RNA-Binding Protein HuD
Controls Insulin Translation
Eun Kyung Lee,1,4,8Wook Kim,2,8Kumiko Tominaga,1Jennifer L. Martindale,1Xiaoling Yang,1Sarah S. Subaran,2
Olga D. Carlson,2Evi M. Mercken,3Rohit N. Kulkarni,5Wado Akamatsu,6Hideyuki Okano,6Nora I. Perrone-Bizzozero,7
Rafael de Cabo,3Josephine M. Egan,2and Myriam Gorospe1,*
1Laboratory of Molecular Biology and Immunology
2Laboratory of Clinical Investigation
3Laboratory of Experimental Gerontology
National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA
4Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea
5Department of Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center and Department of Medicine, Harvard Medical School,
Boston, MA 02215, USA
6Department of Physiology, Graduate School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan
7Department of Neurosciences, School of Medicine, University of New Mexico, Albuquerque, NM 87131, USA
8These authors contributed equally to this work
Although expression of the mammalian RNA-binding
protein HuD was considered to be restricted to
neurons, we report that HuD is present in pancreatic
b cells, where its levels are controlled by the insulin
receptor pathway. We found that HuD associated
with a 22-nucleotide segment of the 50untranslated
region (UTR) of preproinsulin (Ins2) mRNA. Modu-
but HuD overexpression decreased Ins2 mRNA
translation and insulin production, and conversely,
HuD silencing enhanced Ins2 mRNA translation and
HuD rapidly dissociated from Ins2 mRNA and
enabled insulin biosynthesis. Importantly, HuD-
knockout mice displayed higher insulin levels in
pancreatic islets, while HuD-overexpressing mice
exhibited lower insulin levels in islets and in plasma.
In sum, our results identify HuD as a pivotal regulator
of insulin translation in pancreatic b cells.
Changes in circulating glucose modulate insulin production by
the b cells of the pancreatic islets of Langerhans. In turn, insulin
influences glucose uptake in insulin-sensitive peripheral tissues
such as fat and muscle, and maintains glucose homeostasis
(Rhodes and White, 2002). As a key metabolic factor, insulin
levels are tightly regulated by different mechanisms. Insulin is
produced by proteolytic cleavage of preproinsulin in pancreatic
b cells. Preproinsulin is encoded by insulin mRNA, a highly
abundant transcript in b cells (>30% of total mRNA) with an
exceptionally long half-life (>24 hr) due to the presence of a
pyrimidine-rich stretch in its 30untranslated region (UTR) (Itoh
and Okamoto, 1980; Goodge and Hutton, 2000). Tillmar and
Welsh (2002) identified the RNA-binding protein (RBP) polypyri-
midinetract-binding protein(PTB) asbeing responsible for asso-
ciating with the pyrimidine-rich stretch in insulin mRNA and
contributing to its high stability. Increased glucose availability
enhanced PTB binding to insulin mRNA and elevated its levels;
hours later, insulin mRNA was also transcriptionally upregulated
(Jahr et al., 1980).
However, in response to acute elevations in circulating
glucose, the necessary and timely rise in insulin production is
primarily controlled by rapid increases in the translation of insulin
mRNA in b cells. Wicksteed and coworkers (2001) reported that
insulin translation was regulated through the cooperative action
of a stem loop in the 50UTR and the conserved UUGAA
sequence in the 30UTR. A 9 nt element present in the insulin 50
UTR was shown to be responsible for the glucose-dependent
translational increase in insulin production (Wicksteed et al.,
2007). A 29 nt long element within the rat insulin 50UTR was
also found to contribute to the glucose-triggered translational
upregulation (Muralidharan et al., 2007). However, the specific
factor(s) that associate with these elements were unknown.
Here, we identify HuD (human antigen D) as an RBP that binds
to insulin mRNA and controls its translation. Like two other Hu
family members (HuB and HuC), HuD was believed to be ex-
pressed specifically in neurons, while the remaining member,
HuR was ubiquitous (Hinman and Lou, 2008). However, a recent
survey of HuD expression in different tissues (Abdelmohsen
et al., 2010), unexpectedly revealed HuD expression in pancre-
atic b cells. Hu proteins have three RNA recognition motifs
(RRMs) through which they associate with mRNAs bearing
specific sequences that are often AU- and U-rich. HuD bound
to the 30UTR of target mRNAs and stabilized them, as shown
for p21, tau and GAP-43 mRNAs (for review, see Hinman and
Lou, 2008). HuD also modulated target mRNA translation; for
826 Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc.
Figure 1. The IR Pathway Controls HuD Expression
(A) Immunostaining of sections of human and mouse pancreata to detect insulin (green, b cells), HuD, (red), and glucagon (blue, a cells); scale bar, 50 mm.
(B) Western blot analysis of HuD levels in mouse tissues.
(C–E) Western blot analyses of the levels of HuD, IR b subunit (IRb), and loading control b-actin in bIRWT and bIRKO cells cultured for 16 hr in 2 mM glucose, and
further cultured for 6 hr with the indicated glucose concentrations (C), in bIRKO cells transfected with either an control plasmid (vector) or with a plasmid that
expressed IR (D), and in bIRWT cells cultured in 2 mM glucose + 0.1% FBS for 24 hr before insulin treatment for the indicated times (E).
HuD Represses Insulin Translation
Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc. 827
example, interaction of HuD with the p27 mRNA disrupted an
internal ribosome entry site (IRES) and inhibited p27 translation
(Kullmann et al., 2002), whereas HuD enhanced the stability
and translation of Nova1 mRNA (Ratti et al., 2008). Despite the
short and unstructured 50UTRs of the human insulin (INS)
mRNA and the mouse ortholog (Ins2 mRNA), HuD binding to
the Ins2 50UTR repressed Ins2 mRNA translation and decreased
insulin production. Accordingly, HuD knockout mice expressed
higher levels of insulin in b cells, whereas HuD-overexpressing
mice expressed lower insulin levels in b cells and in the
HuD Is Expressed in Pancreatic b Cells
Immunostaining of human and mouse pancreatic sections de-
tected HuD in insulin-producing, b cells (Figure 1A); HuD was
also expressed in brain, but not in other mouse tissues (Fig-
ure 1B; Figures S1A and S1C available online). By western blot
analysis, HuD levels in immortalized b cells isolated from the
pancreas of wild-type (bIRWT) mice were significantly higher
and more glucose-inducible than those in b cells isolated from
an insulin receptor (IR) null (bIRKO) mouse (Figure 1C; Assmann
et al., 2009; Kim et al., 2011); ectopic IR re-expression in bIRKO
cells restored HuD abundance under conditions of low glucose
and low serum (Figure 1D). Treatment of bIRWT cells with insulin
similarly elevated HuD levels in a dose-dependent manner (Fig-
ure 1E). In mouse insulinoma bTC6 cells, silencing the insulin
lowered HuD levels (Figure 1F). Likewise, silencing or inhibiting
Akt, a kinase that functions downstream of Irs2, also lowered
HuD levels (Figures 1G and 1H), as did inhibiting the Akt kinase
PI3K using LY294002 (Figure 1H). Akt phosphorylates and
thereby inactivates the transcriptional repressor FoxO1; in
keeping with the putative FoxO1 binding site in the HuD
promoter (492 bp from the transcription start site), chromatin
immunoprecipitation (ChIP) analysis in bTC6 cells revealed an
interaction of FoxO1 with the HuD promoter. FoxO1 showed
greater association with the HuD promoter in conditions of low
glucose and reduced association in high glucose (Figure 1I; Fig-
ure S1B). As shown, silencing FoxO1 in bIRWT and bTC6 cells
augmented HuD expression levels (Figures 1J and 1K).
Conversely, overexpression of FoxO1 reduced HuD production,
O1(H215R-537), bearing a mutation in the DNA binding domain
(H215R) and C-terminal deletion in the transactivation domain
(Zhang et al., 2011) did not (Figure 1L). Together, these data indi-
cate that HuD is expressed in b cells under control of the
HuD Binds to Insulin 50UTR, Represses Insulin
We tested the interaction of HuD with the mRNAs encoding
mouse insulin (Ins1 and Ins2) in bTC6 cells. By ribonucleoprotein
(RNP) immunoprecipitation analysis (RIP) of bTC6 cells, Ins2
mRNA (the most abundant insulin-encoding transcript) was
To map the RNA region of association, biotinylated segments
spanning the50UTR,CR, and 30UTR of Ins2mRNA were synthe-
sized (Figure 2B), and the RNP complexes of HuD and bio-
tinylated RNAs was detected by biotin pull-down analysis using
streptavidin-coated beads. As shown in Figure 2C, HuD inter-
acted with the 50UTR of the Ins2 mRNA, but not with the Ins2
CR or 30UTR or with a negative control transcript (spanning
the 30UTR of GAPDH mRNA). Biotin pull-down analysis of other
RBPs,including HuR,TIAR, hnRNP K, NF90 (Figure S2A), aswell
as hnRNP C and FMRP (not shown), did not identify other Ins2
mRNA-interacting RBPs. The fact that argonaute IP failed to
show interaction with Ins2 mRNA by either biotin pull-down or
RIP (not shown) suggested that microRNAs were unlikely to be
major regulators of insulin production. Further subdivision of
the 50UTR revealed that segment 50D, spanning positions
52–73 and showing high conservation in human and rodents,
mutant biotinylated 50D (mut1-mut4) RNAs, none of which
showed an affinity for HuD (Figure 2E); similarly, incubation
with recombinant, purified GST-HuD showed selective binding
to50D,butnotto mutant(mut1-mut4)biot-Ins2RNAs (Figure2E).
To test if the interaction of HuD with the Ins2 50UTR was func-
tional, we prepared reporter constructs derived from plasmid
pEGFP in which the entire 50UTR [p(50)EGFP], a single copy of
fragment D [p(50D)EGFP] or three copies of fragment D [p(3 3
50D)EGFP] were inserted before the translation start site of the
EGFP coding region (Figure 2F, left). After transfection of each
reporterconstructinto bTC6 cellsexpressingnormal(CtrlsiRNA)
or silenced HuD (HuD siRNA), EGFP expression was assessed
by western blotting. As observed, silencing HuD selectively
increased EGFP production in p(50)EGFP, p(50D)EGFP, and in
p(3350D)EGFP, but not in the control reporter group (pEGFP).
This increase in EGFP protein (Figure 2F, ‘‘relative to each
Ctrl’’) did not arise from changes in EGFP mRNA (Figure 2G).
Moderate differences in transfection of each plasmid led to
differences in basal expression of reporter EGFP mRNA and
EGFP protein (Figure 2F ‘‘relative to pEGFP Ctrl,’’ Figure 2G,
inset graph). The ‘‘translation efficiency index’’ (which compares
changes in protein relative to changes in mRNA) supports the
view that translation was robustly increased in HuD siRNA cells
(F–H) Expression of the indicated proteins in bTC6 cells was studied by western blot analysis 48 hr after transfecting siRNAs directed to Irs2 (F) or Akt (G) or after
treatment for 24 hr with inhibitors of PI3K (LY294002) or Akt (H).
(I) Chromatin IP analysis of the interaction of FoxO1 with the HuD promoter in bTC6 cells cultured in 2 or 25 mM glucose for 30 min.
(J and K) Western blot analysis of the levels of HuD, FoxO1 and b-actin in bIRWT cells (J) and bTC6 cells (K) 48 hr after siRNA transfection.
(L) Western blot analysis of the expression of the indicated proteins in bTC6 cells 30 hr after transfecting a control plasmid (Vector) or plasmids to express Flag-
tagged FoxO1, either WT or the transcription-deficient mutant (H215R-537). Bottom, diagram of constructs for Flag-tagged WT and H215R-537.
(M) Schematic of the proposed regulation of HuD expression by the IR signaling pathway in b cells. In (C)–(H) and (J)–(L), signals on western blots (WB) were
quantified by densitometry and the errors (SEM) calculated. WBs are representative of at least three independent experiments.
HuD Represses Insulin Translation
828 Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc.
Figure 2. HuD Binds to the Ins2 50UTR and Represses Translation
(A) HuD interaction with Ins2 mRNA was studied by RIP analysis using anti-HuD or control IgG antibodies. RNA was isolated, and Ins2 mRNA levels measured by
reverse transcription-quantitative PCR analysis (RT-qPCR) and normalized to Gapdh mRNA levels.
HuD Represses Insulin Translation
Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc. 829
Likewise, endogenous Ins2 mRNA levels in bTC6 cells did not
change after silencing HuD, while expression of the encoded
insulin precursor (proinsulin) increased markedly (Figure 2H
versus 2I) without changes in proinsulin protein stability (Fig-
ure S2C). These data indicate that HuD associates with the
Ins2 mRNA 50UTR, and that this interaction represses insulin
production, likely by inhibiting its translation.
More direct evidence that HuD represses translation of the
Ins2 mRNA was obtained by polysome analysis in bTC6 cells ex-
pressing different HuD levels. Cytoplasmic extracts from control
ents, with the lightest components sedimenting at the top (frac-
somes, ranging from low- to high-molecular-weight (LMWP,
HMWP) in fractions 6–12 (Figure 2J, left). While in Ctrl siRNA
cells, polysome-associated Ins2 mRNA peaked in fractions 6
and 7,silencing HuDincreased the peak size of Ins2mRNA poly-
somes to fraction 8 (Figure 2J, right). The distribution of the
housekeeping Actin mRNA largely overlapped between the
two groups (Figure 2J, right). Additional evidence that HuD
modulated insulin translation was gained from nascent transla-
tion analysis. The incorporation of
newly synthesized proinsulin during a brief time period
(15 min), immediately followed by IP using anti-insulin antibody,
showed enhanced translation of proinsulin in the HuD siRNA
group, whereas translation of the housekeeping protein GAPDH
was unchanged between the two groups (Figure 2K; signals
were characteristically faint, reflecting the short labeling time).
Together, these findings strongly indicate that HuD represses
Ins2 production by reducing Ins2 mRNA translation.
HuD-Ins2 mRNA RNP Complexes Reduced after
Analysis of HuD-Ins2 mRNA complexes in bTC6 cells revealed
a rapid and robust release of Ins2 mRNA from HuD complex
within 30 min of glucose stimulation (25 mM glucose; Figure 3A).
In keeping with the view that HuD represses insulin production,
glucose treatment (15 mM, 20 min) elevated proinsulin abun-
dance in control cells (vector), but this increase was largely lost
in cells overexpressing HuD (Figure 3B, left; Figure S3A). These
changes occurred without changes in Ins2 mRNA levels (Fig-
ure 3B, right) but were accompanied by changes in nascent
translation of proinsulin (Figure 3C). Conversely, glucose treat-
ment (15 mM, 20 min) elevated proinsulin abundance in cells
transfected with Ctrl siRNA, while HuD-silenced cells expressed
significantly higher levels of proinsulin, as assessed by western
blotting (Figure 3D) and by ELISA measurement of intracellular
and secreted insulin (Figure 3E). Again, changes in proinsulin
and insulin production occurred without changes in Ins2 mRNA
levels (Figure 3D, graph). Similar trends were observed for
C-peptide (a linker between the A and B chains of insulin) in
bTC6 cells (Figure 3F) and for insulin and Ins2 mRNA measured
in other pancreatic b cells (Figures S3B and S3C).
The influence of HuD on insulin expression was further studied
by immunofluorescence. HuD subcellular distribution was visu-
alized relative to the distribution of two subcellular RNA granules
associated with gene repression, processing bodies (PBs), de-
tected using an antibody that recognizes the decapping protein
Dcp1, and stress granules (SGs), detected using an antibody
in the cytoplasm of bTC6 cells, in keeping with its distribution in
were detected; interestingly, by 15 and 30 min after glucose
stimulation, HuD colocalized in part with cellular PBs and with
a few visible SGs (Figure 3G; Figures S3D and S3E).
Insulin Levels in HuD Knockout and Transgenic Mice
Mice bearing with deletions in both HuD alleles (HuD?/?) or one
HuD allele (HuD+/?) showed impaired neuronal differentiation
(Pascale et al., 2004; Akamatsu et al., 2005), in keeping with
the neuronal presence of HuD. Analysis of pancreas from these
(B) Schematic of mouse insulin (Ins2) mRNA (top) and biotinylated segments (50UTR, CR, 30UTR) and subfragments of the 50UTR (50A–50D) tested (bottom).
(C) Biotin pull-down analysis of the interaction of HuD and biotinylated RNAs in (B). Biotinylated GAPDH RNA was included as negative control. IN, input (2 mg of
bTC6 whole-cell lysate).
(D) Sequence alignment of the last 16 nt of fragment 50D in different species.
(E) Biotinylated RNAs (1 mg each, top), including 50D and mutants mut1-mut4 (white, mutated nucleotides) were incubated with bTC6 cell lysates (HuD
MWM, molecular weight marker; IN, input lysate or purified protein.
(F) Left: Reporter plasmids: parent vector (pEGFP), plasmid expressing full-length Ins2 50UTR [p(50)EGFP], plasmid expressing one copy of fragment 50D [p(50D)
EGFP], and plasmid expressing three copies of 50D [p(3 3 50D)EGFP]. Right: 24 hr after siRNA transfection, each plasmid was transfected and EGFP expression
levels assessed24hrafter that.Westernblotanalysisofthelevels ofEGFP, HuD,and loadingcontrolproteins. Afterquantification by densitometry, EGFPsignals
were expressed relative to EGFP levels in the Ctrl siRNA sample in each pair (top row) and relative to EGFP levels in the Ctrl siRNA, pEGFP transfection group
(bottom row). Quantified WB signals (±SEM) are shown.
(G) Cells weretransfected asexplained in(F) andthelevels ofreporterEGFP mRNAswerequantifiedby RT-qPCR and plotted relative tothoseintheCtrlsiRNAof
each group (main graph) and relative to those in the Ctrl siRNA of the pEGFP transfection group (inset graph).
(H and I) Ins2 mRNA levels in bTC6 cells transfected with either control or HuD-directed siRNA were quantified by RT-qPCR analysis (H) and the levels of protein
expressed from Ins2 mRNA (proinsulin), as well as HuD and b-actin were assessed by western blot analysis and quantified (I).
(J) Polysome analysis of Ins2 mRNA. Lysates prepared from bTC6 cells (2 mM glucose) as explained in (H) were fractionated through sucrose gradients to
generate polysome profiles (left). Arrow: direction of sedimentation; –, no ribosomal components; LMWP (fractions 6–8) and HMWP (fractions 9–12). The relative
distribution of Ins2 mRNA and Actin mRNA on polysome gradients was studied by RT-qPCR analysis of the RNA present in each of 12 gradient fractions, and
represented as percentage of total mRNA (right). Data are representative of three independent experiments.
(K) Nascent translation of GAPDH and proinsulin was assessed by incubation of bTC6 cells in the presence of35S-Met/Cys. After IP using IgG (control), anti-
GAPDH, and anti-insulin antibodies, the newly translated proteins were visualized by SDS-PAGE, transfer, and PhosphorImager analysis of the blots (top);
bottom, quantification of35S-proinsulin in three different experiments.
HuD Represses Insulin Translation
830 Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc.
HuD Represses Insulin Translation
Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc. 831
from HuD?/? mice, moderately lower in HuD+/? mice, and
lowest in HuD+/+ mice, as assessed by immunofluorescence
(Figure 4A), and by western blot and ELISA analysis of insulin
production (Figures 4B–4E; Figure S4A). Thus, in HuD?/?
mice, the absence of HuD contributed to elevating constitutive
insulin and proinsulin levels in b cells.
To further test the possibility that HuD controls insulin produc-
tion in pancreatic b cells in vivo, and to extend the analysis to
circulating insulin plasma levels, transgenic mice (HuD Tg)
were used in which HuD was transcribed from the CamKII
promoter and hence was overexpressed in pancreatic b cells
(as well as brain [Bolognani et al., 2006], Figure S4B). Compared
pancreatic b cells of HuD Tg mice expressed higher levels of the
myc-tagged HuD transgene and correspondingly lower insulin
levels, as assessed by immunofluorescence in islets and by
western blot analysis (Figures 4G and 4H). The response to
changing circulating glucose levels was then assessed in HuD
WT and HuD Tg mice by using the intraperitoneal glucose toler-
ance test (IPGTT). Importantly, HuD Tg mice displayed impaired
glucose clearance from blood, as shown by the markedly higher
levels of plasma glucose following the glucose challenge (Fig-
ure 4I, top); at the same time, plasma insulin levels in HuD Tg
mice were lower throughout the IPGTT period (Figure 4I,
bottom). These results reveal that HuD Tg mice had less readily
releasable insulin in b cells compared with HuD WT mice, which
contributed to defective glucose homeostasis. Taken together,
our data indicate that HuD represses insulin production in b cells
of plasma insulin.
We report that HuD, previously considered a neuronal protein
(Hinman and Lou, 2008), is also present in mammalian pancre-
atic b cells, where it regulates insulin levels through the Ins2 50
UTR. By interacting with the p27 50UTR, a ?400 nt long, highly
structured, IRES-containing RNA, HuD repressed p27 transla-
tion, likely by disrupting the IRES (Kullmann et al., 2002). In
contrast, the 50UTR of mouse Ins2 mRNA is short (73 nt) as is
the 50UTR of human INS mRNA (59 nt), suggesting that HuD
does not repress insulin translation by blocking IRES activity. It
is also plausible that simply by occupying the Ins2 50UTR HuD
blocks translation initiation.
The release of Ins2 mRNA from HuD upon treatment with
glucose is reminiscent of the release of HuR-associated mRNAs
kinase C (PKC) was reported to phosphorylate HuD (Pascale
et al., 2005), but neither the specific phosphorylation site of
HuD nor its influence on binding to target mRNAs is known. It
will be important to elucidate whether PKC or other kinases are
responsible for releasing HuD-bound mRNAs and mobilizing
HuD to SGs and PBs following glucose challenge.
It is interesting that basal HuD expression is positively regu-
lated by IR signaling, whereas Ins2 mRNA is rapidly released
from HuD after acute exposure to glucose (Figure 3A), enabling
translation of Ins2 mRNA. The distinct mechanisms of HuD and
insulin regulation underscore the complexity of posttranscrip-
tional processes, where RBP levels, binding to target mRNA,
and localization are governed separately. These multiple events
are likely necessary to respond with appropriate kinetics and
magnitude of stimulation. Accordingly (Figure 4J), we propose
that positive regulation (green arrows) triggered by glucose or
insulin can facilitate insulin production post-transcriptionally in
an acute time frame (minutes) by releasing HuD from Ins2
mRNA and/or relocalizing HuD. The same trigger can balance
these effects via the negative regulation of insulin biosynthesis
byenhancing HuDlevels transcriptionally (redarrows). Thecoor-
dination of these positive and negative influences helps to
restore insulin to homeostatic levels (Figure 4J).
The findings that HuD?/? mice express higher insulin while
HuD Tg mice express lower insulin (Figure 4) support the notion
it will be important to test if HuD function is aberrant in patients
with type 2 diabetes. The constitutive levels of HuD, its subcel-
lular distribution, the release of HuD from the INS mRNA in
response to glucose or insulin stimulation, all of these parame-
ters warrant study in diabetic and nondiabetic patient popula-
tions. For example, in type 2 diabetes, insulin secretion from
b cells is severely defective in response to rising circulating
glucose, especially in the first 10 min after a glucose load, and
therefore it will be important to test if HuD release from INS
mRNA is also slow.
HuD production by lowering both the levels and translation rates
of HuD mRNA (Abdelmohsen et al., 2010). Interestingly, miR-375
regulates insulin secretion in pancreatic b cells, and miR-375
knockout (375KO) mice have a significantly reduced pancreatic
b cell mass compared with WT mice (Poy et al., 2009). The
authorsproposethatthe hyperglycemia of 375KOmiceislargely
due to the relative larger a cell compartment (which secretes
glucagon) in 375KO mice. However, in 375KO mice, b cells
may have higher HuD levels, which lowers insulin production,
Figure 3. Glucose Challenge Dissociates Ins2 mRNA from HuD and Mobilizes Cytoplasmic HuD
(A) RIP analysis of HuD-Ins2 mRNA complexes in bTC6 cells. Glucose-starved (2 mM glucose, 16 hr) bTC6 cells were incubated for with 25 mM glucose (15 or
30 min), whereupon RIP analysis was performed to assess the enrichment of Ins2 mRNA in HuD IP compared with IgG IP.
(B and C) The effect of glucose stimulation (15 mM, 20 min) on insulin expression by western blot analysis (B, left) or by RT-qPCR analysis of Ins2 mRNA (B, right)
was studied in bTC6 cells 48 hr after transfection with control plasmid (vector) or plasmid pMyc-HuD to express Myc-tagged HuD. (C) De novo proinsulin
translation was assessed by incorporation of35S-amino acids as explained in Figure 2K.
(D–F) Forty-eight hours aftertransfection withCtrlsiRNA or HuD siRNA, proinsulin levels were assessed by western blot analysis (D left), Ins2 mRNA levels by RT-
qPCR (D right), intracellular and secreted mature insulin by ELISA (E), and C-peptide by ELISA (F). Graphs depict the means of three experiments and p values.
Data were normalized to protein concentration.
(G) Immunofluorescent detection of HuD, PB marker Dcp1a, and SG marker TIAR in bTC6 cells that were stimulated with glucose as explained in (A). Bar, 10 mm.
Graphs depict the quantification of signal intensities in the segment indicated (red arrows). In the graphs of (B) and (D)–(F), errors represent SEM.
HuD Represses Insulin Translation
832 Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc.
Figure 4. Higher Insulin in HuD–/– Mice; Lower Insulin and Diminished Glucose Tolerance in HuD-Overexpressing Mice
(A) Left, immunofluorescent detection of insulin and HuD in pancreatic islets from HuD?/?, HuD+/?, and HuD+/+ mice; nuclei were visualized by TO-PRO-3
staining. Right, insulin fluorescence was assessed from more than ten sections per animal; n = 3 animals per genotype, >118 islets per genotype.
(B) Western blot analysis of HuD levels in whole pancreas (200 mg) and brain (40 mg) of HuD+/+ and HuD?/? mice.
(C–E) Proinsulin levels were assessed by western blot analysis (C), mature insulin levels by ELISA (D), and Ins2 mRNA levels by RT-qPCR (E) in total pancreatic
lysates from HuD+/+, HuD+/?, and HuD?/? mice.
HuD Represses Insulin Translation
Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc. 833
consistent with the hyperglycemic phenotype of 375KO mice.
Whether miR-375/HuD levels contribute to diabetes also
deserves future investigation.
Cell Culture, Transfection, and Treatment
bIRWT and bIRKO cells (Kulkarni et al., 1999; Assmann et al., 2009; Kim et al.,
2011) and mouse insulinoma bTC6 cells were cultured in high-glucose DMEM
(Invitrogen) supplemented with 10% FBS. Cells were cultured for 16 hr in low-
glucose (2 mM) DMEM before stimulation with the indicated glucose concen-
trations and in low glucose with 0.1% FBS before stimulation with insulin for
24 hr. Chemicals and transfection of siRNA and plasmids are described in
Analysis of RNPs (RIP and Biotin Pull-Down) and Chromatin (ChIP)
Immunoprecipitation (IP) of endogenous RNP complexes from whole-cell
extracts and biotin pull-down analysis were performed as described (supple-
mental text) using anti-HuD or control IgG antibodies (Santa Cruz Biotech-
nology). The RNA isolated from IP was further assessed by quantitative
real-time PCR (RT-qPCR) analysis. Chromatin immunoprecipitation (ChIP)
from bTC6 cells was performed using the EZ-ChIP chromatin IP kit (Millipore,
Billerica, MA) following the manufacturer’s protocol, using anti-FoxO1 anti-
body (Abcam) and HuD promoter-specific primers (SA Bioscience). Further
description is provided in Supplemental Information.
Pancreatic sections of normal (wild-type) C57BL/6J mice were used for detec-
tion of insulin, HuD, and glucagon. HuD?/?, HuD+/? and HuD?/? mice (Aka-
matsu et al.,2005)wereusedat 14weeks ofage. HuD Tgmicewere described
(Bolognani et al., 2006). Mice were anesthetized and tissues were quickly
excised and frozen in liquid nitrogen for western blot analysis and ELISA,
or fixed in 4% paraformaldehyde for immunostaining. For IPGTT, glucose
(1 g/kg body weight) was given intraperitoneally to fasted, 3-month-old female
mice for measurement of blood glucose and plasma insulin levels at the indi-
cated times (details in Supplemental Information). All animal protocols were
approved by the Gerontology Research Center animal care and use
committee of the National Institute on Aging, National Institutes of Health.
Quantitative dataarepresented asthemean±SEMandcompared statistically
by Student’s t test, using Graphpad Prism (GraphPad Software). A p value
of < 0.05 was considered statistically significant.
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at doi:10.1016/
We thank H.J. Okano, M. Igarashi, R. Selimyan, D. Nines, and D. Boyer for
assistance and H. Huang for reagents. This work was supported in part by
the National Institute on Aging-Intramural Research Program, National Insti-
tutes of Health (NIH). R.N.K. is supported by NIH grants RO1 DK 67536 and
68721. H.O. and W.A. are funded by the Japanese Ministry of Education,
Science, Sports, Culture and Technology. E.K.L. is funded by the Korean
Ministry of Education, Science and Technology (5-2011-A0154-00046).
Received: August 8, 2011
Revised: November 21, 2011
Accepted: January 3, 2012
Published online: March 1, 2012
Abdelmohsen, K., Pullmann, R., Jr., Lal, A., Kim, H.H., Galban, S., Yang, X.,
Blethrow, J.D., Walker, M., Shubert, J., Gillespie, D.A., et al. (2007).
Phosphorylation of HuR by Chk2 regulates SIRT1 expression. Mol. Cell 25,
(2010). miR-375 inhibits differentiation of neurites by lowering HuD levels. Mol.
Cell. Biol. 30, 4197–4210.
Akamatsu, W., Fujihara, H., Mitsuhashi, T., Yano, M., Shibata, S., Hayakawa,
Y., Okano, H.J., Sakakibara, S., Takano, H., Takano, T., et al. (2005). The RNA-
Acad. Sci. USA 102, 4625–4630.
Assmann, A., Ueki, K., Winnay, J.N., Kadowaki, T., and Kulkarni, R.N. (2009).
Glucose effects on b-cell growth and survival require activation of insulin
receptors and insulin receptor substrate 2. Mol. Cell. Biol. 29, 3219–3228.
Bolognani, F., Tanner, D.C., Merhege, M., Desche ˆnes-Furry, J., Jasmin, B.,
and Perrone-Bizzozero, N.I. (2006). In vivo post-transcriptional regulation of
GAP-43 mRNA by overexpression of the RNA-binding protein HuD.
J. Neurochem. 96, 790–801.
Goodge, K.A., and Hutton, J.C. (2000). Translational regulation of proinsulin
biosynthesis and proinsulin conversion in the pancreatic b-cell. Semin. Cell
Dev. Biol. 11, 235–242.
Hinman, M.N., and Lou, H. (2008). Diverse molecular functions of Hu proteins.
Cell. Mol. Life Sci. 65, 3168–3181.
Itoh, N., and Okamoto, H. (1980). Translational control of proinsulin synthesis
by glucose. Nature 283, 100–102.
Jahr, H., Schro ¨der, D., Ziegler, B., Ziegler, M., and Zu ¨hlke, H. (1980).
Transcriptional and translational control of glucose-stimulated (pro)insulin
biosynthesis. Eur. J. Biochem. 110, 499–505.
Kim, W., Doyle, M.E., Liu, Z., Lao, Q., Shin, Y.K., Carlson, O.D., Kim, H.S.,
Thomas, S., Napora, J.K., Lee, E.K., et al. (2011). Cannabinoids inhibit insulin
receptor signaling in pancreatic b-cells. Diabetes 60, 1198–1209.
Kulkarni, R.N., Bru ¨ning, J.C., Winnay, J.N., Postic, C., Magnuson, M.A., and
Kahn, C.R. (1999). Tissue-specific knockout of the insulin receptor in pancre-
atic b cells creates an insulin secretory defect similar to that in type 2 diabetes.
Cell 96, 329–339.
Kullmann, M., Go ¨pfert, U., Siewe, B., and Hengst, L. (2002). ELAV/Hu proteins
inhibit p27 translation via an IRES element in the p27 50UTR. Genes Dev. 16,
Muralidharan, B., Bakthavachalu, B., Pathak, A., and Seshadri, V. (2007). A
minimal element in 50UTR of insulin mRNA mediates its translational regulation
by glucose. FEBS Lett. 581, 4103–4108.
(F andG)Immunofluorescent detectionofHuD-mycand insulininpancreaticisletsfrom HuDTgandWT mice.(F) HuD-mycwasvisualized withanti-myc (left)and
anti-HuD (right) antibodies. (G) Insulin fluorescence was quantified and plotted (187 islets from HuD WT mice and 169 islets from HuD Tg mice); n = 5 animals per
(H) Western blot analysis of whole pancreas lysates prepared from HuD WT and HuD Tg mice (200 mg, two mice per group).
(I) Intraperitoneal glucose tolerance test (IPGTT) in 3-month-old female HuD WT and HuD Tg mice, injected with 1g/kg (i.p.). Plasma glucose (top) and insulin
(bottom) levels were measured at the times shown. *p < 0.05, **p < 0.01.
(J) Schematic of HuD expression and influence—positive (green) and negative (red)—on insulin biosynthesis. Bars in (A), (F), and (G) = 50 mm. In the graphs of (A),
(D), (E), (G), and (I), errors represent SEM.
HuD Represses Insulin Translation
834 Molecular Cell 45, 826–835, March 30, 2012 ª2012 Elsevier Inc.
Pascale, A., Amadio, M., Scapagnini, G., Lanni, C., Racchi, M., Provenzani, A., Download full-text
Govoni, S., Alkon, D.L., and Quattrone, A. (2005). Neuronal ELAV proteins
enhance mRNA stability by a PKCalpha-dependent pathway. Proc. Natl.
Acad. Sci. USA 102, 12065–12070.
Pascale, A.,Gusev,P.A., Amadio, M.,Dottorini, T.,Govoni, S.,Alkon,D.L.,and
Quattrone, A. (2004). Increase of the RNA-binding protein HuD and posttran-
scriptional up-regulation of the GAP-43 gene during spatial memory. Proc.
Natl. Acad. Sci. USA 101, 1217–1222.
Poy, M.N., Hausser, J., Trajkovski, M., Braun, M., Collins, S., Rorsman, P.,
Zavolan, M., and Stoffel, M. (2009). miR-375 maintains normal pancreatic a-
and b-cell mass. Proc. Natl. Acad. Sci. USA 106, 5813–5818.
Ratti, A., Fallini, C., Colombrita, C., Pascale, A., Laforenza, U., Quattrone, A.,
and Silani, V. (2008). Post-transcriptional regulation of neuro-oncological
ventral antigen 1 by the neuronal RNA-binding proteins ELAV. J. Biol. Chem.
Rhodes, C.J., and White,M.F.(2002).Molecularinsights intoinsulin actionand
secretion. Eur. J. Clin. Invest. 32 (Suppl 3), 3–13.
Tillmar, L., and Welsh, N. (2002). Hypoxia may increase rat insulin mRNA
levels by promoting binding of the polypyrimidine tract-binding protein (PTB)
to the pyrimidine-rich insulin mRNA 30-untranslated region. Mol. Med. 8,
Wicksteed, B., Herbert, T.P., Alarcon, C., Lingohr, M.K., Moss, L.G., and
Rhodes, C.J. (2001). Cooperativity between the preproinsulin mRNA untrans-
lated regions is necessary for glucose-stimulated translation. J. Biol. Chem.
Wicksteed, B., Uchizono, Y., Alarcon, C., McCuaig, J.F., Shalev, A., and
Rhodes, C.J. (2007). A cis-element in the 50untranslated region of the pre-
proinsulin mRNA (ppIGE) is required for glucose regulation of proinsulin trans-
lation. Cell Metab. 5, 221–227.
Zhang, H., Pan, Y., Zheng, L., Choe, C., Lindgren, B., Jensen, E.D.,
Westendorf, J.J., Cheng, L., and Huang, H. (2011). FOXO1 inhibits Runx2 tran-
scriptional activity and prostate cancer cell migration and invasion. Cancer
Res. 71, 3257–3267.
HuD Represses Insulin Translation
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