Journal Club

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Journal Club
Moslemi AR, Lindberg C, Nilsson J, Tajsharghi H,
Andersson B, Oldfors A. Glycogenin-1 deficiency
and inactivated priming of glycogen synthesis. N
Engl J Med. 2010 Apr 1362(13)1203-10. Lee IM,
Djoussé L, Sesso HD, Wang L, Buring JE. Physical
activity and weight gain prevention. JAMA. 2010
Mar 24303(12)1173-9.
2010?4?8? 830-855 8? ??

• ?????? ???????? ????????
• Department of Endocrinology and Diabetes,
• Saitama Medical Center, Saitama Medical
  University
• ?? ??
• Matsuda, Masafumi

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From the Department of Pathology, Institute of
Biomedicine (A.-R.M., J.N., H.T., A.O.),
Department of Neurology, Institute of Physiology
and Neurological Sciences (C.L.), and Department
of Cardiology, Institute of Medicine (B.A.),
University of Gothenburg, Gothenburg, Sweden.
N Engl J Med 20103621203-10.
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Aim
Glycogen, which serves as a major energy reserve
in cells, is a large, branched polymer of glucose
molecules. We describe a patient who had muscle
weakness, associated with the depletion of
glycogen in skeletal muscle, and cardiac
arrhythmia, associated with the accumulation of
abnormal storage material in the heart.
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Schematic illustration of glycogen synthesis.
Unglucosylated dimers of apoglycogenin- 1 are
autoglucosylated by an initial glucose-1-O-tyrosin
e linkage at Tyr195 (NM_004130), followed by
addition of approximately 10 glucose molecules.
This glycogenin oligosaccharide molecule
constitutes the primer for synthesis of glycogen
catalyzed by glycogen synthase and branching
enzyme.
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Methods
Analysis of genomic DNA Total DNA was extracted
using the DNeasy Tissue Kit or the DNA Blood Mini
Kit (Qiagen, Hilden, Germany). For PCR
amplification we used primers that amplified the
8 exons of GYG1 (GenBank accession number
NM_004130) with flanking intronic sequences
(Supplementary Appendix, Table 2). The conditions
for PCR were as follows an initial denaturing
step at 94C for 3 min, followed by 35 cycles of
94C denaturing for 1 min, 57C primer annealing
for 1 min, 72C primer extension for 1 min and a
final extension step of 72C for 10 min. PCR
amplifications were performed with a GeneAmp PCR
system 2700 (Applied Biosystems, Foster City,
CA). Sequencing was performed using an ABI Prism
377 DNA sequencer and the Big Dye Terminator Kit
v. 1. 1. (Applied Biosystems, Foster City, CA)
and analyzed with MacVector software 8.1.1. The
coding sequences of GYS1, GBE1, GYG2 and PRKAG2
were amplified and sequenced in the same fashion.
The missense mutation 248CgtT in exon 3 of GYG1
eliminated a restriction site for endonuclease
Tsp45I (New Englands Biolabs, Beverly, MA). For
RFLP analysis of this mutation in genomic DNA, a
301-bp fragment was amplified using the forward
and reverse exon 3 primers. Digestion was
performed with 10 units of the enzyme at 37C for
two hours. The amplified 301-bp fragment of
genomic DNA was digested into three fragments of
139, 154 and 8 bp, respectively, of normal DNA
and into two fragments of 293 and 8 bp,
respectively, of DNA carrying the 248CgtT
mutation. The fragments were then separated on a
2.5 agarose gel stained with GelStar and
visualized on a Dark Reader Blue light
transilluminator (Clare Chemical Research,
Dolores, CO). Analysis of cDNA For RFLP
analysis of the GYG1 248CgtT mutation in cDNA,
total RNA was extracted using RNAqueous-4PCR kit
(Ambion, Austin, TX) and complementary DNA (cDNA)
was synthesized using Ready-To-Go You-Prime
First-Strand Beads (Amersham Biosciences,
Buckinghamshire, UK). A 395-bp PCR product was
amplified from GYG1 cDNA from the patient,
parents and controls by using forward (F112) and
reverse (R507) primers (GenBank accession number
NM_004130). Restriction enzyme cleavage with
Tsp45I and visualization of the results were
performed as described above for genomic DNA. The
amplified 395-bp fragment of cDNA was cleaved
into two fragments of 243 and 152 bp,
respectively, of wild type cDNA.
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Tissue culturing Muscle tissue specimens were cut
into small pieces and seeded in flasks with
Dulbeccos Modified Eagles Medium (DMEM),
containing high glucose and glutamine levels and
supplemented with 10 fetal calf serum and 1
penicillin. After 8-15 days the cells were
detached enzymatically and plated in Petri dishes
for proliferation. After expansion entailing 2-3
subcultures during two weeks, the cells were
grown to 80 confluence and fusion was induced by
switching to DMEM supplemented with 2 horse
serum. The myoblasts and myotubes were then
incubated overnight at 37C in chamber slides.
Western blot analysis For Western blot analysis,
cultured muscle cells or cryostat sections of
skeletal muscle and cardiac muscle, each ten µm
thick, were homogenized in 100 µl Laemmli sample
buffer supplemented with 5 ß-mercaptoethanol. To
remove sugar residues from glycogenin, samples
were treated with alpha-amylase (Sigma, St.
Louis, MO), which hydrolyzes the internal a-1,4
glycosidic linkages of glycogen and
autoglucosylated glycogenin. Total protein
samples were loaded and separated on 3-8
Tris-Acetate or 10 Bis-Tris gels (Invitrogen,
Carlsbad, CA) followed by electroblotting onto
Invitrolon PVDF filters at 20 V for 10 minutes.
The membrane was incubated with primary
monoclonal mouse anti-human glycogenin-1 antibody
(Abnova, Taipei City,Taiwan) 1500, for one hour.
Western Breeze (Invitrogen, Carlsbad, CA) was
used for antibody detection. The same protocol
was used with the following primary antibodies
for Western blot analysis of other enzymes
associated with glycogen synthesis Anti-PRKAG2
(Atlas Antibodies, Stockholm, Sweden) 160, GBE1
(B01) (Abnova, Taipei City, Taiwan) 1200 and
Anti- Glycogen Synthase (Millipore, Temecula, CA)
11000. Recombinant protein expression and
purification The full-length muscle isoform of
glycogenin-1 was amplified from wild type and
patient II4s muscle cDNA (with the 248CgtT
mutation) with the forward primer 5-
GGATCCATGACAGATCAGGCCTT-3 and the reverse primer
5- CTCGAGCTGGAGGTAAGTGTCA-3. The fragment was
cloned into the pCDNA6/His Myc vector
(Invitrogen, Carlsbad, USA) using BamHI and XhoI
restriction enzymes. The final constructs were
sequenced to confirm the expected sequences.
Chinese-hamster-ovary (CHO K1) cells were
cultured in Iscoves Modified Dulbeccos Medium
(Lonza Biologicals, Basel, Switzerland),
supplemented with 10 Fetal Bovine Serum (Lonza
Biologicals, Basel, Switzerland) in 6-well
culture plates (Falcon). Transfection of the
vector was performed using Lipofectamine 2000
(Invitrogen, Carlsbad, USA), according to the
manufacturer's instructions. The cells were
harvested after 48 h and the His6-tagged
recombinant glycogenin- 1 was purified using
Ni-NTA spin columns (Qiagen, Hilden, Germany), in
accordance with the manufacturers instructions.
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A 27-year-old man had dizziness and palpitations
shortly after exercising, and emergency medical
services were called. When the ambulance arrived,
the patient was noted to have ventricular
fibrillation, which was converted to sinus rhythm
by cardiac defibrillation. After admission to the
hospital, the patient had several short bursts of
non sustained ventricular tachycardia.
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Cardiac magnetic resonance imaging in short axis
view, showing a late enhancement area in
mid-septum (arrow head) and an extensive area of
late enhancement in the inferior wall
(arrow). Courtesy Dr Carl Lamm, MD, PhD.
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Panel A demonstrates DNA sequence chromatograms
from genomic DNA and cDNA with the identified
mutations in exon 3, 248CgtT, which results in the
amino acid change Thr83Met, and in exon 5,
487delG, which results in a frame shift and a
premature stop codon at amino acid position 167,
Asp163ThrfsX5. Only the allele with the 248CgtT
mutation is expressed at the mRNA level, as
determined by the cDNA sequence. Panels B
(Normal control) and C (patient II4) illustrate
myoblasts and myotubes in tissue culture after
staining with PAS reagent for glycogen. There is
a deficiency of glycogen in the cells of patient
II4.
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In Panel E the presence of glycogenin-1 in
cultured myoblasts and myotubes is identified by
Western blot analysis. In myoblasts from a normal
control, a weak band of normal autoglucosylated
glycogenin-1 can be identified. After
alpha-amylase treatment to remove the
polysaccharide chains from glycogen and
glycogenin, a strong band of unglucosylated
glycogenin-1 appears in the control. These
glycogenin-1 molecules are approximately 1 kD
smaller than the glycogenin-1 molecules
identified without alpha-amylase treatment. In
cultured myoblasts from patients with glycogen
deficiency due to lack of glycogen synthase,11
normal autoglucosylated glycogenin-1 is
identified without alpha-amylase treatment.
Alphaamylase treatment reduces the size of the
molecule by approximately 1 kD. Analysis of
glycogenin-1, without alpha-amylase treatment, in
cultured myoblasts from patient II4 demonstrates
accumulation of glycogenin-1 with a molecular
weight equal to that of normal glycogenin-1 after
alpha-amylase treatment. The cultured cells of
the father I2 contain a larger proportion of
unglucosylated glycogenin-1, compared to control
myoblasts, which is consistent with the
heterozygous expression of the Thr83Met mutation.
Panel F illustrates the results of
immunoblotting of three enzymes associated with
glycogen synthesis. Patient II4, who has the
glycogenin-1 mutation, does not show any obvious
upregulation of these enzymes. This is also the
case for the patient with glycogen depletion due
to glycogen synthase deficiency.
Panel D illustrates results from RFLP analysis of
cDNA from cultured myoblasts, using endonuclease
Tsp45I that cleaves wild-type DNA, leaving DNA
with the 248CgtT (Thr83Met) mutation uncleaved.
Only the mutated allele is expressed in the
patients cultured myoblasts, whereas his father
(I2 in the pedigree) is heterozygous for the
Thr83Met mutation. The myoblasts of the mother
(I1 of the pedigree), who does not carry this
mutation, shows the same RFLP pattern as cultured
myoblasts from a normal control.
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Assessment of cardiac wall thickness and chamber
dimensions by magnetic resonance imaging.
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Figure 1. Histochemical Images of Muscle-Biopsy
Specimens. Muscle-biopsy specimens stained with
periodic acidSchiff reagent show strikingly less
glycogen in the muscle fibers of the patient
(Panel A) than in the fibers of the father (Panel
B) and mother (Panel C), which contain normal
levels. Panel D (with staining for myosin ATPase
at pH 4.3) shows a marked predominance of dark,
slow-twitch, oxidative (type 1) fibers in a
specimen from the patient Panel E shows fibers
from a normal control. Panel F (with staining for
succinate dehydrogenase) shows mitochondrial
accumulation, especially in the subsarcolemmal
region, in a specimen from the patient Panel G
shows a specimen from a normal control. The bars
represent 30 µm.
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Figure 2. Histochemical and Ultrastructural
Images of Myocardial-Biopsy Specimens. A
myocardial-biopsy specimen from the patients
right ventricle, shown in Panels A (hematoxylin
and eosin), B (periodic acidSchiff PAS reagent
for glycogen), D, and E, is characterized by
myocyte hypertrophy and large vacuoles (arrows)
with PAS-positive material lacking the normal
ultrastructural appearance of glycogen. Panel C
(PAS reagent) shows evenly distributed glycogen
in the intermyofibrillar network in a specimen
from a normal control. The electron micrograph in
Panel D shows one myocyte with a large vacuole
(arrow), including scattered mitochondria
(arrowheads) Panel E shows a mitochondrion (Mit)
and the predominantly unstructured appearance of
the storage material in the vacuole, which also
contains lipid droplets (Lip) and small
membrane-bound structures (arrowheads). An
electron micrograph of a myocardial-biopsy
specimen from a control subject, in Panel F,
shows normal glycogen granules (arrows).
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Figure 4. Protein Analyses. Panel A shows the
results of Western blot analysis of glycogenin-1
in skeletal muscle from a normal control, a
patient with glycogen storage disease (Pompes
disease due to alpha-glucosidase deficiency), a
patient with a lack of glycogen due to glycogen
synthase deficiency,11 and the index patient in
this study (Patient II-4). Without alpha-amylase
treatment of the sample (-), glycogenin-1 was
detectable only in the two persons who lacked
glycogen. The size of the glycogenin-1 in these
two persons differed by approximately 1 kD, since
the glycogenin-1 was not autoglucosylated in the
patient, who had the GYG1 Thr83Met mutation.
Panel B shows the presence of glycogenin-1 in
muscle identified by Western blot analyses
performed with () and without (-) alpha-amylase
treatment. In normal skeletal muscle,
glycogenin-1 cannot be seen unless the sample is
treated with alpha-amylase to remove the sugar
residues from the huge glycogen molecules. This
treatment will also hydrolyze the internal
a-1,4-glycosidic linkages between the
autoglucosylated residues. In patients with
deficient glycogen due to a lack of glycogen
synthase,11 normal autoglucosylated glycogenin-1
can be shown without alpha-amylase treatment.
Alpha-amylase treatment will reduce the size of
the molecule by approximately 1 kD. Analysis of
glycogenin-1 in the muscle of the patient (II-4),
without alpha-amylase treatment, showed
accumulation of glycogenin-1 with a molecular
weight corresponding to that of normal
glycogenin-1 after alpha-amylase treatment,
demonstrating that the patients glycogenin-1 was
unglucosylated. The muscle tissue from the father
(I-2) contained a small proportion of
unglucosylated glycogenin-1, visible without
alpha-amylase treatment, which is compatible with
the heterozygous expression of the allele
carrying the Thr83Met mutation. In Panel C,
Western blot analysis performed without treatment
with alpha-amylase (-) shows accumulation of
unglucosylated glycogenin-1 in cardiac tissue
from the patient (II-4). In myocardium from
control subjects, a small proportion of free
autoglucosylated glycogenin-1 could be detected
without alpha-amylase treatment, since the gel
was loaded with a large amount of protein (as
revealed by the myosin heavy-chain band). Panel D
shows the results of Western blot analyses of
human recombinant glycogenin-1. Wild-type and
mutant (Thr83Met) GYG1 was expressed in
Chinese-hamster-ovary cells. In this system,
autoglucosylation will occur, but only of
wild-type glycogenin-1, as shown by alpha-amylase
treatment, which hydrolyzes any existing
a-1,4-glycosidic linkages in autoglucosylated
glycogenin-1. Treatment with alpha-amylase (),
as compared with no treatment (-), reduced the
molecular weight of wild-type glycogenin-1 but
not of Thr83Met mutant glycogenin-1, which
demonstrates the inability of the mutant protein
to autoglucosylate. The recombinant glycogenin-1
was tagged with 6-histidine for purification,
resulting in an estimated molecular weight of
approximately 40 kD.
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After examination and diagnostic testing, the
patient underwent placement of an implantable
cardioverterdefibrillator. Pharmacologic
treatment was initiated with a ß1-adrenergic-recep
tor blocker and an angiotensin-convertingenzyme
inhibitor. The patient has had no further cardiac
arrhythmias and no clinical signs or symptoms of
heart failure. At follow-up 1 year after the
acute episode, his physical capacity was
categorized as New York Heart Association
functional class I or II.
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Results
The skeletal muscle showed a marked predominance
of slow-twitch, oxidative muscle fibers and
mitochondrial proliferation. Western blotting
showed the presence of unglucosylated
glycogenin-1 in the muscle and heart. Sequencing
of the glycogenin-1 gene, GYG1, revealed a
nonsense mutation in one allele and a missense
mutation, Thr83Met, in the other.
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Conclusion
In summary, we have described a metabolic disease
that is due to a deficiency of glycogenin-1. The
disease affects the priming of glycogen synthesis
and results in glycogen depletion and
accumulation of abnormal storage material in the
heart. The missense mutation resulted in
inactivation of the autoglucosylation of
glycogenin-1 that is necessary for the priming of
glycogen synthesis in muscle.
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Division of Preventive Medicine (Drs Lee, Sesso,
Wang, and Buring) and Aging (Drs Djousse ,
Sesso, and Buring), Department of Medicine,
Brigham and Womens Hospital, Harvard Medical
School Department of Epidemiology, Harvard
School of Public Health (Drs Lee and Buring)
Department of Ambulatory Care and Prevention,
Harvard Medical School (Dr Buring), and
Massachusetts Veterans Epidemiology and Research
Information Center, Boston Veterans Affairs
Healthcare System (Dr Djousse ) Boston,
Massachusetts.
JAMA. 2010303(12)1173-1179
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MET metabolic equivalent
Physical Activity MET Light Intensity
Activities lt 3 sleeping 0.9 watching
television 1.0 writing, desk work,
typing 1.8 walking, less than 2.0 mph (3.2 km/h),
level ground, strolling, very slow 2.0 Moderate
Intensity Activities 3 to 6 bicycling,
stationary, 50 watts, very light
effort 3.0 calisthenics, home exercise, light or
moderate effort, general 3.5 bicycling, lt10 mph
(16 km/h), leisure, to work or for
pleasure 4.0 bicycling, stationary, 100 watts,
light effort 5.5 Vigorous Intensity Activities gt
6 jogging, general 7.0 calisthenics (e.g.
pushups, situps, pullups,jumping jacks), heavy,
vigorous effort 8.0 running jogging, in place 8.0
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Background
Context The amount of physical activity needed
to prevent long-term weight gain is unclear. In
2008, federal guidelines recommended at least 150
minutes per week (7.5 metabolic equivalent MET
hours per week) of moderate-intensity activity
for substantial health benefits. Objective To
examine the association of different amounts of
physical activity with long-term weight changes
among women consuming a usual diet.
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Method
Design, Setting, and Participants A prospective
cohort study involving 34 079 healthy US women
(mean age, 54.2 years) from 1992-2007. At
baseline and months 36, 72, 96, 120, 144, and
156, women reported their physical activity and
body weight. Women were classified as expending
less than 7.5, 7.5 to less than 21, and 21 or
more MET hours per week of activity at each time.
Repeated measures regression prospectively
examined physical activity and weight change over
intervals averaging 3 years. Main Outcome
Measure Change in weight.
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Results
Women gained a mean of 2.6 kg throughout the
study. A multivariate analysis comparing women
expending 21 or more MET hours per week with
those expending from 7.5 to less than 21 MET
hours per week showed that the latter group
gained a mean (SD) 0.11 kg (0.04 kg P.003) over
a mean interval of 3 years, and those expending
less than 7.5 MET hours per week gained 0.12 kg
(0.04 P.002). There was a significant
interaction with body mass index (BMI), such that
there was an inverse dose-response relation
between activity levels and weight gain among
women with a BMI of less than 25 (P for
trend.001) but no relation among women with a
BMI from 25 to 29.9 (P for trend.56) or with a
BMI of 30.0 or higher (P for trend.50). A total
of 4540 women (13.3) with a BMI lower than 25 at
study start successfully maintained their weight
by gaining less than 2.3 kg throughout. Their
mean activity level over the study was 21.5 MET
hours per week (60 minutes a day of moderate
intensity activity).
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Conclusion
Among women consuming a usual diet, physical
activity was associated with less weight gain
only among women whose BMI was lower than 25.
Women successful in maintaining normal weight and
gaining fewer than 2.3 kg over 13 years averaged
approximately 60 minutes a day of
moderate-intensity activity throughout the study.
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