# HIV-1 Infection and the PPARγ-Dependent Control of Adipose Tissue Physiology

Authors: Marta Giralt; Pere Domingo; Francesc Villarroya
Journal: PPAR Research
 (2009)

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## Abstract

PPARγ is a ligand-dependent master transcription factor controlling adipocyte differentiation as well as multiple biological processes taking place in other cells present in adipose tissue depots such as macrophages. Recent research indicates that HIV-1 infection-related events may alter adipose tissue biology through several mechanisms involving PPARγ, ranging from direct effects of HIV-1-encoded proteins on adipocytes to the promotion of a proinflammatory environment that interferes with PPARγ actions. This effect of HIV-1 on adipose tissue cells can occur even in the absence of direct infection of adipocytes, as soluble HIV-1-encoded proteins such as Vpr may enter cells and inhibit PPARγ action. Moreover, repression of PPARγ actions may relieve inhibitory pathways of HIV-1 gene transcription, thus enhancing HIV-1 effects in infected cells. HIV-1 infection-mediated interference of PPARγ-dependent pathways in adipocytes and other cells inside adipose depots such as macrophages is likely to create an altered local environment that, after antiretroviral treatment, leads to lipodystrophy in HIV-1-infected and HAART-treated patients.


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## Body

## 1. Introduction

A  complex set of metabolic alterations,  preferentially involving adipose
tissue,  has emerged in recent years in a substantial number of HIV-1-infected
patients under highly-active antiretroviral treatment (HAART). This is the
so-called HIV-1/HAART-associated lipodystrophy syndrome. Disturbances in
adipose tissue of these patients range from lipoatrophy of subcutaneous adipose
tissue to visceral adipose accumulation or lipomatosis [1]. Moreover, during
recent years, basic research on adipose tissue biology has succeeded in
identifying major molecular players in the differentiation and function of
adipose tissue. Among them, the peroxisome
proliferator-activated receptor γ (PPARγ)subtype
of PPARs has emerged as a master transcriptional regulator of adipose cells
[2]. Although we are still far from a full understanding of the molecular basis
of HIV-1 lipodystrophy, research has been actively undertaken in this area and
has examined the role of alterations in PPARγ-dependent pathways in eliciting the
syndrome [3, 4]. Regardless of the potential effects of antiretroviral drugs on
PPARγ, several recent findings suggest that
HIV-1 infection-related events may cause disturbances in the PPARγ-dependent pathways of control of
adipose tissue physiopathology, and they are summarized in the present review.


## 2. Adipose Tissue, HIV-1 Infection, and Lipodystrophy

The concepts concerning the causal basis of HIV-1 associated
lipodystrophy have evolved substantially in the last few years. The initial
identification of HIV-1 lipodystrophy was coincident with the introduction of
drugs from the protease inhibitor family for use in antiretroviral treatment,
and these drugs were at first considered to be causative of the syndrome [5].
After it was recognized that patients without protease inhibitor treatment
could also develop lipodystrophy, the syndrome was associated with HAART
overall. Although several drugs of HAART regimes are known at present to be
more prone to induce the appearance of lipodystrophy or of some of their
particular features (abdominal fat hypertrophy versus peripheral lipoatrophy)
than others [6], no single class of drugs can account for elicitation of the
overall syndrome. On the other hand, there have been suggestions that
antiretroviral treatment causes lipodystrophy only when acting upon
HIV-1-infected patients, and that events related to HIV-1 infection are
intrinsically associated with the development of the syndrome. Evidently, there
are no data on long-term antiretroviral treatment of non-HIV-1-infected
patients that could establish the specific role of HAART independent of the
HIV-1 infection, and a single two-week study of nucleotide-analog reverse
transcriptase inhibitor treatment of non-infected volunteers indicated the
appearance of only a few features of the lipodystrophy syndrome [7].Some data
have indicated that mild alterations of adipose tissue biology are already
present in nontreated HIV-1-infected patients [8]. The studies of body
composition in the era before HAART reported a disproportionate loss of body
fat mass in men with advanced HIV-1 disease or AIDS [9]. This was attributed to the progression towards AIDS-related wasting
and associated diseases. However, some data suggested that weight loss and
depletion of body fat may precede the progression to AIDS, even in adults with
normal CD4+ lymphocyte counts [10]. Further studies confirmed an indirect
effect of HIV-1 viremia leading to effects similar to AIDS wasting. Studies by
Visnegarwala et al. of HIV-1-infected men with CD4+ lymphocyte counts >200
cells/mm3 suggested that malnutrition due to decreased caloric
intake and increased  energy demands associated with an active opportunistic
infection was not contributing
factors in explaining reduced body weight [11]. It was concluded that reduction
in fat mass is related to HIV-1 infection itself, independent of the additive
effects of opportunistic illnesses. Similar conclusions were obtained from the
analysis of dyslipemia. Before the introduction of HAART, the early appearance
of hypertriglyceridemia and enhanced lipolysis was observed in HIV-1-infected patients before the
onset of overt illness [12, 13]. Recent studies have confirmed that HIV-1
infection-induced changes in lipolysis are unrelated to the further effects of
HAART leading to full-blown lipodystrophy [14]. Finally, several rodent models
relevant to HIV-1 infection events have shown metabolic disturbances in the
absence of any exposure to antiretroviral drugs. For instance, transgenic mice
expressing the HIV-1 accessory
viral protein R (Vpr) in liver and adipose tissue exhibit altered systemic fat
metabolism [15]. Disturbances of PPARγ, as a master regulator of adipose tissue
differentiation and function, may play a major role in the alterations of
adipose mass and lipid metabolism elicited by HIV-1 infection, and this issue
is summarized in the present review.


## 3. PPARγ and Its Pivotal Role in Adipose Tissue Function

PPARγ is highly expressed in adipose tissue, where
it plays a key role in adipose tissue development and function. There are two
major splice variants of PPARγ, γ1 and γ2, which differ in their N-terminal region
(PPARγ2 contains an additional 30 amino acids) and in
their tissue-specific expression; PPARγ2 is found almost exclusively in white and
brown adipose tissues whereas PPARγ1 is also relatively abundant in macrophages
and endothelial cells [16–18]. The activity
of both isoforms is regulated by posttranscriptional modifications and by
ligand-dependent transactivation and recruitment of coactivators. For instance,
phosphorylation inhibits the transcriptional activity of PPARγ [19] and promotes sumoylation, which further
reduces its transcriptional activity [20]. PPARγ forms heterodimers with retinoid X receptors
(RXRs) to bind to specific DNA sequences in its target genes. In the absence of
ligands, corepressors such as nuclear receptor corepressor (N-CoR) or silencing
mediator of retinoid and thyroid (SMRT) receptors bind to these heterodimers
and recruit histone deacetylases to repress transcription (reviewed in [21]).
Binding of ligands to PPARγ triggers conformational changes that allow the
recruitment of transcriptional coactivators, including members of the steroid
receptor coactivator (SRC) family [22] and PPARγ-coactivator 1α (PGC-1α) [23] that ultimately recruit histone
acetyltransferase coactivators such as p300/CBP or PCAF [21].However, the natural ligands for PPARγ remain unknown. Recent studies have provided
functional evidence for an unidentified natural ligand that is produced
transiently during adipogenesis [24]. There is also evidence that small
lipophilic compounds, such as polyunsaturated fatty acids and fatty acid
derivatives (eiocosanoids) bind and activate this receptor [25], thus
supporting the concept that PPARγ is a nutrient sensor that finely regulates
metabolic homoeostasis in response to different nutritional states.Regarding synthetic ligands, it is clear that members of the
thiazolidinedione (TZD) family of antidiabetic drugs are high-affinity agonists
for PPARγ [26]. TZDs have been reported to enhance insulin sensitivity in
animals and humans [27]. Furthermore, cellular, genetic, and pharmacological
studies have provided strong evidence both that TZDs function via PPARγ, and that adipose tissue is the main site
where the insulin-sensitizing effects of PPARγ are produced (reviewed in [28]).It was reported that TZDs induce adipocyte differentiation even before
they were known to be ligands of PPARγ [29]. By now, the key role of PPARγ as a
master regulator of adipogenesis has been clearly established, and
gain-of-function experiments have demonstrated that PPARγ is sufficient to
induce adipocyte differentiation in the presence of an appropriate ligand [30].
However, loss-of-function experiments to prove that PPARγ is required for this
process have been more difficult, since PPARγ homozygous inactivation results
in embryonic death due to placental alteration, in a developmental stage before
there is any adipose tissue development [31]. Later, however, studies utilizing
chimeric mice [32] and adipose-specific PPARγ knockout mice [33] confirmed the
essential role of PPARγ in adipose tissue differentiation.PPARγ also plays an important role in regulation of
lipid metabolism in mature adipocytes. Activation of PPARγ increases both fatty acid uptake and its
storage into adipocytes by promoting the transcription of genes such as those
encoding lipoprotein lipase, fatty acid binding protein-4 (aP2/FABP4),
phosphoenolpyruvate carboxykinase (PEPCK) [34–37], and also glucose transporter
GLUT-4, in order to increase fatty acid synthesis [38]. These effects of PPARγ may underlie its insulin-sensitizing effects.
Thus, together with the proadipogenic role of PPARγ (glucose homeostasis requires adequate amounts
of adipose tissue), the improvement of lipid storage in this tissue will
prevent ectopic lipid accumulation in nonadipose tissues such as liver,
skeletal muscle, and β-cells. Furthermore, PPARγ has been reported to induce transcription of
the PGC-1α gene in adipose tissue [39]. The coactivator
PGC-1α promotes mitochondrial biogenesis, thus
leading to an increase in fatty acid oxidation in adipose tissue, which may
protect against adipocyte hypertrophy [40]. Finally, adipose tissue has
endocrine functions, and PPARγ regulates expression of genes encoding
adipokines such as adiponectin, leptin, resistin, or cytokines such as TNFα. Activation of PPARγ promotes the expression of a
pro-insulin-sensitizing adipokine profile (i.e., induction of adiponectin and
reduction of TNFα gene expression) thus involving the cross-talk
between adipose tissue and other insulin-sensitive organs (liver, skeletal
muscle) in the insulin-sensitizing effects of PPARγ [41].Evidence from human mutations in PPARγ has further underlined the importance of PPARγ in the development of adipose tissue, in the
maintenance of glucose and lipid homeostasis and more generally in the control
of energy balance (reviewed in [42]). Patients harboring mutations in the
ligand-binding domain of PPARγ have a stereotyped phenotype characterized by
partial lipodystrophy, severe insulin resistance, dyslipidemia, hepatic
steatosis, and hypertension, thus identifying PPARγ as playing a molecular role in the
pathogenesis of the metabolic syndrome [43, 44].PPARγ is also expressed in macrophages
and endothelial cells, that is, cells that are present in adipose tissue
[17, 18, 45]. In endothelial cells, activation of PPARγ has antiproliferative,
antiangiogenic, and anti-inflammatory effects [45]. PPARγ is induced during
macrophage differentiation, and its activation increases the expression of
macrophage-specific markers, such as CD14 and CD11b [17, 46]. However,
loss-of-function approaches have demonstrated that PPARγ is not essential for
monocyte/macrophage differentiation either in vivo or in vitro [47, 48] but
selective deletion of PPARγ in macrophages results in increased insulin
resistance [49]. Recently, macrophage-mediated inflammation in adipose tissue
has been proposed to play a central role in the pathogenesis of insulin
resistance [50]. Two types of macrophages, proinflammatory M1 and
anti-inflammatory M2, are present in adipose tissue and their relative
abundance may change dynamically through recruitment of polarized monocytes
from the blood (macrophage infiltration) or through the effects of local
cytokines on macrophages in adipose tissue. Activation of PPARγ by TZDs has now
been reported to increase the proportion of anti-inflammatory M2 macrophages in
adipose tissue [51]. Furthermore, TZDs also act through PPARγ to inhibit the
expression of inflammatory mediators in macrophages, and as reported above, to
negatively regulate expression of cytokines such as IL-6, TNF-α, and monocyte
chemoattractant protein—1 (MCP-1/CCL-2)
in adipocytes [52]. In summary, activation of PPARγ improves adipose tissue
function by having a beneficial effect on the adipocyte—macrophage
relationship, which may result in prevention of insulin resistance.


## 4. HIV-1 Infection and PPARγ

Recent studies revealed that expression of marker genes of adipogenesis,
such as those encoding GLUT-4, adiponectin, or lipoprotein lipase is already
altered in subcutaneous adipose tissue from HIV-1-infected patients in the
absence of treatment [53]. These genes are known targets of PPARγ, and the expression of PPARγ itself is also reduced in HIV-1-infected
patients without treatment, relative to healthy controls [53]. In fact, in this
same sense, it has been observed that PPARγ expression is lower in HIV-1-infected and HAART-treated
patients with lipodystrophy relative to healthy controls [54], but similar when
compared to levels in antiretroviral-naive patients [55]. These findings point
to the possibility that HIV-1 infection and associated events interfere with
the action of PPARγ as a master transcriptional controller of
adipogenesis and, in a broader sense, of adipose tissue biology, thus
contributing to the appearance of lipodystrophy. Recent experimental evidence
is supportive of this possibility (see below). However, a relevant role of HAART
in worsening potential HIV-1-mediated alterations in PPARγ expression
cannot be excluded.  In this sense, it has been recently reported that a 6-month
interruption of antiretroviral treatment results in a significant amelioration
of PPARγ levels in patients formerly under HAART
containing protease inhibitors [56]. On the other hand, one of the features of
HIV-1-associated lipodystrophy is insulin resistance. It cannot be excluded
that PPARγ impairment in HIV-1-infected patients may
contribute to reduced insulin sensitivity, as PPARγ is a known target of drugs with
insulin-sensitizing properties (see above).It must be taken into account that the action of HIV-1 infection in adipose tissue and PPARγ activity should not be considered only in relation to adipocytes. As stated above, adipose tissue depots contain, in addition to adipocytes, other cells such as preadipocytes, macrophages, or
endothelial cells. Recently, even lymphocytes have been reported to be present
inside adipose depots [57, 58]. As these other cell types (e.g., macrophages or endothelial cells) also express PPARγ, they could themselves be sensitive to
HIV-1-mediated disturbances in PPARγ expression and activity. Moreover, cells
inside adipose tissue can release regulatory factors (such as adipokines or
cytokines) or metabolites (free fatty acids) capable of influencing surrounding
cells, for instance preadipocytes and adipocytes, and the overall pathways of
gene regulation dependent on PPARγ.


## 5. How May HIV-1 Infection Affect PPARγ Pathways in Adipose Tissue?

The possibility
that HIV-1 infection could influence PPARγ activity leads to the first consideration;
whether the cells present in adipose tissue depots can be infected by HIV-1.
The capacity of HIV-1 to infect adipocytes is controversial. Whereas some
initial reports indicated that adipocytes could be infected [59, 60], later
data appeared to exclude this possibility [61]. However, more recently, it was
reported that substantial infection of adipocytes could take place when TNFα was present in the medium [62]. High levels of TNFα are found in adipose tissue from
HIV-1-infected patients even before treatment and are part of a proinflammatory
environment already present in adipose tissue as a consequence of long-term
HIV-1 infection [53, 63]. It should be noted that studies on the capacity of
HIV-1 to infect adipose cells have focused on mature adipocytes and less is
known concerning preadipocytes. In adipose depots, resident preadipocytes are
found and they can differentiate into mature adipocytes. Lipoatrophic
situations combine a loss of adipose cells (primarily via apoptosis) and an
incapacity of preadipocytes to differentiate and replenish the depot; thus any
direct or indirect effect of HIV-1 infection that interferes with PPARγ could lead to this impaired differentiation.
Recent identification of PPARγ gene mutations causative of lipodystrophies of
genetic origin supports the notion that abnormal PPARγ function can lead to lipodystrophy [42, 64].Evidently, if a
cell of the adipocyte lineage is infected by HIV-1, it will be exposed to gene
products of HIV-1 and to their potential effects on PPARγ actions. A reported example of this is Nef, a
27 kDa HIV-1-encoded protein that localizes in the cytoplasm as well as nucleus
of infected cells [65]. It has been shown that nuclear Nef results in a
reduction in the expression of PPARγ and of PPARγ gene targets in human T cells and macrophages
as well as interfering with fat accumulation in cell lines [66]. The effects of
Nef were specific in impairing PPARγ-dependent, but not PPARα-dependent,
transcriptional activity.However, the
most relevant evidence of interference of PPARγ pathways by HIV-1-encoded products concerns
the HIV-1 accessory protein Vpr. Kopp and collaborators have recently shown
that Vpr suppresses the differentiation of adipocytes in cell culture by
interfering with PPARγ-dependent transactivation of target genes
[67]. Vpr acts as a corepressor of PPARγ by interacting with the ligand-binding domain
of the receptor in an agonist-dependent manner. Remarkably, this effect could
be observed when Vpr was added to the adipose cell culture media thus
indicating that, as already shown in other cell types [68], exogenous Vpr can
enter the cell and interfere with metabolic pathways. These findings are highly
relevant to an understanding of the etiopathogenesis of lipodystrophy in
HIV-1-infected patients. Thus, it is expected that adipose cells can be
affected by Vpr either due to direct infection by HIV-1, or indirectly, because
Vpr is known to be present as a soluble protein in the circulation of HIV-1
infected patients [69]. Moreover, adipocytes and preadipocytes may be exposed
to local concentrations of Vpr higher than those in the overall circulation,
given the proximity of cells such as macrophages (or even resident lymphocytes)
that can be infected and release Vpr.Finally, recent
research has revealed several features of the biology of PPARγ, in relation to adipogenesis, in which HIV-1
infection may be hypothesized to interfere. Thus, several players in cell cycle
control, such as cyclin D3 and CDK4, have been reported to promote adipogenesis
through interaction with PPARγ [70, 71], whereas E2F4 represses PPARγ during adipogenesis [72]. HIV-1 infection may
interfere with the cell cycle machinery, and specifically the HIV-1-encoded
proteins Vpr and Tat have been recognized as being capable of interacting with
CDK4, cyclin D3, and E2F4 [73–75]. However, the
capacity of these interactions to influence PPARγ-dependent pathways in adipocytes or in other
cells present in adipose depots, and their consequences for metabolism, remains
to be explored. Similarly, a number of reports have indicated that
HIV-1-encoded Tat or Vpr can interact with known coactivators of PPARγ required for its transcriptional activity,
such as p300/CBP or PCAF [76, 77]. It cannot be
excluded that HIV-1 infection-mediated events affect PPARγ activity in adipose tissue through these
interactions, although experimental evidence for this is lacking at present.


## 6. The Reciprocal Issue: The Effect of PPARγ on HIV-1 Biology, and the Implications of this for Adipose Tissue Pathophysiology

What we have described up to now provides evidence that HIV-1 infection may alter PPARγ activities. However, several reports also indicate the occurrence of reciprocal events, that is, the action of PPARs and particularly PPARγ on HIV-1 biology, especially on the replication and transcription of the HIV-1 genome. The capacity of nuclear receptors to interact with the long-terminal repeat of HIV-1 was recognized several years ago [78]. It was observed that heterodimers of RXR and PPARα were capable of binding a region between −356
to −320 in the long terminal repeat. Accordingly, PPARα agonists such as clofibric acid were shown to activate HIV-1 transcription [79]. This effect, which may be relevant in tissues such as liver which express high levels of PPARα, is not expected to be involved in alterations of white adipocytes and preadipocytes which are almost devoid of PPARα [80].In contrast, it was reported recently that the PPARγ agonist ciglitazone inhibits HIV-1 replication in a dose-dependent manner in acutely-infected human monocyte-derived macrophages and in latently-infected and viral entry-independent
U1 cells, suggesting an effect at the level of HIV-1 gene expression [81].
Cotransfection of PPARγ wild-type vectors and treatment with PPARγ agonists inhibited HIV-1 promoter activity in
U937 cells, and activation of PPARγ also decreased HIV-1 mRNA stability following
actinomycin D treatment. Thus, natural and synthetic PPARγ agonists may play a role in controlling HIV-1
infection in macrophages [81, 82]. Similar observations were
obtained by Skolnik et al. who observed that activation of PPARγ, and also of PPARα, by specific agonists also decreased HIV-1 replication in peripheral blood mononuclear cells
acutely infected with HIV-1, in a chronically-infected monoblastoid cell line
and in alveolar macrophages from HIV-1-infected subjects and uninfected
controls [83]. The precise mechanisms of action of PPARγ on HIV-1 are not
fully known and, in addition to the potential direct interaction with specific
regions of the long-terminal repeat mentioned above, indirect effects via nuclear
factor κB have also been proposed on the basis of the effects of PPARγ and its ligand rosiglitazone impairing nuclear
factor κB-mediated enhancement of HIV-1 replication in macrophages [84].All these findings indicate the occurrence of a potential cross-talk between PPARγ and HIV-1 that could reinforce the activity of HIV-1 proteins in cells harboring PPARγ. Thus, a
reduction in PPARγ levels and/or activity as a consequence of HIV-1 infection may lead to depression of such a pathway of potential inhibition of HIV-1 transcription and could create an environment prone to enhancement of HIV-1 gene expression, establishing a “vicious cycle” further augmenting adipose pathogenesis.


## 7. Conclusions

In summary, research to date indicates that HIV-1 infection-related events may
alter adipose tissue and contribute to development of the full-blown
lipodystrophy syndrome after antiretroviral treatment. The role of HIV-1
infection of cells in adipose tissue, of soluble proteins released by infected
cells as well as of the indirect effects elicited by the mild proinflammatory
environment associated with viral infection, is issues expected to be the
subject of intense research in the near future. For all these aspects, PPARγ appears as a main candidate for the mediation of pathogenic
events. Moreover, a full understanding will be required of the relationships
among the complex set of cell types that, in addition to adipocytes, are
present in adipose tissue depots. Macrophages, endothelial cells, preadipocytes,
and perhaps even lymphocytes are present in adipose depots and establish a
complex regulatory cross-talk that is altered by HIV-1 infection, and which may
ultimately lead to disturbances in adipocytes and in the whole adipose mass (see Figure 1).
All of these cell types express PPARγ, and the pivotal role of this receptor in
adipogenesis, insulin sensitivity, lipid metabolism, and inflammatory pathways
point to this receptor as a key target of future research on adipose tissue
disturbances in the HIV-1/HAART-associated lipodystrophy syndrome.Figure 1
Schematic representation of
the potential effects of HIV-1 infection on PPARγ-mediated pathways in adipocytes and
macrophages. HIV-1 infection of macrophages may lead to the synthesis of HIV-1
encoded proteins, that is, Vpr, with negative effects on the expression of PPARγ target genes. This may lead to reduced
expression of anti-inflammatory genes as well as promotion of HIV-1
replication. Release of HIV-1-encoded proteins as well as enhanced production
of inflammatory cytokines, that is, TNFα and other, by macrophages as a consequence of
HIV-1 infection may lead to impaired PPARγ action in adipocytes and preadipocytes, thus
impairing adipogenesis and fat accretion. Direct effects of HIV-1 infection in
line with what happens in macrophages cannot be excluded. Similar events to
those depicted for macrophages could be considered to occur in endothelial
cells or even lymphocytes present in adipose tissue depots as a consequence of
HIV-1 infection.



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