The introduction of combination antiretroviral therapy (cART) has significantly changed the course of human immunodeficiency virus (HIV) infection, increasing life expectancy but also leading to complex clinical and metabolic changes [1]. The term lipodystrophy was initially used to summarize these clinical and metabolic modifications [2]. The most important metabolic abnormalities observed in HIV-patients during cART include: insulin resistance (IR) with hyperinsulinemia and glucose intolerance [3], hypercholesterolemia with low levels of HDL-cholesterol, hypertriglyceridemia, lactic acidemia and hypercoagulopathy [4].

Lipodystrophy also associates fat redistribution: subcutaneous lipid stores wasting (Bichat’s fat pad, buttocks and extremities), central adiposity and fat accumulation in the dorsocervical region (lipohypertrophy) [2]. As patients with these metabolic abnormalities have relatively high rates of experiencing a cardiovascular event, there is growing concern about the potential damaging impact of cART in HIV-positive patients. Metabolic abnormalities may result from various and combined effects of cART antiretroviral drugs. Several studies have reported that almost all protease inhibitors (PIs) can cause IR, effects likely caused by the inhibition of glucose transport mediated by glucose transporter type 4 (GLUT-4) [5, 6]. Antiretroviral drugs may also impair endocrine functions of adipocytes [7]. Adipose tissue synthesizes various adipokines and hormones, such as adiponectin, leptin, resistin, tumour necrosis factor-alpha (TNF-α), interleukins and release free-fatty acids [8]. Adiponectin and leptin may be involved in fat distribution and insulin sensitivity in HIV-infected patients [9]. Nucleoside reverse transcriptase inhibitors (NRTIs) may impair the secretion of adiponectin and thus may cause IR [10].

The aim of the study was to evaluate the prevalence of the IR and the association of the IR with clinical and biological variables, including serum adipokines, in a Caucasian HIV-infected non-diabetic population of men and women, undergoing cART.

A cross-sectional research was performed in a Caucasian cohort of HIV-1-positive patients attending Prof. Dr. Matei Bals National Institute of Infectious Diseases from Bucharest, between June 2009 and June 2010. The protocol was approved by the Prof. Dr. Matei Bals National Institute of Infectious Diseases ethics committee (protocol no. 190/15.01.2009). An informed consent was obtained from each of the research subjects. The study was part of a national research grant (SLD-ART no. 62077/2008) on HIV infected patients. All consecutive non-diabetic patients who signed a written informed consent to participate in the study were enrolled. We included HIV-infected men and women, aged 18 years and over, undergoing stable cART for at least 6 months. The patients completed during the medical visit a standard questionnaire for demographic parameters and for personal and family medical histories. Body mass index (BMI) was defined according to WHO criteria [11]. Overweight was defined as a BMI ≥ 25 kg/m² and obesity as BMI ≥ 30 kg/m².

Blood samples were tested for: Adiponectin (BioSource ELISA, KAPME09), Resistin (BioSource ELISA, KAPME 50), Leptin (BioSource EASIA KAP2281), Tumor necrosis factor alpha - TNF-alpha (BioSource EASIA KAP 1751), Interleukin-6 (IL-6) (BioSource EASIA KAP 1261), CD4 T cells count (BD FACSCanto II flow cytometer), HIV viral load (Roche LightCycler® 480 Real-Time PCR System), fasting triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and fasting plasma glucose (dry slide technology, Vitros 950, Johnson & Johnson), fasting insulin (ELx50 Bioelisa Washer BioTek).

IR was determined using the Quantitative Insulin Sensitivity Check Index (QUICKI), calculated as 1/(log(fasting insulin μU/mL) + log(fasting glucose mg/dL)[12].

In normal populations, mean QUICKI levels range from 0.37 to 0.39 [13]. Consistent with previous studies, QUICKI values were dichotomized at the cut-off of 0.33, lower levels signifying IR [13–15].

We also assessed the homeostatic model assessment – insulin resistance (HOMA-IR) index, first described by Matthews et al. in 1985 [16]. The formula used to calculate HOMA-IR was:

HOMA-IR = fasting insulin (μU/mL) × fasting plasma glucose (mmol/L)/22.

We assumed a HOMA-IR value of > 3 to define IR, consistent with previous studies investigating insulin resistance in Caucasian HIV populations [17].

A total of 89 patients (age 18–65, median: 28 years) including 51 men (57.3%, age range: 18–63 years, median: 32 years) and 38 women (42.7% age range: 19–65 years, median: 21 years) were included in the study. The mean BMI was 23.6 (± 3.9) kg/m². The median time from HIV diagnosis was 80.5 months and the median time on antiretroviral therapy was 72.5 months. Sixty five patients (73%) had a current PI-based cART regimen. Median serum levels of adiponectin, leptin, resistin, TNF alpha and IL-6 were 10.2 μg/mL, 1.4 ng/mL, 5.5 ng/mL, 10.3 pg/mL and 26.5 pg/mL, respectively.

In order to determine the relationship between laboratory parameters and IR, first we assessed the relationship of all individual variables with QUICKI score by fitting a linear regression model. The variables with univariate association to QUICKI were further tested to improve the model predictivity. After all variables were tested in the univariate analysis for the entire population, including BMI and age, only log transformed triglycerides serum levels (LOGTriglycerides) were significantly associated with QUICKI (R=0.24, p=0.01).

IR appears when maintaining normal physiological values of glycaemia require insulin levels that exceed normal concentrations [18]. IR is associated with the development of diabetes and obesity [19]. Up to 20% of non-HIV western population may have IR [20, 21]. Several studies reported a two-fold increase of cardiovascular risk compared to non-insulin resistant controls [22, 23]. In the pre-cART era several cross-sectional studies reported slightly increased [24] or normal [25] insulin sensitivity when compared to uninfected controls. cART introduction led to increases in fasting insulin and decreases in insulin sensitivity, effects dependent both on the class of the antiretroviral used and the different antiretrovirals within each class [26, 27]. Different studies have evaluated the prevalence of IR at 20 to 50%, in HIV-positive patients [28]. We noted higher levels of IR than reported by previous studies, independent of current treatment with PIs and of BMI, which may be attributed in part to our method of assessment. QUICKI has a very good correlation with the glucose clamp index of insulin sensitivity, which is considered the gold standard method for IR assessment [15]. IR presence in our population of HIV-positive young adults was not influenced either by the time from HIV diagnosis or to the duration of cART. This finding may suggest that the effect of cART on IR may not have a cumulative or long-term response, a similar result being reported also by other studies [29]. As IR presence is associated with an increased risk of diabetes, cardiovascular events, stroke and death, the reduction of its prevalence must be considered an essential therapeutic target in HIV-patients undergoing retroviral therapy.

Adiponectin is exclusively secreted from adipose tissue and has an important role as a mediator of peroxisome proliferator-activated receptor gamma action [30]. Adiponectin may increase insulin responsiveness of the glucose transport system in adipocytes through increased GLUT4 gene expression and increased GLUT4 recruitment to the plasma membrane [31]. Several studies have reported that adipocyte differentiation was perturbed both in vitro and in vivo as a direct effect of cART and the consecutive decrease in serum adiponectin levels was associated with insulin resistance [32, 33]. We found an inverse correlation between adiponectin and IR in men, but not in females which partially concurs with previous studies. Vigouroux et al. (2003) have also reported that adiponectin was positively correlated with insulin sensitivity in a cohort of 131 HIV-infected men with PI-based cART regimens [9]. In a study on 237 Afro-Caribbean HIV-1-infected patients, published in 2011, Deloumeaux et al. found also an inverse correlation between adiponectin and IR in both men and women. After adjusting for clinical and metabolic parameters, the correlation between adiponectin and insulin resistance was observed only in women [34], a conclusion that contradicts our results and the previously mentioned study. Alterations of serum adiponectin concentration in relation to insulin resistance was also observed in a cohort of HIV-infected youths (aged 5.0 - 19.4 years), in a study published by Viganò et al. in 2011, which did not take into account sex differences but evaluated lipodystrophy. Low adiponectin concentration was associated to central fat and mixed lipohypertrophy and to signs of IR [35]. Other studies have also shown that adiponectin was inversely associated with IR, type 2 diabetes and obesity in HIV and non HIV-patients in without adjusting for gender differences [36, 37].

Leptin is a 167-amino-acid adipokine secreted mainly by white adipose tissue. The levels of leptin are positively correlated with the amount of body fat [37]. Leptin regulates energy homeostasis and various neuroendocrine functions [38]. Our data showed higher levels of leptin in women with IR vs women without IR and a weak correlation between IR presence and leptin, observed in the linear regression model, with QUICKI as dependent and LOGLeptin as independent variable. Leptin did not contribute to an improvement of our linear model of IR in men. Other studies investigating this association have reported inconclusive results but very few have based their analyses on gender differences. Azzoni et al. in 2010 observed that serum leptin levels are inversely associated with HIV replication, independent of disease progression. In the same study, the authors reported a weak association between leptin levels and the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) [39]. Mynarcik et al. (2002) found that levels of leptin did not correlate with insulin sensitivity, in HIV-infected subjects with body fat redistribution [37]. In other study investigating adipokines and antiretroviral therapy related lipodystrophy, Tao et al. (2009) observed that leptin was positively correlated with the levels of total body fat, limb fat, and trunk fat, both in lipodystrophic and patients without body fat redistribution, but did not correlate with IR, and might serve only as a biomarker for total body fat mass [40]. Nagy et al. (2003) found a significant association between leptin concentrations and IR in patients with different morphological phenotypes. The lowest mean leptin levels were observed in patients with lipoatrophic changes. Hypoleptinemia was independently associated with IR in patients with lipoatrophy. Highest leptin concentrations were observed in patients with lipohypertrophy. Leptin levels in between the two extremes were observed in subjects with no body shape changes [41]. The role of leptin alterations in IR pathogenesis remains to be clarified by future long-term and prospective studies.

Despite the relative low number of patients, our study has several strengths. This is the first study in Romania to investigate adipokine dysregulation in association with HIV metabolic abnormalities during cART. The young age of our cohort is a distinct feature for the HIV epidemiology in Romania, compared with western European countries. The patients were probably infected parenterally in their childhood and adolescence between 1980–1990. QUICKI was used in order to diagnose IR, which is one of the most accurate surrogate indexes for determining insulin sensitivity in humans. Very few studies have evaluated untill now IR risk factors based on sex differences. Factors associated with IR, which have been lacking in previous studies, including the time from HIV diagnosis and the time on cART were measured and evaluated in our analyses.

Our study has some limitations. Correlations were small to moderate in magnitude. The association between IR and adiponectin/triglycerides in men and IR and leptin in women disappeared after including age and BMI in the regression models. This may be caused by BMI variations and changes in adiponectin with age that may contribute to IR pathogenesis and also by the low number of enrolled patients, with loss of statistical power as more variables were added. QUICKI was used to diagnose IR, a surrogate method, which has some limitations, such as the absence of a definite threshold for IR in Caucasian populations. QUICKI requires the quantification of fasting plasma insulin. Similar observations in clinical practice may be predicted with the use of International Diabetes Federation (IDF) or Adult Treatment Panel III (ATPIII) definitions of metabolic syndrome, which may provide cheaper ways for detecting patients at risk of diabetes and cardiovascular events. As leptin may be positively correlated with the levels of total body fat, limb fat, and trunk fat [41], the predictive value of our model might have been improved if we had included body fat redistribution in our analysis.

We report a significant prevalence of IR in a young non-diabetic Caucasian population with HIV infection undergoing antiretroviral therapy that was independent of the time of the HIV diagnosis. Hypoadiponectinemia in men and hyperleptinemia in women was demonstrated to be associated with IR presence which brings new data to support the role of these cytokines in IR pathogenesis. As adipokines play major roles in regulating glucose homeostasis with levels varying according to sex, we suggest that further studies investigating adipokines should base their analyses on gender differences. In addition, new studies are needed to investigate the mechanisms contributing to the significant differences observed between men and women.

SLD-ART Study Group: Sorin Petrea, Otilia Benea, Mariana Mărdărescu, Monica Luminos, Dan Oţelea, Ana Maria Tudor, Simona Paraschiv, Viorica Leoveanu (deceased), Mihaela Rădulescu, Mihai Lazăr, Sergiu Costoiu, Ioana Olaru, Cristina Popescu, Cristina Loredana Benea, Ruxandra Moroti, Violeta Molagic, Viorica Poghirc.