# Antiretroviral-Related Adipocyte Dysfunction and Lipodystrophy in HIV-Infected Patients: Alteration of the PPARγ-Dependent Pathways

Authors: Martine Caron; Corinne Vigouroux; Jean-Philippe Bastard; Jacqueline Capeau
Journal: PPAR Research
 (2009)

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## Abstract

Lipodystrophy and metabolic alterations are major complications of antiretroviral therapy in HIV-infected patients. In vitro studies using cultured murine and human adipocytes revealed that some protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) were implicated to a different extent in adipose cell dysfunction and that a chronic incubation with some PIs decreased mRNA and protein expression of PPARγ. Defective lamin A maturation linked to PI inhibitory activity could impede the nuclear translocation of SREBP1c, therefore, reducing PPARγ expression. Adipose cell function was partially restored by the PPARγ agonists, thiazolidinediones. Adverse effects of PIs and NRTIs have also been reported in macrophages, a cell type that coexists with, and modulates, adipocyte function in fat tissue. In HIV-infected patients under ART, a decreased expression of PPARγ and of PPARγ-related genes was observed in adipose tissue, these anomalies being more severe in patients with ART-induced lipoatrophy. Altered PPARγ expression was reversed in patients stopping PIs. Treatment of patients with agonists of PPARγ could improve, at least partially, the subcutaneous lipoatrophy. These data indicate that decreased PPARγ expression and PPARγ-related function, resulting from ART-induced adipose tissue toxicity, play a central role in HIV-related lipoatrophy and metabolic consequences.


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## Body

## 1. Introduction

HIV-associated lipodystrophy (LD) is a disorder characterized by a selective damage of the adipose tissue
resulting in part from antiretroviral drugs [1, 2]. The LD syndrome includes progressive
subcutaneous fat loss and/or central fat accumulation along with dyslipidemia,
glucose alterations, and insulin resistance, altogether generating
cardiovascular dysfunctions [3, 4]. Recent studies have hypothesized that HIV
itself could play a role in the LD phenotype (see Giralt et al. [5]). However, the risk of developing fat tissue redistribution
has been related in priority to the antiretroviral treatment  (ART) and mainly to the use of two classes of
drugs, protease inhibitors (PIs) and nucleoside reverse transcriptase
inhibitors (NRTIs) [6–8]. Lipoatrophy in the face and extremities has been
linked repeatedly to the use of stavudine (and to a lesser extend zidovudine)
among NRTIs [7, 9, 10] and increases with long-term exposure [11]. PIs have been mainly associated with central fat
accumulation along with insulin resistance. However, nelfinavir or indinavir
can independently decrease limb fat level in patients cotreated with NRTIs [7, 12]. Peripheral fat loss and
central fat accumulation can occur simultaneously, though lipoatrophy may
emerge as the more dominant feature on prolonged treatments [12, 13]. Recently, a role for the
nonnucleoside analog efavirenz in lipoatrophy has been reported but needs to be
confirmed [14].The pathogenesis
of adipose cell dysfunction includes the mitochondrial toxicity of NRTIs [15–19] and the adverse effects of
PIs and NRTIs on the adipocyte differentiation status [17, 20–26], insulin sensitivity [27, 28], survival [17, 18, 23, 29], ability to secrete a variety
of adipokines [30–33], and longevity [19, 34]. The oxidative stress induced
by both PIs and NRTIs at the fat cell level [19, 28, 33–35] probably plays a major role
in the setup of lipodystrophy.Severe adipose
tissue alterations have been reported in HIV-infected patients with ART-related
lipodystrophy. Lipoatrophic adipose tissue biopsies present major histological
alterations with decreased and heterogeneous size of adipocytes, increased
fibrosis, altered mitochondria, and macrophage infiltration [1, 2, 36–38], consistent with a profound
remodeling of subcutaneous fat tissue. The presence of isolated fat droplets,
macrophages, and apoptotic cells in the enlarged vascular stroma argues for a
progressive destruction of subcutaneous adipocytes [1, 2, 29, 37, 39, 40].PPARγ is expressed in priority in
adipocytes. It is also expressed in different other cell types including macrophages and
regulates genes associated with growth, differentiation, insulin sensitivity,
inflammation, and immunity [41–46] (see [5]). PPARγ plays an essential role in the development and
normal function of white adipocytes, where it mediates part of the regulatory
effect of dietary fatty acids on gene expression [43, 47], regulates the differentiation program [48] and insulin sensitivity [45]. PPARγ also controls the production and secretion of
adipokines such as leptin and adiponectin, which are important mediators of
insulin action in peripheral tissues [42]. In brown adipocytes, PPARγ also controls the adipogenic program and the
switch from white to brown adipocytes [49]. In macrophages, PPARγ controls alternative activation and improves
insulin resistance [50]. It plays an important role in macrophage
inflammation and cholesterol homeostasis and inhibits the production of
proinflammatory cytokines through inhibition of the NFκB
and AP-1 pathways [48, 51–54].Loss-of-function or
dominant-negative mutations in the PPARG gene in humans (see [5]), and genetically-induced PPARγ
deficiency in mice [55, 56] are responsible for lipodystrophic syndromes
with insulin resistance, showing the primarily involvement of PPARγ
defects in adipose tissue development and metabolic roles. Alternatively, other
causes of adipocyte differentiation defects lead to a secondary decreased PPARγ
expression and/or function, that further contribute to adipose tissue
dysfunction, as shown in vivo in murine models [57] or in vitro [58–60].In that setting, the implication of
PPARγ in the ART effect has been demonstrated both
in vitro, in cultured adipocytes and macrophages, and ex vivo, in adipose
tissue samples from patients, and has been confirmed by the beneficial effects,
at least partial, of the PPARγ
agonists, thiazolidinediones. PPARγ
defects, although probably secondary to the multiple deleterious consequences
of ART on adipose tissue, play a central role in ART-related lipodystrophy and
metabolic alterations.


## 2. Effects of ART on PPARγ Expression and Signaling in Cultured Adipocytes

PPARγ contributes to the setup of the
differentiation program and to insulin sensitivity. PIs and NRTIs, the two
major classes of antiretrovirals associated with lipodystrophy in HIV-infected patients,
may interfere at several steps of PPARγ signaling in adipose cells, such as
differentiation, insulin action, oxidative stress, inflammation, and
mitochondrial function.A number of studies have clearly
shown that the first generation PIs, indinavir, nelfinavir, and ritonavir, used
at concentrations comparable to their Cmax in patients’ serum or at
suprapharmacological concentrations, impaired adipocyte differentiation [20, 21, 23, 25, 26, 32, 61–67]. They were also shown to induce insulin
resistance [21, 23, 27, 33, 62, 67–70] in murine and human cultured adipocytes. This
was associated with a reduced protein and mRNA expression of PPARγ in both murine [20, 21, 25, 26, 64] and human adipocytes [24, 66, 71, 72]. Interestingly, decreased PPARγ expression was also observed in mature
adipocytes chronically incubated with PIs, consistent with PI-induced adipose
cell dedifferentiation.Most PIs (nelfinavir, indinavir,
saquinavir, ritonavir, and amprenavir) were shown to acutely inhibit insulin
activation of glucose uptake in cultured adipocytes, via a direct inhibition of
the glucose transporter Glut4 [73]. Indinavir and nelfinavir also altered the
activation of proximal steps in insulin signaling as revealed by decreased
phosphorylation of extracellular-regulated kinase (ERK) 1/2 and Akt/protein
kinase B. Accordingly, distal events in insulin signaling pathways, glucose
transport, and lipogenesis were also affected [21, 30, 74]. Regarding PPARγ, cell imaging studies revealed that indinavir
and nelfinavir but not amprenavir severely decreased nuclear expression of PPARγ [21], indicating for the first time that the
transcriptional activity of PPARγ may be defective in PI-treated cells. The
beneficial effect of rosiglitazone [21, 23, 32] confirmed the implication of PPARγ in PI action, and indicated that PIs act
upstream of PPARγ in its signaling cascade to alter adipocyte
differentiation and insulin sensitivity. Recent data of our laboratory further
support the implication of PPARγ in PI action by showing that two angiotensin
II-receptor blockers (telmisartan and irbesartan), that display partial PPARγ agonist activity [75], prevented the PI effects on lipid
accumulation and insulin response in murine and human adipocytes (Boccara F. et al., unpublished results).The effect of ritonavir on insulin
signaling has been particularly studied since this commonly prescribed PI is
associated with dyslipidemia and metabolic disorders in HIV-infected patients [67, 76, 77]. Ritonavir induced insulin resistance in
cultured adipocytes [24, 32, 64]. Another study reported that ritonavir reduced
differentiation and insulin sensitivity in human preadipocytes and adipocytes
but surprisingly without decreasing PPARγ2 gene expression [68]. However, the protein expression and the
activation of PPARγ have not been evaluated in this study.The mechanism whereby PIs alter
adipose cell differentiation and insulin sensitivity is obviously complex and
multifactorial. Impaired SREBP-1 nuclear penetration [21, 22] may inhibit the activation of PPARγ or related adipogenic transcription factors
thus leading to defective adipogenesis and insulin resistance. When going
further into the mechanism of PI action, we and others demonstrated that some
PIs prevented the maturation of lamin A/C [22, 34, 78], a nuclear membrane protein essential for
normal nuclear membrane folding and for nuclear penetration of SREBP-1 [59, 79, 80]. Defective SREBP-1c signaling may explain the decreased differentiation
and insulin resistance of PI-treated cells and the ability of PPARγ agonists to overcome the PI effects on fat
cell differentiation and insulin response [21].NRTI therapy is also associated
with fat tissue disease in HIV-infected patients. In murine adipose cell lines
and primary cultured human adipocytes, stavudine and zidovudine, but not other
NRTIs (tenofovir, abacavir, didanosine, and lamivudine), alter lipid storage [23, 31, 33, 81]. They also decrease the expression and
secretion of adiponectin in cultured human and murine adipocytes [23, 32, 33, 82] and induce oxidative stress, suggesting that
they could secondarily participate to the insulin resistance setup [33]. The negative effect of NRTIs on PPARγ expression and signaling has been reported
only in a few studies. Stavudine or zidovudine have a modest, or no effect, on
adipose cell differentiation assessed by the gene expression profile of
differentiating adipocytes [25] and by protein and mRNA expression of
adipogenic transcription factors, among them PPARγ [20, 25, 31, 32, 82]. Altered adipocyte lipid phenotype and insulin
sensitivity resulting from NRTI treatment are suspected to result from their
mitochondrial toxicity [15–18]. We recently reported that stavudine or
zidovudine, but not other NRTIs, triggers mitochondrial oxidative stress and
premature senescence in cultured fibroblasts and adipocytes [19]. Stavudine also altered in human preadipocytes
[72] the expression of the PPARγ coreceptor 1-alpha (PGC1-α) a transcriptional coactivator upregulated by
thiazolidinediones which controls mitochondrial function and biogenesis, and
metabolic pathways and integrates insulin signaling and mitochondrial function [83, 84]. Stavudine
increased its expression together with mitochondria number [72]. Thus, conversely to PIs, in vitro, thymidine analogs
have no or mild detrimental effect on PPARγ function.The non-NRTI class
of antiretrovirals has not yet, as a class, been associated with long-term
toxicity [7] even if efavirenz was shown in one study to be
associated with lipoatrophy [14]. Very few studies report
experimental in vitro findings
on the effects of the non-NRTIs efavirenz or nevirapine on white adipose cell
functions. Efavirenz but not nevirapine induced a delayed and moderate
reduction in lipid accumulation in both murine and human cultured adipocytes,
and decreased SREBP-1c and PPARγ expression [85].


## 3. Effect of ART on PPARγ Expression and Function in Animal Models

Ritonavir was shown to increase
lipogenesis [86] and to induce insulin resistance in animal
models [87]. In mouse fat tissue, it partially inhibits
the function of PPARγ as shown by the decreased induction of PPARγ target genes by rosiglitazone [88]. Lopinavir-ritonavir but not atazanavir
decreased by 25% the weight of peripheral inguinal fat in mice treated for 8
weeks, while the profound epididymal adipose tissue depot was not affected. The
expressions of SREBP-1c and of its target gene fatty acid synthase were
increased in the peripheral inguinal fat while that of PPARγ tended to be decreased in the two depots and
that of its target gene adiponectin was not modified [89]. Even if not entirely conclusive, these data
are in favor of an altered expression and/or function of PPARγ induced by some PIs in murine models.


## 4. Effect of ART on PPARγ Expression and Function in Patients’ Adipose Tissue

Studies performed on human adipose
tissue samples studied ex vivo concerned, at first, healthy controls treated
with ART. Mallon et al. [90] reported that a 2-week treatment with
stavudine/lamivudine or zidovudine/lamivudine resulted in an increased
expression of PGC1α
and PPARα
and a decreased expression of PPARγ without any modification in the expression of
SREBP1. Altered expression of PGC1α
was correlated with upregulation of nuclear genes involved in transcription
regulation of mtRNA and oxidation of fatty acids suggesting a central role for
PGC1 in nuclear response to mitochondrial dysfunction.Several studies
evaluated the expression of PGC1α and PPARγ in adipose tissue from long-term ART treated
HIV-infected patients with lipodystrophy. A decreased expression of the two
factors was reported in abdominal fat from lipodystrophic patients as compared
to controls [36, 37] and to non-lipodystrophic
patients [91]. A decreased expression of
PPARδ was also found in this latter study. Accordingly, a
decreased expression of the transcription factor SREBP-1 was also reported [36, 91, 92]. PPARγ adipose tissue expression was found decreased in HIV-infected
patients as compared to noninfected controls by Giralt et al. [5] but the major decrease was
observed in naïve versus ART-treated patients, without differences between
lipodystrophic and nonlipodystrophic patients, arguing for a major role for the
virus itself. The expression of PGC1α was increased. The group of D. Nolan and S. Mallal
observed that the PPARγ2 mRNA level was similar in fat from treatment-naïve
patients and in patients under PI or zidovudine but lower in patients under
stavudine. However, noninfected controls were not evaluated in that study [93]. Interestingly, adipose
tissue dysfunction appears more severe in peripheral than in abdominal
subcutaneous adipose tissue, as shown by the decreased expression of PPARγ, C/EBPα, and adiponectin in adipose tissue from thigh
versus abdomen [94]. Therefore, a strong alteration in PPARγ expression was found in most studies using
HIV-infected patients’ subcutaneous fat samples.To examine the
reversibility of adipose tissue alterations in HIV-infected patients, adipose
tissue biopsies were studied before and after a 6-month interruption of ART in
the Lipostop study. Adipose tissue inflammation improved markedly, with fewer
infiltrating macrophages and fewer TNFα- and IL6-expressing cells. mRNA expression of PPARγ and of markers of mitochondrial function and
biogenesis (cytochrome oxidase subunit 2 and PGC1α) improved after PI withdrawal. In patients who
stopped taking stavudine or zidovudine, adipose tissue inflammation,
mitochondrial status, and SREBP-1 expression were improved [95]. Since PGC1α is playing a leading role in mitochondria function [84], this indicates that altered
PGC1α and PPARγ expression induced by some ART may be involved in
mitochondria dysfunction observed in patients’ fat [90, 95].Decreased PPARγ expression was
also strongly correlated with increased expression of inflammatory cytokines
such as IL-6 and TNF-α and decreased expression and circulatory
levels of adiponectin which is involved in liver and muscle insulin sensitivity
[1, 36, 37, 91, 96]. These data confirm that
altered PPARγ function in adipose tissue plays a role in overall
insulin resistance associated with lipodystrophy, as reported in
genetically-determined PPARγ dysfunctions [45]. In accordance, the study from Sutinen et al. [97] reported the effects on adipose tissue of a
24-week treatment with the PPARγ
agonist rosiglitazone compared with placebo in HIV-infected patients with
lipodystrophy. The expression of adiponectin, PPARγ, and PGC1α
significantly increased while that of IL-6 decreased. Expression of other genes
involved in lipogenesis, fatty acid metabolism, or glucose transport, such as
PPARδ,
and SREBP-1, remained unchanged. Rosiglitazone also significantly induced an
increase in serum adiponectin concentration, which was inversely correlated
with the changes in fasting serum insulin concentration and liver fat content.
Such data have led to conduct clinical trials using thiazolidinediones to try
to reverse peripheral fat loss. Even if the results obtained with rosiglitazone
were disappointing (see [97]), possibly
due to the ongoing presence of stavudine in the ART regimen, recent data
obtained with pioglitazone are more promising and reveal, in patients not
treated with stavudine, an improvement of peripheral fat [98] further supporting a role for PPARγ dysfunction in lipoatrophy.


## 5. PPARγ Expression and Fat Hypertrophy in HIV-Infected Patients

The lipodystrophic phenotype
observed in HIV-infected patients associates, to
different extent, peripheral lipoatrophy and fat hypertrophy in different fat
depots. In particular, a buffalo hump has been observed in a number of
patients. The group of F. Villaroya showed that buffalo humps from HIV-infected
patients displayed a brown adipose tissue phenotype with both specific
uncoupling protein 1 (UCP1) expression and mitochondrial dysfunctions [99]. However, there were no significant changes in
the expression of other UCP genes or of that of markers of adipogenesis including PPARγ, PGC1α, and adiponectin relative to controls. A
more extensive analysis indicated that buffalo hump tissue does not express a
complete brown adipocyte phenotype but rather a distorted brown-versus-white
phenotype associated with enhanced proliferation [2]. In addition, buffalo humps failed to show
increased expression of TNFα
or the macrophage marker CD68 indicating the absence of a local inflammatory
status. Since adipose tissue inflammation and the presence of proinflammatory
cytokines has been presumed to play a role in subcutaneous fat lipoatrophy in
HIV-infected patients, this absence of inflammation could explain, at least in
part, the absence of fat loss observed in that depot.The effect of antiretrovirals on
brown adipocytes has been evaluated in two studies. In primary culture of
differentiated murine brown adipocytes, neither the cell differentiation nor
the level of PPARγ was modified by the treatment with a series of
NRTI including stavudine and zidovudine. By contrast, regarding the NNRTI,
nevirapine increased and efavirenz decreased brown adipocyte differentiation
and PPARγ expression. PGC1α
expression was not modified by the drugs except for its increase in response to
stavudine and nevirapine [100]. In the T37i brown adipocyte cell-line,
indinavir, stavudine, and zidovudine alone or in association impaired PPARγ2 and
UCP1 expression together with a strong inhibition of cell differentiation and
mitochondrial functions, although the 3T3-F442A white adipocyte cell line,
studied under similar conditions, was less severely affected [26]. Therefore, brown fat can also be a target of
antiretrovirals. Since the presence of brown adipose tissue in normal humans
has been recently reassessed [101], it would be important to further evaluate its
alterations in HIV-infected patients under ART.Increased visceral fat is also a
characteristic feature of HIV-related lipodystrophy. However, samples from
patients are difficult to obtain and no study, up to now, has reported specific
data obtained with HIV-infected patients’ visceral fat. A few studies compared
the effect of antiretrovirals on adipocytes issued from subcutaneous and
visceral fat from noninfected subjects but the expression of PPARγ or PGC1 was not evaluated.


## 6. PPARγ and Macrophages

PPARγ plays an important role in macrophage function
and phenotype and exerts an overall anti-inflammatory function (see [5]). Recent data have shown that adipose tissue
from obese individuals presents macrophage infiltration as well as increased
number of “M1” or “classically activated” macrophages. Importantly, the
agonists of PPARγ have been shown to alter macrophage phenotype
to “M2” or an “alternatively activated” anti-inflammatory
phenotype and may induce macrophage specific cell death [102]. PIs could alter PPARγ in macrophages by increasing PPARγ mRNA
expression resulting in foam cell formation [103]. In the Lipostop study [95], we observed that stopping
ART resulted in an improvement of adipose tissue function associated with a
decreased number of M1 but not M2 macrophages together with an increased
expression of PPARγ. This can result from modified PPARγ expression both in adipocytes and macrophages.


## 7. Conclusion

In vitro and in vivo data strongly
suggest that altered PPARγ function plays a role in HIV-related
lipodystrophy as a result of a multifactorial toxicity of ART on adipose
tissue. In vitro studies investigating the effect of individual antiretrovirals
have clearly revealed that some PIs inhibit PPARγ functions,
probably at the earlier step of SREBP1c activation. Ex vivo studies of adipose
tissue, both in healthy volunteers and in HIV-infected patients, confirmed
these data but also point to a possible toxicity of NRTI, principally stavudine
and to a lesser extent, zidovudine. Since PPARγ is playing a central role in adipose tissue
differentiation and function, decreased PPARγ expression could be expected to be involved in
the pathophysiology of lipodystrophy. Importantly, both adipocytes and
macrophages present in patients’ adipose tissue can be affected at the PPARγ level. Adipose tissue dysfunction could induce
insulin resistance and deregulate adipokine secretion with increased release of
proinflammatory cytokines and decreased adiponectin, alterations which will
impact on the liver and muscles.Most studies in that setting
evaluated the expression and function of PPARγ and only scarce data are available for PPARα
and PPARδ.Using thiazolidinediones to reverse
fat lipoatrophy was a logical proposition. However, trials using rosiglitazone
were disappointing, in part due to the absence of discontinuation of stavudine.
Pioglitazone was more promising and resulted in some recovery of limb fat
further arguing for a role for PPARγ in initial fat alteration.



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