in the dephosphorylation of PKA
and HSL and suppresses isoproterenol-stimulated glycerol release by 3T3-L1 cells.
The recently discovered adipose triglyceride lipase (ATGL) does not seem to be involved in
the catecholamine resistance of lipolysis observed in abdominal subcutaneous adipose tissue of
obese subjects [
]. Interestingly, we showed that ATGL phosphorylation (similar to HSL
phosphorylation) is suppressed in 3T3-L1 adipocytes by different doses of insulin (1, 10, 100
nM) and 1 ?M isoproterenol. At a dose of 100 nM, insulin significantly reduced ATGL
phosphorylation to 45% at 24 h. However, AKT siRNA transfection did not affect ATGL expression
or phosphorylation. These findings indicate that insulin-mediated ATGL downregulation is
independent of the AKT signaling pathway (Fig 3A and 3C).
Effects of PID1 on lipolysis and phosphorylation of the AKT/PKA/HSL
signaling pathway and ATGL molecules
We sought to determine the underlying mechanism by which PID1 affected lipolysis and
whether this mechanism was involved in the AKT/PKA/HSL signaling pathway. Preadipocytes
were transfected with PID1 plasmids and allowed to differentiate; the differentiated 3T3-L1
adipocytes were treated with 1 ?M isoproterenol and 100 nM insulin for 24 h. The expression
of PID1 was verified by immunofluorescence and RT-PCR (Fig 4A?4C). We found that
glycerol release in cells overexpressing PID1 was approximately 2 fold higher than that in control
cells (Fig 4D). Additionally, PID1 overexpression resulted in a noticeable inhibition of AKT
phosphorylation and PDE3B expression in the presence of insulin (Fig 4E?4H). We also
evaluated the phosphorylation of PKA and HSL, downstream signaling molecules of AKT in the
insulin antilipolytic signaling pathway. We found that PKA and HSL phosphorylation levels
were significantly increased in cells overexpressing PID1 (Fig 4E?4H). We also evaluated the
phosphorylation of ATGL and found no significant change in the phosphorylation of ATGL
(Fig 4E?4H). We further explored whether PID1 knockdown could reverse the antilipolytic
effect of insulin. As shown in Fig 4I?4N, PID1 knockdown increased AKT phosphorylation
and PDE3B expression and resulted in a noticeable inhibition of cAMP levels and the
phosphorylation of PKA and HSL, which in turn led to the inhibition of isoproterenol-stimulated
glycerol release in 3T3-L1 cells. However, PID1 knockout did not affect the phosphorylation of
ATGL. This result indicates that PID1 promotes lipolysis in the presence of insulin, which
involves the inhibition of AKT phosphorylation and the phosphorylation of PKA and HSL.
Our animal experiments showed that the mRNA levels of PID1 in adipose tissue from HFD
rats were higher than those in adipose tissue from ND rats at 20 weeks and showed a positive
correlation with insulin levels and lipolysis in blood. Mechanistically, in 3T3-L1 adipocytes, we
found that the antilipolytic effect of insulin is mediated by AKT and that AKT phosphorylation
by insulin can result in the inhibition of PDE3B expression and an elevation of cAMP levels,
which in turn leads to the dephosphorylation of PKA and HSL and the suppression of glycerol
release. In addition, it is possible that, as has been demonstrated for HSL, some degree of
ATGL phosphorylation occurs to coordinately regulate triglyceride hydrolysis. Isoproterenol
stimulation results in ATGL phosphorylation, but in contrast to HSL, this modification are
apparently not mediated by AKT. However, the overexpression of PID1 and treatment with
1 ?M isoproterenol and 100 nM insulin for 24h resulted in an increased release of glycerol and
a noticeable inhibition of AKT phosphorylation, PDE3B expression and the phosphorylation
of PKA/HSL in 3T3-L1 cells but did not affect the phosphorylation of ATGL.
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Fig 4. Effects of PID1 expression on lipolysis and the phosphorylation of AKT/PDE3B/PKA/HSL signaling molecules and
ATGL. Preadipocytes were subjected to PID1knockout or upregulation and allowed to differentiate into 3T3-L1 adipocytes; these
cells were treated with 1 ?M isoproterenol and 100 nM insulin for 24 h(in triplicate). (a-b)Immunofluorescence analysis was
performed to assess the expression of the PID1 gene in empty vector cells, PID1-overexpressing cells, and control cells.(c)RT-PCR
analyses of the mRNA expression of PID1 in empty vector cells, PID1-overexpressing cells, and control cells. (d)Glycerol released
into the medium after the upregulation of PID1.(e-f)Protein expression of AKT, PDE3B, PKA, HSL and ATGL in empty vector
cells, PID1-overexpressing cells, and control cells. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated
HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control.(g)
The phosphorylated protein/total protein ratios for AKT, PKA, HSL, and ATGL in 3T3-L1 adipocytes after transfection with the
PID1 overexpression plasmid. (h)The expression of PDE3B was determined by Western blot after the PID1 overexpression plasmid.
(i-j)Protein expression of AKT, PDE3B, PKA, HSL and ATGL after transfection with PID1 shRNA. Phosphorylated AKT (p-AKT),
phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to
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their total protein level as a loading control. (k)RT-PCR analyses of mRNA after transfection with PID1 shRNA. (l) Glycerol was
released into the medium after knockdown of PID1. (m)Phosphorylated protein/total proteinratios for AKT, PKA, HSL, and ATGL
after PID1 knockdown. (n)The expression of PDE3B was determined by Western blot after transfection with PID1 shRNA.
High-fat diets have long been used to study fat storage, insulin sensitivity, and glucose
tolerance and metabolism in experimental animals. In general, diets containing 60% fat and 20%
carbohydrate for 4?8 weeks resulted in an approximately 40?50% increase in body weight, fat
stores and insulin level compared to a normal diet. However, there was no significant
difference with respect to the plasma levels of glucose or FFAs [
]. In these models, we believe
that FFA levels were not elevated because the duration of high-fat feeding was not long
enough. We extended the duration of the high-fat diet and found that a long-term high-fat
diet led to the accumulation of visceral fat and an increase in plasma insulin and FFA levels.
The levels of circulating FFAs depend primarily on the rates of lipolysis in adipose tissue.
One of the key physiological functions of insulin as the major anabolic hormone in the body is
to restrain lipolysis and promote fat storage in adipose tissue in the postprandial state [
Insulin, the most important physiological inhibitor of catecholamine-induced lipolysis, was
shown to induce the phosphorylation and activation of phosphodiesterase type 3B (PDE3B),
which led to a decrease in PKA and HSL activity [
]. In our in vitro experiments, we found
that insulin inhibited isoproterenol-induced lipolysis in a dose-dependent manner, in parallel
with the decreased release of glycerol, promotion of AKT phosphorylation and PDE3B
expression, and dephosphorylation of PKA, HSL and ATGL. Pretreatment with PKA inhibitors also
caused a dose-dependent decrease in HSL expression and glycerol release in 3T3-L1 cells.
These results indicate that insulin inhibits lipolysis not only by dephosphorylating HSL but
also by reducing ATGL phosphorylation. Our findings are consistent with those of previous
studies. However, whether the insulin-induced inhibition of lipolysis depends on AKT is not
clear. Findings from experimental studies suggest that the insulin-induced inhibition of
lipolysis involves the phosphorylation of AKT, which in turn leads to the phosphorylation of
PDE3B, thus stimulating lipolysis [
]. Other studies suggest that insulin antagonizes
triglyceride hydrolysis via a mechanism that is independent of AKT . We showed that AKT
depletion inhibited PDE3B expression, activated PKA and HSL phosphorylation, and
ameliorated the inhibitory effect of insulin on lipolysis, but AKT depletion had no effect on ATGL
phosphorylation. These data confirm that AKT is essential for the antilipolytic effect of insulin
and that insulin modulates AKT activity via phosphorylation, but these results also show that
the phosphorylation of ATGL is not required for AKT.
A wide body of evidence has implicated a defect in antilipolysis as the critical etiological
abnormality that initiates the positive amplifying circuit that characterizes insulin resistance
]. However, the molecular mechanism of the impaired control of lipolysis in obesity has
yet to be elucidated.
Many studies have indicated that PID1 may play an important role in the development of
obesity-related IR [
]. Zhao et al found that PID1 can impair insulin signal transduction
. In adipocytes and muscle cells, PID1 also inhibits the insulin-mediated phosphorylation
of insulin receptor substrate-1(IRS-1) and the insulin-mediated translocation of the GLUT-4
glucose transporter, which results in decreased glucose uptake [
]. PID1?/? mice exhibited
improved glucose tolerance and insulin sensitivity under a chow diet, with increased AKT
phosphorylation in WAT .Therefore, we hypothesized that PID1 may impair the
phosphorylation of insulin signaling molecules that inhibit lipolysis. Many studies have
demonstrated increased expression of PID1 in adipose tissues in obesity [
]. In the present study, we
also found increased mRNA expression of PID1 in adipose tissue from HFD rats. Moreover,
12 / 15
Fig 5. A hypothetical model showing how PID1 promotes lipolysis by regulating the AKT/PDE3B/PKA/HSL signaling pathway, which is supported by
the results of this study. (a)Insulin inhibits isoproterenol-induced lipolysis by increasing PDE3B expression via AKT. The elevation of PDE3B catalyzes the
hydrolysis of cAMP, which reduces the cellular level of cAMP. The lowering of cAMP further dephosphorylates PKA and thereby results in a decrease in
hormone-sensitive lipase (HSL) and lipolysis. (b)PID1 promotes lipolysis and induces a noticeable inhibition of the phosphorylationof AKT and PDE3B
expression, which further increases cAMP levels and the phosphorylation of PKA and HSL, leading to increased lipolysis.
our results show that PID1 promoted lipolysis and induced a noticeable inhibition of AKT
phosphorylation and PDE3B expression and the phosphorylation of PKA/HSL in 3T3-L1 cells
but did not affect the phosphorylation of ATGL.
Since this study merely tested lipolysis in the presence of insulin, further studies on the
expression of PID1 and the phosphorylation of AKT in 3T3-L1 cells without insulin treatment
are needed to determine whether PID1 expression and AKT phosphorylation are dependent
on insulin. In addition, this study did not conclusively show that the inhibition of AKT and
induction of the phosphorylation of PKA and HSL are the mechanisms by which PID1
promotes lipolysis. Proof of this mechanism awaits the generation and analysis of the appropriate
loss- and gain-of-function mediators downstream of PID1using in vivo models.
In conclusion, our results demonstrate that PID1 promotes lipolysis in the presence of insulin,
which is mediated via the inhibition of AKT phosphorylation and PDE3B expression and the
phosphorylation of PKA and HSL (Fig 5). These findings provide new insights into the
mechanisms of lipolysis in obesity. These findings may lead to new therapeutic avenues to ameliorate
adipocyte lipolysis and improve insulin sensitivity.
Conceptualization: Yanfeng Xiao.
Data curation: Wei hua Liu.
Formal analysis: Wei hua Liu.
13 / 15
Funding acquisition: Chunyan Yin.
Investigation: Chunyan Yin.
Methodology: Yuesheng Liu.
Project administration: Yuesheng Liu, Yanfeng Xiao.
Resources: Li Wang.
Validation: Li Wang.
Writing ? original draft: Chunyan Yin.
Writing ? review & editing: Yanfeng Xiao.
14 / 15
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