THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 48, pp. 34627–34637, November 29, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Deficiency of a Glycogen Synthase-associated Protein,
Epm2aip1, Causes Decreased Glycogen Synthesis andHepatic Insulin Resistance*□
S

Received for publication, May 6, 2013, and in revised form, October 7, 2013 Published, JBC Papers in Press, October 18, 2013, DOI 10.1074/jbc.M113.483198

Julie Turnbull‡§1, Erica Tiberia‡, Sandra Pereira¶, Xiaochu Zhao‡, Nela Pencea‡储, Anne L. Wheeler**, Wen Qin Yu¶,
Alexander Ivovic¶, Taline Naranian‡, Nyrie Israelian‡, Arman Draginov‡, Mark Piliguian‡, Paul W. Frankland**,
Peixiang Wang‡, Cameron A. Ackerley储, Adria Giacca¶, and Berge A. Minassian‡ ‡‡2
From the ‡Program in Genetics and Genome Biology, 储Department of Pathology and Laboratory Medicine, **Program in
Neurosciences and Mental Health, and ‡‡Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario M5G 1X8,
Canada and the §Department of Molecular Genetics and ¶Department of Medicine and Physiology, University of Toronto,
Toronto, Ontario M5S 1A1, Canada

Glycogen synthesis is a major component of the insulin
response, and defective glycogen synthesis is a major portionof insulin resistance. Insulin regulates glycogen synthase(GS) through incompletely defined pathways that activate the
enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs
allosteric activation of GS by glucose 6-phosphate, decreases
hepatic glycogen synthesis, increases liver fat, causes hepatic
insulin resistance, and protects against age-related obesity. Our
work identifies a novel GS-associated GS activity-modulating
component of insulin resistance.

Under physiological conditions, the entirety of carbohydrate
from a meal is rapidly absorbed and, except for what is directly
consumed, is immediately packaged into glycogen (1). Glycogen synthase (GS)3, encoded by the GYS2 gene in liver and theGYS1 gene in muscle and other tissues, is the rate-limiting
enzyme of glycogen synthesis. GS is activated allosterically by
glucose 6-phosphate (G6P), the main intracellular form of glucose, and much less potently by dephosphorylation (2). Activation of GS is a principal function of insulin, and in insulinresistant states, a major portion of the resistance stems from theimpaired ability of insulin to activate GS (3–5). Insulin activates* This work was supported in part by the Canadian Institutes of HealthResearch.
This article contains supplemental Figs. S1–S4, Methods, Data, andReferences.
1
Supported by a Canada Graduate Scholarship from the National Sciencesand Engineering Research Council.
2
Holder of the Michael Bahen Chair in Epilepsy Research, University ofToronto. To whom correspondence should be addressed. Tel.: 416-8137721; E-mail: berge.minassian@sickkids.ca.
3
The abbreviations used are: GS, glycogen synthase; G6P, glucose 6-phosphate;
LGSKO, liver glycogen synthase knock-out; MGSKO, muscle glycogen synthase knock-out.
□S

NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

GS through both the allosteric and dephosphorylation routes,
the former through raising the levels of G6P. In skeletal muscle,
insulin raises intracellular G6P mainly by translocating GLUT4
to the sarcolemma to increase glucose import and conversion
to G6P. In liver, glucose transport is not regulated by insulin
and is unrestricted, equivalent to free diffusion (6, 7). In thatorgan, insulin raises intracellular G6P transcriptionally, including by increasing expression of glucokinase and decreasing
expression of glucose 6-phosphatase (6). Insulin activation of
GS via dephosphorylation proceeds through inhibition of glycogen synthase kinase 3 (GSK3) (insulin receptor 3 Irs1/2 3
PI3K 3 Akt ⵬ GSK3) and other GS kinases (8, 9), and promotion of GS dephosphorylation by targeting protein phosphatase
1 (PP1) to GS (10) through adaptor proteins (PTG, GL, GM, RGL,or R6) that bind GS and PP1 (11).
Although GS determines the amount of glycogen, glycogenbranching enzyme confers glycogen its unique spherical structure. Every six glucose units added by GS to a glycogen strand
are detached by the branching enzyme as a hexamer and reattached upstream to the side of the strand through an ␣1– 6
linkage. Repetitions of GS and branching enzyme actions
expand the molecule radially into a dense soluble sphere containing up to 55,000 glucosyl residues. Branching enzyme deficiency (type IV glycogenosis) results in malformed glycogenmolecules (polyglucosans) that resemble plant starch, with long
strands and poor branching, and that, like starch, are insoluble,
and precipitate and aggregate into large cytoplasmic masses
that lead to hepatic cirrhosis and/or cardiac, skeletal muscle,
and neurological disease (2, 12). Polyglucosans also form in asecond disease, Lafora disease, in which the liver, muscle, heart,and brain exhibit increased amounts of glycogen along with thepolyglucosan masses (13, 14). In the brain, polyglucosans overtake neuronal dendritic cytoplasms and provoke intractableneurodegeneration, epilepsy, and death because of massive
convulsions (13). Lafora disease is caused by mutations in the
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Background: Impaired activation of glycogen synthesis is a major component of insulin resistance.
Results: We identified a protein, Epm2aip1, that associates with glycogen synthase (GS) and whose absence impairs allostericactivation of GS and causes hepatic insulin resistance.
Conclusion: Epm2aip1 is a modulator of GS activity under insulin control.
Significance: This study uncovers a novel component of glycogen regulation and hepatic insulin resistance.

Epm2aip1 Regulates Glycogen Synthesis
EPM2A gene, encoding a glycogen binding phosphatase (laforin), or the EPM2B gene that encodes an E3 ubiquitin ligase(malin) that interacts with laforin (13, 15). The pathogenesis of
Lafora disease remains unsettled. The results to date support
two main hypotheses. 1) The laforin-malin complex regulates
PTG and GS, and the absence of laforin or malin leads toincreased GS activity, excessive elongation of glycogen strands,
and conversion of GS to polyglucosan (16, 17). 2) The laforinmalin complex regulates glycogen phosphorylation, its deficiencies resulting in glycogen hyperphosphorylation and consequent precipitation and gradual conversion to polyglucosan(14, 18).
Here, we characterize Epm2a-interacting protein 1 (Epm2aip1),
a laforin-interacting protein (19) of unknown function. We findthat Epm2aip1 associates with GS and that its absence results in
reduced allosteric activation of GS by G6P and in hepatic insulin
resistance.

EXPERIMENTAL PROCEDURES

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Generation of Epm2aip1⫺/⫺ Mice—The Epm2aip1 genetrapped embryonic stem cell line (BA0314) was obtained fromthe Sanger Institute Gene Trap Resource (SIGTR) (Fig. 1A). ES
cells were injected into C57/BL6 blastocysts. Chimeras werebred to C57/BL6 mice and screened for positive transmission.
Heterozygous mice were interbred to produce Epm2aip1⫺/⫺
progeny (KO mice). Except where indicated, mice were 4 – 6
months old in all experiments. All glycogen metabolic studies
were performed at 6 h refeeding following a 24-hour fast, except
where noted. Animal procedures were approved by The Hospital for Sick Children and The Toronto Centre for Phenogenomics Animal Care Committees.
Microscopy, Western Blotting, and Immunoprecipitation—
Standard electron and light microscopic methods were used as
described previously (20). For immunofluorescence, 3T3-L1
cells were grown on glass coverslips to confluence and fixed
briefly in phosphate-buffered 4% paraformaldehyde followedby permeabilization with 0.1% Triton X-100. Cells were thenwashed thoroughly in PBS and blocked in 0.5% BSA prior to
incubation with the Epm2aip1 antibody for 1 h. Cells were thenincubated with goat anti-mouse IgG antibody conjugated to
Cy3 (Jackson ImmunoResearch Laboratories). Nuclei were
stained with DAPI (Sigma), coverslips were mounted on glass
slides in n-propyl gallate (Sigma). Images were acquired on a
Quorum spinning disk microscope (Zeiss Axiovert 200) using a
63⫻ water objective (Zeiss) with a numerical aperture of 1.2
equipped with a Hamamatsu C9100-13 electron multiplying
charge-coupled device camera. Images were collected and processed with Volocity 4.4.1. (Improvision Ltd.)
Monoclonal Epm2aip1 antibody was from Abnova, and GS
and p641GS antibodies were from Cell Signaling Technology.
Lysates for Western blot analyses were prepared from tissues
using homogenization buffer and protease/phosphatase inhibitors (Roche). To assess the amount of glycogen-bound GS,
lysates were centrifuged at 200,000 ⫻ g to obtain the glycogenpellet after an initial 1,500 ⫻ g centrifugation to remove cellulardebris. Protein ratios in each fraction were calculated by normalizing to GAPDH levels using TotalLab Quant.

Immunoprecipitation was performed as described previously (20) using an HA-tagged Epm2aip1 construct (19) and a
Myc-tagged GS construct containing the full-length coding
sequence of Gys1 in pcDNA3.1 (Invitrogen).
Biochemical Assays—Glycogen levels were measured as
described previously (18). Glycogen synthase activity wasmeasured in the presence of varying concentrations of its allosteric activator, G6P, as listed for each experiment. Protein samples were added to a reaction mixture containing 50 mM TrisHCl (pH 8.0), 20 mM EDTA, 25 mM potassium fluoride, 10
mg/ml glycogen, 7.2 mM UDP-glucose, 0.5 ␮Ci/ml UDP[14C]glucose, and the stated concentration of G6P. Reactions
were incubated at 37°C for 10 –30 min and purified on G50
columns (21). The amount of radiolabeled glucose incorporation into glycogen was calculated using a scintillation counter.
Fractional activity was calculated as the ratio between low G6P
and high G6P. For GS assays using skeletal muscle after exhaustive exercise, mice were trained for 4 days (3 min/day) on atreadmill. On the first training day, the treadmill incline was set
to 5°, and the speed was set at 8 m/min. Speed was increased by
1 m/min during the training session to 10 m/min. For eachfollowing training day, the incline was increased by 5°, and theinitial speed was increased by 1 m/min. Following training,mice were fasted for 16 h and then placed on the treadmill to
run at 12 m/min at an incline of 20° for 2.5 h or until exhaustion,
as judged by refusal to remain on the treadmill. A mild electricalstimulus (16 –28 V) was applied to mice that stepped off thetreadmill belt occasionally, or a plastic straw was used to brushthe mice.
Insulin and Glucose Tolerance Tests—For insulin tolerance
tests, mice were fasted for 4 h before receiving intraperitoneal
injection of insulin at time 0. Male mice were given 0.75 milliunit/g body weight insulin and female mice 0.5 milliunit/g bodyweight insulin (Humulin, Eli Lilly). Blood glucose levels were
measured at 0, 15, 30, 60, and 120 min (Abbott). For glucose
tolerance tests, mice were fasted overnight (14 –16 h). Glucose
was injected intraperitoneally at 2 mg/g body weight (18, 22),and blood glucose levels were measured at 0, 15, 30, 60, and 120
min. Areas under or above the curve were calculated as
described previously (23).
Hyperinsulinemic-Euglycemic Clamps—Hyperinsulinemiceuglycemic clamps were performed as described previously
described (24). Mice fasted for 3 h were given a primed infusionof [3-3H] glucose at ⫺120 min, and basal glucose production
was measured from ⫺30 to 0 min. At time 0, a continuous
infusion of insulin (3 milliunits/kg/min) was started, andplasma glucose was maintained constant via a variable glucose
infusion adjusted according to plasma glucose levels obtained
every 10 min. The glucose infusion was radiolabeled to minimize changes in plasma glucose-specific activity. Endogenous
glucose production and utilization were determined in the last
30 min of the 120-min clamp.
Statistical Analysis—Data are shown as mean ⫾ S.E. Significance was evaluated using an unpaired Student’s t test, andvalues were considered significant at p ⬍ 0.05 unless statedotherwise stated.

Epm2aip1 Regulates Glycogen Synthesis

RESULTS
Epm2aip1⫺/⫺ Mice Exhibit Hepatic Glycogen Deficiency—
To probe for Epm2aip1 functions, we generated mice lacking
Epm2aip1 using a gene-trapped embryonic stem cell line. The
trap (␤geo vector) had inserted into the extreme 5⬘ end of thesingle exon of Epm2aip1, disrupting the gene (Fig. 1A).
Epm2aip1⫺/⫺ pups are born at expected Mendelian frequencies and are fertile. DNA analysis by PCR confirms the insertionand the absence of a WT Epm2aip1 sequence (Fig. 1B). Immunohistochemistry and Western blotting show a complete lackof the protein (Fig. 1C), indicating a null allele.
Epm2a⫺/⫺ and Epm2b⫺/⫺ mice replicate human Lafora
disease, exhibiting increased glycogen, profuse polyglucosan
accumulations in the brain and other tissues, epilepsy, ataxia,
motor deficits, and anxiety (18, 25, 26). We tested whether
Epm2aip1⫺/⫺ mice also have symptoms of Lafora disease. The
mice did exhibit heightened anxiety on the basis of responses in
the open field and startle tests but none of the cardinal symptoms of the disease (epilepsy, motor deficits, or ataxia). They, infact, outperformed WT animals on the rotarod (supplemental
Figs. S1–S3). Pathologically, periodic acid-Schiff staining, thestandard histochemical stain for glycogen and polyglucosan
bodies, showed no increase in glycogen and no polyglucosan
bodies in any tissues, even at 12 months of age (supplemental
Fig. S4). Collectively, these results indicated that Epm2aip1⫺/⫺
mice do not have Lafora disease. Rather than increased, liver
glycogen content was actually decreased (Figs. 2, A and B). Fatcontent was also abnormal in Epm2aip1⫺/⫺ livers, in this caseincreased (Figs. 2, C–F). Skeletal muscle, the other major glyNOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

FIGURE 2. Decreased glycogen and increased lipid in livers of
Epm2aip1ⴚ/ⴚ mice. A, periodic acid-Schiff staining of wild-type liver. Notethe relatively homogenous staining of the acid mucosubstances (glycogen)in the majority of the liver parenchymal cells. B, periodic acid-Schiff staining of
Epm2aip1⫺/⫺ mouse liver. Large areas of the liver parenchyma have little or
no staining (asterisks). These areas are predominantly in zone 1 (periportal) of
the liver lobule (note the portal triad in the center of the main glycogen-poor
region), which is the zone that is repleted last and depleted first during normal cycles of liver glycogen formation-consumption (29). C, hematoxylin andeosin staining of WT liver at 2 months of age. D, a similar area in Epm2aip1⫺/⫺
mouse liver at the same age as above. Note the focal patches of steatosis (lipid
accumulation) (asterisks). E, electron micrograph of WT liver. F, a similar areafrom an Epm2aip1⫺/⫺ mouse liver. Note the numerous lipid droplets (asterisks) throughout the cell.

cogen storing organ, had normal glycogen content and no lipid
accumulation.
Epm2aip1⫺/⫺ Mice Have Reduced Glycogen Synthesis in
Liver—To investigate the reduced liver glycogen, we asked
whether it was due, at least in part, to reduced glycogen synthesis. We fasted Epm2aip1⫺/⫺ and WT mice for 24 h, which
depletes liver glycogen, and then refed them for 6 h, which
normally fully replenishes the glycogen (27). In WT mice, liver
glycogen fully replenished, whereas in Epm2aip1⫺/⫺ mice it did
not. Epm2aip1⫺/⫺ livers resynthesized less than half the quantity of glycogen generated by WT animals (Fig. 3A), confirming
the presence of reduced glycogen synthesis in Epm2aip1⫺/⫺
livers.
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FIGURE 1. Generation and characterization of the Epm2aip1ⴚ/ⴚ nullmouse line. A, schematic of the Epm2aip1 gene trap allele. The En2-SA␤geo-pA trap (En2, 1.5 kb of the En2 gene; SA, splice acceptor site; pA, poly(A)tail; ␤geo, ␤-galactosidase-neomycin resistance fusion) inserts near the startof the single Epm2aip1 exon. Ab, epitope region of the Epm2aip1 antibody
used throughout this study; IP1-F, IP1-R, and ␤geo-R, locations of PCR primers
used in experiment in B. B, confirmation of Epm2aip1 gene disruption. C,
immunohistochemical (brain, hippocampal region) and Western blot confirmation of Epm2aip1 absence. Three different mice per genotype are shownin the Western blots. Scale bars ⫽ 500 ␮m.

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FIGURE 3. Reduced hepatic glycogen synthesis, increased blood glucose clearance, hepatic insulin resistance, and other metabolic results. A, liver
glycogen levels at base line, 24 h fasted, and after 6 h of refeeding (␮mol glucose/g tissue). n ⫽ 3–5 mice/genotype. B, blood glucose levels after a 16-hour fast
(mmol/liter). n ⫽ 15–17 mice/genotype. C, insulin tolerance test. n ⫽ 8 –11 mice/genotype. Inset, area above the curve (AUC). D, glucose tolerance test. Glucose
was injected immediately after time 0. Note the much lower blood glucose in Epm2aip1⫺/⫺ mice 15 min following injection. n ⫽ 15–17 mice/genotype. ***, p ⬍0.001; *, p ⬍ 0.05. Body weights were not significantly different between genotypes. Inset, area under the curve (AUC). p ⬍ 1 ⫻ 10⫺4. E, endogenous (hepatic)
glucose production (EGP) during the hyperinsulinemic-euglycemic clamp. Note lack of reduction in EGP in Epm2aip1⫺/⫺ mice following insulin infusion (after
time 0 min). Left inset, insulin values at the end of the clamp (120 min). Right inset, percent suppression of EGP (percentage drop in EGP during the clamp(average of the last three time points) from basal EGP levels (average of the first three time points). Lack of suppression was significant at p ⬍ 0.007. n ⫽ 5– 6
mice/genotype. F, whole-body glucose disposal during the hyperinsulinemic-euglycemic clamp. Basal glucose disposal and disposal during insulin infusion
are shown. n ⫽ 5– 6 mice/genotype. Avg, average.

Base-line blood glucose, cholesterol, triglycerides, and insulin were normal in Epm2aip1⫺/⫺ mice. Blood glucose following
a 16-hour fast was reduced significantly (Fig. 3B), consistent
with the low liver glycogen stores, the main stores consumed tomaintain blood glucose during overnight fasts in mice (22). The
insulin tolerance test (the degree of drop in blood glucose following insulin injection) was normal (Fig. 3C). The rate of clear-

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ance of injected glucose in the glucose tolerance test wasincreased (Fig. 3D).
Epm2aip1⫺/⫺ Mice Have Hepatic Insulin Resistance—Reduced glycogen synthesis is a major component of insulin
resistance (3–5). To determine whether Epm2aip1⫺/⫺ mice
are insulin-resistant, we performed a euglycemic-hyperinsulinemic clamp experiment. In this experiment, euglycemia
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FIGURE 4. The absence of Epm2aip1 impairs hepatic GS activation by G6P. A, example Western blot analysis of hepatic GS. Note higher-phosphorylation(top) and lower-phosphorylation (bottom) bands. B, densitometric quantification of total hepatic GS (sums of higher-phosphorylation and lower-phosphorylation bands). Protein levels were normalized by dividing with GAPDH. n ⫽ 5–7 mice/genotype. C, hepatic GS activity at low and high G6P. n ⫽ 8 mice/
genotype. n.s., not significant. D, the activity ratio (GS activity at low G6P divided by GS activity at high G6P), a standard measure for reporting the active GS
fraction of total possible GS activity in a sample, on the basis of the usually invariant full activation of GS by high G6P. In this situation, the latter fails, and theactivity ratio is inapplicable. The ratio is high, not because GS activity at low G6P (numerator) is high but because GS activity at high G6P (denominator) is
impaired. E, densitometric quantification of the higher-phosphorylation GS band. n ⫽ 5–7 mice/genotype. F, Western blot analysis of hepatic GS using a
specific antibody against GS phosphorylated at serine 641. This antibody also is known to detect two bands, likely GS proteins with different degrees ofphosphorylation outside of serine 641. Equal amounts of protein were run on two separate gels for phospho-GS (p641GS) and GS. G, Western blot analysis ofhepatic GS, using the antibody against total GS, run long in a low percentage (10%) polyacrylamide gel. Note that the upper band migrates faster in
Epm2aip1⫺/⫺ mice.

(achieved by infusing varying amounts of glucose) and a constant amount of insulin (3 milliunits/kg/min) are maintained, and whole-body glucose utilization and endogenous
glucose production are measured (28). Insulin resistancecomprises impaired whole-body glucose disposal (mainly amuscle and liver function) and impaired endogenous glucose
production (mainly a liver function). Of note, endogenous
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glucose production is highly dependent on GS activity. The
liver constantly breaks down glycogen (glycogenolysis) andconstructs glucose (gluconeogenesis) to supply blood glucose while, at the same time, removing excess glucose into
glycogen (29, 30). The amount of glucose supplied to theblood by the liver is regulated through the rate of glycogensynthesis. How much glucose reaches the blood (endogeJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 5. Epm2aip1 subcellular locations. A, confocal image of an immunofluorescent Epm2aip1-stained 3T3-L1 cell. Note the widespread punctate
staining throughout the cytoplasm as well as in a reticular and perinuclear
fashion (arrows). Note also the presence of the protein within the nucleus(arrowheads). B, electron micrograph of an immunogold-labeled ultrathinliver cryosection using the Epm2aip1 antibody. Note the clusters of gold particles (8 nm) in close proximity to nuclear pores (arrows). A cluster is also seen
within the nucleus (arrowhead). C, immunogold labeling in the cytoplasm of ahepatocyte at the endoplasmic reticulum (asterisk). D, immunogold labelingin a glycogen-rich region of liver (arrows).

Epm2aip1⫺/⫺ livers. Phosphorylation does not appear to be
involved. If anything, the enzyme is hypophosphorylated, possibly a compensatory response in the face of the impaired allosterism, allowing achievement of WT GS activity levels at low
G6P, but not at high G6P (Fig. 4C).
Epm2aip1 Associates with GS—Having shown that Epm2aip1
affects GS activity, we investigated whether it does so throughassociation with the enzyme. Toward this end, we first determined the subcellular location of Epm2aip1. The protein localizes mainly in the cytoplasm (Figs. 5 and 6, C–F) but also in the
nucleus (Fig. 5, A and B). In the cytoplasm, it is not present
inside any organelle. Instead, it is distributed in glycogen-richregions (Figs. 5D (from liver) and 6, C–F (from skeletal muscle)), and it is present at the endoplasmic reticulum (Fig. 5C)
and at the nuclear membrane (Fig. 5, A and B), including inclusters at the nuclear membrane pores (Fig. 5B). Both the glycogen-rich cytoplasm and the nucleus are cell compartments inwhich GS is also located (2, 16).
To determine whether Epm2aip1 associates with GS, we
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Downloaded from http://www.jbc.org/ by guest on January 1, 2016nous glucose production) depends, in a major part, on GS
activity, regulated by insulin (30).
Whole-body glucose utilization of Epm2aip1⫺/⫺ mice in theclamp experiment was normal (Fig. 3F). Therefore, despite therapid rate in clearing excess blood glucose in the glucose tolerance test, the overall quantity of blood glucose they are able toremove in response to insulin is unchanged.
Normally, endogenous glucose production drops precipitously during the clamp experiment (i.e. glucose production by
the liver is suppressed) because the external glucose and insulin
infusions drive hepatic GS maximally. Hepatic insulin resistance is diagnosed when this response does not occur (7, 28, 30).
Our WT controls exhibited the expected normal response,
whereas the Epm2aip1⫺/⫺ mice had complete failure of endogenous glucose production suppression (Fig. 3E) and, therefore,
have hepatic insulin resistance.
Hepatic GS Activation by G6P Is Greatly Reduced in
Epm2aip1⫺/⫺ Mice—Blocked hepatic glycogen synthesis at atime when it should normally be highly activated, i.e. during
feeding after fasting or during the hyperinsulinemia of theclamp, could be explained by a deficiency in the quantity of GS
or a defect in its activity or activation. GS Western blot analyses
detect a double band composed of higher-phosphorylation (lessactive) upper and lower-phosphorylation (more active) lower
bands (Fig. 4A) (31). We quantified total hepatic GS by quantifying the two bands together densitometrically and found nodifference between Epm2aip1⫺/⫺ and WT mice (Fig. 4B). We
also quantified glycogen-bound GS following ultracentrifugation of the glycogen pellet and, again, found no differences
between genotypes. GS amounts in Epm2aip1⫺/⫺ livers are,therefore, not reduced.
We next examined whether there is a defect in GS activity or
activation. We measured GS activity from liver extracts in thepresence of low (0.3 mM) and high (6.7 mM) G6P. Activity at low
G6P reflects GS activity as set by the degree of GS phosphorylation. Activity at high G6P, which overrides any inhibition
through phosphorylation, indicates maximal GS activity in aparticular sample (2). At low G6P, there was no difference in GS
activity between Epm2aip1⫺/⫺ and WT mice, but at high G6P,
Epm2aip1⫺/⫺ mice exhibited a major deficit in GS activity (Fig.
4, C–D), indicating that Epm2aip1⫺/⫺ mice have a major defectin allosteric activation of GS by G6P.
We next queried whether the second arm of GS regulation,
phosphorylation, is affected. The quantity of the higher-phosphorylation GS band was not increased in the Epm2aip1⫺/⫺
mice (Fig. 4E). GS phosphorylated at serine 641, the main GSK3
phosphorylation site of GS, which reflects overall phosphorylation of the enzyme (32), was normal (Fig. 4F). Differences inlevels of phosphorylation can be distinguished by running protein samples for longer periods of time in low-concentration
gels, with more highly phosphorylated proteins migratingslower (8, 33). When we ran our Western blot analyses in thisfashion we observed that the higher-phosphorylation GS
band of Epm2aip1⫺/⫺ mice runs faster, not slower, than its
WT counterpart (Fig. 4G), indicating that it has reduced, notincreased, phosphorylation.
The results in this section support a role for a defect in GS
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FIGURE 6. Epm2aip1 and GS associate. A, immunoprecipitation (IP) of Epm2aip1 by GS, and vice versa, from transfected cells. B, immunoprecipitation ofendogenous Epm2aip1 by GS, and vice versa, from mouse brain and liver. WB, Western blot. C, ultrathin cryosection of skeletal muscle from WT muscle
double-labeled with the Epm2aip1 (15-nm particles) and GS (8-nm particles) antibodies. Note the intense colocalization in particle clusters in the perinuclear
cytoplasm and in the spaces between the sarcomeres. D, subsarcolemmal colocalization of Epm2aip1 and GS. E, colocalization of Epm2aip1 and GS in thesarcoplasm. F, ultrathin cryosection of skeletal muscle from an Epm2aip1⫺/⫺ mouse. No Epm2aip1 signal was detected. GS was found in similar clusters to thoseseen in the WT mouse muscle.

tion using antibodies against one coprecipitated the other (Fig.
6A). Likewise, without overexpression, immunoprecipitating
endogenous GS from mouse tissues coprecipitated Epm2aip1
and vice versa (Fig. 6B), indicating that the two proteins interact
or closely associate in a complex and that, therefore, the resistance of GS to G6P activation in Epm2aip1⫺/⫺ mice is likely
due to loss of a direct or near-direct effect of Epm2aip1 on GS.
Electron microscopy following immunogold labeling of endogenous Epm2aip1 and GS confirmed the close physical association of the two proteins (Figs. 6, C–F).
Impaired Activation of GS by G6P Is Also Present in Skeletal
Muscle—Analyzing Epm2aip1⫺/⫺ skeletal muscle revealed nodiminution in glycogen at the base line or upon refeeding after
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fasting, the latter not unexpected because fasting alone does not
significantly reduce muscle glycogen (34) and did not do so inthese animals. The total quantity of GS and the fraction of GS
associated with glycogen were also normal.
GS activation by G6P was not impaired, although there
appeared to be a possible trend (Figs. 7, A and B). Noting thelatter, we took an additional step and asked whether G6P activation of GS might be significantly abnormal under conditions
where GS is maximally active. We fasted the mice for 16 h, ran
them on a treadmill until exhaustion, which greatly reduces muscle glycogen (34), and then refed them for 1 h before sacrifice, i.e.
the mice were sacrificed in the midst of maximal glycogen resynthesis. Under these conditions, we observed a robustly significant
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reduction in the ability of G6P to activate GS in Epm2aip1⫺/⫺
mice (Fig. 7C), confirming that the effect of Epm2aip1 absence on
GS activation in liver is also present in skeletal muscle.
Epm2aip1⫺/⫺ Mice Are Resistant to Age-dependent Obesity—
As mentioned, GS has two isoforms, one specific to liver(GYS2), the other to skeletal muscle and other tissues (GYS1)(2). As discussed below, many features of the Epm2aip1⫺/⫺
mice are shared between mice lacking Gys2 (LGSKO) (22) andmice lacking Gys1 (MGSKO) (35). Normally, adult laboratory mice fed standard chow gain weight and become obesewith age, which is true of LGSKO mice (22) but not of MGSKO
mice. The latter did not gain weight in adult life, despite
consuming normal amounts of food (35). To determine
whether adult Epm2aip1⫺/⫺ mice gain weight with age inadulthood, we weighed a group of mice every 2 months over
1 year. Between 2 and 12 months, WT animals gained anaverage of 22.44 ⫾ 3.60 g, whereas Epm2aip1⫺/⫺ mice
gained only 9.85 ⫾ 1.24 g (Fig. 8). The amount of food consumed by the two genotypes was the same (Epm2aip1⫺/⫺,0.21 ⫾ 0.03; WT, 0.23 ⫾ 0.07 g/day/g body weight).
Epm2aip1⫺/⫺ mice are therefore, like MGSKO mice, resistant to age-related weight gain in adulthood.

DISCUSSION
Insulin moves glucose entering the circulation into appropriate storage and processing. At the same time, and equally

34634 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 8. Epm2aip1ⴚ/ⴚ mice exhibit an absence of weight gain with age
in adulthood. n ⫽ 5– 6 mice/genotype. *, p ⬍ 0. 05; **, p ⬍ 0.01.

importantly, it suppresses endogenous glucose production andsecretion into the bloodstream, primarily by increasing hepatic
glycogen synthesis. Insulin resistance comprises impaired glucose disposal and impaired suppression of glucose secretion.
Identifying the proteins involved in these processes is, therefore, critical to understanding insulin resistance. The two principal glucose disposal tissues are skeletal muscle and liver.
Knockout of the insulin receptor, or of GS, from mouse skeletal
muscle alone has little or no impact on whole-body glucose
disposal even while affecting muscle glucose disposal (35–38).
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FIGURE 7. GS activation by G6P is also impaired in skeletal muscle. A, skeletal muscle GS activity with low and high G6P. n ⫽ 6 mice/genotype. n.s., notsignificant. B, GS activity ratio of the results in A: GS activity at low G6P divided by that at high G6P. Note that the activity ratio is high, not because the activity
at low G6P is high but because the activity at high G6P, i.e. the ability of G6P to activate GS, is low. n ⫽ 6 mice/genotype. C, skeletal muscle GS activity at 1-h
refeeding following glycogen depletion by fasting and exhaustive exercise. n ⫽ 6 mice/genotype.

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on glycogen synthesis likely requires non-transcriptional
mechanisms. GS in tissues of patients with type 2 diabetes has
reduced activity and is hyperphosphorylated (2, 8 –10, 47). The
latter observation focused searches for pathomechanisms oftype 2 diabetes on disturbances affecting the phosphorylationof the enzyme. Although part of the answer to insulin resistancemust involve GS phosphorylation, the literature is equally
replete with evidence that, in type 2 diabetics, allosteric activation of GS by G6P is also impaired to a major degree (48 –52).
Uncovering the molecular components underlying this failure
is important in understanding insulin resistance. The micestudied here, lacking the Epm2aip1 protein, have failed GS
allosteric activation and hepatic insulin resistance. Epm2aip1,
which associates with GS, may, therefore, be a modulator of GS
allosterism under insulin control. Its precise mode of action andregulation awaits further study.
Acknowledgments—We thank Drs. J. Rommens and S. Meyn forreview of our manuscript.

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Removal of the insulin receptor, or GS, from the liver alone
impairs the suppression of endogenous glucose provision to theblood and causes hepatic insulin resistance (22, 39). We identified a ubiquitously expressed protein, Epm2aip1, that associates with GS and whose absence impairs GS activation andcauses hepatic insulin resistance.
Epm2aip1⫺/⫺ mice share features of LGSKO mice (22),including reduced hepatic glycogen synthesis, hepatic insulin
resistance, fasting hypoglycemia, and hepatic fat accumulation,
the latter occurring earlier in Epm2aip1⫺/⫺ (at 2 months) thanin LGSKO (at 7 months). Reduced hepatic glycogen synthesis,
hepatic insulin resistance, and hepatic fat accumulation also
occur with disturbances of hepatic insulin signaling. When theinsulin receptor, or its downstream signaling substrates Irs1
and Irs2, were completely removed from mouse liver, glycogensynthesis was greatly reduced, with no associated lipid accumulation (39, 40), but when Irs1 and Irs2 were down-regulated but
not eliminated, glycogen synthesis was partially reduced, andhepatic lipid accumulation occurred at 2 months of age (41).
Thus, the effect of absent Epm2aip1 on hepatic glycogenesis
and lipogenesis is most similar to that of partial impairment of
hepatic insulin signaling.
Epm2aip1⫺/⫺ mice also share features of MGSKO mice (35),
namely resistance against age-dependent obesity and a rapid
rate of clearance of exogenous glucose. The mechanisms
underlying the former remain unknown. Results in MGSKO
mice preliminarily suggest heightened oxidative capacity (35).
Uncovering these mechanisms in MGSKO and Epm2aip1⫺/⫺
mice is of obvious importance. The rapid rate of glucose clearance in response to a rise in glucose is also unexplained but may
reflect an increased ability to dispose of glucose via insulinindependent mechanisms, i.e. increased glucose effectiveness(42). Neither MGSKO (35) nor Epm2aip1⫺/⫺ or LGSKO mice(22) have reduced whole-body glucose utilization in response
to insulin, indicating that partial loss of glycogen synthesis
through complete loss in only muscle (MGSKO) or in only
liver (LGSKO) or through partial loss across both organs(Epm2aip1⫺/⫺) does not, alone, suffice to affect the overall
ability of insulin to clear glucose from the blood.
Although it has long been known that allosteric activation of
GS is far more potent than dephosphorylation in vitro, it was
not confirmed in vivo until recently. Mutating a key amino acidin the GS allosteric site led to 80% loss of insulin-mediatedglycogen synthesis without alteration of the phosphorylation of
the enzyme (36). In this work, we identify a protein, Epm2aip1,that modulates the allosteric response of GS to G6P, possibly
through direct action given its association with GS. Its action
appears to not involve phosphoregulation because the inhibited
enzyme in its absence is hypophosphorylated. Finally, in theabsence of Epm2aip1, the sensitivity of the liver to insulin, in
which GS is a principal actor, is impaired. Until now, the only
known way by which insulin regulates the allosteric action of
GS by G6P is through increasing the amount of G6P. In liver,
although glucokinase and glucose 6-phosphatase are subject tonon-transcriptional regulation during refeeding (43– 45), theonly known mechanism whereby insulin itself affects these
enzymes and increases G6P are transcriptional, e.g. increasing
glucokinase expression (6, 46). The immediacy of insulin actionEpm2aip1 Regulates Glycogen Synthesis

34636 JOURNAL OF BIOLOGICAL CHEMISTRY

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Supplementary methods and data
Supplementary methods – Behavioural tests
For open field test, mice were placed in the center for 20 min, tracked by camera linked to a computer
system using LimeLight software (Coulbourn Instruments, Allentown, PA), and the time in each zone (s):1- outside, 2-middle, 3-center & total distance travelled (cm) measured.
To measure startle response, an acoustic startle reflex system (Med Associates Inc. St. Albans,VT) was used. Four standard cages were placed in sound-attenuated chambers with each cage being a
Plexiglas cylinder 3cm in diameter, mounted on a platform connected to an analogue-digital converter.
Background noise and acoustic bursts were conveyed through two speakers placed in proximity to thestartle. Both speakers and startle cages were connected to a main PC, which detected and analyzed allchamber variables with custom software. Mice were acclimatized to apparatus for 5 minutes thenpresented with 80 startle stimuli (120 dB noise bursts) with an inter-stimulus interval of 15 s (backgroundnoise was 68 dB throughout session) and startle amplitude (V) measured.
Pre pulse inhibition apparatus was same as used in startle response tests. A 5-minute acclimation
period, during which only the 68 dB background noise was presented, then a set of 20 startle stimuli (120dB, 40 ms long, 15 s interval) followed by the prepulse session consisting of 90 trials each having a 20 ms
pre-pulse tone, a 40 ms startle tone of 120 dB and a 100 ms inter-tone interval (onset to onset). There
were 3 different pre-pulse tone amplitudes: 70, 75, 80 dB as well as no stimulus (0 dB) trials and startle
alone (120 dB) trials, each presented 10 times. Inter-trial interval was 15 sec. The maximum startle
response (Vmax) was measured during a 40-ms sampling window after presentation of the 120 dB startle
tone. The percent of prepulse inhibition was calculated as 100 – [(average Vmax of prepulse trials/
average Vmax of startle alone trials) x 100] for each prepulse level.
For rotarod tests, mice were habituated to the procedure on day 0, each trained until all met thecriteria of 1 min at a constant speed of 4rpm (if not reached, mice were removed from the study). 3 daysof testing followed with 3 trials per day using an accelerated rotarod protocol (4 to 40 rpm over 10 min).
Mean time to fall (s) on each test day was measured.
To measure foot splay, mouse feet were dipped in ink and the mouse was dropped from 30 cm
onto a blank paper (1). The distance between feet (cm) was measured from 2 trials averaged.
To test gait, mice trained to run through a passage to a goal box for 2 days. The next day, they
were tested by dipping feet in ink (forepaws and hindpaws tested separately) and the base width and stride
length measured (4 largest measurements for each averaged) (2).
The grid test was performed by placing mice on a 40cmx40cm grid with 15x15mm squares, andvideotaping while walking on grid for 3 minutes. The number of hind paw slips were counted for eachmouse (3).
For hanging grip, mice were held at the base of the tail and allowed to grasp the rod with theirfront paws. They were suspended 30 cm, and 3 trials were performed on 1 day (max time per trial 60 sec).
The time to drop (s) was measured for each mouse (4).
For negative geotaxis, mice were placed on a platform at a 45 degree incline. The mean time toturn around 180 degrees was measured (4).
Fear conditioning experiments were conducted in a windowless room containing 4 stainless steel
conditioning chambers (31 cm × 24 cm × 21 cm; Med Associates, St. Albans, VT), containing a stainless
1steel shock-grid floor. Shock grid bars (diameter 3.2 mm) were spaced 7.9 mm apart. The grid floor was
positioned over a stainless-steel drop-pan, which was lightly cleaned with 70% ethyl alcohol to provide abackground odour. The front, top, and back of the chamber were made of clear acrylic and the two sides
made of modular aluminum. Day 1- training consisted of placing mice in a conditioning chamber and,two min later, presenting a tone (2800 Hz, 30 s, 85 dB) that co-terminated with a shock (2 s, 0.6 mA).
Mice remained in the chamber for 30 s after shock delivery. Day 2- context test: returned to context for 5
minutes. Day 3- cue (tone) test: Mice were placed in a novel chamber and two min later, the tone waspresented for 3 min. The index of memory (freezing behaviour) was monitored via four overheadcameras. Freezing was assessed using an automated scoring system (Actimetrics, Wilmette, IL), which
digitized the video signal at 4 Hz and compared movement frame by frame to determine the amount offreezing.
For the Morris water maze test, a circular water maze tank (120 cm in diameter, 50 cm deep),
located in a dimly-lit room was used. The pool was filled to a depth of 40 cm with water made opaque byadding white, nontoxic paint. Water temperature was maintained at 28 ± 1°C by a heating pad beneath thepool. A circular escape platform (10 cm diameter) was submerged 0.5 cm below the water surface, in a
fixed position in one quadrant. The pool was surrounded by curtains, at least 1 m from the perimeter ofthe pool. The curtains were white with distinct cues painted on them. Prior to commencing training, mice
were individually handled for 2 min each day over seven consecutive days. Mice were trained over fivedays. On each training day, mice received six training trials (presented in two blocks of three trials;
interblock interval was 1 h, intertrial interval was 15 s). On each trial they were placed into the pool,facing the wall, in one of four start locations. The order of these start locations was pseudorandomly
varied throughout training. The trial was complete once the mouse found the platform or 60 s had elapsed.
If the mouse failed to find the platform on a given trial, the experimenter guided the mouse onto theplatform. Following the completion of training, spatial memory was assessed in a probe test the next day.
In this test the platform was removed from the pool, and the mouse was allowed 60 s to search for it.
Behavioral data from training and the probe tests were acquired and analyzed using an automated tracking
system (Actimetrics, Wilmette, IL). Measurements were made of time to reach platform (a measure oflearning across days) during training. During in probe testing, performance was quantified by the amountof time mice searched the target zone (20 cm radius, centered on the location of the platform during
training) vs. the average of three other equivalent zones in other areas of the pool. These zones each
represent approximately 11% of the total pool surface (a measure of memory for platform location) (5).

Supplementary References
1.
2.
3.
4.
5.

Mohammad, F. K., Faris, G. A., Rhayma, M. S., and Ahmed, K. (2006) Neurotoxicology 27, 153157
Iancu, R., Mohapel, P., Brundin, P., and Paul, G. (2005) Behav Brain Res 162, 1-10
Starkey, M. L., Barritt, A. W., Yip, P. K., Davies, M., Hamers, F. P., McMahon, S. B., andBradbury, E. J. (2005) Exp Neurol 195, 524-539
Rogers, E. H., Hunter, E. S., 3rd, Moser, V. C., Phillips, P. M., Herkovits, J., Munoz, L., Hall, L.
L., and Chernoff, N. (2005) J Appl Toxicol 25, 527-534
Stone, S. S., Teixeira, C. M., Zaslavsky, K., Wheeler, A. L., Martinez-Canabal, A., Wang, A. H.,
Sakaguchi, M., Lozano, A. M., and Frankland, P. W. (2010) Hippocampus Epub: 2010/09/09

Supplementary figure 1. Neurobehavioral testing of Epm2aip1-/- mice.
2

(A–B) Open field test. Measure of anxiety by measuring time spent in each zone (seconds) and total
distance traveled in cm; n = 10 per genotype.
(C) Acoustic startle response. Reflex to protective startle in response to a sudden acoustic stimulus,
displayed as mean startle amplitude (V). n = 10 per genotype.
(D) Prepulse inhibition. Sensorimotor gating measurement using % of prepulse inhibition; n = 10 per
genotype.
(E–F) Fear conditioning. Context and cue (tone) fear memory to determine amount of freezing (% timespent freezing); n = 10 per genotype.
(G–H) Morris water maze. Spatial memory test showing time to reach platform (training) and amount oftime spent searching for target zone (probe); n = 10 per genotype.

Supplementary figure 2. Neuromotor testing in Epm2aip1-/- mice.
(A) Rotarod. Motor coordination and/or fatigue shown by mean time to fall on each test day (seconds).
Parametric ANOVA statistical analyses also showed significant differences among genotypes; p <0.05; n = 10 per genotype.
(B) Grid test. Deficits in voluntary motor control shown by number of hind paw slips per mouse; n = 10
per genotype.
(C) Hanging grip test. Muscle strength measured by time to drop (seconds); n = 10 per genotype.
(D) Negative geotaxis. Neuromotor coordination tested by calculating time for each mouse to turn around180 degrees after being placed facing downwards; n = 10 per genotype.
(E) Foot splay analysis. Evaluation of peripheral neuropathy and/or central nervous system depression bymeasuring distance between splayed feet after dropping from a height of 30 cm; n = 10 per genotype.
Supplementary figure 3. Gait testing in Epm2aip1-/- mice.
(A–B) Forelimb gait analysis. Motor impairment tested by measuring stride length and base width(distance between foot prints in cm); n = 10 per genotype.
(C–D) Hindlimb gait analysis. Motor impairment tested by measuring stride length and base width(distance between foot prints in cm); n = 10 per genotype.

Supplementary figure 4. Epm2aip1-/- mice do not have Lafora disease (12 months-old).
(A) Hippocampus from an Epm2aip1-/- mouse stained with PASD(B) Hippocampus from a mouse with LD (malin-deficient) stained with PASD; note abundant
polyglucosan bodies (numerous PASD-positive granules in the neuropil around asterisks).
(C) Muscle cross section from an Epm2aip1-/- mouse stained with PAS-D(D) Muscle from a mouse with LD (laforin-deficient) stained with PAS-D. Note abundant polyglucosan
bodies.

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Metabolism:
Deficiency of a GlycogenSynthase-associated Protein, Epm2aip1,
Causes Decreased Glycogen Synthesis andHepatic Insulin Resistance

J. Biol. Chem. 2013, 288:34627-34637.
doi: 10.1074/jbc.M113.483198 originally published online October 18, 2013

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