NATAP: Leptin levels Not Lipoatrophy Related, study finds

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Plasma leptin levels in men are not related to the development of lipoatrophy during antiretroviral therapy

AIDS: Volume 19(16) 4 November 2005

Wunder, Dorotheaa; Bersinger, Nick Aa; Fux, Christophb; Weber, Rainerc; Bernasconi, Enosd; Cavassini, Matthiase; Bucher, Heiner Cf; Schiffer, Veroniqueg; Schmid, h; Furrer, Hansjakobb; the Swiss HIV Cohort Study

From the aDepartment of Obstetrics and Gynaecology, University Hospital of Berne

bKlinik und Poliklinik für Infektiologie, University Hospital of Berne, Berne

cDivision of Infectious Diseases, University Hospital of Zurich, Zurich

dDivision of Infectious Diseases, Hospital of Lugano, Lugano

eInfectious Diseases Services, University Hospital of Lausanne, Lausanne

fDivision of Infectious Diseases and Basel Institute for Clinical Epidemiology, University Hospital of Basel, Basel

gDivision of Infectious Diseases, University Hospital of Geneva, Geneva

hDivision of Infectious Diseases, Cantonal Hospital of St Gall, St Gall, Switzerland.

Abstract

Objectives: To assess the correlations between the hormone leptin and lipoatrophy in HIV-positive, treatment-naive patients on combination antiretroviral therapy (cART).

Design: Case-control study nested in a multicentre cohort of HIV-infected adults. Cases were patients that developed lipoatrophy and controls those who did not.

Patients and methods: Clinical parameters and plasma leptin determinations were studied in 97 HIV-1-infected, treatment-naive Caucasian men (10 cases and 87 controls) on an unchanged and virologically successful drug regimen with a zidovudine/lamivudine backbone at baseline and after 2 years of cART. The association of plasma leptin levels and the development of lipoatrophy was investigated.

Results:

o Two years of cART was not associated with a change in plasma leptin levels.

o Plasma leptin levels remained sensible to changes in body mass index.

o There was no difference in leptin levels between patients who developed lipoatrophy and controls, neither before nor after cART.

o The only predictor of development of lipoatrophy was a higher age (P = 0.02).

Conclusions:

Leptin as measured in plasma is unlikely to play a major role in the genesis of lipoatrophy.

Introduction

Lipodystrophy is a fat redistribution abnormality occurring in several diseases such as diabetes or HIV infection treated with combination antiretroviral therapy (cART) [1]. It is characterized by peripheral lipoatrophy and central fat accumulation and either of these two features may be found alone [2-4]. In HIV-infected patients, these manifestations were initially attributed to the prescription of protease inhibitors (PI), but this syndrome occurs also in patients treated with PI-sparing antiretroviral double nucleoside reverse transcriptase inhibitor (NRTI)-based therapies.

The aetiology of lipodystrophy in HIV is not fully understood. Possible influences of cART on cytokines (e.g. tumour necrosis factor-α) and their receptors [5,6], appetite regulators [5], inhibition of sterol regulatory enhancer-binding protein 1 mediated activation of the heterodimer consisting of adipocyte retinoid X receptor and peroxisome proliferator-activated receptor (PPARγ) [7] and mitochondrial function [8,9] on the one hand, and hormone alterations [10,11] on the other hand were proposed. It is likely that different mechanisms account for fat accumulation and lipoatrophy in lipodystrophy associated with cART. Lipoatrophy has been related mainly to the mitochondrial toxicity of NRTIs, and those which exert a more profound inhibition of the mitochondrial DNA polymerase γ are associated with a higher risk of development of lipoatrophy [12,13].

Leptin is a hormone which has an important function in regulating energy homeostasis and is encoded by the obesity gene [14]. Leptin is not only produced in white adipose tissue but also in other tissues [15-17]. Leptin levels are indicative of whether nutritional stores are adequate. They are decreased in conditions in which the nutritional status is suboptimal [18] and increased in the case of excess energy stores such as obesity [19].

Serum leptin levels are influenced by a variety of growth factors and cytokines which regulate its transcription [20]. It has been demonstrated that metabolic abnormalities develop as a consequence of fat loss [21]. Based on the finding that leptin may play a critical role in preventing insulin resistance and hypertriglyceridemia in lipodystrophy in a mouse model [22], Oral et al. conducted a study on leptin replacement in lipodystrophic diabetic patients and showed that leptin replacement improved glycemic control and decreased triglyceride levels in these patients with leptin deficiency [23].

Severe HIV-associated lipoatrophy is caused by a deficiency or destruction of adipose cells and was found to be associated with low leptin levels [24]. Moreover, the expression of adipocyte-specific markers, including leptin, was lower in fat from patients with lipoatrophy than in fat from controls [7]. One study [25] has proposed a link between CD4 T lymphocytes, serum leptin and highly active antiretroviral therapy in children.

Lipoatrophy, especially the common facial lipoatrophy, is distressing and potentially stigmatizing and can lead to poor adherence to cART [26]. Only through understanding the aetiology of lipodystrophy it will be possible to establish its optimal management. The aim of this study was to assess the changes of leptin during successful cART and their correlations with development of lipoatrophy.

Patients, materials and methods

The Swiss HIV Cohort Study (SHCS; www.shcs.ch ) is a prospective cohort study enrolling HIV-infected adult (16 years or older) persons. Data are collected in seven study centres according to a standardized protocol [27] at registration and 6-monthly follow-up visits. Every 6 months blood plasma samples are stored at -80°C for future research questions.

Study design

This was a case-control study evaluating the prospectively collected clinical data and plasma samples in men with 2 years of continuous unchanged cART. Women were not included in this analysis because leptin levels in women have been reported to show fluctuations between the different phases of the menstrual cycle. Similarly, because leptin levels may be influenced by ethnicity [28] a homogeneous population with respect to ethnicity was evaluated in this study.

Caucasian patients with a good virological response (plasma HIV RNA levels below 50 copies/ml) after 2 years of treatment who did (cases) or did not (controls) develop lipoatrophy within 24 months after the start of cART were included. As the development of lipoatrophy is associated with certain NRTIs, patients on a uniform and stable NRTI-backbone with zidovudine and lamivudine were chosen.

Patients

All 11 patients on the defined NRTI backbone who developed lipoatrophy after 2 years of cART were identified within the SHCS database and defined as cases. Out of 128 participants on the same nucleoside backbone regimen 89 patients who had no development of lipoatrophy were randomly chosen as controls out of the SHCS database. The patients had to be treatment naive before starting the predefined regimen. Exclusion criteria were treatment with hormones, diabetes mellitus before starting cART, treatment interruptions within the study period, and active AIDS-defining illness at baseline.

In one of the controls, the hormone determinations failed in one plasma sample. In addition, source data verification in patients' charts revealed that one case and one control had been on antiretroviral drugs before entering the Swiss HIV Cohort Study. These two patients were also withdrawn from analysis. Therefore, the studied was performed with 10 cases and 87 controls.

Definition of lipoatrophy

In the SHCS, lipoatrophy is diagnosed when fat loss occurs in one or more of the following locations: face (buccal, periorbital, temporal), arm, leg or buttocks. The presence of at least one sign of fat wasting (lipoatrophy) is considered indicative for lipoatrophy if the patient and the treating physician agree on the finding.

Measurements of plasma viral load and CD4 cell counts in the plasma

Plasma viral load was measured using the Roche Amplicor Monitor assay (Roche Diagnostics, Rotkreuz, Switzerland; limit of detection < 50 copies/ml). The CD4 cell counts were determined by flow cytometry in the reference laboratories.

Measurement of hormones in the plasma

Leptin levels in plasma were determined using a microplate enzyme-linked immunometric method (ELISA) which was developed and validated in our laboratory and is described elsewhere in detail [29]. Sample dilution was 1: 50 in Blotto (Pierce, USA, obtained from Socochim, Lausanne, Switzerland). The limited diurnal variation in leptin secretion [30] was not expected to influence our study results since the plasma samples were randomly chosen and unbiased.

The measurement of leptin after frozen storage has been validated [31]. All assays were performed in duplicate.

Statistical analysis

Wilcoxon signed-rank test and two-sample Wilcoxon rank-sum test were used for numerical analysis and chi-squared statistics for categorical data. Possible associations of parameters with the development of lipoatrophy were evaluated in a logistic regression model. A two-sided P-value of 0.05 or less was accepted as significant.

We used the plasma leptin levels in the study of Estrada et al. [24] for sample size calculation. In the mentioned study the leptin level in lipoatrophy was 2.85 ng/ml with a standard deviation (SD) of 1.5. By analogy, we took the leptin levels in cART-naive HIV patients of 4.57 (SD 3.2) ng/ml as substitute for leptin level on ART without lipoatrophy after 2 years. With these assumptions we had a power of 0.83 to detect a difference between cases and controls with regard to leptin levels.

RESULTS

Plasma leptin levels after 2 years of cART did not differ significantly from baseline levels, neither in the whole study group (P = 0.4), nor in cases (P = 0.7) or controls (P = 0.5).

There were no significant differences in leptin levels between cases and controls either before or after 2 years of cART. There were no differences in the changes in leptin levels during cART between cases and controls.

We performed a logistic regression for the development of lipoatrophy during cART that included age older than 40 years, body mass index (BMI), leptin levels before cART, changes of leptin levels during cART, the use of protease inhibitors in the regimen, baseline CD4 cell count (below against above 200 cells/μl) and the log of plasma HIV-RNA levels at baseline. The only variable significantly associated with the development of lipoatrophy was age over 40 years [odds ratio (OR), 8.5; 95% confidence interval (CI), 1.4-52; P = 0.02]. However, baseline leptin levels (OR, 0.9; 95% CI, 0.7-1.3; P = 0.7) or change in leptin levels during cART (OR, 0.9; 95% CI, 0.6-1.3; P = 0.5) showed no association with development of lipoatrophy.

There was a significant positive correlation between the changes in BMI and changes in leptin levels (P < 0.001), and this correlation did not differ significantly between cases and controls.

Discussion

In our study we did not find an influence of cART on leptin levels. In addition, our results did not show significantly lower leptin levels in HIV-infected, Caucasian men who developed lipoatrophy on cART, either before starting cART or after 2 years of treatment. Thus, according to our results, it appears that leptin deficiency as measured with plasma levels is not a sensitive biomarker of the metabolic abnormalities in lipoatrophy. Our data also argue against the hypothesis of leptin being a major aetiologic factor of lipoatrophy observed during cART. Consequently, systemic replacement with leptin seems unlikely to improve the symptoms in lipoatrophic HIV-positive patients.

This is in contrast to the findings of Oral et al. [23], who treated diabetic patients with lipodystrophy and low basal leptin levels with recombinant leptin. Results from this patient population should therefore not be transferred to HIV patients.

The relation of leptin levels and lipoatrophy in HIV-infected patients is controversial. Whereas some studies found lower leptin levels in patients with lipoatrophy [24,32] others found the contrary [33] or no difference [34] as our study.

In addition, we could not verify the findings of Paganelli et al. [35] who demonstrated a substantial increase of plasma leptin levels after 24 months of cART irrespective of lipoatrophy.

We found a significant positive correlation between changes of BMI and changes in leptin levels during cART. Therefore, it seems that the physiological function of leptin is maintained during cART.

In contrast to most of the cited studies, we have investigated a very homogenous population of Caucasian men starting their first cART on a specific and stable nucleoside analogue backbone and being very adherent to their regimen. By using the case-control design with randomly chosen controls the potential reporting bias is smaller than in other studies. We therefore think that our study has fewer confounders than most others, and our findings point against a major correlation between lipoatrophy on cART and leptin levels. However, given the still limited numbers of individuals in our study we cannot rule out minor associations between both findings. A further limitation of our study is the purely clinical diagnosis of lipoatrophy.

In conclusion, even taking into account the limited power of our study, our data argue against the support of the hypothesis of leptin being a major contributor to the lipoatrophy observed in HIV patients on cART. If, however, the adipocyte hormone leptin is unlikely to be a major aetiologic factor or contributor to the development of lipoatrophy, implementation of trials evaluating the expensive therapy with recombinant leptin does not seem to be warranted.