From the *Department of Medicine, Division of Endocrinology, †Department of Preventive Medicine, and ‡Department of Surgery, State University of New York at Stony Brook, Stony Brook, NY.
".....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..."
HIV-associated lipodystrophy is characterized by a loss of adipose tissue from the subcutaneous compartment. Previously reported data suggested that this loss of adipose tissue was the result of an increased rate of apoptosis in subcutaneous adipose tissue. The present study examined the rate of apoptosis in subcutaneous adipose tissue with a sensitive ligase-mediated polymerase chain reaction technique to amplify DNA ladders. Individuals with HIV lipodystrophy were compared with HIV-infected subjects without lipodystrophy and subjects without HIV disease. Although apoptosis was observed in subjects with HIV lipodystrophy, there was no difference in the frequency of individuals with apoptosis among those with HIV lipodystrophy (10/22), those with HIV but no lipodystrophy (13/25), and subjects without HIV disease (13/27).
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. The mechanism(s) underlying the decline in the abundance of subcutaneous adipose tissue (SAT) are not completely understood, but both HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) 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. This elevation in sTNFR2 is often indicative of cumulative exposure to TNF-α. Since TNF-α is capable of inducing apoptosis in adipocytes, 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. 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. The study by Domingo et al 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.
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 use as well as NRTI usage. 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,and also elevated levels of sTNFR, indicative of TNF-α activity. Since in vitro studies of adipocytes indicate that TNF-α induces apopotosis, 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. 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 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 al 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 expression 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. This regional fat loss is associated not only with dysregulation of carbohydrate and lipid 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.
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).
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 (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).