Glucotoxicity and α cell dysfunction: involvement of the PI3K/Akt pathway in glucose-induced insulin resistance in rat islets and clonal αTC1-6 cells.

Abstract

The objective of this study was to assess how long-term exposure to high glucose affects the α cell function and whether the increased glucagon secretion is mediated via insulin resistance.
We established a β cell-depleted rat model to obtain pure primary α cells. Furthermore, isolated rat islets and TC1-6 cells (a clonal α cell line) were exposed to high glucose (25 or 30 mmol/L) and low glucose (5.5 mmol/L) for up to 5 days to evaluate the influence of chronic glucose toxicity on glucagon secretion and glucagon gene expression. Moreover, we added insulin and/or Wortmannin to examine if the inhibitory effect of insulin on glucagon secretion was impaired by high glucose via the phosphatidylinositol 3 kinase/PKB protein kinase B pathway.
Both glucagon secretion and glucagon gene expression were increased in response to 5 days exposure to high glucose. While a moderate insulin concentration slightly inhibits glucagon secretion from rat islets and α TC1-6 cells at high glucose, a pronounced increase in glucagon secretion was observed at low glucose. We found that the insulin-mediated activity of the phosphatidylinositol 3 kinase/PKB protein kinase B pathway in the α cell was markedly impaired by chronic exposure to high glucose.
The hypersecretion of glucagon induced by glucotoxicity may be secondary to insulin resistance of the α cell induced by impaired activity of the insulin signaling pathway.

Full text

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EndocrineResearch,37:12–24,2012
Copyright C ?InformaHealthcareUSA,Inc.
ISSN:0743-5800print/1532-4206online
DOI:10.3109/07435800.2011.610855
Glucotoxicity and α Cell Dysfunction: Involvement
of the PI3K/Akt Pathway in Glucose-Induced Insulin
Resistance in Rat Islets and Clonal αTC1-6 Cells
Xiao-xia Shen1, Hong-liang Li2, Lin Pan2, Jing Hong2, Juan Xiao2,
Kjeld Hermansen3, Per Bendix Jeppesen3, and Guang-Wei Li1
1EndocrinologyandCardiacDiseaseClinicalCenter,FuwaiHospital,PekingUnionMedical
CollegeandChineseAcademyofMedicalSciences,Beijing,PRChina,2Endocrinologyand
MetabolicDiseaseClinicalCenter,China-JapanFriendshipHospital,Beijing,PRChina,
3DepartmentofEndocrinologyandMetabolismC,AarhusUniversityHospital,AarhusC,
Denmark
Aim/hypothesis.Theobjectiveofthisstudywastoassesshowlong-termexposuretohighglucose
affects the α cell function and whether the increased glucagon secretion is mediated via insulin
resistance. Materialsandmethods. We established a β cell-depleted rat model to obtain pure pri-
mary α cells. Furthermore, isolated rat islets and TC1-6 cells (a clonal α cell line) were exposed
to high glucose (25 or 30 mmol/L) and low glucose (5.5 mmol/L) for up to 5 days to evaluate
the influence of chronic glucose toxicity on glucagon secretion and glucagon gene expression.
Moreover, we added insulin and/or Wortmannin to examine if the inhibitory effect of insulin on
glucagon secretion was impaired by high glucose via the phosphatidylinositol 3 kinase/PKB pro-
tein kinase B pathway. Results. Both glucagon secretion and glucagon gene expression were
increased in response to 5 days exposure to high glucose. While a moderate insulin concen-
tration slightly inhibits glucagon secretion from rat islets and α TC1-6 cells at high glucose, a
pronounced increase in glucagon secretion was observed at low glucose. We found that the
insulin-mediated activity of the phosphatidylinositol 3 kinase/PKB protein kinase B pathway in
the α cell was markedly impaired by chronic exposure to high glucose. Conclusion. The hyper-
secretion of glucagon induced by glucotoxicity may be secondary to insulin resistance of the α
cell induced by impaired activity of the insulin signaling pathway.
Keywords High glucose, α Cell, Insulin resistance, PI3K/Akt pathway
INTRODUCTION
Type 2 diabetes is a heterogeneous metabolic disorder characterized by hyper-
glycemia resulting from defects in both insulin action and insulin resistance
(1). Thirty years ago, Unger and Orci (2) proposed the bihormonal-abnormality
hypothesis, which highlighted that both deficient insulin secretion and excessive
glucagon secretion contribute to the hyperglycemic state. Currently, the existence of
X. Shen and H. Li contributed equally to this work.
Address correspondence to X. Shen, Endocrinology and Cardiac Disease Clinical Center, Fuwai
Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing
100037, PR China. E-mail: shenxx09@yahoo.cn
12
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GlucotoxicityandαCellDysfunction 13
αcelldysfunctionindiabetesisgenerallyaccepted,becausesustainedhyperglycemia
can also induce abnormalities in α cell function such as relative or absolute fasting
hyperglucagonemia,hyper-responsivenesstoanaminoacidchallenge,failuretosup-
press glucagon release in response to hyperglycemia, and inadequate responses to
hypoglycemia (3). In both type 1 and type 2 diabetes, the α cell function becomes
abnormal, and glucagon secretion is no longer suppressed by hyperglycemia (4).Two
possible explanations for this α cell dysfunction in diabetes mellitus have been pro-
posed. One suggests that the α cell abnormality is entirely secondary to insulin
deficiency (5,6) while the other indicates that diabetes mellitus is a bihormonal dis-
ease with a primary defect in α cells (7,8). Diabetic hyperglucagonemia is often
found in combination with normal or elevated insulin levels (9,10). Raskin et al. (11)
reported that a 10-fold greater dose of insulin is needed to modify α cell secretion
in subjects with diabetes compared with subjects without diabetes. Consequently,
the inability of insulin to suppress glucagon secretion in diabetes may be caused
by insulin resistance of α cells. Tsuchiyama et al. (12) also found that insulin resis-
tance in α cells occurs concomitantly with increasing systemic insulin resistance in
patients with type 2 diabetes. However, the mechanism of insulin resistance in α cells
is unclear and remains to be explored.
In this study, we elucidated the effect of high glucose on glucagon secretion and
synthesis in α cells and explored whether exogenous insulin is able to rescue the
abnormal α cell function. Furthermore, we investigated the activity of the phos-
phatidylinositol 3 kinase/PKB protein kinase B (PI3K/Akt) pathway to clarify the
possible mechanisms involved in the glucotoxicity-induced insulin resistance in α
cells.
MATERIALS AND METHODS
ReagentsandAntibodies
Male Sprague–Dawley rats were purchased from the Animal Center of Vital River
Company (BJ, CHN) and the TC1-6 cell were a kind gift from Jie Wang (University of
Chicago, Chicago, IL, USA). Collagenase P was purchased from Roche Products Lim-
ited(WelwynGardenCity,Herts,UK),primarymonoclonalrabbitantihumaninsulin
and polyclonal rabbit antihuman glucagon antibodies were from Santa Cruz (CA,
USA) and the bicinchoninic acid (BCA) kit was from Bio-Rad (Hercules, CA, USA).
Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s Modified Eagle
Media (DMEM) medium, fetal bovine serum (FBS), TRIzol and DNAzol reagents and
nitrocellulose membranes were obtained from Invitrogen (Grand Island, NY, USA).
The glucagon Radio Immunoassay (RIA) kit was from Linco (St. Charles, MO, USA).
TheRT-PCRkitand SYBPGreen PCRmaster mix were fromTOYOBO(Osaka,Japan).
The PI3-kinase ELISA kit was from Echelon Biosciences (UT, USA) and antibodies
against Akt and phosphorylated Akt were purchased from Cell Signaling Technology
(Beverly, MA, USA). The Electro Chemi Luminescence (ECL) chemiluminescence kit
was from Amersham Pharmacia Biotech (UK). Wortmannin (W1628) was purchased
from Sigma Chemicals (St. Louis, MO, USA).
EstablishmentoftheβCell-DepletedRatModelandIsolationofRatIslets
Sprague–Dawley (SD) rats received a single intraperitoneal injection of 100 mg/kg
streptozotocin and we administered the diabetic rats with Neutral Protamine Hage-
dorn (NPH) insulin to maintain their glucose levels at 5–10 mmol/L. Five days later,
theratswerekilled.Weexcised100mgoftissuefromtheheadofpancreasandmixed
it with hydrochloric acid alcohol to form a pancreatic tissue homogenate, which
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14X.Shenetal.
was centrifuged at 4◦C and the insulin and glucagon content in the supernatant was
determined by radioimmunoassay kit.
Islets were isolated by collagenase digestion from the rat pancreas as previously
described (13,14). Briefly, the bile duct of the β cell-depleted SD rats was punctured
and the pancreas was infused with 8–10 mL of 1 mg/mL collagenase type P. After
it was surgically excised, the pancreas was incubated in the collagenase solution at
38◦C for 10 min. The islets were collected by handpicking to remove any remaining
exocrine tissue.
ImmunohistochemistryandImmunofluorescenceofthePancreas
The tail of the pancreas was fixed by Bolins liquid overnight at 4◦C, embed-
ded in paraffin wax, and dewaxed in graded alcohols as described previously
(15). The expression of insulin and glucagon in the pancreas was determined by
immunofluorescence.
CultureofRatIsletsandαTC1-6Cells
After handpicking, the rat islets were seeded in six-well plates at 50 islets per well for
the determination of glucagon secretion and gene expression, or at 200 islets per well
for PI3K activity, or at 100 islets per culture dish for western blotting. Islets were cul-
turedinRPMI1640mediumcontaining11.1mmol/Lglucoseandsupplementedwith
10%FBS,pH7.4,at37◦Cinahumidifiedatmosphere(5%CO2,95%air)for24hbefore
use in the following experiments.
TC1-6 cells were cultured in DMEM medium containing 25 mmol/L glucose
supplemented with 10% FBS. Cells were passaged weekly after trypsinization and
mediumwasreplacedevery48h.Cellswerethenseededinsix-wellplatesatadensity
of 2×105cells/well and used in the following experiments.
GlucagonSecretionofRatIsletsandαTC1-6Cells
Ratisletswereculturedfor1,3,or5daysat37◦CinRPMI1640mediumcontaining5.5,
11.1,or25mmol/LglucoseandαTC1-6cellswereculturedfor1,3,or5daysinDMEM
containing 5.5, 11.1, 25, or 30 mmol/L glucose. The next day, cells were washed twice
with warm KRB and preincubated for 30 min in Krebs-Ringer phosphate (KRB) (pH
7.4) and subsequently incubated for 2 h with KRB containing 2.8 mmol/L glucose
and 0.5% BSA (1 mL/well) (16). Aliquots from each treatment group were removed
and stored at −20◦C until assayed for glucagon by RIA. After the secretion study, the
intracellular protein concentration was measured as previously described (16) and
the glucagon levels were adjusted to the protein concentration.
To determine the effect of insulin on glucagon secretion, rat islets were cultured
with 5.5 or 25 mmol/L glucose and αTC1-6 cells were cultured with 5.5 or 30 mmol/L
glucose for 5 days. The next day, the media were replaced with KRB containing 0.5%
BSA, as described above, and incubated for 30 min. The islets were then stimulated
with1,10,or100nmol/Linsulinfor2handcellsfor4h.Afterinsulinstimulation,the
culture media were collected and frozen until required for the glucagon assay.
GeneExpressionofRatIsletsandαTC1-6Cells
Rat islets and αTC1-6 cells were cultured using the same incubation protocol as
described above. Total cellular RNA was extracted using Trizol reagent. RNA (2 μg)
was reverse transcribed with RT-PCR kits using random primers in a total reac-
tion volume of 20 μL. The real-time PCR amplification was performed in a volume
of 20 μL containing 2.5 mmol/L MgCl2, 0.5 mmol/L of forward/reverse primers, 2
μL SYBP Green PCR master mix, and 2 μL of cDNA. PCR was performed for 40
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GlucotoxicityandαCellDysfunction 15
cycles at 95◦C for 15 s, 60◦C for 15 s, and 72◦C for 45 s. The relative quantifica-
tion of gene expression was analyzed by the 2−??Ctmethod (17) and was presented
as the fold change versus the control. The primer sequences for the genes and
expectedproductsizeswereasfollows:5?-GGAATTCATTGCTTGGCTGGT-3?(sense)
and 5?-CGTGTTCATCTCATCAGAG AA-3?(antisense) for glucagon (121 bp), and
5?-TGCGTGTGAACCACGAGAA-3?(sense) and 5?-GGCATG GACTCTGG TCATGA-
3?(antisense) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (145 bp).
Beforeuse,theoptimalannealingtemperaturesandpredictedsizeofthePCRproduct
for glucagon and GAPDH gene were verified by gradient tests and electrophoresis.
To determine whether insulin affects glucagon mRNA expression in rat islets and
αTC1-6cells,theratisletsandTC1-6cellsweregrownasdescribedabove.After5days,
the culture medium was replaced with KRB for 30 min and followed by stimulation
with or without (100 nmol/L) insulin for 6 or 12 h as to rat islets and αTC1-6 cells.
After insulin stimulation, the glucagon gene expression was determined as above.
PI3KActivityAssay
Rat islets were cultured with 5.5 or 25mmol/l glucose and αTC1-6 cells were cultured
with 5.5 or 30mmol/l glucose for 5 days. After 5 days, the media were replaced with
KRB for 30 min and the cells were stimulated with or without 100 nmol/L insulin for
30min,fortheindicatedgroups,andWortmanninwasadded30minbeforethestim-
ulation of insulin. The cells were then lysed for 20 min with 1 mL of ice-cold lysis
bufferPhenylmethanesulfonylfluoride(PMSF).Theproteincontentofthelysateswas
quantified, and equal amount of proteins was used for immunoprecipitation assays,
which were performed as previously described (18), using the anti-PI3K Ab. The
immunoprecipitatedenzymesweresubjectedtoakinaseassaybycompetitiveELISA,
inaccordancewiththemanufacturer’sinstructions(19).Thedataforthekinaseactiv-
ity are expressed as the fold induction for treated cells compared with the activity in
untreated cells.
WesternBlottingAnalysisofPhospho-Akt473
Rat islets and αTC1-6 were cultured as described above for 5 days. Then they were
scrapedandlysedinradioimmunoprecipitationassaybuffersupplementedwithpro-
teaseinhibitors(1mmol/LPMSF)andphosphataseinhibitors(phosphataseinhibitor
mixture I). The total cellular protein level was quantified by Coomassie blue G stain-
ing, subjected to SDS-PAGE (10%), and transferred to nitrocellulose membranes. The
membranes were blocked for 2 h at room temperature in 5% nonfat dried milk. The
membranes were the incubated overnight at 4◦C with antibodies against phospho-
Akt 473 or Akt (both: 1:1,000). The bound antibody was visualized using anti-rabbit
secondary antibody (1:5000) and the bands were developed onto X-ray film using the
ECL chemiluminescence method.
StatisticalAnalysis
Each experiment was repeated four times. All data are expressed as mean ± SD, Sta-
tistical significance of differences among groups was evaluated by one-way ANOVA.
p < 0.05 was considered as significant.
RESULTS
EvaluationoftheβCell-DepletedRatModel
Immunohistochemistryshowedthatthetotalpancreasislandareasofβcell-depleted
rats were approximately one-seventh of those of normal control rats. β cell areas
accounted for 6% of the total pancreas island areas of β cell-depleted rats, while it
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16X.Shenetal.
TABLE 1 The contents of Insulin and Glucagon in Pancreas Tissue Homogenate, The Pancrease
Island Areas and the Percentages of β Cell Areas and α Cells in NC and DB Groups
Group Insulin content
(mU/L)
Glucagon
content ( ng/L)
Pancreas island
areas (μ m2)
Percentages of β
cell areas (%)
Percentages of α
cell areas (%)
NC
DB
198.32 ± 21.42
5.74 ± 0.82∗
291 ± 48
312 ± 25
42863 ± 3462
6093 ± 547∗
75.6 ± 5.3
6.1 ± 0.8∗
17.2 ± 2.6
78.4 ± 4.8∗
Abbreviations: NC, control group; DB, deleting β cell group.
Mean ± SEM of eight separate experiments.
∗P < 0.01 compared with NC group.
accountedfor76%inthenormalcontrolrats.Incontrast,theαcellareaaccountedfor
78% of the total pancreas island areas, while it only accounted for 17% in the normal
control rats. Furthermore, the insulin content in the pancreatic tissue homogenate of
the β cell-depleted rats was less than 3% of that of the normal rats, while the glucagon
contentincreasedasweexpected(Table1andFigure1).Immunofluorescenceassays
revealedthepresenceofαcellsintheperipheryofthepancreasislandsofthenormal
control rats. In contrast, in the β cell-depleted rats, the α cells had moved from the
periphery to the center of the pancreas islands (Figure 1).
EffectsofHighGlucoseonGlucagonSecretionfromRatIsletsandαTC1-6Cells
Therewasnosignificantdifferenceinglucagonsecretioninresponsetothethreeglu-
coselevelsintheratisletsorinTC1-6cellsafter1day,Atday3,theglucagonsecretion
inratisletsexposedto25mmol/Lglucosewasmarkedlyincreasedby42%(2252.64±
41.71 vs. 3205.97 ± 45.53 pg/mg protein; p < 0.05) compared with the control group
(5.5 mmol/L), but there was no difference in αTC1-6 cells. At day 5, the glucagon
secretioninratisletswasincreasedby52%inresponseto11mmol/Lglucose(2452.22
±67.89vs.3735.44±54.89pg/mgprotein;p<0.05)andby1.17-foldinresponseto25
mmol/Lglucose(p<0.05)comparedwiththecontrolgroup.However,withrespectto
αTC1-6 cells, glucagon secretion was remarkably increased only at day 5 in response
to25mmol/Lglucosecomparedwith5mmol/Lglucose.Glucagonsecretionwas32%
greaterinαTC1-6cellsexposedto30mmol/Lglucosecomparedwithcellsexposedto
25 mmol/L glucose (p < 0.05; Figure 2A and 2C).
EffectsofHighGlucoseonGlucagonGeneExpressioninRatIsletsandαTC1-6Cells
TheeffectsofhighglucoseonglucagonmRNAlevelsinratisletsandTC1-6cellswere
investigated. Acute (1 day) exposure of αTC1-6 cells and rat islets to high glucose had
no effect on glucagon mRNA levels measured by RT-PCR relative to cells incubated
with 5.5 mmol/L glucose (control group). However, at day 3, the glucagon mRNA lev-
els of rat islets incubated with 25 mmol/L glucose increased by 125% compared with
thecontrolgroup(p<0.05),whereastherewaslittlechangeinglucagonmRNAlevels
inαTC1-6cells.Meanwhile,atday5,theglucagonmRNAlevelsofratisletsandαTC1-
6 cells exposed to 25 mmol/L glucose increased by 272% and 164%, respectively,
relative to the control groups (both: p < 0.05). Furthermore, the glucagon mRNA lev-
elsofTC1-6cellsincubatedwith30mmol/Lglucoseincreasedby43%comparedwith
cells incubated with 25 mmol/L glucose (p < 0.05) (Figure 2B and 2D).
EffectofInsulinonGlucagonSecretionandGeneExpressioninRatIslets
andαTC1-6Cells
We found that 1 nmol/L insulin was unable to decrease glucagon secretion from rat
islets and TC1-6 cells cultured with 5.5 mmol/L glucose or 25/30 mmol/L glucose (25
mmol/L for rat islets and 30 mmol/L for αTC1-6 cells), whereas 10 nmol/L insulin
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GlucotoxicityandαCellDysfunction17
FIGURE 1 Immunohistochemistry of islets with insulin and glucagon antibodies (×40/×20). (A)
The α cell-positive areas in the pancreas from normal rats were distributed in the periphery of the
pancreatic islets. (B) The β cell-positive area in the pancreas from normal rats comprised about
80% of the whole islet. (C) The α cell-positive areas in the pancreas from β cell-depleted rats were
irregularly distributed in the pancreatic islets and expression of glucagon antibodies was stronger
than in the pancreas from normal rats. (D) The β cell-positive areas in the pancreas from β cell-
depletedratshadmarkedlydecreased.Immunofluorescencestainingfortheinsulinantibody(red)
and the glucagon antibody (green) (×40). Expression of glucagon (E) and insulin (F) in the pan-
creas from normal rats. Glucagon expression in α cells in the pancreas from β cell-depleted rats
was greater than that in normal rats (G) and the insulin expression in β cells from β cell-depleted
rats was markedly less than that in normal rats (H).
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18X.Shenetal.
5.5 mM
11.1 mM
25 mM
30 mM
*
*,***
TC 1-6 cell glucagon
secretion (pg/mg protein) 3000
2000
1000
0
Glucose (mM) 5.5 11.1 25
Culture period (d)
1
5.5 11.1 25
3
5.5 11.1 25
5
303030
5.5 mM
11.1 mM
25 mM
30 mM
*
*,***
TC 1-6 cell glucagon
mRNA expression (%)
500
400
300
200
100
0
Glucose (mM)
Culture period (d)
5.5 11.1 25
1
5.5 11.1 25
3
5.5 11.1 25 303030
5
(C) (D)
5.5 mM
11.1 mM
25 mM
*
*
*,**
Islets glucagon
secretion (pg/mg protein) 6000
(A)(B)
5000
4000
3000
2000
1000
0
Glucose (mM)
Culture period (d)
5.5 11.1 25
1
5.5 11.1 25
3
5.5 11.1 25
5
400
300
200
100
0
5.5 mM
11.1 mM
25 mM
*
*
*,**
Islets glucagon
mRNA expression (%)
Glucose (mM)
Culture period (d)
5.5 11.1 25
1
5.5 11.1 25
3
5.5 11.1 25
5
FIGURE 2 Time- and dose-dependency of glucose-induced glucagon secretion and gene expres-
sion in rat islets and TC1-6 cells. Rat islets (A, B) and TC1-6 cells (C, D) were exposed to glucose
for 1, 3, or 5 days and glucagon secretion and gene expression were measured (n = 4). All values
are expressed as mean ± SD.∗p < 0.05 versus 5.5 mM glucose (control group) for 1 day;∗∗p < 0.05
versus rat islets exposed to 11.1 mM glucose for 5 days;∗∗∗p < 0.05 versus TC1-6 cells exposed to 25
mM glucose for 5 days.
significantly inhibited glucagon secretion from rat islets and TC1-6 cells cultured
in 5.5 mmol/L glucose by 61% (2315.02 ± 48.12 vs. 915.99 ± 32.54 pg/mg protein;
p < 0.05) and by 62% (1381.04 ± 40.13 vs. 527.74 ± 22.32 pg/mg protein; p < 0.05),
respectively, but did not decrease glucagon secretion from rat islets or TC1-6 cells
incubated in 25/30 mmol/L glucose (p > 0.05). When the insulin concentration was
increased to 100 nmol/L, the glucagon secretion of rat islets cultured with 5.5 and 25
mmol/L glucose was decreased by 62% (2543.17 ± 66.96 vs. 975.04 ± 42.48 pg/mg
protein; p < 0.05) and 66% (3872.34 ± 29.66 vs. 1307.65 ± 23.34 pg/mg protein; p <
0.05), respectively. Similarly, the glucagon release from αTC1-6 cells incubated with
5.5 or 30 mmol/L glucose was decreased by 75% (1539.05 ± 27.27 vs. 383.38 ± 41.38
pg/mg protein; p < 0.05) and 59% (3444.7 ± 18.97 vs. 1421.21 ± 48.56 pg/mg protein;
p < 0.05), respectively (Figure 3A and 3C). Meanwhile, 100 nmol/L insulin inhibited
glucagon mRNA expression in rat islets and in αTC1-6 cells irrespective of the glu-
coseconcentration. Theglucagon mRNAexpression wassuppressedby63% and42%
(both: p < 0.05) in rat islets exposed to 5.5 or 25 mmol/L glucose, respectively, and by
66% and 65% (both: p < 0.05) in αTC1-6 cells exposed to 5.5 or 30 mmol/L glucose,
respectively (Figure 3B and 3D).
EffectofHighGlucoseonPI3KActivityinRatIsletsandαTC1-6Cells
When exposed to 5.5 mmol/L glucose for 5 days, PI3K activity in rat islets and TC1-
6 cells increased by 5.2- and 3.8-fold, respectively, while those incubated with 25/30
mmol/Lglucoseonlyincreasedby3.4-and2.1-fold,respectively.Evidentlytheability
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GlucotoxicityandαCellDysfunction19
**
*
TC 1-6 cell glucagon
m RNA expression (%)
(D)
500
400
300
200
100
0
–
–
–
+
10
+
–
10
+
+
1010
Insulin (100 nm)
Time (h)
Glucose (25 mm)
*
*
**
LGB
LGA
HGB
HGA
TC 1-6 cell glucagon
secretion (pg/mg protien)
(C)
4000
3000
2000
1000
0
Insulin (nm)
Time (h)
1
4
10
4
100
4
*
**
Islets glucagon
m RNA expression (%)
(B)
5000
4000
3000
2000
1000
0
Insulin (100 nm)
Time (h)
Glucose (30 mm)–
–
6
–
+
6
+
–
6
+
+
6
Islets glucagon
secretion (pg/mg protien)
(A)
*
*
**
LGB
LGA
HGB
HGA
5000
4000
3000
2000
1000
0
Insulin (nm)
Time (h)
1
2
10
2
100
2
FIGURE3 Effects of insulin on glucagon secretion and gene expression in rat islets (A, B) and TC1-
6 cells (C, D) incubated for 1 or 5 days in DMEM media containing 5.5 mM glucose (LG) or 25/30
mM glucose (HG) and stimulated with 1, 10, or 100 nM insulin for 2 or 4 h for glucagon secretion,
or with 100 nM insulin for 4 or 10 h for glucagon gene expression (n = 4). LGB, LG without insulin;
LGA, LG plus insulin; HGB, HG without insulin; HGA, HG plus insulin. Values are mean ± SD.
∗p < 0.05 versus LGB;∗∗p < 0.05 versus HGB.
for high glucose to increase insulin-mediated PI3K activity was less than that for low
glucose. However, pretreatment with 100 nmol/L Wortmannin, the blocker of PI3K,
decreased PI3K activity in rat islets and αTC1-6 cells exposed to low glucose by 63%
and 60%, respectively, and by 45% and 46% in islets and cells exposed to high glucose
(Figure 4A and 4B).
EffectsofHighGlucoseonPhosphorylationofser473-AktProteinExpressioninRat
IsletsandαTC1-6Cells
In the absence of insulin, the phospho-Akt 473 in rat islets and TC1-6 cells exposed
to 25/30 mmol/L glucose was not significantly difference from that in islets and cells
exposed to 5.5 mmol/L glucose. However, when 100 nmol/L insulin was added for 30
min,thelevelofphospho-Akt473inratisletsandαTC1-6cellsexposedto5.5mmol/L
glucose increased by 200% and 180%, respectively (both: p < 0.05). Meanwhile, the
levelofphospho-Akt473wasincreasedinratisletsandαTC1-6cellsexposedto25/30
mmol/L glucose by 137% and 70%, respectively, compared with that in islets/cells
exposed to 5.5 mmol/L glucose for 5 days (both: p < 0.05). Pretreatment with 100
nmol/L Wortmannin reduced the level of phospho-Akt 473 in rat islets and αTC1-6
cells by 50% and 56% (both: p < 0.05), respectively, at 5.5 mmol/L glucose, and by
22% and 31%, respectively, at 25/30 mmol/L glucose (Figure 5A and 5B).
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20X.Shenetal.
Insulin (100 nm)
Glucose (25 mm)
Islet PI3K Activity (fold induction)
0
1
2
3
4
5
*
*
*,**
*,***
(A)
–
–
–
–
–
+
+
–
–
+
–
+
–
+
–
–
+
+
+
+
–
+
+
+Wortmannin (100 nm)
*,**
*,***
*
*
TC 1-6 cell PI3K Activity (fold induction)
0
1
2
3
4
5
(B)
Insulin (100 nm)
Glucose (25 mm)–
–
–
–
–
+
+
–
–
+
–
+
–
+
–
–
+
+
+
+
–
+
+
+ Wortmannin (100 nm)
FIGURE 4 Effect of high glucose on PI3K activity in rat islets and TC1-6 cells. Rat islets and TC1-6
cellswerepretreatedwithorwithoutWortmannin(100nM)for30minandstimulatedwithorwith-
out insulin (100 nM) for 30 min. PI3K activity in rat islets (A) and TC1-6 cells (B) was determined
usingaPI3KELISAkit(n=4).Kinaseactivityisexpressedasthefoldinductionrelativetotheactiv-
ity in the untreated LG group. Values are mean ± SD.∗p < 0.05 versus the corresponding LG or HG
group without insulin;∗∗p < 0.05 versus LG plus insulin;∗∗∗p < 0.05 versus HG plus insulin.
Islet P-Akt (% of control)
3
3.5
2
2.5
1
1.5
*
*
*,**
*,***
0
0.5
(A)
P-ser473Akt
T-Akt
Insulin (100 nm)
Wortmannin (100 nm)
Glucose (25 mm)–
–
–
–
–
+
+
–
–
+
–
+
–
+
–
–
+
+
+
+
–
+
+
+
*,***
*
*,**
*
TC 1-6 cell P-Akt (% of control)
3
3.5
2
2.5
1
1.5
0
0.5
(B)
P-ser473Akt
T-Akt
Insulin (100 nm)
Wortmannin (100 nm)
Glucose (25 mm)–
–
–
–
–
+
+
–
–
+
–
+
–
+
–
–
+
+
+
+
–
+
+
+
FIGURE 5 Effects of insulin and Wortmannin on phospho-Akt 473 in rat islets and TC1-6 cells. Rat
islets and TC1-6 cells were pretreated with or without wortmannin (100 nM) for 30 min and simu-
lated with or without insulin (100 nM) for 30 min. Phospho-Akt 473 in rat islets (A) and TC1-6 cells
(B) was measured by western blotting and the results are expressed as the fold increase over con-
trol (phosphorylated/total) in arbitrary units (n = 3). The levels of phospho-Akt 473 (P-Akt; upper
panel) and total Akt (lower panel) are shown. Values are mean ± SD.∗p < 0.05 versus the corre-
spondingLGorHGgroupwithoutinsulin;∗∗p<0.05versusLGpluswithinsulin;∗∗∗p<0.05versus
HG plus insulin.
DISCUSSION
The bihormonal-abnormality hypothesis highlighted that both deficient insulin
secretionandexcessiveglucagonlevelcontributedtothehyperglycemia.Itconvinced
us that the inability of insulin to suppress glucagon secretion from α cells may play
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GlucotoxicityandαCellDysfunction 21
an important role in the development of hyperglycemia in diabetes. It is well known
that the insulin level is higher in subjects with impaired glucose tolerance and that
the plasma glucagon level is also higher than in normal subjects; this indicates that
the insulin is unable to correct the exaggerated α cell response in these subjects
(8). At overt hyperglycemia in diabetes, the abnormal α cell secretion even worsens.
This suggests that insulin resistance in α cells may exist. However, the mechanism
leading to insulin resistance of α cells remains to be explored.
This study showed that high glucose led to increased glucagon release and a
high glucagon mRNA expression from both rat islets and TC1-6 cells. Moreover, a
moderate concentration of insulin (10 nmol/L) was unable to inhibit the glucagon
secretion and gene expression in rat islets and in αTC1-6 cells exposed to high glu-
cosefor5days.However,thisinsulinlevelcouldsuppresstheglucagonsecretion and
gene expression in α cells exposed to 5.5 mmol/L glucose. Interestingly, the insulin-
mediated PI3K/ser473-Akt pathway was suppressed at high glucose concentrations.
This indicates that the ability of insulin to inhibit glucagon secretion is impaired in
the presence of high glucose and suggests that the α cell dysfunction to high glucose
at least in part is due to the inhibition of PI3K activity in the impaired insulin signal
pathway of α cells.
Glucose is a major physiological regulator of glucagon secretion (20). The acute
(1 h) and chronic (5 days) response of α cells to glucose differs. A U-shaped dose–
response relationship in glucose-regulated acute glucagon secretion (24 h) was
described in In-R1-G9 cells (21,22), while chronic exposure to high glucose levels
stimulatesthereleaseandsynthesisofglucagon,aswellasproglucagonmRNAlevels.
Our study demonstrated that glucagon secretion and gene expression in α TC1-
6 cells incubated for 5 days in media containing 25 mmol/L glucose increased to
approximately 230% and 264%, respectively, compared with that in cells incubated
at 5 mmol/L glucose. In view of the fact that the usual concentration of glucose in
the medium for αTC1-6 cells is 25 mM, the glucose level was raised to 30 mM in
our study to reflect the hyperglycemic diabetic condition. As a result, this increased
glucagon secretion and gene expression in αTC1-6 cells to almost 272% and 278%,
respectively, compared that in with cells incubated with 5.5 mM glucose. It showed
clearly that the high glucose level was responsible for the paradoxical response of the
αTC1-6 cells.
The αTC1-6 cells and InR1-G9 are clonal α cell lines from tumors; they may
not have a normal α cell response to glucose perturbations. To validate our results
obtained in clonal α cells, it is warranted to perform comparative experiments in pri-
mary α cells. We established a β cell-depleted rat model via injection streptozotocin
100 mg/kg to rats. Results from immunohistochemistry showed that after injection
of this high dose streptozotocin, the β cells were deleted in rat islets and the α cell
area accounted for 78% of the islet, indicating that our β cell deleting rat model
was successfully established. We found in both αTC1-6 cells and rat islets exposed
to 25mmol/L glucose that the glucagon secretion and glucagon gene expression
increasedclearlyinbothmodelscomparedwithisletsexposedto5.5mmol/Lglucose.
These results showed that chronic high glucose stimulates α cells to secrete more
glucagon,whichiscorrelatedwithhighglucagonmRNAsynthesis.Toourknowledge,
the ability of exogenous insulin to inhibit glucagon secretion and proglucagon gene
expression reported both in vivo and in vitro is controversial (23–30). The reason for
thedifferingresultsmaybeascribedtodifferencesinlevelsofinsulin,concentrations
of glucose, and/or the exposure time adopted in the different studies.
Insulin, Zn2+, ATP, γ-aminobutyric acid, and possibly glutamate may suppress
glucagon secretion (31,32). Of these molecules, insulin is considered to be the most
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22X.Shenetal.
likely candidate to be involved in the response to high glucose by inhibiting α cell
activity (33,34). Our data revealed that pretreatment with 1 nmol/L insulin did not
suppress glucagon secretion or glucagon gene expression in both rat islets and αTC1-
6 cells at 5.5 mmol/L glucose. However, the higher insulin concentration (10 nmol/L)
inhibited glucagon secretion and gene expression in rat islets and αTC1-6 cells
exposed to 5.5 mmol/L glucose. It is noteworthy that 10 nmol/L insulin is not able
to suppress glucagon secretion and gene expression when the α cells are exposed
to 25/30 mmol/L glucose. Thus, the higher insulin level (10 nmol/L) is only partially
able to overcome the severe insulin resistance in α cells induced by very high glucose
levels (25/30 mmol/L), and sometimes very high doses of insulin may be needed to
completely overcome the severe insulin resistance in α cells.
Chronic hyperglycemia impairs pancreatic β cell insulin secretion by affecting
insulin receptor expression and insulin receptor-associated PI3K activity (1).Sus-
tained hyperglycemia can lead to the glucotoxicity in β cell and the glucotoxicity is
responsibleforaggravatinginsulinresistancebyinhibitinginsulinsignalingpathway.
Itisreportedthattranscriptsencodingtheinsulinreceptorarehighlyabundantinrat
α cells and prolonged exposure to high glucose concentrations increased glucagon
secretion and proglucagon mRNA levels in the islet cell line InR1-G9 and αTC1-6
(35,36). Clearly, glucose toxicity causes α cell dysfunction. Kankeo et al. (37) found
thatinsulincouldactivatePI3KinInR1-G9cells.However,itremainsunclearwhether
glucotoxicity-induced insulin resistance in α cells is mediated by the PI3K/Akt path-
way.
Inordertoexplorethepossiblemechanismsofinsulinresistanceinαcellsinduced
byglucotoxicity,weevaluatedtheeffectsofhighglucoseonthelevelsofphospho-Akt
473inratαcellsandαTC1-6cellsandfoundthathighglucoseattenuatedtheabilityof
insulintoactivatethelevelsofphospho-Akt473inαcells.Furthermore,wemeasured
PI3K, which is the upstream molecule of the Akt pathway, and found high glucose
could reduce PI3K activity in rat α cells and αTC1-6 cells. Moreover, Wortmannin,
a PI3K inhibitor, markedly decreased PI3K activity in cells exposed to low glucose,
while slightly decreased PI3K activity in cells exposed to high glucose. PI3K activity
playsanimportantroleinαcelldysfunctioninducedbyhighglucose,thusweconfirm
that the insulin-mediated PI3K/Akt pathway involved in glucose toxicity in α cells,
which could be a significant reason to cause insulin resistance in α cells. We hope
thatstudiesonamelioratingglucosetoxicityinαcellsinthefuturewillshedlightona
new clinical therapeutic direction for diabetes mellitus.
In summary, this study demonstrates that chronic exposure to high glucose stim-
ulates glucagon secretion and gene expression in α cells (rat islets and αTC1-6 cells)
andthattheinsulin-mediatedPI3K/Aktsignalpathwaywasimpairedinαcellsbyglu-
cose toxicity. This indicates that high glucose caused glucagon hypersecretion from
α cells which may be caused by insulin resistance in α cells and impairment of the
insulin signaling pathway in α cells.
ACKNOWLEDGMENTS
This work was supported by generous research grants from the European Foun-
dation for the Study of Diabetes (EFSD). We thank Changzheng Wang and Jie
Wang (the Medical Research Center of Chicago.) for presenting αTC1-6 cells as
a gift and we are deeply grateful to Professors Zhe Cai, Lan Zhang, and Jun Shu
(the Clinical Research Institute of China-Japan Friendship Hospital) for excellent
technical assistance.
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GlucotoxicityandαCellDysfunction23
DeclarationofInterest
The authors report no conflicts of interest. The authors alone are responsible for the
content and writing of this article.
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