Protective Effects of GLP-1 on Glomerular Endothelium and Its

Inhibition by PKC Activation in Diabetes

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Citation

Mima, A., J. Hiraoka-Yamomoto, Q. Li, M. Kitada, C. Li, P.

Geraldes, M. Matsumoto, et al. 2012. Protective Effects of
GLP-1 on Glomerular Endothelium and Its Inhibition by PKC
Activation in Diabetes. Diabetes 61 (11): 2967-2979.
doi:10.2337/db11-1824. http://dx.doi.org/10.2337/db11-1824.

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doi:10.2337/db11-1824

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September 14, 2014 1:19:17 AM EDT

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ORIGINAL ARTICLE

Protective Effects of GLP-1 on Glomerular Endothelium

and Its Inhibition by PKCb Activation in Diabetes
Akira Mima,1 Junko Hiraoka-Yamomoto,1 Qian Li,1 Munehiro Kitada,1 Chenzhong Li,1
Pedro Geraldes,2 Motonobu Matsumoto,1 Koji Mizutani,1 Kyoungmin Park,1 Christopher Cahill,1
Shin-Ichi Nishikawa,3 Christian Rask-Madsen,1 and George L. King1

To characterize glucagon-like peptide (GLP)-1 signaling and its

effect on renal endothelial dysfunction and glomerulopathy. We
studied the expression and signaling of GLP-1 receptor (GLP-1R)
on glomerular endothelial cells and the novel nding of protein
kinase Adependent phosphorylation of c-Raf at Ser259 and its
inhibition of angiotensin II (Ang II) phosphoc-Raf(Ser338) and
Erk1/2 phosphorylation. Mice overexpressing protein kinase
C (PKC)b2 in endothelial cells (EC-PKCb2Tg) were established.
Ang II and GLP-1 actions in glomerular endothelial cells were
analyzed with small interfering RNA of GLP-1R. PKCb isoform
activation induced by diabetes decreased GLP-1R expression and
protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phosphoc-Raf(Ser338)
and Ang II activation of phospho-Erk1/2. EC-PKCb2Tg mice
exhibited decreased GLP-1R expression and increased phospho
c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic
EC-PKCb2Tg mice exhibited greater loss of endothelial GLP-1R
expression and exendin-4protective actions and exhibited more
albuminuria and mesangial expansion than diabetic controls.
These results showed that the renal protective effects of GLP-1
were mediated via the inhibition of Ang II actions on cRaf
(Ser259) and diminished by diabetes because of PKCb activation
and the increased degradation of GLP-1R in the glomerular endothelial cells. Diabetes 61:29672979, 2012

ndothelial pathologies such as thrombotic microangiopathy and mesangiolysis are parts of glomerulopathy because of insulin resistance and
diabetes, which are leading causes of clinical renal disease (1,2). Endothelial dysfunction is postulated to
accelerate the progression of diabetic glomerulopathy as
a result of the inhibition of endothelial nitric oxide (NO)
synthesis (eNOS) and its product, NO (3).
We have reported that activation of the b isoform of
protein kinase C (PKC) by hyperglycemia can cause glomerular endothelial dysfunction and reduce eNOS activation
partially owing to inhibition of insulin action on glomerular
endothelial cells (4,5). Clinically, ruboxistaurin (RBX),
a specic inhibitor of PKCb, has been reported to improve
endothelial dysfunction induced by hyperglycemia (4,6).
Further, studies have associated PKCb activation with
From the 1Research Division, Joslin Diabetes Center, Harvard Medical School,
Boston, Massachusetts; the 2Department of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada; and the 3Laboratory for Stem Cell
Biology, RIKEN Center for Developmental Biology, Kobe, Japan.
Corresponding author: George L. King, [email protected].
Received 23 December 2011 and accepted 12 May 2012.
DOI: 10.2337/db11-1824
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1824/-/DC1.
2012 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for prot,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
diabetes.diabetesjournals.org

glomerular pathology induced by hyperglycemia possibly

due to the enhancement of angiotensin action (7). However,
the biochemical mechanism by which PKCb enhances angiotensin II (Ang II) action to accelerate the progression of
diabetic glomerulopathy has not been claried.
Recently, glucagon-like peptide-1 (GLP-1) has been reported to biologically improve endothelial function and
prevent some renal pathologies in diabetic rodents (8,9).
However, a mechanistic explanation regarding GLP-1
protective action on the endothelial cell is unknown.
GLP-1 is a gut incretin hormone that augments glucosedependent insulin responses in the b cells (10). GLP-1
receptor (GLP-1R) is present abundantly in the gastrointestinal tract but has also been reported in endothelium
and kidney and may stimulate NO production (8,11,12). In
this study, we have identied a new biochemical mechanism for GLP-1 to inhibit Ang II inammatory action via the
c-Raf/extracellular signalrelated kinase (Erk)1/2/plasminogen
activator inhibitor (PAI)-1 pathway in glomerular endothelial cells. Further, we have demonstrated a dual signaling
mechanism by which diabetes, via PKCb activation, can increase Ang II action by increasing the inammatory cytokines
and extracellular matrix and inhibiting GLP-1protective
effects by reducing GLP-1R expression in the glomerular
endothelium.
RESEARCH DESIGN AND METHODS
Generation of endothelial cellspecic PKCb2overexpressing mice. All
animal protocols were approved by the Joslin Diabetes Center committee in
accordance with NIH guidelines. The pVECD-PKCb2 vector was constructed
by inserting mouse PKCb2 cDNA into pVECD vector (13). Transgenic mice
expressing PKCb2 were generated from C57BL/6J mice. Diabetes was induced
by ve consecutive days of injections of streptozotocin (STZ) (55 mg/kg
body wt; Sigma) in 0.05 mol/L citrate buffer (pH 4.5). Blood glucose levels
were determined by glucose analyzer (Yellow Spring Instruments). Glycemic
levels .16.7mmol/L were dened as having diabetes. Two weeks after diabetes, exendin-4 (1.0 nmol/kg/day; Sigma) or diluents were administrated
intraperitoneally to mice for 6 months. Regular human insulin (10 mU/g; Lilly)
or diluents were injected into the inferior vena cava for 10 min to study insulin
signaling. Kidneys were harvested and procedures were performed within 30 min.
Measurement of blood pressure. Blood pressure was determined in conscious animals using a noninvasive computerized automated tail-cuff system
(Vistech Systems). After the mice were trained for ve consecutive days,
they were placed on a heated platform and studied for three 10-cycle
measurements.
Measurement of urinary albumin, creatinine, and cAMP. Urinary albumin
was measured from 24-h urine collection with mice housed in individual
metabolic cages and assessed by Albuwell (Exocell). Creatinine levels were
measured by colorimetric detection kit (Assay Designs), and urinary cAMP was
measured after injection with exendin-4 or vehicle by using ELISA kit (Cell
Biolab).
Isolation of glomeruli and cell culture. Isolation of mouse glomeruli was
performed as previously described (14). Rat glomerular and lung endothelial
cell were also cultured as previously described (4).
Immunoblot analysis. Samples were dissolved in 0.5% Nonidet P-40 and
immunoprecipitated with antibody to GLP-1R (Santa Cruz Biotechnology) and
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PKCb INHIBITS GLP-1 ACTION IN DIABETIC NEPHROPATHY

protein A/G-Sepharose beads. The proteins were separated by SDS-PAGE and

subsequently blotted with antibodies as indicated.
Immunohistochemistry and real-time PCR analysis. Immunohistochemistry and its analysis were performed as previously described (4). Real-time
PCR was also performed as previously described (4) (Supplementary Table 1).
Data analysis. Data are expressed as means 6 SD. Comparisons were made
between groups using either two-sample and paired t tests for two-way
comparisons or one-way ANOVA for multiple groups to establish statistically
signicant differences. All analyses were performed using StatView (SAS Institute). Statistical signicance was dened as P , 0.05.

RESULTS

Effect of diabetes and PKCb2 activation on GLP-1R

expression. Double immunostaining studies showed that
GLP-1R was mainly expressed by glomerular endothelial
cells and that expression levels were decreased by 43 6
12% in diabetic mice compared with nondiabetic mice (Fig.
1A). Immunoblot study of renal cortex lysate showed that
GLP-1R protein expression was decreased by 35 6 9%
after 6 months of diabetes (Fig. 1B). In the glomeruli, GLP1R protein expression was decreased by 37 6 7% (Supplementary Fig. 1) after 3 months of diabetes. In contrast,
GLP-1R mRNA expression was not changed by diabetes
(Fig. 1C). To evaluate the mechanisms of insulin receptor
substrate 1 diabetes-inhibitory actions, we studied the effect of PKC activation on GLP-1R expression in glomerular
endothelial cells (RGECs). PKC activation by phorbol
12-myristate 13-acetate (PMA) decreased protein expression
of GLP-1R by 46 6 4% (Fig. 1D) without altering GLP-1R
mRNA levels (Fig. 1E). Addition of GF109203X (GFX),
a general PKC inhibitor, and RBX reversed the inhibitory
effect of PMA by 29 6 8 and 17 6 7%, respectively. Since
diabetes and PKC activation appear to affect GLP-1R by a
posttranscriptional mechanism, the addition of proteasome
inhibitor MG132 in RGECs increased GLP-1R protein levels
by 29 6 10% during PMA treatment (Fig. 1D). To conrm
that increases in the degradation of GLP-1R were induced
by PKC activation, RGECs were exposed to 100 nmol/L
PMA for 4 h, which increased the ubiquitinated GLP-1R
levels signicantly by 8.9 6 1.1-fold; levels were inhibited by
GFX or RBX by 37 6 6 and 22 6 6%, respectively (Fig. 1F).
Adenovirus-mediated overexpression of PKCb2 (AdPKCb2) in RGECs decreased GLP-1R protein levels by 29 6
3% compared with a control construct (Fig. 1G) without
changing mRNA expression (Fig. 1H). Overexpression of
PKCa and PKCd isoforms by infection with Ad-PKCa and
Ad-PKCd did not decrease GLP-1R protein expression
(Fig. 1G).
To determine whether PKCb2 activation in the endothelial cells can decrease the expression of GLP-1R, we
created mice overexpressing mouse PKCb2 in endothelial
cells (EC-PKCb2Tg) using a transgene consisting of
a fragment of the vascular endothelial-cadherin promoter
and mouse PKCb2 cDNA (Supplementary Fig. 2A).
Transgenic founder lines carrying the mouse PKCb2
transgene were identied by Southern blot analysis (Supplementary Fig. 2B). In situ PKC activity increased by 53 6
16% in the glomeruli of EC-PKCb2Tg mice (Supplementary
Fig. 2C). RT-PCR analysis for PKCb2 revealed that mRNA
expression of the transgene in the glomeruli increased by
32 6 8-fold compared with wild-type (WT) mice (Supplementary Fig. 2D). Immunoblot analyses showed that
expression of PKCb2 in cytosolic and membrane fractions from glomeruli increased by 2.8 6 0.3- and 7.5 6 0.3fold, respectively, in EC-PKCb2Tg compared with WT
controls (Supplementary Fig. 2E). In contrast, the levels of
PKCa, PKCd, and PKC were not changed in cytosolic or
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membrane fractions in the renal cortex of transgenic mice

(Supplementary Fig. 2F). Immunoblot analyses of primary
lung endothelial cells showed that the expression of PKCb
increased by 2.6 6 0.9-fold greater in transgenic than in
WT mice (Supplementary Fig. 2G). Double immunostaining of the renal glomeruli showed that the increases of
PKCb2 were detected mainly in the glomerular endothelial
cells (Supplementary Fig. 2H).
GLP-1R expression in the renal glomeruli, measured by
immunohistochemistry, was reduced by 45 6 19% in the
EC-PKCb2Tg mice versus WT mice. After 6 months of diabetes, GLP-1R expression in diabetic EC-PKCb2Tg mice
was reduced by 64 6 19% compared with nondiabetic WT
mice and by 37 6 14% versus diabetic WT mice (Fig. 2A).
Immunoblot analysis showed that GLP-1R protein expression decreased in nondiabetic and diabetic ECPKCb2Tg mice by 35 6 4 and 51 6 3%, respectively,
compared with nondiabetic WT mice and reduced by 22 6
3% when diabetic EC-PKCb2Tg mice were compared with
diabetic WT mice (Fig. 2B). GLP-1R protein expression
also decreased in glomeruli of nondiabetic and diabetic
EC-PKCb2Tg mice by 34 6 7 and 59 6 6%, respectively,
compared with nondiabetic WT mice and reduced by 34 6
10% when diabetic EC-PKCb2Tg mice were compared with
diabetic WT mice (Supplementary Fig. 2I). No changes in
GLP-1R mRNA expression levels were observed in any
group (Fig. 2C). The levels of polyubiquitination of GLP-1R
were increased signicantly in diabetic WT mice by 6.5 6
1.4-fold and in nondiabetic EC-PKCb2Tg mice by 5.3 6 1.1fold compared with nondiabetic WT mice. Further, polyubiquitination of GLP-1R in diabetic EC-PKCb2Tg mice
was more prominent by 8.4 6 1.0-fold versus nondiabetic
WT mice and by 1.3 6 0.1-fold compared with nondiabetic
EC-PKCb2Tg mice (Fig. 2D).
Nuclear factor-kB activation and inammation in
renal cortex. We also characterized the effect of PKCb2
activation on nuclear factor-kB (NF-kB) and inammatory
cytokine expression, which are markers of diabetic glomerulopathy and angiotensin activation (15,16). NF-kB
activation was observed to be 1.3 6 0.3-fold higher in the
renal cortex of EC-PKCb2Tg mice than in diabetic WT
mice after 6 months of diabetes (Supplementary Fig. 3A).
Similarly, NF-kB, as measured by DNA binding, increased
in both diabetic EC-PKCb2Tg and WT mice compared with
their nondiabetic controls after 3 and 6 months of diabetes
(by 1.3 6 0.1-fold in WT and by 1.5 6 0.2-fold in ECPKCb2Tg mice, respectively) (Supplementary Fig. 3B). For
inammatory cytokines, expression of tumor necrosis
factor-a mRNA levels was increased by 3.2 6 1.1- and
1.4 6 0.2-fold after 3 and 6 months of diabetes in diabetic
versus nondiabetic WT mice. After 6 months of diabetes,
tumor necrosis factor-a mRNA levels were 1.4 6 0.2-fold
higher in EC-PKCb2Tg mice compared with WT mice (Supplementary Fig. 3C). Similarly, expression of interleukin-6
mRNA levels in the renal cortex of diabetic WT mice was
increased by 2.9 6 0.6- and 2.0 6 0.5-fold after 3 and
6 months of diabetes compared with WT mice. For ECPKCb2Tg mice, diabetes increased interleukin-6 mRNA expression by 1.5 6 0.5-fold compared with diabetic WT mice
after 6 months of diabetes (Supplementary Fig. 3D). Diabetes
also increased the expression of CD68 mRNA levels in WT
mice by 6.1 6 2.9-fold and in EC-PKCb2Tg mice by 6.1 6
2.8-fold at 3 months (Supplementary Fig. 3E).
Effect of diabetes on renal function and pathology.
After 6 months, body weight, blood glucose, and plasma
insulin concentrations were not different between diabetic
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A. MIMA AND ASSOCIATES

FIG. 1. Diabetes and PKCb2 activation decreases GLP-1R. A: Immunostaining for GLP-1R and CD31 and merge images in the glomeruli and
morphometric analysis of glomerular expression of GLP-1R and CD31. For quantication of the expression of GLP-1R and CD31, the positive
staining area/glomerular area (%) was measured using ImageJ (NIH). For each animal, 50 glomeruli were evaluated. Bar = 50 mm. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. n = number of mice. DM, mice with STZ-induced
diabetes; NDM, nondiabetic mice. *P < 0.05. Magnication 3400. B: Immunoblots of GLP-1R from renal cortex of mice with STZ-induced diabetes
for 6 months. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. *P < 0.05. C: GLP-1R
mRNA expression in the renal cortex of mice with STZ-induced diabetes for 6 months. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in
nondiabetic transgenic and diabetic transgenic mice. D: Immunoblots of GLP-1R in RGECs. RGECs were incubated with PMA (4 h) with or without
a PKC-specic inhibitor (GFX), PKCb-specic inhibitor (RBX), or proteasome inhibitor (MG132). **P < 0.001 vs. PMA2, GFX2, RBX2, and

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EC-PKCb2Tg and WT mice or between nondiabetic ECPKCb2Tg and WT mice (Table 1 and Supplementary Table
2). Diabetic EC-PKCb2Tg mice exhibited more albuminuria compared with diabetic WT mice by 1.5 6 0.5-fold
(Fig. 3A), but they did not differ in creatinine clearance
and glomerular ltration rates (Supplementary Figs. 3F
and G). The width of glomerular basement membrane
(GBM) was signicantly increased by 1.3 6 0.4-fold in diabetic EC-PKCb2Tg mice compared with diabetic WT littermates of the same age (Fig. 3B). Mesangial matrix expansion
was 1.4 6 0.4-fold greater in diabetic EC-PKCb2Tg mice
than in diabetic WT mice. Diabetic EC-PKCb2Tg mice also
exhibited a greater fraction of glomerular area stained
for type IV collagen (Col4) and bronectin than diabetic
WT mice (1.3 6 0.3- and 1.8 6 0.5-fold higher, respectively)
(Fig. 3C). Protein expression of Co14 and bronectin in
the renal cortex were increased signicantly in both
groups of diabetic mice, but the increases were much
greater in diabetic EC-PKCb2Tg mice by 1.4 6 0.1-fold
compared with diabetic WT mice (Supplementary Fig.
3H). This was also reected in their mRNA levels (Supplementary Fig. I).
Evaluation of PAI-1, tissue plasminogen activator,
and eNOS activation in EC-PKCb2Tg mice. In immunohistochemistry studies, diabetes increased PAI-1positive
areas in the glomeruli of diabetic EC-PKCb2Tg mice by
1.3 6 0.4-fold more than in diabetic WT mice (17) (Fig. 3D).
Expression of PAI-1 mRNA level was also increased in the
renal cortex of both diabetic WT mice and EC-PKCb2Tg
mice in concordance with histological studies (Supplementary Fig. 3J). In contrast, areas positive for tissue
plasminogen activator and its mRNA did not change in ECPKCb2Tg or WT mice compared with nondiabetic controls
(Supplementary Figs. 3K and J).
Analysis of eNOS expression and activation in the renal
cortex showed that insulin increased both phospho-Akt
and phospho-eNOS signicantly in WT mice, but its effect
was decreased in EC-PKCb2Tg mice compared with WT
mice by 63 6 8 and by 72 6 7%, respectively (P , 0.05)
(Supplementary Fig. 3L) (3).
Effect of exendin-4 in diabetic EC-PKCb2Tg mice. To
determine whether GLP-1 can prevent endothelial dysfunction induced by diabetes, we determined the effect of
treatment with exendin-4, an analog of GLP-1. Infusion
with exendin-4 signicantly increased urinary cAMP in
nondiabetic WT mice by 1.9 6 0.6-fold, which was decreased by 29 6 13% in diabetic WT mice and by 28 6 12%
in nondiabetic EC-PKCb2Tg mice. Exendin-4 did not signicantly increase urinary cAMP in diabetic EC-PKCb2Tg
mice (Fig. 4A). Treatment with exendin-4 did not prevent
the reduction of GLP-1R protein (Supplementary Fig. 4A)
or mRNA levels (Supplementary Fig. 4B) in the renal cortex
or affect body weight, blood glucose, blood pressure, urine
volume, food intake, plasma insulin, or creatinine clearance
(Supplementary Table 2 and Supplementary Fig. 4C). In
contrast, exendin-4 signicantly decreased albuminuria in
both diabetic WT and EC-PKCb2Tg mice by 27 6 10 and

44 6 20%, respectively (Fig. 4B). Quantitative analysis of

immunohistochemistry showed that exendin-4 treatment
reduced mesangial matrix fraction in both diabetic WT and
EC-PKCb2Tg mice by 38 6 10 and 31 6 8%, respectively
(Fig. 4C and Supplementary Fig. 4D). Col4- and bronectinpositive staining areas were reduced by exendin-4 treatment (Col4 by 26 6 8% in diabetic WT mice and 27 6 9% in
diabetic EC-PKCb2Tg mice and bronectin by 26 6 11% in
diabetic WT mice and 29 6 13% in diabetic EC-PKCb2 Tg
mice, respectively) (Supplementary Fig. 4D). Similar
reductions in the mRNA were observed when EC-PKCb2Tg
mice were treated with exendin-4 (Supplementary Fig. 5A).
Immunostaining analysis showed that exendin-4 decreased diabetes-induced elevation of PAI-1 protein expression in diabetic WT and EC-PKCb2Tg mice (by 32 6 11
and 31 6 10%, respectively) (Fig. 4D and Supplementary
Fig. 5B). Expression of PAI-1 mRNA in renal cortex was
decreased by exendin-4 treatment both in diabetic WT and
EC-PKCb2Tg mice by 24 6 9 and 32 6 10%, respectively
(Supplementary Fig. 5C).
Exendin-4 decreased mRNA expression of macrophage
markers CD68 and CXCL2 in diabetic WT (CD68 by 52 6 7
and 31 6 10%, respectively) (Supplementary Fig. 5D) and
EC-PKCb2Tg (CXCL2 by 36 6 11 and 35 6 14%, respectively) mice (Supplementary Fig. 5D and E) (9).
Exendin-4mediated inhibition of c-Raf/Erk1/2/PAI-1
pathway by angiotensin II. Activation of c-RafErk1/2
pathways by Ang II can mediate the expression of renal
inammatory cytokines (1820). In RGECs, Ang II signicantly increased phosphoc-Raf(Ser338), phospho-Erk1/2,
and PAI-1 expression by 9.9 6 2.4-, 9.2 6 2.2-, and 2.2 6
0.6-fold, respectively. Exendin-4 increased phosphoc-Raf
(Ser259) signicantly by 13 6 1-fold and decreased Ang II
effects on phosphoc-Raf(Ser338), phospho-Erk1/2, and
PAI-1 by 37 6 2, 52 6 6, and 22 6 2%, respectively. H89,
a selective protein kinase A inhibitor, decreased the effect
of exendin-4 on phosphoc-Raf(Ser259) by 43 6 5% and
reduced exendin-4inhibitory effects on Ang II-induced
phosphoc-Raf(Ser338), phospho-Erk1/2, and PAI-1 by
38 6 4, 35 6 3, and 11 6 2%, respectively. mitogenactivated protein kinase kinase inhibitor PD98059 decreased Ang II action on phospho-Erk1/2 and PAI-1 by
85 6 22 and 38 6 4%, respectively, yet it did not affect
Ang II action on phosphoc-Raf(Ser338), but p38 mitogenactivated protein kinase inhibitor SB203580 did not have
any effect on these markers (Fig. 5A and Supplementary Fig. 6A). However, MDL12330A, a cAMP-selective
inhibitor in RGECs, reduced exendin-4induced phosphoc-Raf(Ser259) by 68 6 7% (Fig. 5B) and partially
reduced exendin-4inhibitory effects on Ang II action
[phosphoc-Raf(Ser338), phospho-Erk1/2, and PAI-1 by
23 6 7, 45 6 14, and 29 6 9%, respectively] (Fig. 5B and
Supplementary Fig. 6B). These results strongly suggest
that GLP-1protective action on endothelial cells is
due to cAMP-induced phosphorylation of c-Raf(Ser259),
which inhibits Ang II activation of phosphoc-Raf
(Ser338).

MG1322. P < 0.05 vs. PMA+, GFX2, RBX2, and MG1322. E: GLP-1R mRNA expression in the renal cortex of mice with STZ-induced diabetes for 6
months. F: Immunoprecipitation and immunoblots of ubiquitin-targeted GLP-1R. RGECs were incubated with PMA (4 h) with or without GFX or
RBX. Whole-cell lysates were immunoprecipitated with antiGLP-1R antibody, subjected to SDS-PAGE, and blotted with ubiquitin antibody. **P <
0.001 vs. PMA2, GFX2, and RBX2. P < 0.05 vs. PMA+, GFX2, and RBX2. G and H: Immunoblot analyses (G) and mRNA expression (H) of GLP-1R.
RGECs were transfected with Adgreen uorescent protein (GFP), Ad-PKCa, Ad-PKCb2, or Ad-PKCd as indicated. *P < 0.05 vs. Adgreen
uorescent protein. One of three independently performed experiments is shown. Comparisons were made between groups using either twosample and paired t tests for two-way comparisons or one-way ANOVA for multiple groups to establish statistically signicant differences. Results
are means 6 SD. AU, arbitrary units; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblot; IP, immunoprecipitation; NS, not
signicant. (A high-quality digital representation of this gure is available in the online issue.)
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A. MIMA AND ASSOCIATES

FIG. 2. Decreases of GLP-1R in the glomeruli of EC-PKCb2Tg mice. A: Immunostaining for GLP-1R and CD31, merge images in the glomeruli, and
morphometric analysis of glomerular expression of GLP-1R and CD31. Bar = 50 mm. DM, mice with STZ-induced diabetes; NDM, nondiabetic mice;
Tg, EC-PKCb2Tg mice. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. *P < 0.05 vs.
nondiabetic WT mice. P < 0.05 vs. diabetic WT mice. Magnication 3400. B: Immunoblots of GLP-1R from renal cortex of mice with STZ-induced
diabetes for 6 months. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. *P < 0.05 vs.
nondiabetic WT mice. P < 0.05 vs. diabetic WT mice. C: GLP-1R mRNA expression in the renal cortex of mice with STZ-induced diabetes for 6
months. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. D: Immunoprecipitation and
immunoblots of ubiquitin-targeted GLP-1R in each group of mice. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and
diabetic transgenic mice. **P < 0.001 vs. nondiabetic WT mice. Results are expressed as means 6 SD. One of three independently performed
experiments is shown. Comparisons were made between groups using either two-sample and paired t tests for two-way comparisons or one-way
ANOVA for multiple groups to establish statistically signicant differences. AU, arbitrary units; NS, not signicant; IP, immunoprecipitation; IB,
immunoblot. (A high-quality digital representation of this gure is available in the online issue.)

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TABLE 1
Physiological characteristics of nondiabetic and diabetic WT and EC-PKCb2Tg mice
Nondiabetic WT
n
Body weight (g)
Blood glucose (mg/dL)
SBP (mmHg)
rKW/BW (mg/g)

33.5
109
99.9
5.2

6
6
6
6
6

1.3
8
7.8
0.8

Diabetic WT
25.2
510
102.8
9.4

6
6
6
6
6

1.4*
54*
8.0
1.3*

Nondiabetic Tg
7
31.9 6
101 6
101.6 6
5.0 6

1.3
7
10
0.7

Diabetic Tg
23.5
507
103.4
11.2

7
6
6
6
6

3.0*
57*
8.5
2.6*

Data are means 6 SD unless otherwise indicated. rKW/BW, right kidney weight/ body weight; SBP, systolic blood pressure; Tg, EC-PKCb2Tg
mice. *P , 0.05 vs. nondiabetic WT mice.

Effect of PKC activation on c-Raf, mitogen-activated

protein kinase, and PAI-1 activity. PKC activation by
PMA increased phosphoc-Raf(Ser338) and phospho-Erk1/2
in RGECs by 2.7 6 0.7- and 7.0 6 3.3-fold, respectively,
but did not alter phosphoc-Raf(Ser259). In contrast, the
addition of exendin-4 increased phosphoc-Raf(Ser259) by
8.9 6 1.0-fold in parallel with decreasing PMA-induced
phosphoc-Raf(Ser338) and phospho-Erk1/2 by 58 6 8 and
43 6 5%, respectively (Fig. 6A). Addition of GFX or RBX
decreased PMA-induced phosphoc-Raf(Ser338) by 55 6 4
and 34 6 4% and phospho-Erk1/2 by 53 6 2 and 31 6 8%,
respectively (Fig. 6B). Similar to PMA, Ang II also increased phosphoc-Raf(Ser338) and phospho-Erk1/2 by
6.0 6 2.1- and 10 6 2-fold, respectively, which were reduced signicantly by inhibitors GFX and RBX by 43 6 2
and 26 6 7%, respectively (Fig. 6C). Lastly, Ang II increased mRNA levels of PAI-1 by 5.4 6 2.4-fold, which was
also inhibited by GFX and RBX by 45 6 21 and 36 6 15%,
respectively (Supplementary Fig. 6C).
Role of GLP-1R on exendin-4 and Ang II signaling. For
ascertainment of whether GLP-1R was mediating exendin4inhibitory effect, the expression of GLP-1R was reduced
by GLP-1R small interfering RNA in RGECs. The addition
of GLP-1R small interfering RNA in RGECs reduced its
expression by 55 6 22% (Supplementary Fig. 6D), inhibited
exendin-4induced phosphoc-Raf(Ser259) by 47 6 8%
(Fig. 6D), and decreased exendin-4inhibitory effects on
Ang II signaling of phosphoc-Raf(Ser338), phospho-Erk1/2,
and PAI-1 mRNA by 47 6 6, 16 6 5, and 18 6 3%, respectively (Fig. 6D and Supplementary Fig. 6E and F). Ang
II induced production of PAI-1 by 2.3 6 0.4-fold; production
was decreased by exendin-4. In contrast, knockdown of
GLP-1R in RGECs reduced exendin-4inhibitory effects by
23 6 5% (Supplementary Fig. 6G). Lastly, blocking GLP-1R
with exendin-3, a GLP-1R antagonist, inhibited exendin-4
induced phosphoc-Raf(Ser259) by 57 6 4% and increased
phosphoc-Raf(Ser338) and phospho-Erk1/2 by 35 6 3 and
56 6 2%, respectively (Fig. 6E and Supplementary Fig. 6H)
in the presence of Ang II and exendin-4.
Evaluation of Ang II and effect of GLP-1 on c-Raf in
the glomeruli. Intravenous infusing of Ang II (100 ng/kg/
min) increased phosphoc-Raf(Ser338) and phospho-Erk1/2
by 8.1 6 0.8- and 6.8 6 0.6-fold, respectively, in the
glomeruli of WT mice compared with saline infusion.
Further, Ang II increased phospho-Erk1/2 signicantly
more in the EC-PKCb2Tg mice than WT mice by 1.4 6 0.2fold. In both WT and EC-PKCb2Tg mice, treatment with
exendin-4 increased phosphoc-Raf(Ser259) by 8.6 6
0.2- and 8.8 6 1.0-fold, respectively, and decreased Ang
II-induced phosphoc-Raf(Ser338) by 41 6 11% in WT mice
and by 31 6 9% in EC-PKCb2Tg mice (Fig. 7A).
Similar to the results in the glomeruli, infusing Ang II
increased phosphoc-Raf(Ser338) (by 6.4 6 0.2-fold),
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phospho-Erk1/2 (by 7.9 6 3.1-fold), and PAI-1 mRNA (by

11 6 2-fold) levels in the renal cortex compared with levels
in the renal cortex of WT mice without Ang II. Further, Ang
II increased phosphoc-Raf(Ser338), phospho-Erk1/2, and
PAI-1 expression signicantly more in the EC-PKCb2Tg
mice compared with WT mice with Ang II (by 1.3 6 0.1-,
1.4 6 0.1-, and 1.5 6 0.2-fold, respectively) (Supplementary
Fig. 7A and B). In both WT and EC-PKCb2Tg mice, treatment with exendin-4 increased phosphoc-Raf(Ser259)
and signicantly decreased Ang IIinduced phosphoc-Raf
(Ser338), phospho-Erk1/2, and PAI-1 expression (Supplementary Fig. 7A and B).
As previously reported (4,21), diabetes increased phosphoErk1/2 in the renal cortex of both WT and EC-PKCb2Tg
mice, and this increase was 1.8 6 0.3-fold greater in diabetic EC-PKCb2Tg mice than diabetic WT mice. Diabetes
also increased phosphoc-Raf(Ser338) in the renal cortex
of diabetic EC-PKCb2Tg mice, and this increase was 1.4 6
0.1-fold greater than the increase in diabetic WT mice.
Lastly, exendin-4 treatment decreased diabetes-induced
increases of phospho-Erk1/2 by 37 6 10% and phospho
c-Raf(Ser338) by 25 6 3% in WT mice and by 20 6 9 and
16 6 4% in EC-PKCb2Tg mice (Fig. 7B).
DISCUSSION

The current study provides a biochemical pathway by

which GLP-1 mediates its protective action in the glomerular endothelial cells to inhibit Ang II signaling and its
proinammatory action. These data demonstrate that hyperglycemia via the activation of PKCb can cause endothelial dysfunction through a dual pathway including
reduction of GLP-1R and enhancement of Ang II signaling
and action.
Previously, GLP-1R activation by exendin-4 was shown
to inhibit Ang II activation in renal proximal tubular cells
(22). Our results demonstrate that GLP-1 is partly mediating its protective action via its own receptor by the activation of PKA. The elevation of cAMP levels increased
phosphoc-Raf(Ser259), which may inhibit phosphoc-Raf
(Ser338)/phospho-Erk1/2, which are activated by Ang II to
induce its inammatory action such as PAI-1 expression.
Several studies have shown that phosphorylation of c-Raf
(Ser259) induced by cAMP can inhibit the action of
phosphoc-Raf(Ser338), leading to inammatory action
(23,24). The inhibitory effect of cAMP is likely to be direct,
since Dhillon et al. (24) and others have reported a direct
interaction between PKA and c-Raf (2526). We have
shown for the rst time in vivo that GLP-1 only increased Ser259 phosphorylation and Ang IIincreasing
Ser338 phosphorylation on c-Raf in renal glomeruli.
However, when the two were infused together, GLP-1
clearly decreased Ang IIinduced phosphoc-Raf(Ser338).
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FIG. 3. Functional and histological examinations of diabetic EC-PKCb2Tg and WT mice. A: Albuminuria in mice with STZ-induced diabetes (DM),
nondiabetic mice (NDM), WT mice, and EC-PKCb2Tg mice is shown. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic
(Tg) and diabetic transgenic mice. **P < 0.001 vs. nondiabetic WT mice. P < 0.05 vs. diabetic WT mice. B: Representative views of GBM in each
group of mice and quantication of GBM thickness in each group of mice. Bar = 1 mm. n = 6 in nondiabetic WT and diabetic WT mice; n = 7 in
nondiabetic transgenic and diabetic transgenic mice. *P < 0.05 vs. nondiabetic WT mice. P < 0.05 vs. diabetic WT mice. C: Representative light
microscopic appearance of glomeruli (periodic acidSchiff [PAS] and periodic acidmethenamine-silver [PAM] staining), immunohistochemistry of
Col4 and bronectin, and morphometric analysis of periodic acidmethenamine-silver, Col4-, and bronectin-positive staining area. The glomerular periodic acidmethenamine-silver, Col4-, and bronectin-positive staining area was measured. Bar = 50 mm. n = 6 in nondiabetic WT and
diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. *P < 0.05 vs. nondiabetic WT mice. P < 0.05 vs. diabetic WT mice.
Magnication 3400. D: Immunostaining for PAI-1 in each group of mice and analysis of glomerular expression of PAI-1. Bar = 50 mm. n = 6 in
nondiabetic WT and diabetic WT mice; n = 7 in nondiabetic transgenic and diabetic transgenic mice. *P < 0.05 vs. nondiabetic WT mice. P < 0.05
vs. diabetic WT mice. Magnication 3400. One of three independently performed experiments is shown. Results are expressed as means 6 SD. For
all the studies above, comparisons were made between groups using either two-sample and paired t tests for two-way comparisons or one-way
ANOVA for multiple groups to establish statistically signicant differences. (A high-quality digital representation of this gure is available in the
online issue.)

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FIG. 4. Effect of exendin-4 (Ex-4) treatment in EC-PKCb2Tg mice. A: Urinary cAMP excretions in each group are shown: mice with STZ-induced diabetes
(DM), nondiabetic mice (NDM), WT mice, and EC-PKCb2Tg mice. n = 6 in nondiabetic WT plus vehicle, nondiabetic WT plus exendin-4, diabetic WT plus
vehicle, diabetic WT plus exendin-4, nondiabetic transgenic (Tg) plus exendin-4, and diabetic transgenic plus exendin-4; n = 7 in nondiabetic transgenic
plus vehicle and diabetic transgenic plus vehicle. *P < 0.05 vs. WT/nondiabetic/exendin-42; P < 0.05 vs. WT/nondiabetic/exendin-4+. B: Albuminuria in
each test group is shown. n = 6 in nondiabetic WT plus vehicle, nondiabetic WT plus exendin-4, diabetic WT plus vehicle, diabetic WT plus exendin-4,
nondiabetic transgenic plus exendin-4, and diabetic transgenic plus exendin-4; n = 7 in nondiabetic transgenic plus vehicle and diabetic transgenic plus
vehicle. *P < 0.05 vs. WT/nondiabetic/exendin-42; P < 0.05 vs. WT/diabetic/exendin-42; P < 0.05 vs. transgenic/diabetic/exendin-42. C: Representative
light microscopic appearance of glomeruli periodic acidSchiff (PAS) and periodic acidmethenamine-silver (PAM) staining. Bar = 50 mm. n = 6 in nondiabetic WT plus vehicle, nondiabetic WT plus exendin-4, diabetic WT plus vehicle, diabetic WT plus exendin-4, nondiabetic transgenic plus exendin-4, and
diabetic transgenic plus exendin-4; n = 7 in nondiabetic transgenic plus vehicle and diabetic transgenic plus vehicle. Magnication 3400.
D: Immunostaining for PAI-1 in each group of mice is shown. Bar = 50 mm. n = 6 in nondiabetic WT plus vehicle, nondiabetic WT plus exendin-4, diabetic WT
plus vehicle, diabetic WT plus exendin-4, nondiabetic transgenic plus exendin-4, and diabetic transgenic plus exendin-4; n = 7 in nondiabetic transgenic
plus vehicle and diabetic transgenic plus vehicle. Magnication 3400. One of three independently performed experiments is shown. Results are expressed
as means 6 SD. Comparisons were made between groups using either two-sample and paired t tests for two-way comparisons or one-way ANOVA for
multiple groups to establish statistically signicant differences. (A high-quality digital representation of this gure is available in the online issue.)
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FIG. 5. Exendin-4 (Ex-4) decreased effect of Ang II on Erk1/2/PAI-1 signaling. A: Immunoblots of phosphoc-Raf (p-c-Raf)(Ser259), phospho
c-Raf(Ser338), and phospho-Erk1/2 (p-Erk1/2) in RGECs stimulated with Ang II in the presence or absence of PD98059, SB203580, exendin-4,
or H89. **P < 0.001 vs. Ang II2/PD980592/SB2035802/exendin-42/H892; P < 0.05 vs. Ang II2/PD980592/SB2035802/exendin-4+/H892; P < 0.05,
P < 0.001 vs. Ang II+/PD980592/SB2035802/exendin-42/H892; P < 0.05 vs. Ang II+/PD980592/SB2035802/exendin-4+/H892. B: Immunoblots of
phosphoc-Raf(Ser259), phosphoc-Raf(Ser338), and phospho-Erk1/2 in RGECs stimulated with Ang II (10 nmol/L) in the presence or absence of
exendin-4 or MDL12330A. *P < 0.05, **P < 0.001 vs. Ang II2/exendin-42/MDL12330A2; P < 0.05 vs. Ang II2/exendin-4+/MDL12330A2; P < 0.05
vs. Ang II+/exendin-42/MDL123302; P < 0.05 vs. Ang II+/exendin-4+/MDL123302. One of three independently performed experiments is shown.
Comparisons were made between groups using either two-sample and paired t tests for two-way comparisons or one-way ANOVA for multiple
groups to establish statistically signicant differences. Results are expressed as means 6 SD. AU, arbitrary units.

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PKCb INHIBITS GLP-1 ACTION IN DIABETIC NEPHROPATHY

FIG. 6. Effect of PKC activation (PMA), knockdown, or blocking of GLP-1R on exendin-4 (Ex-4)-stimulated inhibition of Ang II on phosphoc-Raf
(p-c-Raf)(Ser338), phospho-Erk1/2 (p-Erk1/2), and PAI-1 activity in RGECs. A: Immunoblots of phosphoc-Raf(Ser259), phosphoc-Raf(Ser338),
and phospho-Erk1/2 in RGECs stimulated with PMA (100 nmol/L) in the presence or absence of exendin-4 are shown. **P < 0.001 vs. PMA2/
exendin-42 and P < 0.05 vs. PMA+/exendin-42. B: Immunoblots of phosphoc-Raf(Ser338) and phospho-Erk1/2 in RGECs stimulated with PMA in
the presence or absence of GFX or RBX. *P < 0.05 vs. PMA2/GFX2/RBX2; P < 0.05 vs. PMA+/GFX2/RBX2. C: Immunoblots of phosphoc-Raf
(Ser338) and phospho-Erk1/2 in RGECs stimulated with Ang II in the presence or absence of GFX or RBX. *P < 0.05 vs. Ang II2/GFX2/RBX2; P <
0.05 vs. Ang II+/GFX2/RBX2. D: Immunoblots of phosphoc-Raf(Ser259), phosphoc-Raf(Ser338), and phospho-Erk1/2 in RGECs transfected with
small interfering GLP-1R or small interfering control, stimulated with Ang II in the presence or absence of exendin-4. **P < 0.001 vs. small interfering control/Ang II2/exendin-42; P < 0.05 vs. siControl/Ang II2/exendin-4+; P < 0.05 vs. siControl/Ang II+/exendin-42; P < 0.05 vs.
siControl/Ang II+/exendin-4+. E: Immunoblots of phosphoc-Raf(Ser259), phosphoc-Raf(Ser338), and phospho-Erk1/2 in RGECs stimulated with
Ang II in the presence or absence of exendin-4 or exendin-3(9-39) [Ex-3(9-39)] are shown. **P < 0.001 vs. Ang II2/exendin-42/exendin-3(9-39)2;
P < 0.05 vs. Ang II2/exendin-4+/exendin-3(9-39)2; P < 0.05 vs. Ang II+/exendin-42/exendin-3(9-39)2; P < 0.05 vs. Ang II+/exendin-4+/exendin-3
(9-39)2. One of three independently performed experiments is shown. Comparisons were made between groups using either two-sample and paired
t tests for two-way comparisons or one-way ANOVA for multiple groups to establish statistically signicant differences. Results are expressed as
means 6 SD. AU, arbitrary units.

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FIG. 7. Exendin-4 (Ex-4) decreased angiotensin II or diabetes-induced increases of phosphoc-Raf (p-c-Raf)(Ser338) in the glomeruli and renal
cortex. A: Immunoblots of phosphoc-Raf(Ser259), phosphoc-Raf(Ser338), and phospho-Erk1/2 (p-Erk1/2) in the glomeruli. Exendin-4 (1.0
nmol/kg) or diluents were administrated intraperitoneally to mice. After 2-h administration of exendin-4, Ang II (100 ng/kg/3 mL/min) or saline
was infused through the jugular vein. After 3-h continuous infusion, glomeruli were corrected. n = 35. Tg, transgenic. *P < 0.05 vs. WT/Ang II2/
exendin-42; P < 0.05 vs. WT/Ang II+/exendin-42; P < 0.05 vs. transgenic/Ang II2/exendin-42; P < 0.05 vs. WT/Ang II+/exendin-42; #P < 0.05
vs. transgenic/Ang II+/exendin-42. B: Immunoblots of phosphoc-Raf(Ser259), phosphoc-Raf(Ser338), and phospho-Erk1/2 in the renal cortex.
n = 6 in nondiabetic WT plus vehicle, nondiabetic WT plus exendin-4, diabetic WT plus vehicle, diabetic WT plus exendin-4, nondiabetic
transgenic plus exendin-4, and diabetic transgenic plus exendin-4; n = 7 in nondiabetic transgenic plus vehicle and diabetic transgenic plus
vehicle. DM, mice with STZ-induced diabetes; NDM, nondiabetic mice. *P < 0.05 vs. WT/nondiabetic/exendin-42; P < 0.05 vs. WT/diabetic/
exendin-42; P < 0.05 vs. transgenic/diabetic/exendin-42. One of three independently performed experiments is shown. Comparisons were
made between groups using either two-sample and paired t tests for two-way comparisons or one-way ANOVA for multiple groups to establish
statistically signicant differences. Results are expressed as means 6 SD. C: Schematic diagram of the inhibitory effects of PKCb2 on the
protective action of GLP-1 signaling against the effects of Ang IImediated glomerular pathology. AU, arbitrary units; ECM, extracellular
matrix.

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Interestingly, Ang II did not inhibit GLP-1 effect on phospho

c-Raf(Ser259). The reduction of c-Raf phosphorylation at
Ser338 by GLP-1 caused decreases in phospho-Erk1/2 and
PAI-1 in the renal glomeruli.
Diabetes of long duration also inhibited GLP-1protective
action by decreasing GLP-1R via increases in ubiquitination
of GLP-1R. However, Zhou et al. (27) reported that highglucose conditions did not decrease GLP-1R protein expression or increase its ubiquitination in the islet cells after
24 h. Further, Kodera et al. (9) reported that GLP-1R
was not decreased in the glomeruli of rats with STZ-induced
diabetes after several weeks of disease. However,
Park et al. (8) reported that GLP-1R was decreased in the
glomeruli of db/db mice and exendin-4 treatment ameliorated this decrease. These differences are likely due to
using different cell types and mice strains and length of
diabetes.
Thus, our results suggest that PKCb activation induced
by hyperglycemia can inhibit GLP-1protective action and
enhance inammatory effects on the endothelium by at
least two signaling pathways. First, our results expand on
previous ndings and show that PKCb activation can enhance Ang II effect by phosphorylating c-Raf at Ser338 to
activate phospho-Erk1/2 and cause increased production
of PAI-1 (28). Second, we found that PKC activation can
also inhibit the protective actions of GLP-1 by reducing the
expression of its receptors in the endothelial cells. One
potential mediator of Ang II adverse effect in endothelial
cells is the induction of PAI-1 (7), which may impair brinolysis and promote increased extracellular matrix
(29,30).
Our study identies the biochemical mechanisms by
which PKC activation can increase the actions of Ang II.
The results demonstrate that PKC activation, especially
PKCb, increased phosphoc-Raf(Ser338), which leads to
the activation of phospho-Erk1/2. This activation is selective for PKCb isoform, since RBX, a PKCb isoformspecic
selective inhibitor, inhibited phosphoc-Raf(Ser338). In
addition, infusion of Ang II in EC-PKCb2Tg mice increased
phosphoc-Raf(Ser338) to a greater extent than in WT
mice both in vivo and in endothelial cells. In parallel with
changes in phosphoc-Raf(Ser338), EC-PKCb2Tg mice
exhibited greater expression of PAI-1 than did controls
after Ang II infusion. It is interesting to note, however, that
endothelial dysfunction induced by the overexpression of
PCKb alone, specically in the endothelial cells, without the
presence of diabetes did not exhibit signicant pathology as
measured by mesangial matrix expression and PAI-1. Thus,
endothelial dysfunction alone without diabetes will only
induce minimal albuminuria and extracellular matrix production. Studies using long-duration treatment of Ang II
will be needed to correlate changes in phosphoc-Raf and
glomerular pathology.
The second new nding shows that diabetes and hyperglycemia via PKCb activation will blunt the protective
actions of GLP-1 by decreasing GLP-1R protein expression
in the endothelium. The reduction in GLP-1R is manifested
by a decrease in urinary cAMP with infusion of exendin-4
and less reduction in phosphoc-Raf(Ser338)/phosphoErk1/2 in the glomeruli of EC-PKCb2Tg mice. However, even
with the reduction in GLP-1R, treatment with exendin-4
in vivo was still partially effective to reduce glomerular
pathology of both diabetic WT and EC-PKCb2Tg mice. It is
surprising that exendin-4 still had positive effects on the
glomerular pathology, although it did not increase urinary
cAMP signicantly. This could be due to the difculty of
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measuring cAMP as a result of degradation in urine that

took 24 h to collect and the dilution of samples by the
increases of urine volume induced by diabetes. It is also
possible that the benecial effects of exendin-4 could
be partially mediated via cAMP-independent pathways.
All of the protective actions of exendin-4 occurred without changes in glucose, insulin level, blood pressure, and
body weight.
Thus, our study has identied mechanisms by which
GLP-1 can induce protective actions on the glomerular
endothelial cells by inhibiting the signaling pathway of Ang
II at phosphoc-Raf(Ser338) via phosphoc-Raf(Ser259).
Further, we have demonstrated, in vivo and in vitro, that
hyperglycemia can activate PKCb isoforms, which enhance Ang II toxic effect in glomerular endothelial cells.
These studies suggest that effective therapeutic agents
could be designed to enhance GLP-1R on the endothelium,
which may prevent glomerular endothelial dysfunction
and slow the progression of diabetic nephropathy.
ACKNOWLEDGMENTS

This work was supported by National Institutes of Health/

National Eye Institute (EY016150) and the Diabetes and
Endocrinology Research Center (P30DK036836). A.M. is
the recipient of a Research Fellowship (Manpei Suzuki
Diabetes Foundation, Kanzawa Medical Research Foundation, and the NOVARTIS Foundation) (Japan) for the Promotion of Science.
No potential conicts of interest relevant to this article
were reported.
A.M. designed research, researched data, and wrote,
reviewed, and edited the manuscript. J.H.-Y., Q.L., M.K.,
C.L., P.G., M.M., K.M., K.P., and C.C. researched data. S.-I.N.
contributed to discussion. C.R.-M. researched data. G.L.K.
designed research and wrote, reviewed, and edited the
manuscript. G.L.K. is the guarantor of this work and, as
such, had full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy
of the data analysis.
Parts of this study were presented in abstract form at the
72nd Scientic Sessions of the American Diabetes Association, Philadelphia, Pennsylvania, 8-12 June 2012.
The pVECD vector was generously provided by
S. Nishikawa (RIKEN, Japan). The authors thank Patti
Muehter, Joslin Diabetes Center, for preparing the manuscript.
REFERENCES
1. Goldberg RJ, Nakagawa T, Johnson RJ, Thurman JM. The role of endothelial cell injury in thrombotic microangiopathy. Am J Kidney Dis 2010;56:
11681174
2. Nakagawa T, Tanabe K, Croker BP, et al. Endothelial dysfunction as a potential contributor in diabetic nephropathy. Nat Rev Nephrol 2011;7:3644
3. Nakagawa T, Sato W, Glushakova O, et al. Diabetic endothelial nitric oxide
synthase knockout mice develop advanced diabetic nephropathy. J Am
Soc Nephrol 2007;18:539550
4. Mima A, Ohshiro Y, Kitada M, et al. Glomerular-specic protein kinase
C-b-induced insulin receptor substrate-1 dysfunction and insulin resistance in rat models of diabetes and obesity. Kidney Int 2011;79:883896
5. Naruse K, Rask-Madsen C, Takahara N, et al. Activation of vascular protein
kinase C-beta inhibits Akt-dependent endothelial nitric oxide synthase
function in obesity-associated insulin resistance. Diabetes 2006;55:691698
6. Gould CM, Newton AC. The life and death of protein kinase C. Curr Drug
Targets 2008;9:614625
7. Feener EP, Northrup JM, Aiello LP, King GL. Angiotensin II induces
plasminogen activator inhibitor-1 and -2 expression in vascular endothelial
and smooth muscle cells. J Clin Invest 1995;95:13531362
diabetes.diabetesjournals.org

A. MIMA AND ASSOCIATES

8. Park CW, Kim HW, Ko SH, et al. Long-term treatment of glucagon-like

peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice. J Am Soc Nephrol 2007;18:
12271238
9. Kodera R, Shikata K, Kataoka HU, et al. Glucagon-like peptide-1 receptor
agonist ameliorates renal injury through its anti-inammatory action
without lowering blood glucose level in a rat model of type 1 diabetes.
Diabetologia 2011;54:965978
10. Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3:153165
11. Bullock BP, Heller RS, Habener JF. Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor. Endocrinology 1996;137:29682978
12. Erdogdu O, Nathanson D, Sjholm A, Nystrm T, Zhang Q. Exendin-4
stimulates proliferation of human coronary artery endothelial cells through
eNOS-, PKA- and PI3K/Akt-dependent pathways and requires GLP-1 receptor.
Mol Cell Endocrinol 2010;325:2635
13. Hisatsune H, Matsumura K, Ogawa M, et al. High level of endothelial cellspecic gene expression by a combination of the 59 anking region and the
59 half of the rst intron of the VE-cadherin gene. Blood 2005;105:4657
4663
14. Takemoto M, Asker N, Gerhardt H, et al. A new method for large scale
isolation of kidney glomeruli from mice. Am J Pathol 2002;161:799805
15. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of
NF-kappa B. Annu Rev Cell Biol 1994;10:405455
16. Goodfriend TL, Elliott ME, Catt KJ. Angiotensin receptors and their antagonists. N Engl J Med 1996;334:16491654
17. Nicholas SB, Aguiniga E, Ren Y, et al. Plasminogen activator inhibitor-1
deciency retards diabetic nephropathy. Kidney Int 2005;67:12971307
18. Duckworth BC, Cantley LC. Conditional inhibition of the mitogenactivated protein kinase cascade by wortmannin. Dependence on signal
strength. J Biol Chem 1997;272:2766527670
19. Bentires-Alj M, Kontaridis MI, Neel BG. Stops along the RAS pathway in
human genetic disease. Nat Med 2006;12:283285

diabetes.diabetesjournals.org

20. Sharma K, Ix JH, Mathew AV, et al. Pirfenidone for diabetic nephropathy.
J Am Soc Nephrol 2011;22:11441151
21. Jiang ZY, Lin YW, Clemont A, et al. Characterization of selective resistance
to insulin signaling in the vasculature of obese Zucker (fa/fa) rats. J Clin
Invest 1999;104:447457
22. Hirata K, Kume S, Araki S, et al. Exendin-4 has an anti-hypertensive effect
in salt-sensitive mice model. Biochem Biophys Res Commun 2009;380:4449
23. Cook SJ, McCormick F. Inhibition by cAMP of Ras-dependent activation of
Raf. Science 1993;262:10691072
24. Dhillon AS, Pollock C, Steen H, Shaw PE, Mischak H, Kolch W. Cyclic
AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation
of serine 259. Mol Cell Biol 2002;22:32373246
25. Hagiwara M, Brindle P, Harootunian A, et al. Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is
rate limited by nuclear entry of protein kinase A. Mol Cell Biol 1993;13:
48524859
26. Doucas V, Shi Y, Miyamoto S, West A, Verma I, Evans RM. Cytoplasmic
catalytic subunit of protein kinase A mediates cross-repression by
NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci USA 2000;
97:1189311898
27. Zhou J, Livak MF, Bernier M, et al. Ubiquitination is involved in glucosemediated downregulation of GIP receptors in islets. Am J Physiol Endocrinol Metab 2007;293:E538E547
28. Koyanagi T, Wong LY, Inagaki K, Petrauskene OV, Mochly-Rosen D. Alteration of gene expression during progression of hypertension-induced
cardiac dysfunction in rats. Am J Physiol Heart Circ Physiol 2008;295:
H220H226
29. Toer GH, Massaro J, Levy D, et al. Relation of the prothrombotic state to
increasing age (from the Framingham Offspring Study). Am J Cardiol 2005;
96:12801283
30. Paueksakon P, Revelo MP, Ma LJ, Marcantoni C, Fogo AB. Microangiopathic injury and augmented PAI-1 in human diabetic nephropathy.
Kidney Int 2002;61:21422148

DIABETES, VOL. 61, NOVEMBER 2012

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