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Departments of HIV and Chemical Pathology (M.Sh., S.D., R.C., P.N.), University Hospitals Birmingham, Birmingham B29 6JF, United Kingdom; and Department of Diabetes and Endocrinology (M.St., F.S.-M., N.C.J., W.J., A.M.U.), St. Thomas’ Hospital, GKT School of Medicine, Kings College, London, SE1 7EH, United Kingdom
Address all correspondence and requests for reprints to: Margot Umpleby, Department of Diabetes and Endocrinology, 4th Floor, N Wing, St. Thomas Hospital, Lambeth Palace Road, London SE1 7EH, United Kingdom. E-mail: margot.umpleby{at}kcl.ac.uk.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Compared with the controls, high-density lipoprotein cholesterol was reduced in all groups (P < 0.01). Plasma triglyceride was increased in patients taking protease inhibitors (P < 0.05). VLDL and IDL apoB fractional catabolic rate (FCR) was lower in all treatment groups (P < 0.05) compared with controls. Trunk fat, VLDL apoB absolute secretion rate, and insulin resistance were not different between groups. Peripheral fat was lower in the treated patients (P < 0.05) and correlated with duration of therapy (r = –0.55; P < 0.001). There was a positive correlation between peripheral fat and VLDL apoB FCR (P = 0.002) and IDL apoB FCR (P = 0.002) and a negative correlation with VLDL apoB pool size, VLDL cholesterol, and triglyceride (P < 0.03; P < 0.01; P < 0.002).
These results suggest that mild dyslipidemia resulting from antiretroviral therapy is caused by a decrease in VLDL and IDL apoB FCR, which is associated with a loss of peripheral fat.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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HIV infection itself has been reported to increase triglyceride levels and impair triglyceride metabolism (6, 7) and lipoprotein lipase activity (7). HIV-infected patients also exhibit reduced plasma cholesterol, low-density lipoprotein (LDL) cholesterol (7) and apolipoprotein-B 100 (apoB100). Additionally, they have reduced high-density lipoprotein (HDL) and apoA-1 associated with this lipoprotein (7, 8), higher LDL triglyceride, and higher total cholesterol-HDL cholesterol ratio (6).
Treatment with HIV protease inhibitors for very short periods causes hypertriglyceridemia and impaired insulin sensitivity (9) even in HIV-negative subjects (10, 11). Finally, the fat redistribution, which becomes apparent only after several months on treatment (2), is associated with the dyslipidemia and insulin resistance (2), although the dyslipidemia may occur in the absence of obvious lipodystrophy (12). Both the nucleoside reverse transcriptase inhibitor (NRTI) and the protease inhibitor (PI) component of highly active antiretroviral therapy (HAART) (2, 13) contribute in an as yet undefined way to the lipid abnormalities and body fat distribution. The nonnucleoside reverse transcriptase inhibitor (NNRTI) component of HAART may also contribute to the dyslipidemia (14, 15), although its relation to the lipodystrophy is unclear (16).
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A cross-sectional study was performed on 40 HIV-positive patients who were taking two NRTIs plus either a PI (n = 15) or the NNRTIs nevirapine (n = 11) or efavirenz (n = 14) for between 1 and 6 yr. Patients on PI were taking nelfinavir (n = 6), lopinavir/ritonavir (n = 3), ritonavir alone (n = 2) or with indinavir (n = 2) or saquinavir (n = 1), and indinavir alone (n = 1). There was no difference in the nucleoside backbone between treatment groups (Table 1
). Fifteen patients naive to antiretroviral therapy (TN) were also studied as well as 12 presumed HIV-negative controls. A negative HIV test within the last 3 months was required if risk history revealed a risk of HIV (two subjects).
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Study protocol
After an overnight fast, patients were admitted to the Wellcome Trust Clinical Research Facility, and an iv line was placed in the antecubital vein of each arm, one for blood sampling and one for administration of [1-13C]leucine (13C enrichment, 99%; Cambridge Isotope Laboratory, Andover, MA) as a primed (1 mg/kg) constant infusion (1 mg/kg·h) for 9 h. A baseline blood sample was taken before the infusion, and then samples were taken at 30 min and then every hour for 9 h. Body fat distribution was measured by whole-body dual-energy x-ray absorptiometry (DEXA) scan within 1 month of the study. Fifty-seven subjects were scanned using a Hologic QDR 4500A (version 11.2:3, Hologic Inc., Waltham, MA) and nine using a Lunar DPX-L (version1.3 g, GE Medical Systems, Waukesha, WI). One patient refused a scan. Results are presented as leg fat, peripheral fat (arm plus leg), or trunk fat (g), divided by the body mass index (BMI, kg/m2).
Experimental methods
Very-low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) were separated by sequential flotation ultracentrifugation (Beckman Coulter Optima LE80-K ultracentrifuge, High Wycombe, UK). ApoB-100 was precipitated from VLDL and IDL fractions with tetramethylurea, delipidated, and hydrolyzed with 6 M hydrochloric acid. The isotopic enrichment of leucine in VLDL and IDL apoB was determined by selected ion monitoring of the t-butyldimethylsilyl derivative at mass-charge ratios (m/z) of 302 and 303 on a gas chromatograph-mass spectrometer (Agilent 5973 network MSD, Bracknell, UK) in the electron impact ionization mode. The isotopic enrichment of 
-ketoisocaproic acid was determined by selected ion monitoring of the quinoxalinol-tert-butyldimethylsilyl derivative at m/z 259 and 260 on a gas chromatograph-mass spectrometer employing electron impact ionization.
ApoB-100 VLDL and IDL concentrations were determined by an in-house specific sandwich ELISA using a polyclonal antibody against apoB (The Binding Site Ltd., Birmingham, UK) as a capture antibody and a biotinylated anti-apoB-100, 4G3 antibody (Ottawa Heart Institute, Ottawa, Canada) as a detection antibody (interassay coefficient of variation, 8%). Enzymatic methods were used to measure plasma total, VLDL, and IDL cholesterol and triglyceride (ABX Diagnostics, Shefford, UK) and HDL cholesterol (Roche Diagnostics Ltd., Lewes, UK). LDL was separated by ultracentrifugation and cholesterol content measured by enzymatic methods (ABX Diagnostics). Nonesterified fatty acid (NEFA) was measured enzymatically (Wako Chemicals, Neuss, Germany) and lipoprotein(a) with an immunoturbidimetric method (Diasorin Ltd., Wokingham, UK) using a Cobas Fara II analyzer (Roche Diagnostics). Serum insulin was measured by ELISA (Mercodia, Uppsala, Sweden) and glucose concentrations using a glucose analyzer (Roche Diagnostics).
Data analysis
Insulin resistance was calculated using the homeostasis assessment model (HOMA) (17). The fractional catabolic rate (FCR) and absolute secretion rate (ASR) of VLDL and IDL were calculated using a multicompartmental model with an intrahepatic delay function as previously described by Duvillard et al. (18) using simulation analysis and modeling (SAAM II software, SAAM Institute, Seattle, WA). Patients were in a steady state in the study as shown by the constant VLDL and IDL apoB concentration. In this case, the fractional secretion rate equals the FCR. The VLDL and IDL apoB ASR (mg/kg·day) were calculated from the product of the fractional secretion rate (pools/d) and the pool size (mg) divided by body weight. The VLDL or IDL pool size was calculated from the product of the mean VLDL or IDL apoB concentration (mean concentration of apoB in four pooled samples) and the plasma volume. Plasma volume was calculated using the formula of Pearson et al. (19).
Because of problems with the sample analysis, it was not possible to model IDL data from one subject in the TN group, two subjects in the PI group, and five subjects in the NNRTI group.
Initial comparison between the five groups was by one-way ANOVA or Kruskal-Wallis followed by Bonferroni’s or Dunn’s multiple comparison test (SPSS 10.0.7 for Windows). Fisher’s exact test was used for categorical data between groups, and associations were analyzed by Spearman’s rank correlation test. A stepwise linear regression model examined the effect of variables on VLDL and IDL metabolism. Variables entered were age, sex, ethnicity, smoking, family history of diabetes, family history of cardiovascular disease, alcohol intake, peripheral fat/BMI, trunk fat/BMI, glucose, HOMA, and NEFA. In a separate model, only patients on treatment were included.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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There was no significant difference between groups with regard to age, ethnicity, and BMI (Tables 1
and 2). Family history of diabetes was, however, different between controls and HIV TN patients. All had normal liver function tests. There was no difference between the groups in serum AST, 
-glutamyltransferase (GGT), alkaline phosphatase, or hemoglobin. No patient had hepatitis C or was a carrier for hepatitis B. Current CD4 counts were not different between HIV groups, nor were current or pretreatment viral loads or pretreatment CD4 counts among the treatment groups. Two PI-treated patients on HAART had a detectable viral load (800 and 1100 copies/ml). The remaining patients had viral loads below the detection limit of the assay (<50 copies/ml). Length of treatment was lower in the NNRTI group than the PI group (P < 0.001).
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, 3, and 4)
Only five patients had baseline cholesterol plasma levels above 6 mmol/liter (three PI and two NNRTI), and 13 had fasting plasma triglyceride over 2.3 mmol/liter (two TN, four PI, and seven NNRTI). Severe hyperlipidemia (cholesterol > 7 mmol/liter and/or triglyceride > 5 mmol/liter) was observed in only two patients, both on PI. In the PI group, total cholesterol was significantly greater than the TN group (P < 0.05), and plasma triglyceride, IDL triglyceride, VLDL cholesterol, and IDL cholesterol were significantly greater than the control group (P < 0.05). IDL triglyceride was also greater in the TN group than the control group (P < 0.05). HDL cholesterol was lower in TN, PI, and NNRTI group compared with the control group (P < 0.01) (Table 2). There was no difference in the lipid profile in the nevirapine- and efavirenz-treated patients in the NNRTI group.
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and 4)
In the PI and NNRTI groups, VLDL and IDL apoB FCR were lower (P < 0.001; P < 0.05) and VLDL and IDL residence time were higher than the control group (P < 0.001; P < 0.05) but were not different from the HIV TN patients. There was no significant difference in VLDL apoB ASR, VLDL apoB pool size, or IDL apoB ASR between groups, although IDL apoB pool size was higher in the PI-treated patients compared with the control subjects (P < 0.05). There was no difference in VLDL and IDL apoB kinetics in the nevirapine- and efavirenz-treated patients in the NNRTI group. VLDL and IDL apoB kinetics in the HIV TN patients showed a trend in the same direction as the antiretroviral-treated groups. Patients on ritonavir-containing regimens had a lower VLDL apoB FCR (median, 4.6 pools/d; interquartile range, 2.6–7.1; P < 0.03) and increased residence time [5.21 h (3.43–9.2); P < 0.03] compared with non-ritonavir-containing PI regimens [8.4 pools/d (6.1–9.4) and 2.86 h (2.5–4.0), respectively]. Antiretroviral-treated patients with dyslipidemia (cholesterol 
6 mmol/liter or triglyceride 2.3 mmol/liter) had lower VLDL FCR (P < 0.001), larger VLDL and IDL apoB pool size (P < 0.003), increased VLDL residence time (P < 0.001), and increased trunk fat/BMI (P < 0.04), HOMA (P < 0.04), and NEFA (P < 0.003) compared with those without dyslipidemia.
Fat distribution (Table 2)
Peripheral fat/BMI and leg fat/BMI were significantly reduced in the PI and NNRTI groups compared with the control group (P < 0.05). Trunk fat was not significantly different between groups. Combining all groups, peripheral fat/BMI was correlated with VLDL apoB FCR (Fig 1A; r = 0.37; P = 0.002) and IDL apoB FCR (Fig 1B; r = 0.4; P = 0.002) and inversely with duration of treatment (r = –0.55; P < 0.001), VLDL apoB pool size (r = –0.28; P < 0.03), VLDL cholesterol (r = –0.31; P = 0.01), VLDL triglyceride (r = –0.38; P = 0.002), and IDL cholesterol (r = –0.34; P = 0.006). Trunk fat/BMI was negatively correlated with VLDL (r = –0.25; P < 0.05) and IDL (r = –0.29; P < 0.03) apoB FCR and positively with VLDL apoB pool size (r = 0.32 P < 0.01), VLDL cholesterol (r = 0.35; P = 0.004) and triglyceride (r = 0.29; P < 0.02), and HOMA (r = 0.49; P < 0.001).
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In a linear regression model, HOMA inversely predicted VLDL apoB FCR (P = 0.005) and IDL ASR (P < 0.02), whereas HOMA predicted VLDL apoB residence time (P = 0.005), VLDL cholesterol (P = 0.006), and VLDL triglyceride (P = 0.008). Peripheral fat predicted VLDL apoB FCR (P < 0.001) and IDL apoB FCR (P < 0.001) but inversely predicted IDL cholesterol (P < 0.001). Trunk fat predicted VLDL apoB pool size (P = 0.006), VLDL cholesterol (P < 0.001), and IDL residence time (P < 0.001) and inversely IDL apoB FCR (P < 0.001).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The lipodystrophy that follows antiretroviral treatment of HIV is a complex interrelation between peripheral fat loss and visceral adipose accumulation (1, 20, 21). The fat loss of antiretroviral therapy is thought to be mainly the result of the NRTI backbone of HAART regimens through their interference with mitochondrial DNA polymerase- (22), accentuated by the addition of PI (23). However, there is also evidence that HIV infection itself may cause some sc fat loss (24). Our study showed peripheral fat was reduced to a similar extent in all the HIV treatment groups including patients on NNRTI-containing antiretroviral regimens despite different time exposure to antiretroviral therapy. Longitudinal studies have shown that peripheral fat loss is progressive in the first 2 yr but slows thereafter (1, 25) in a nonlinear fashion (13). Although PI use has been shown to contribute to the central fat accumulation (20), results from this study show that trunk fat, which on DEXA scan is an amalgam of two opposing trends of sc fat loss and visceral fat gain, was not statistically different between groups.
HDL cholesterol was reduced in the HIV TN patients and both HAART treatment groups as reported previously (26). The dyslipidemia in the patients in the current study was less marked than in many previous studies, many of which were in patients taking PI-containing medication (1, 5). Efavirenz-containing HAART regimens are also reported to raise plasma cholesterol (15) and triglyceride levels (27), whereas nevirapine-containing regimens appear to have a better lipid profile than PI (12) and efavirenz (14). We have shown that PI-containing regimens have a greater dyslipidemic effect than NNRTI, in particular causing an increase in plasma triglyceride, VLDL and IDL cholesterol, and IDL triglyceride concentration. VLDL and IDL cholesterol/apoB and triglyceride/apoB ratios were not different between the controls and HIV treatment groups, suggesting that there was no change in VLDL and IDL particle size in contrast with a study in patients on PI (28) that reported an increased VLDL triglyceride/apoB ratio.
This study demonstrates that the mild dyslipidemia in the treatment groups is predominantly because of a decrease in VLDL and IDL apoB FCR. The resulting increased residence time will allow increased exchange of triglyceride from VLDL and IDL with HDL and LDL. This could explain the reduced HDL cholesterol seen in all our HIV groups. The decrease in VLDL and IDL apoB FCR was strongly associated with peripheral fat loss. In addition, in a regression model, regional fat changes predicted VLDL and IDL apoB FCR. These findings suggest that there may be common mechanisms shared between the regional fat redistribution and the metabolism of apoB-containing lipoproteins. This may be related to an interference of antiretroviral treatment with lipoprotein lipase and hepatic lipase, which is closely linked to the lipodystrophy. This may be a direct effect of antiretroviral treatment on lipoprotein and hepatic lipase or an effect of changes in circulating adipokines resulting from the loss of peripheral fat. This is supported by the finding that lipoprotein lipase mRNA is lower in HIV patients with lipodystrophy compared with patients with no lipodystrophy (29). HIV infection itself, however, has also been reported to reduce lipoprotein lipase and hepatic lipase activity (7, 10). The trend toward a reduction in VLDL and IDL apoB FCR in HIV TN patients compared with controls would be in keeping with these reports. Although short-term exposure of healthy volunteers to PI has no effect on post-heparin lipoprotein lipase activity, it has been shown to reduce hepatic lipase (10). Longer treatment with PI-containing HAART regimens is reported to result in reduced lipoprotein lipase activity (26). PI treatment and lipodystrophy have been previously shown to be associated with insulin resistance (1, 2). This may result in impairment in lipoprotein lipase activity (30). Although, in contrast to many previous studies, we were unable to demonstrate a significant change in insulin resistance as measured by HOMA in any of the treatment groups, in a regression model, insulin sensitivity predicted VLDL apoB FCR, whereas insulin resistance predicted VLDL cholesterol and triglyceride content, possibly through the control of insulin on lipoprotein lipase activity. A decrease in the recently described endothelial lipase could also play a role in the lipoprotein abnormalities in HIV patients. Although it has been shown that this lipase is important for the metabolism of HDL, a recent study in mice suggests it may also have a role in the metabolism of apoB-containing particles (31).
Central obesity and increased visceral fat is strongly associated with insulin resistance (31). The failure to find a significant change in insulin resistance between groups in the present study may be explained by the lack of a significant change in trunk fat, although when groups were combined, there was a significant negative correlation between trunk fat and HOMA. Interestingly, trunk fat, NEFA, HOMA, and VLDL apoB pool size were significantly higher and VLDL apoB FCR was significantly lower in antiretroviral patients with dyslipidemia (cholesterol 6 mmol/liter or triglyceride 2.3 mmol/liter) compared with those without. The association between visceral fat, insulin resistance, and dyslipidemia is well documented (32).
There was no significant difference in VLDL ASR between any groups. This contrasts with a previous study that found an increase in VLDL ASR in PI-treated patients, all of whom had marked abnormalities in serum lipids (33). The authors also reported reduced rates of VLDL transfer into denser lipoproteins, implying a lower rate of lipoprotein lipase-mediated delipidation. Similarly, Reeds et al. (34) reported an increase in VLDL triglyceride synthesis and decreased clearance in six PI-treated patients with lipodystrophy selected to have plasma triglycerides more than 2.8 mmol/liter. The present study together with these studies suggest that a primary abnormality in HAART-treated patients is a decrease in VLDL clearance that leads to mild dyslipidemia, whereas severe dyslipidemia is the result of both a decrease in VLDL clearance and an increase in VLDL ASR. The latter may be associated with an increase in insulin resistance that is characterized by an increase in the production rate of VLDL apoB (35).
The only previous study of the effect of a NNRTI on lipoprotein kinetics compared VLDL and IDL apoB kinetics in six PI-treated patients and five patients treated with nevirapine in the fed state (12). Patients had no lipodystrophy as defined by physical examination. No comparison was made with control subjects or HIV TN patients. As in the present study, patients had only mild dyslipidemia, and VLDL and IDL FCR were not different in the two groups. Unlike the present study, VLDL and IDL apoB production rates were higher in the PI-treated patients. This difference may be because of the small numbers or the fed state.
In conclusion, our data suggest that in patients with mild dyslipidemia, reduced VLDL and IDL apoB FCR may be the primary abnormality in lipoprotein kinetics in HIV infection, possibly consequent to the peripheral fat loss, which is further exacerbated by treatment with either PI or NNRTI-containing HAART regimens. The association of VLDL and IDL apoB FCR with regional fat distribution suggests that the mechanism for decrease in this parameter may be related to a decrease in lipoprotein lipase and hepatic lipase activity. Additional studies on the time course of appearance of these changes may help elucidate the interrelationships between lipodystrophy and dyslipidemia.
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Acknowledgments |
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Footnotes |
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First Published Online November 2, 2004
Abbreviations: ApoB, Apolipoprotein-B; ASR, absolute secretion rate; BMI, body mass index; DEXA, dual-energy x-ray absorptiometry; FCR, fractional catabolic rate; HAART, highly active antiretroviral therapy; HDL, high-density lipoprotein; HOMA, homeostasis assessment model; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; NEFA, nonesterified fatty acid; NNRTI, nonnucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; TN, treatment-naive; VLDL, very-low-density lipoprotein.
Received July 1, 2004.
Accepted October 22, 2004.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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co-activator 1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapy. AIDS 1753–1762
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