Improvements in lipoatrophy, mitochondrial DNA
levels and fat apoptosis after replacing stavudine
with abacavir or zidovudine
Grace A. McComseya,b, Denise M. Paulsenc, J. Tyler Lonergand,
Siegrid M. Hessenthalerc, Charles L. Hoppelb, Vanessa C. Williamsc,
Robin L. Fisherc, Catherine L. Cherrye, Cathy White-Owenb,
Katherine A Thompsone, Steve T. Rossc,
Jaime E. Hernandezcand Lisa L. Rossc
Objective: To determine if stavudine (a4T)-associated mitochondrial toxicity could be
reversed by substitution with another nucleoside reverse transcriptase inhibitor. As
apoptosis and dysfunction of electron transport chain (ETC) activities may underlie
mitochondrial toxicity, these parameters were also evaluated.
Design: The 16 participants (on d4T for >3 years; with lipoatrophy and/or hyperlacta-
temia) substituted abacavir or zidovudine for stavudine in their antiretroviral regimen.
Key parameters including dual-energy X-ray absorptiometry (DEXA) scans, fat apop-
tosis, mitochondrial DNA (mtDNA) content in peripheral blood mononuclear cells
(PBMC), skeletal muscle and fat, as well as skeletal muscle mitochondrial ETC activities
were evaluated at study entry and at 48 weeks after the substitution.
Methods: Quantitative PCR was used to evaluate mtDNA levels and the presence of
deletions/rearrangements; CLIA-validated methods for ETC activities; terminal deox-
ynucleotidyl transferase dUTP-digoxigenin nick-end labeling assays to evaluate adi-
pocyte apoptosis; and DEXA scans to measure changes in body fat.
Results: MtDNA was depleted at study entry in muscle, adipose tissue and PBMC but
levels rebounded with respective mean increases of 141%, 146%, and 369% at week
48. Corresponding fat improvements were noted with DEXA increases of 21%, 11%,
and 16% in arm, leg, and trunk, respectively. Quantitative adipocyte apoptosis were
significantly increased at baseline (P < 0.01 versus HIV-negative controls), with a
significant reduction at week 48 (P < 0.05 versus baseline). Mean values for seven
mitochondrial enzyme activities assays at entry indicated substantial loss of function
(48% to 85% of controls) with significant improvement of complex I activity by
Conclusions: Substitution of stavudine with abacavir or zidovudine improves mito-
chondrial indices and fat apoptosis in the setting of lipoatrophy.
? 2005 Lippincott Williams & Wilkins
AIDS 2005, 19:15–23
Keywords: stavudine, abacavir, mitochondrial DNA, mitochondrial toxicity,
electron transport chain, apoptosis, lipoatrophy
FromaRainbow Babies and Children’s Hospital andbCase School of Medicine Center for AIDS Research, Cleveland, Ohio,
Correspondence to G. McComsey, 11100 Euclid Avenue, mailstop 8A, Cleveland, Ohio 44106, USA.
Received: 29 January 2004; revised: 9 June 2004; accepted: 7 September 2004.
dUniversity of California, San Diego, California, USA; andeMonash University and the Burnet Institute,
ISSN 0269-9370 Q 2005 Lippincott Williams & Wilkins
Nucleoside reverse transcriptase inhibitors (NRTI) have
been shown to cause mitochondrial dysfunction in vitro
[1,2] and several clinical toxicities including lactic
acidosis, hepatic steatosis, peripheral neuropathy, and
myopathy which have been linked to mitochondrial
dysfunction . Recent studies have suggested that
NRTI-related mitochondrial dysfunction plays a major
role in lipoatrophy [4–6] and have identified stavudine
(d4T) as a particular risk factor [7,8]. Indeed, d4T has
been shown to induce a higher degree of mitochondiral
(mt)DNA depletion than other NRTI, such as zidovu-
dine (ZDV) and abacavir (ABC) [9,10]. In addition to
functional mitochondrial abnormalities and fat apoptosis
in subjects with lipoatrophy [4,6,11]. It has been
suggested that fat apoptosis in lipoatrophy could be
linked to cytokine dysregulation .
We and others [13–15] have shown that subcutaneous
lipoatrophy improves after substitution of d4T for ABC
or ZDV. We designed this present study to assess the
of 16 HIV-infected patients who had been on d4T for
over 3 years and who had lipoatrophy and/or hyperlac-
tatemia. Fat apoptosis, mtDNA content in peripheral
muscle mitochondrial electron transport chain (ETC)
activities and cytokine expression in adipose tissue were
evaluated at studyentryand 48 weeks after substitution of
d4Twith ABC or ZDV.
Materials and methods
Subjects in this study were a subgroup of the
study that enrolled 118 HIV-infected subjects with
lipoatrophy and/or hyperlactatemia who had been on
d4T for at least 6 months . Participation in the
substudy was based on the ability to perform biopsies at
the sites and the subjects’ willingness to participate.
Cleveland, Ohio (n ¼ 10), UCSD Antiviral Research
Center in San Diego, California (n ¼ 4), Boriken
Neighborhood Health Center, New York, (n ¼ 1), and
Bronx VA Medical Center, New York, (n ¼ 1). Local
investigators were given the option to substitute either
ABC or ZDV for d4T, and all but two subjects were
switched to ABC. Adipose and muscle tissues were
obtained from HIV-1 negative donors (ZEN-BIO, inc.,
Ardais, ABS) who were otherwise undergoing elective
cosmetic surgery. PBMC were also obtained from HIV-1
negative donors at both Case Western Reserve and
GlaxoSmithKline. No attempt was made to match age
groups for PBMC in the HIV negative volunteers.
Measurements of all indices were taken at baseline (study
entry) during therapy with d4T, and at 48 weeks after
substitution of d4Twith ABC or ZDV. Informed consent
was obtained from patients according to the human
experimentation guidelines of the US Department of
Tissue sampling and DNA extraction
Each subject underwent fat and muscle biopsies at study
entry and had 10 ml of venous blood extracted for
PBMC isolation. Twelve subjects underwent repeat
sampling at week 48 of the study. One subject refused
biopsies but did have PBMC mtDNA performed, and
three discontinued prematurely from the study. The
biopsies were done in the operating room under heavy
sedation with local anesthetic injected only above the
fascia, so not to impair ETC activities. The protocol
called for the incision to be made in the lateral thigh and
the vastus lateralis muscle to be sampled. Subcutaneous
fat was removed during the same surgical procedure. All
tissue samples were stored at ?80 8C and not thawed
prior to analysis, at which time the tissue was carefully
dissected to remove any visible extraneous non-fat tissue.
DNA was extracted from PBMC and biopsies of adipose
and muscle tissue at baseline and at 48 weeks for mtDNA
analysis. As controls, the same tissue types were also
analyzed for mtDNA levels in HIV-uninfected donors in
the same age range. PBMC were isolated from whole
blood using lymphocyte separation media (LSM, ICN/
Cappel, Aurora, Ohio, USA). Cells were then washed
three times in D-PBS at room temperature and
centrifuged at 260 g for 15 min, depleting platelets by
20% prior to extraction. Total DNA was extracted from
approximately 25 mg of tissue or from PBMC isolated
from 2 ml whole blood using the QIAamp DNA Mini
Kit (Qiagen Inc. California, USA).
Assessment of changes in body fat
Changes in physical signs of lipoatrophy were assessed by
full-body dual-energy X-ray absorptiometry (DEXA)
(Hologic QDR-4500A, Hologic, Inc., Waltham, Mas-
sachusetts, USA) at baseline and week 48. At the same
time points, assessments of weight and body mass index
Mitochondrial DNA quantification
Quantitative PCR was performed on serial dilutions (1 to
1:100) of total DNA from PBMC, adipose and muscle
tissue. MtDNA and genomic DNA from experimental
analyzer (Applied Biosystems Inc., California, USA) and
the results compared against standard curves generated
using plasmid controls for both mtDNA and nuclear
genes. The number of mtDNA genomes was calculated
per nuclear genome and converted to mitochondrial
copies per cell for fat and PBMC, and per genomic
equivalent for muscle, which is multi-nucleated .
16AIDS 2005, Vol 19 No 1
Assay for large deletions or rearrangements
Analysis for the presence of large rearrangements and
deletions was performed by amplifying a 120-bp area of
the 16S rRNA gene and a 225-bp area in a region
included in all three of the major mtDNA deletions. Two
primer probe sets: GWmt18F (AAAAGTAAAAGGAA
CTCGGCAAA) / GWmt19R (AGGGTACCGCGGC
CGT) / GWmt20P (FAM-AGCATCACCAGTATTA
GAGGCACCGC-QSY) and GWmt10F (CCTGTAG
CATTGTTCGTTACATGG) / GWmt11R (TGTTGG
CATCTGCTCGGG) / GW14P (FAM-TCATCGGCT
counts were compared to that of pooled putative normal
donors. These results were confirmed using long
extension PCR in separate PCR reactions using three
different mitochondrial primers sets which would result
in PCR products of 6.5, 10.4 and 14.3 kb, respectively if
no rearrangements were present, and 3.9 kb if rearrange-
ments were present.
Adipocyte apoptosis assays
The degree of subcutaneous adipocyte apoptosis was
assessed by terminal deoxynucleotidyl transferase dUTP-
of fat from study subjects and HIV-uninfected controls.
Histology was assessed by a pathologist blinded to date of
specimen collection, as well as to clinical and treatment
details. The degree of apoptosis was graded from 0 (no
TUNEL-positive cells) to 3 (most adipocytes TUNEL
positive). Duplicate slide scores were averaged. In
addition, quantitative analysis of apoptotic adipocytes
per unit area of adipose tissue was performed using video
image analysis by a blinded histologist.
Mitochondrial ETC activities
All tissue samples were stored at ?80 8C. Frozen skeletal
muscle samples were thawed immediately prior to
analysis and homogenates prepared using cholate. ETC
activities were measured using CLIA validated methods
and were performed at the CIDEM laboratory (Case
Western Reserve University, Cleveland, Ohio) which
has a wide experience of more than a decade in
performance of ETC and other mitochondrial assays
Measurements of NADH cytochrome c reductase (NCR)
reflecting activities of complex I and III, NADH
ferricyanide reductase (first three subunits of complex
I), succinate cytochrome c reductase (complex II and III),
succinate dehydrogenase, decylubiquinol cytochrome c
reductase (complex III), cytochrome c oxidase (complex
IV), and citrate synthase (marker of mitochondrial mass)
were performed. In our laboratory, ETC activities
determined by using frozen skeletal muscle were
equivalent to those using fresh tissues (C. Hoppel,
Cytokine expression levels in fat tissues
Levels of interleukin (IL)-1 (a and b), IL-2, IL-4, IL-5,
IL8, IL-10, IL-12 (p35 and p40), IL-15, interferon-g and
tumor necrosis factor (TNF)-a RNA expression were
measured at baseline and week 48. Total RNA was
extracted from 25 mg of adipose tissue using the RNeasy
Mini Kit (Qiagen) and DNA removed using on-column
DNase digestion with the RNase-Free DNase Set
(Qiagen). cDNA was generated from 2 mg RNA from
subject samples and a normal control provided using the
High-Capacity cDNA Archive Kit (Applied Biosystems).
Five-hundred nanograms cDNA was used in each
reaction well of the TaqMan Cytokine Gene Expression
Plate I (Applied Biosystems). Samples were done in
duplicate for each of the 12 cytokines and a normal
human cDNA calibrator was used in each plate. Real-
time PCR was performed on the ABI 7900 and results
analyzed on the Sequence Detection Software v1.7.
The Wilcoxon signed-rank test was used to test whether
the change from baseline to week 48 was different from
zero for the adipose tissue, muscle, and PBMC data. The
95% confidence intervals (CI) were calculated for the
mean mtDNA levels using the normal approximation.
Interquartile ranges were also calculated for the mtDNA
and apoptosis results. These analyseswere not adjusted for
multiple comparisons due to the small sample size and the
descriptive nature of the analyses.
Sixteen subjects participated in the substudy, and 12
underwent repeat tissue sampling at week 48. An
additional subject had PBMC analyzed at week 48.
Demographics for the substudy subjects are presented in
Table 1 along with the 118 subjects who participated in
the main study. At entry, 15/16 (95%) were on a protease
inhibitor (PI) and 7/16 (44%) were on a non-nucleoside
reverse transcriptase inhibitor. Antiretroviral therapy was
maintained throughout the 48 weeks of the study in all
subjects. All 16 substudy subjects and 97% (114/117) of
the main study participants had lipoatrophy. Substudy
subjects were comparable to those in main study with no
notable selection bias. All 16 substudy subjects had been
on d4T for at least 3 years, and seven of them had
hyperlactatemia, with at least one symptom suggestive of
mitochondrial toxicity. Undetectable HIV-1 RNA levels
weremaintainedthroughout thestudywith medianlevels
of 1.69 log10copies/ml for at study entry and week 48.
No subject developed serious adverse events as a result of
the procedures. Most subjects developed self-limited pain
at the site of the incisions. Six subjects developed small
painless seromas at the site of the incision; uncomplicated
needle aspiration was required in one subject.
d4T toxicity improves after switch to ABC McComsey et al.17
Results of serial DEXA scans are shown in Fig. 1. Median
increases from baseline in arm, leg, trunk, and total limb
adipose mass of 21%, 11%, 16%, and 15%, respectively
were seen at week 48 (arm P ¼ 0.101; leg P ¼ 0.101;
trunk P ¼ 0.006; limb fat P ¼ 0.068) from baseline. At
week 48, weight, body mass index, fasting lipids and
pyruvate were not significantly different from baseline.
Lactate levels significantly decreased (change from
baseline ?0.40; P ¼ 0.012).
at study entry week 0 (n ¼ 16) and at week 48 (n ¼ 12).
repeat biopsies at week 48 was lost. The control samples
included PBMC (n ¼ 30), muscle (n ¼ 13), and adipose
tissue (n ¼ 25) from HIV-uninfected donors. Tissue
samples of fat and muscle from uninfected donors were
Fig.2a, themean patient(n ¼ 16) week 0 mtDNA/cellin
PBMC was 64, but rose by week 48 to 256 (n ¼ 13,
P < 0.0001). The mean PBMC mtDNA/cell was 448 in
HIV-negative controls. There is no overlap between the
negative control mtDNA and the week 0 patient mtDNA
As seen in Fig. 2b, mean muscle mtDNA/cellwas 2305 at
week 0 (n ¼ 16), and rose to 3754 at week 48 (n ¼ 12,
P ¼ 0.11 versus week 0). The mean muscle mtDNA/
18 AIDS 2005, Vol 19 No 1
Table 1. Baseline demographics and characteristics.
ESS 40010 main
study (n ¼ 118)
substudy (n ¼ 16)
Age mean years (range)
Median HIV-1 RNA (log10copies/ml)
Median CD4 cell count (?106/l)
Median lactate level (mmol/l)
Median pyruvate (mmol/l)
Median total cholesterol (mg/dl)
Median LDL cholesterol (mg/dl)
Triglycerides (median mg/dl)
Median prior NRTI use duration [months (% of patients)]
Regimen post-baseline switch
Abacavir þ lamivudine þ nelfinavir
Abacavir þ lamivudine þ indinavir
Abacavir þ lamivudine þ efavirenz
Abacavir þ lamivudine þ nevirapine
Abacavir þ lamivudine þ ritonavir þ saquinaivir
Abacavir þ lamivudine þ indinavir þ ritonavir þ didanosine
Abacavir þ amprenavir þ ritonavir þ efavirenz þ didanosine
Abacavir þ indinavir þ efavirenz
Abacavir þ ritonavir þ saquinavir þ didanosine
Combivir þ efavirenz
Combivir þ nelfinavir
LDL, Low density lipoprotein; NRTI, nucleoside reverse transcriptase inhibitor.
percent change from
t a f m
t a f g e l
nu r t
i l l a t o t
t a f k
mt a f b
Fig. 1. Increase in arm, leg, and trunk fat over time from
baseline for patients with symptoms of lipoatrophy who
switched from d4T based therapies to ABC or ZDV based
therapies (n ¼ 13).
nuclei was 3927 for the HIV-negative donors. 95% CI are
shown with the mean value.
The mean week 0 mtDNA/adipose tissue cell ratio was
194 (n ¼ 16), and rose at week 48 to 430 mtDNA/cell
(n ¼ 11, P ¼ 0.01 versus week 0). The mean mtDNA/
adipose tissue cell was 921 copies/cell for the HIV-
negative controls. 95% CI are shown with each mean
value in Fig. 2, and do not overlap.
We conducted an analysis to determine the correlations
between mtDNA levels in the three compartments
examined (muscle, fat and PBMC) at baseline and week
48 as well as changes in levels on-study. The strongest
correlations found were between mtDNA in fat and
mtDNA in PBMC at baseline (correlation coefficient,
0.718; P ¼ 0.0017), between change from baseline in
mtDNA in muscle and change from baseline in mtDNA
in PBMC (correlation coefficient, ?0.664; P ¼ 0.0185),
and between change from baseline in mtDNA in fat
and change from baseline in mtDNA in muscle
(correlation coefficient, ?0.600; P ¼ 0.05). The correla-
(adipose tissue) and mtDNA (PBMC) was 0.218;
P ¼ 0.519.
No large mitochondrial genomic deletions or rearrange-
ments were detected for study subjects or controls.
Theresultsof theTUNEL assayaresummarizedin Fig.3.
Enough adipose tissues for apoptosis assays were available
from 13 subjects at week 0, 10 at week 48, and 20
uninfected controls. The analysis of three additional week
to minimal numbers of adipocytes present in the fixed
sections. Few apoptotic adipocytes were seen in samples
fromHIVuninfectedcontrols(medianscore0.5,n ¼ 20).
Tissue from subjects on d4Twith lipoatrophy was found
to have many apoptotic cells (median score 2, n ¼ 13.)
and this appeared to reduce following cessation of
stavudine, with a median apoptosis score of 1.25 in fat
biopsies collected 48 weeks after switching stavudine to
The results of all skeletal muscle ETC activities are
summarized in Table 2. At study entry, mitochondrial
dysfunction was evident; mean baseline values for ETC
activities (complex I–IV) ranged from 48% to 85% of the
mean levels observed in controls. The most consistent
alterations were noted with complexes I and III.
Depressed citrate synthesis was also observed reflecting
a quantitative loss of mitochondrial mass. Significant
respiratory chain dysfunction was noted in 7/16 subjects
at week 0, and 3 had very marked deficiencies in at least
two enzyme assays. Results of the enzyme activities are
shown as mean ? SD; the unit is nmol/min/g wet
in NCR at week 48 which reflects activity of complex I
d4T toxicity improves after switch to ABC McComsey et al. 19
0 . 0
0 . 05
0 . 005
0 . 0
0 . 0
8 4 k
= n e
n i l e
mtDNA per cell for PBMC
0 . 0
0 . 0
0 . 00
0 . 0003
0 . 0
0 . 0
0 . 0
1= n −
21=n 84 keeW61=n en i l esaB
mtDNA per muscle nuclei
1= n en i l esaB
mtDNA per adipose cell
Fig. 2. mtDNA at weeks 0 and 48 for patients with symptoms of lipoatrophy who switched from d4T based therapies to ABC or
ZDV based therapies, and for the HIV-1 negative control subjects. (a) mtDNA copies/PBMC. (b) mtDNA copies/muscle genome.
(c) mtDNA copies/adipose cell.
t ce j buS
n i l esab
e j bu l or tno
Apoptosis scores (median)
Upper 75% IQ
Lower 25% IQ
Fig. 3. Median apoptosis score at baseline and week 48 for
patients with symptoms of lipoatrophy who switched from
d4T based therapies to ABC or ZDV based therapies and for
the HIV-1 negative control subjects.
and III, from 0.6 ? 0.5 at baseline to 0.9 ? 0.6 at week
48; P ¼ 0.020). In contrast, significant worsening in
complex III activity was seen; 6.0 ? 3.9 at week 48 versus
12.3 ? 5.5 at baseline; P ¼ 0.027. Other changes in
enzyme activities were not significant. These results were
similar when analyzed using absolute values, or relative
values after normalization for citrate synthase, which
adjusts for mitochondrial number.
Immunologic assessments included CD4 cell counts and
measurements of adipose tissue levels of 12 cytokine
mRNAs. All of these indices remained unchanged
compared to week 0 (data not shown).
While several studies have shown that in the setting of
lipoatrophy, the substitution of abacavir or ZDV for d4T
improve subcutaneous fat content [13–15] the potential
for reversibilityof abnormalmitochondrialindicesandfat
apoptosis after such interventions has not been previously
ABC or ZDV because several studies have shown that
d4T has a more deleterious effect on mitochondria than
either ABC or ZDV [1,21]. ZDV was permitted for those
patients who elected not to take ABC. Since only two of
the 16 patients switched to ZDV, the results for these two
patients were not analyzed separately; however, they did
not differ noticeable from those of subjects who switched
to ABC (data not shown).
In this study, we showed a depletion of mtDNA content
and alteration of mitochondrial function in d4T-treated
patients with lipoatrophy, and a partial reversion after
substitution of d4T with ABC or ZDV for 48 weeks.
Unfortunately, only two subjects switched d4T to ZDV,
precluding us from making any separate observation
regarding the d4T to ZDV switch intervention. In the
main TARHEEL study, we had shown that d4T switch to
ZDV led to improvement of subcutaneous fat, although
not to the level seen with d4T to ABC switch .
Due to its non-invasive nature, mtDNA content in blood
ofmitochondrial toxicity withconflicting results[22–25].
In our study, the findings of mtDNA depletion at baseline
are consistent with prior studies which found mtDNA
depletion in bloodand/or fat [4,26,27]. The current study
evaluated three tissue types (muscle, adipose tissue, and
PBMC)concurrently from each patientto provide a more
global assessment of the effect of NRTI on mitochondria
and to determine if there is tissue specificity in mito-
chondrial dysfunction or recovery. In addition, functional
assessment of mitochondria is not standardized in blood
or fat, and therefore sampling of muscle was necessary.
Substantial mean increases in the levels of mtDNA
20AIDS 2005, Vol 19 No 1
Table 2. Summary data of mitochondrial functional assays.
Baseline (n ¼ 16)
(% of normal control)
Week 48 (n ¼ 10)
(% of normal control)
Normal controls (n ¼ 49)
All data are medians unless stated otherwise.
bUnit, mU/g wet weight skeletal muscle.
48 in muscle, adipose tissue, and PBMC, respectively.
on adipose tissue of patients with lipoatrophy [4,26]. The
significant correlation between on-study changes in
muscle mtDNA and on-study changes in both PBMC
and fat mtDNA, the latter two being easier compartments
to access in clinical practice.
Consistent with prior studies [4,25], we found no large
deletions or rearrangements of the mitochondrial
genome at baseline or week 48 for any of the patients
While the minimum mtDNA content (threshold level)
for normal function has not been established, the
substantial increases seen in this study indicate consider-
able recovery and also point to the marked level of
depletion after 3 years on d4T-containing antiretroviral
therapy. In inherited mitochondrial diseases, severe
symptoms usually occur when mtDNA are depleted to
approximately 20% of normal, although this threshold
seems to depend on the tissue examined . This
threshold has not been yet established in fat tissues.
There has been one previous study examining muscle
ETC activities in HIV-infected patients and deficiencies
were noted . Our study was consistent in revealing
similar deficiencies at study entry. Surprisingly, 48 weeks
following the NRTI switch, only the activity of complex
I improved. While the mitochondrial compensatory
mechanisms are robust, these patients may have reached
levels of depletion that could not be compensated.
Alternatively, reversal of mitochondrial dysfunction may
take longer than 48 weeks to occur. Lastly, as had been
previously suggested [25,29], mtDNA depletion may not
be the sole mechanism involved in the NRTI-induced
Another intriguing finding is the lack of correlation
between changes in muscle mtDNA and citrate synthase
activity, the latter being widely used as an indirect
measure of mitochondrial mass [30–32]. Despite imp-
unchanged. This enzyme is coded in the nucleus,
produced in the cytoplasm, and then incorporated into
the mitochondria. Therefore, it is conceivable that the
defect in NRTI-associated mitochondrial dysfunction
could be linked at least partly to a defect in nuclear DNA.
Increases in mitochondrial content were associated with
increases in arm, leg, and trunk adipose tissue of 21%,
11%, and 16%, respectively. This suggests that the
recovery of mitochondrial content may be a factor in
the reversal of lipoatrophy. Moyle had suggested that the
significant increase in limb fat seen in his study after d4T
substitution to ABC may be linked to the observed
decrease in CD4 cells . Our study suggests otherwise,
since the improvement in fat was not associated with
either changes in CD4, HIV RNA, nor with changes in
fat levels of 12 measured cytokine mRNAs. This suggests
that the improvement seen in fat more probably reflects
the consequence of improved mitochondria, rather than
the effect of immune reconstitution.
Recent studies suggested apoptosis, rather than necrosis
to be the primary mechanism involved in lipoatrophy
[11,33]. In this study, increased levels of adipocyte
d4T toxicity improves after switch to ABC McComsey et al.21
0 . 0
0 . 4
0 . 8
0 . 21
0 . 6
0 . 02
e j bu
a l ena
Complex III nmol/min/g wet weight
0 0 . 0
0 5 . 0
0 . 1
0 5 . 1
0 0 . 2
0 5 . 2
e j b
b l e
NCR nmol/min/g wet weight
l o r t n
Fig. 4. Skeletal muscle electron transport chain activities for NCR and decylubiquinone cytochrome creductases (CIII) in nmol/
min/g wet weight skeletal muscle in patients with symptoms of lipoatrophy who switched from d4T based therapies to ABC or
ZDV based therapies with paired samples at week 0 and week 48 (n ¼ 10) compared to HIV-1 uninfected controls (n ¼ 49). By
complex III activity alone declined for most subjects.
apoptosis along with decreased mtDNA were noted in
adipose tissue from d4T-treated subjects with lipoatrophy
relative to uninfected controls. We showed that 48 weeks
after substituting ABC or ZDV for d4T, fat apoptosis was
reduced to levels seen in HIV-negative controls and fat
mtDNA was significantly increased along with improve-
suggest that adipocyte apoptosis secondary to mitochon-
drial toxicity may be an underlying mechanism for
lipoatrophy, and that this toxicity is reversible after
switching patients from d4T to ABC or ZDV. Domingo
the PI to nevirapine . Our study is the first to show
improvement of fat apoptosis after an intervention for
lipoatrophy, and suggest that the increased apoptosis
observed in lipoatrophy is due to the NRTI component.
There are a number of limitations to this study, including
the small numberof subjects and the uncontrolled design.
that involve such invasive procedures.
In conclusion, the observations in this d4T-treated cohort
of patients with evidence of lipoatrophy and/or hyper-
lactatemia suggest that it is possible to at least partially
reverse the mitochondrial dysfunction and fat loss
associated with d4T use by substitution with ABC or
ZDV. Larger and longer studies are needed to elucidate
the mechanisms involved in both the initial depletion and
recovery of mitochondrial function.
manuscript, Steve Wesselingh for technical advice and
support, and the following GSK personnel: Tracey
Sponsorship: Supported by GlaxoSmithKline. ABC and
ZDV were supplied by GlaxoSmithKline. G. McComsey
received grant support, is a consultant and serves on the
Speaker Bureau for GlaxoSmithKline, Bristol-Myers
Squibb, Agouron/Pfizer and Merck Sharp & Dohme.
Sciences and Abbott Laboratories. D. M. Paulsen, V. C.
Williams, R. L. Fisher, S. T. Ross, S. M. Hessenthaler, J. E.
Hernandez and L. L. Ross are/were employees of
GlaxoSmithKline during the conduct of the study.
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d4T toxicity improves after switch to ABC McComsey et al.23