HIV lipodystrophy (HIV-LD) is characterized by the loss of adipose tissue from the subcutaneous compartment with increased visceral adipose tissue and is accompanied by insulin resistance and hypertiglyceridemia.1-4 The mechanism(s) underlying the decline in the abundance of subcutaneous adipose tissue (SAT) are not completely understood, but both HIV protease inhibitors (PIs)5,6 and nucleoside reverse transcriptase inhibitors (NRTIs)7,8 have been implicated in the pathogenesis of the HIV-LD syndrome.
In addition to antiretroviral therapy, the syndrome of lipodystrophy in HIV occurs within the context of active viral infection. Significantly elevated levels of the soluble tumor necrosis factor-α receptor type 2 (sTNFR2) have been demonstrated in individuals with HIV-LD compared with either HIV-infected individuals without HIV-LD or compared with uninfected individuals.9 This elevation in sTNFR2 is often indicative of cumulative exposure to TNF-α.10 Since TNF-α is capable of inducing apoptosis in adipocytes,11 increased apoptosis has been suggested as the mechanism for loss of SAT in individuals with HIV-LD.
There are reports of apoptosis in cells in subcutaneous tissue from individuals with HIV-LD.12 These studies employed the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) approach to identifying nuclei from cells with alterations in DNA suggesting apoptosis.13 The study by Domingo et al12 clearly demonstrates the presence of positively staining nuclei in subcutaneous tissue of individuals with HIV-LD. Whether the extent of SAT apoptosis is elevated in HIV-LD, compared with material from uninfected controls or individuals with HIV but without HIV-LD, remains unresolved.
Because nuclei account for a very small proportion of the volume of adipose tissue, making histologic assessments of apoptosis difficult, we have investigated the role of apoptosis in the loss of SAT with an alternative method that provides a sensitive estimate of a classic marker of apoptosis, the DNA ladder. DNA ladders can be visualized by the ligase-mediated PCR (LM-PCR) amplification of DNA,14 to amplify evidence of internucleosomal DNA cleavage. This amplification enhances the sensitivity of the determination of apoptosis. With the LM-PCR DNA ladder technique, there was evidence of apoptosis in subjects with HIV-LD, but the degree of DNA laddering was not increased above levels in HIV-infected subjects without LD or uninfected control subjects. Using this method, we find no evidence of increased apoptosis in the subcutaneous tissue from subjects with HIV-LD.
MATERIALS AND METHODS
Patients and Samples
Twenty-seven control subjects and 47 individuals with HIV disease participated in this study, which was approved by the Committee on Research Involving Human Subjects at the University Medical Center of the State University of New York at Stony Brook. All subjects gave their informed written consent. Individuals enrolled in the study included 22 HIV-infected individuals with self-reported loss of SAT and increased abdominal fat (HIV-LD). The self-reported alterations in body fat distribution were confirmed by clinical assessment. For comparison, 25 HIV-infected individuals without self-reported loss of SAT and 27 individuals who were seronegative for HIV were also studied. All study subjects had been free of acute illness for the 3 months preceding their participation in the study.
Study subjects were admitted to the General Clinical Research Center on the evening prior to the procedure. Subjects fasted overnight, following a snack at 10 PM. The following morning between 7 and 8 AM, subcutaneous fat biopsy material was harvested from the outer aspect of the thigh, under sterile conditions and local anesthesia. The biopsy material was immediately blotted free of excess blood, frozen in liquid nitrogen, and stored at −140°C.
Body fat distribution was assessed with dual-energy x-ray absorptiometry as described previously.9 The percent of body fat present in the limbs was calculated as the proportion of limb fat to the total of limb and trunk fat multiplied by 100.
Genomic DNA Isolation
Frozen SAT was pulverized under liquid nitrogen. DNA was extracted from frozen tissue powder using the Puregene DNA purification kit from Gentra Systems (Minneapolis, MN), following the manufacturer's protocol. Briefly, 10-20 mg of the frozen tissue powder was extracted with 600 μL of the cell lysis solution by gentle disruption of the tissue in a microcentrifuge tube fitted with a polypropylene pestle (Kontes, Vineland, NJ). The lysates were supplemented with 3 μL of Proteinase K solution (20 mg/mL), mixed, and incubated at 55°C overnight. RNAwas removed by digestion with RNase A for 60 minutes at 37°C. Following precipitation, protein was removed by centrifugation and DNA was precipitated with ispropanol.
The ligase-mediated PCR ladder assay was performed using the Apo-Alert Ladder Assay Kit from Clontech (San Jose, CA). DNA was mixed with ligation mixture, containing the ligation primers. The primer pair consisted of a 12mer with a dephosphorylated 5′ terminus and sequence 5′-TGCGGTGAGAGG-3′ and the intact 24mer 5′-AGCACTCTCGAGCCTCTCACCGCA-3′. The reaction mixture was overlaid with mineral oil and incubated at 55°C for 10 minutes. The reaction mixture was cooled to 10°C gradually over 1 hour, to allow DNA annealing, and then treated with T4 DNA ligase. The reaction transferred to 16°C and incubated overnight to yield the ligated DNA product.
The LM-PCR was carried out following the manufacturer's instructions. Briefly, the amplification mixture, consisting of 150 ng of ligated genomic DNA, dNTPs, ligation primers, and Taq DNA polymerase, was incubated at 72°C for 8 minutes to fill in with complements to the 24mer ligation adapter. Cycle parameters were 94°C for 1 minute and 72°C for 3 minutes for 25 cycles. The reaction products were separated in 1.2% agarose gels, stained with ethidium bromide, and photographed.
Confirmation of DNA Mass Equivalence
The PCR amplification of a single copy gene (En-2) was used to establish DNA mass equivalence in the LM-PCR ladder assay. The materials and protocols were included in the Apo-Alert Kit. En-2 sequence amplification was carried out as recommended by the manufacturer. En-2 primers used in the amplification were 5′-TCTACCCAGTTCCCAGGTCTCG-3′ (forward) and 5′-ACTTGCCCTCCTTGGCTGTGGT-3′ (reverse). The En-2 sequence was amplified from 150 ng of ligated genomic DNA using cycling parameters of 1 minute at 94°C, 1 minute at 70°C, and 1 minute at 72°C for 26 cycles. Reaction products were separated in 1.2% agarose gels, stained with ethidium bromide, and photographed. Evidence of the 290-bp En-2 PCR product was used to confirm that a sufficient ligated genomic DNA was used in the LM-PCR reaction.
Healthy, uninfected control subjects (control), HIV-positive individuals without evidence of lipodystrophy (HIV), and HIV-positive individuals with lipodystrophy (HIV-LD) were all of similar age and body mass index (wt/h2) (Table 1). Both the HIV and the HIV-LD groups were similar in the pattern of antiretroviral medications (Table 1). One subject in both the HIV and the HIV-LD groups was not taking any antiretroviral medication. The numbers of CD4+ cells and viral burden were similar in the HIV and HIV-LD groups. In the individuals with HIV-LD, the percentage of body fat present in the limbs was significantly lower compared with either the control or the HIV group without LD (P < 0.05, Student-Newman-Keuls, Table 1).
Evidence of DNA laddering, suggesting apoptosis, was observed in all groups of subjects. In the control group, 13 of 27 subjects displayed evidence of DNA ladders by LM-PCR. This was similar to the proportion of individuals displaying evidence of LM-PCR DNA ladders in the HIV group (13 of 25) and the HIV-LD group (10 of 22). Individuals in the HIV-LD group displayed no more evidence of apoptotic DNA laddering, following LM-PCR amplification, than individuals in the control or HIV groups (Fig. 1, P = 0.9). There was also no apparent link between subcutaneous apoptosis and HIV medication (Table 1). Adjusting for NRTIs, NNRTIs, and PIs in a multiple logistic regression model, there was no statistical evidence of increased apoptosis in the LD group compared with the non-LD group (odds ratio = 0.88, 95% CI: 0.25, 3.04; P = 0.84). A multivariate logistic regression model combining HIV and HIV-LD subjects and adjusting for PI and NRTI use showed a protective effect of NNRTIs for apoptosis (odds ratio = 5/67, 95% CI, 1.05, 30.76, P = 0.04).
The data from the present study clearly demonstrate that there is no increased incidence of apoptosis in the subcutaneous tissue from individuals with HIV-LD, compared with uninfected control subjects or subjects with HIV disease but without HIV-LD.
The loss of SAT in HIV-LD has been associated with both HIV PI use15 as well as NRTI usage.7,16 Apoptosis has been suggested as a mechanism for the subcutaneous fat loss associated with HIV-LD.12,17,18 This suggestion is supported by the observation of elevated levels of TNF-α,19 and also elevated levels of sTNFR,9 indicative of TNF-α activity.10 Since in vitro studies of adipocytes indicate that TNF-α induces apopotosis,11 this mechanism has been suggested to account for the subcutaneous loss of adipose tissue in HIV-LD. In support of this hypothesis, Domingo et al have provided evidence of apoptosis in subcutaneous tissue from individuals with the HIV-LD syndrome, using the TUNEL procedure. This assay technique adds appropriately labeled deoxynucleotide triphosphates to available 3′-hydroxyl termini, as a way of assessing which nuclei display evidence of internucleosomal DNA cleavage, a hallmark of apoptosis.20 While there appears to be ample evidence of apoptotic nuclei in the subcutaneous tissue samples from individuals with HIV-LD in the work of Domingo et al, there were no data presented to address whether the extent of apoptosis observed in samples from individuals with HIV-LD was increased relative to similar material obtained from uninfected controls or from individuals with HIV but no evidence of lipodystrophy.
The surprising finding of the present study (Fig. 1) is the demonstration that there is no increase in the frequency of HIV-LD individuals displaying apoptotic DNA ladders in subcutaneous tissue compared with uninfected controls or with individuals with HIV but without lipodystrophy. Furthermore, the intensity of DNA ladders in the material from the HIV-LD group is similar to that for material from the other 2 groups. Thus, these data do not support the hypothesis that increased apoptosis is a mechanism for the loss of SAT in HIV-LD.
The discrepancies between the studies of Domingo et al and the present study need to be explored. In addition to the obvious inclusion of control groups in the present study, other technical differences may contribute to the different results. Use of the TUNEL assay, which requires histologic localization of nuclei, is complicated in adipose tissue by the fact that so little of an adipocyte is nucleus. The size discrepancy between the nucleus (∼5 μm in diameter) and the adipocyte (≥100 μm in diameter) means that in a 5-μm section the probability of obtaining a section through an adipocyte with its associated nucleus is ≤5%. This means that many sections will need to be examined to determine the rate of apoptosis in adipose tissue.
Also, neither the studies by Domingo et al12,17,18 nor the present study address the question of constancy in the rate of loss of SAT in subjects with HIV-LD. The assumption in both studies is that measurement of a single point in time can be taken as representative of the rate of apoptosis over time. It is possible that there are periods of very rapid loss of SAT that may have accelerated rates of apoptosis, though varying rates of SAT loss have not been reported.
Although there are limitations of the present study, it seems unlikely that accelerated apoptosis is responsible for the loss of SAT associated with HIV-LD. Interestingly, multivariate logistic regression modeling of the data combining both the HIV non-LD and HIV-LD subjects with adjustment for the current PI and NRTI use indicated a protective effect of NNRTIs for apoptosis (odds ratio = 5/67, 95% CI: 1.05, 30.76, P = 0.04), suggesting a differential effect on apoptosis among antiretroviral therapies. However, there is mounting evidence that a more likely mechanism for the loss of SAT may involve a reduction in the rate of adipose-specific gene expression5,21 and subsequent differentiation of preadipocytes into adipocytes.
In conclusion, the syndrome of HIV-LD is characterized by the loss of adipose tissue mass from the subcutaneous region.1 This regional fat loss is associated not only with dysregulation of carbohydrate9 and lipid22 metabolism, but also with distressing alterations in body fat distribution that may even lead to noncompliance with antiretroviral medication in an attempt to reverse HIV-LD. Establishing the plausible mechanisms for this loss of subcutanteous fat is integral to the design of therapeutic strategies to restore peripheral adipose tissue. It appears that reduced adipocyte differentiation is a more likely mechanism for HIV-LD than accelerated apoptosis.
The authors thank Dr. Mark Kaplan from the Department of Infectious Diseases, North Shore University Hospital, and Roy Steigbigel at Stony Brook for help in recruiting subjects from their clinical practice. We also thank the nursing staff of the General Clinical Research Center for meticulously carrying out this protocol and Ms. Bernice Sealy for coordinating this study.
1. Carr A. HIV lipodystrophy: risk factors, pathogenesis, diagnosis and management. AIDS.
2. Hadigan C, Miller K, Corcoran C, et al. Fasting hyperinsulinemia and changes in regional body composition in human immunodeficiency virusinfected women. J Clin Endocrinol Metab.
3. Lo JC, Mulligan K, Tai VW, et al. "Buffalo hump" in men with HIV-1 infection. Lancet.
4. Saint-Marc T, Partisani M, Poizot-Martin I, et al. Fat distribution evaluated by computed tomography and metabolic abnormalities in patients undergoing antiretroviral therapy: preliminary results of the LIPOCO study. AIDS.
5. Bastard JP, Caron M, Vidal H, et al. Association between altered expression of adipogenic factor SREBP1 in lipoatrophic adipose tissue from HIV-1-infected patients and abnormal adipocyte differentiation and insulin resistance. Lancet.
6. Riddle TM, Kuhel DG, Woollett LA, et al. HIV protease inhibitor induces fatty acid and sterol biosynthesis in liver and adipose tissues due to the accumulation of activated sterol regulatory element-binding proteins in the nucleus. J Biol Chem.
7. Saint-Marc T, Partisani M, Poizot-Martin I, et al. A syndrome of peripheral fat wasting (lipodystrophy) in patients receiving long-term nucleoside analogue therapy. AIDS.
8. Nolan D, Hammond E, Martin A, et al. Mitochondrial DNA depletion and morphologic changes in adipocytes associated with nucleoside reverse transcriptase inhibitor therapy. AIDS.
9. Mynarcik DC, McNurlan MA, Steigbigel RT, et al. Association of severe insulin resistance with both loss of limb fat and elevated serum tumor necrosis factor receptor levels in HIV lipodystrophy. J Acquir Immune Defic Syndr.
10. Dri P, Gasparini C, Menegazzi R, et al. TNF-induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase. J Immunol.
11. Prins JB, Niesler CU, Winterford CM, et al. Tumor necrosis factor-alpha induces apoptosis of human adipose cells. Diabetes.
12. Domingo P, Matias-Guiu X, Pujol RM, et al. Subcutaneous adipocyte apoptosis in HIV-1 protease inhibitor-associated lipodystrophy. AIDS.
13. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol.
14. Staley K, Blaschke AJ, Chun J. Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ.
15. Carr A, Samaras K, Burton S, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS.
16. Brinkman K, Smeitink JA, Romijn JA, et al. Mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. Lancet.
17. Domingo P, Matias-Guiu X, Pujol RM, et al. Switching to nevirapine decreases insulin levels but does not improve subcutaneous adipocyte apoptosis in patients with highly active antiretroviral therapy-associated lipodystrophy. J Infect Dis.
18. Lloreta J, Domingo P, Pujol RM, et al. Ultrastructural features of highly active antiretroviral therapy-associated partial lipodystrophy. Virchows Arch.
19. Johnson JA, Albu JB, Engelson ES, et al. Increased systemic and adipose tissue cytokines in patients with HIV-associated lipodystrophy. Am J Physiol Endocrinol Metab.
20. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature.
21. Mynarcik D, McNurlan M, Steigbiegel R, et al. Elevated triglycerides and loss of limb fat are associated with a decrease in peripheral adipose tissue lipoprotein lipase gene expression. Paper presented at: 82nd Annual Meeting of the Endocrine Society; Toronto, Canada, 2000. Abstract.
22. Hui DY. Effects of HIV protease inhibitor therapy on lipid metabolism. Prog Lipid Res.