# Peroxisome Proliferator-Activated Receptors and Hepatitis C Virus-Induced Insulin Resistance

Authors: Francesco Negro
Journal: PPAR Research
 (2009)

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## Abstract

Insulin resistance and type 2 diabetes are associated with hepatitis C virus infection. A wealth of clinical and experimental data suggests that the virus is directly interfering with the insulin signalling in hepatocytes. In the case of at least one viral genotype (the type 3a), insulin resistance seems to be directly mediated by the downregulation of the peroxisome proliferator-activated receptorγ. Whether and how this interaction may be manipulated pharmacologically, in order to improve the responsiveness to antivirals of insulin resistant chronic hepatitis C, patients remain to be fully explored.


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## Body

## 1. Introduction

Hepatitis C virus (HCV) infection is associated with an increased risk of developing glucose intolerance and diabetes. This is in part due to a direct interference of HCV with the insulin signalling pathway. The
mechanisms involved seem to be HCV genotype-specific, and this suggests that HCV may profit from the insulin resistant phenotype to establish and/or maintain a persistent infection. Since the peroxisome proliferator-activated
receptors (PPARs) are nuclear factors involved—among others—in the regulation of glucose homeostasis, the relationship between HCV replication and protein expression and PPARs has been
the focus of some recent studies. However, the data available so far are quite
scanty and concern only the HCV genotype 3a. In fact, although most viral
genotypes seem to activate members of the suppressors of cytokine signalling
(SOCS) family in order to inhibit insulin signalling, in the case of genotype
3a, some in vitro observations
are consistent with a downregulation of the PPARγ. If confirmed, these
observations may be relevant to the treatment of chronic hepatitis C, since
insulin resistance is a factor of poor response to antivirals. This article
will briefly review the role of PPARs in insulin resistance, the interactions
between HCV and PPARs, and their clinical significance.


## 2. PPARs in Insulin Resistance and Diabetes

PPARs are the established targets of several
classes of drugs used in the management of the metabolic syndrome, like the
fibrates and the thiazolidindiones. A new class of pan-PPAR agonists, the
glitazars, is presently under investigation. This article will describe the
role and significance of PPARαand γin glucose metabolism, since the few data from
HCV models have addressed the deregulation of only these two factors. No data
are currently available about the involvement of PPARδ in the HCV-associated
insulin resistance.PPARα is principally expressed
in tissues exhibiting high rates of β-oxidation such as liver,
kidney, heart, and muscle, and can be activated by dietary fatty acids and
eicosanoids or by specific drugs such as fibrates. Activation of PPARα results in increased fatty acid β-oxidation in the liver [1],
increased expression of HDL apolipoproteins Apo A-I and Apo A-II [2], inhibited
expression of Apo C-III [3], increased lipoprotein lipase activity [4], and,
therefore, increased VLDL and remnants clearance. As to insulin resistance, PPARα seems to improve—indirectly—insulin sensitivity by increasing hepatic
catabolism of lipids, and thus reducing lipid supply to skeletal muscle [5].PPARγ is expressed at very high
levels in adipose tissue, and much less in the liver and other organs. Apart
from its natural ligands, that is, fatty acids, it is activated by drugs of the
class of thiazolidindiones such as rosiglitazone and pioglitazone. Hyperglycaemia
and hyperlipidaemia in obese and diabetic animals are improved by pioglitazone
through reduction of both peripheral and hepatic insulin resistance [6]. In the
animal model, treatment with PPARγ agonists lowers plasma
level of free fatty acids and insulin, and increase the phosphorylation level
of Akt at both threonine 308 and serine 473 in the liver and both the adipose
and muscle tissues [7]. This, in turn, is correlated with tyrosine phosphorylation
of insulin receptor β subunit and insulin receptor substrate-1, and serine phosphorylation of glycogen synthase kinase-3α/β [7]. Additional hepatic effects
include increased insulin sensitivity via G-protein subunits downregulation,
leading to reduced glucose production by approximately 30%, accompanied by a
significant increase of glucose-induced insulin suppression in β-cells [8].In addition to the direct effects on factors
involved in lipid and glucose homeostasis, PPARs may have insulin sensitizing
effects via their anti-inflammatory activity. PPARγ reduces the expression of
activator protein-1 and nuclear factor-κB, reduces specific subsets of lipopolysaccharide and interferon
(IFN) target genes in macrophages [9, 10], and reduces tissue expression of tumor necrosis factor-α (TNFα), IFN-γ, C-reactive protein, and induction of other proinflammatory cytokines,
including hepatocytes [11].Thus, treatment with PPARs agonists results in
improved insulin sensitivity via diverse mechanisms, both direct and indirect,
and both at the level of the liver and at the level of extrahepatic tissues.


## 3. Hepatitis C Virus Infection and Insulin  Resistance

Insulin resistance and type 2 diabetes are common
complications of all liver diseases, especially at the advanced stage. In the
case of HCV infection, however, both clinical and experimental observations
suggest that HCV may directly interfere with glucose homeostasis.
Cross-sectional, case-control, and longitudinal studies—performed in both
large unselected cohorts and in patients with liver or kidney transplantation—have suggested
that type 2 diabetes may be more prevalent in chronic hepatitis C patients,
especially if aged more than 40 and if other major risk factors of glucose
intolerance are present [12]. The confounding effect of liver disease stage can
be eliminated if studies are conducted in patients with little or no liver
fibrosis, and if—instead of looking for cases with overt
diabetes—glucose homeostasis is assessed as level of
insulin sensitivity. Hui et al. [13] found that 121 HCV-infected patients with
stage 0 or 1 liver fibrosis had higher levels of HOMA score compared with 137
healthy volunteers matched by sex, body mass index, and waist-to-hip ratio.
This work proved that HCV may induce insulin resistance at early stages of
liver diseases, and provided, in addition, the first evidence that this effect
may be due to genotype-specific sequences.Some recent work suggests a trend between HCV replicative level and level of insulin
resistance [14]. The low-level correlation may be due to the fact that the
global level of insulin resistance is likely to depend on the contribution from
the adipose tissue and the muscle, two extrahepatic compartments that are not
infected by HCV.The
effect of antiviral therapy is another classical way to prove an association
between infection and disease. Romero-Gómez et al. have shown that both the
level of insulin resistance [15] and the incidence of ex novo glucose
intolerance over time [16] are reduced after successful therapy in chronic
hepatitis C patients, whereas no improvement is observed in nonresponders. However,
independent confirmation of these observations is warranted.The association between HCV and insulin
resistance has noteworthy consequences, clinically and conceptually. From the
clinical standpoint, insulin resistance accelerates fibrogenesis [13, 17–19] and impairs
response to IFN-α-based antiviral therapy
[15, 20, 21]. In addition, HCV infection is an interesting example of insulin
resistance not necessarily associated with other components of the metabolic
syndrome, thus providing the framework for longitudinal studies on specific
risk assessment. Last, but not least, the question arises what may be the advantage
for HCV to increase insulin resistance; it is apparent that all HCV genotypes
studied so far induce insulin resistance, albeit to a different extent [13, 14],
suggesting some evolutionary constraints aimed at maintaining the insulin-resistant
phenotype despite the viral genome sequence divergence over time.Since HCV appears to directly interfere with the glucose homeostasis,
several studies have tried to analyze in detail the potential interactions
between viral products and insulin signalling. Experimental data suggest a
direct interference of HCV with the insulin signalling cascade via proteasome
degradation of the insulin receptor substrates -1 and -2 [22, 23]. HCV may also impair insulin
signalling transduction indirectly, that is, through increased levels of
proinflammatory cytokines such as TNF-α [24, 25]. The interference with the insulin signalling
seems to proceed via HCV genotype specific mechanisms (see below) and insulin
resistance levels vary according to the infecting HCV genotype, although all
genotypes induce insulin resistance. Interestingly, intracellular factors
dysregulated by HCV and responsible for the insulin resistant phenotype may
play promiscuous effects as they are also involved in regulating IFN-α signalling. These factors include some members of the
suppressor of cytokine signalling (SOCS) family [22, 23, 26] and the protein
phosphatase 2A [27]. Thus, modulating the levels and/or the activity of these
factors may not only reverse hepatic insulin resistance but also help establishing
the IFN-α-induced
antiviral state at the site of HCV replication. This is one of the reasons for
trying to restore insulin sensitivity in chronic hepatitis C patients failing
to respond to therapy (see below).


## 4. HCV Infection and PPARs Expression

Very little data is available concerning
the interaction between HCV products and PPARs expression (Figure1). PPARγ
mRNA level was measured in Huh-7 hepatoma cells transfected with the HCV genotype
3 core encoding sequence, and was found significantly decreased compared to
untransfected cells. No changes were observed in cells transfected with the core
protein 1b [28]. Incidentally, PPARα
was undetectable, even in untransfected cells, precluding further evaluations
of an interaction with HCV, if any.Figure 1
Reported effects of HCV on some of the relevant
physiological functions of PPARα and PPARγ. The inhibitory effect on PPARγ has
been described only for the genotype 3a. For further explanations, please refer
to the text.Cells transfected with the
genotype 3a had both increased content of triglycerides [29] and reduced levels
of IRS-1, leading to insulin resistance, as measured by reduced Akt
phosphorylation following incubation with insulin [23]. The role of PPARγ in HCV 3a-associated insulin resistance in vitro was further assessed by
treating transfected cells with a PPARγ agonist, rosiglitazone. Both IRS-1 protein and insulin-stimulated Akt phosphorylation
levels increased after treatment with rosiglitazone of cells transfected with
the core protein 3a. The recovery of the IRS-1 protein expression and Akt
phosphorylation levels was, however, rather modest, that is, about 20% and 26%,
respectively [23]. Additional recovery of both IRS-1 level and Akt activation was
obtained by inhibiting SOCS-7 upregulation induced by the same viral genotype
in this model [23]. Although cells expressing the HCV core 1b had reduced IRS-1
content and insulin resistance, the effect seemed to be mediated by both SOCS-
and PPARγ-independent
mechanisms. Thus, based on this isolated observation, it seems that PPARγ downregulation may be responsible, at least in part, for
the insulin resistance observed in
vitro upon expression of HCV genotype 3a core protein expression.By analyzing total RNA extracted from the liver of chronic
hepatitis C patients, de Gottardi et al. found that the transcription level of
both PPARα and PPARγ was decreased in genotype 3 infection as compared to genotype 1 [28]. When
patients were stratified according to the presence of steatosis, PPARα
and γmRNA were reduced only in steatotic livers
from patients infected with genotype 1. In those with genotype 3, both PPAR
mRNA levels were always low, independently of the presence of steatosis. In
this study, the two groups of patients with genotype 3 and 1 were comparable as
to gender distribution, age, BMI, liver disease activity, and fibrosis stage. However, in this study, a
direct correlation between PPARs levels and IR was not evaluated.The level of PPARα mRNA has been measured in the liver of chronic
hepatitis C patients also in another study [30], and found to be profoundly
suppressed (about 85% compared to control livers). The inhibition of PPARα
was paralleled by a significant decrease of the carnitine palmitoyl acyl-CoA
transferase 1 mRNA, a key enzyme in the mitochondrial β-oxidation of fatty acids, and confirmed
in an in vitro model expressing the HCV core protein [30]. These authors
focused their discussion on the role of PPARα as anti-inflammatory mediator, and on the consequences
of its suppression in the pathogenesis of hepatitis C. It is important to remember that the
impaired transcriptional activity of PPARα associated with HCV infection may indirectly worsen
IR via increased expression of TNF-α; thus,
the role of inflammation in the pathogenesis of HCV-associated IR should not be
overlooked [31]. On the contrary, it is unlikely that HCV-induced steatosis,
due to decreased expression of PPARα
and several other mechanisms [32], may aggravate insulin resistance, since the
latter has been convincingly shown to precede steatosis, and not vice versa
[33].


## 5. Perspectives for Treatment

As said above, insulin resistance reduces the
rate of response to antivirals in chronic hepatitis C [15, 20, 21]. A sustained
virological response (SVR) was observed in 23 of 70 (32.8%) of patients with
genotype 1 and insulin resistance (i.e., with a HOMA > 2) versus 26 of 43
(60.5%) of genotype 1 patients without insulin resistance (P=.007) [15].
These findings were independently confirmed [20] and extended to nonresponders
with genotypes 2 and 3 [21]. Thus, it was suggested that insulin resistance
should be corrected in patients with chronic hepatitis C not responding to IFN-α-based treatment, in order to improve response upon
retreatment. The modalities of this intervention, however, have not been
established. In addition, the optimal HOMA-IR score to be reached has not been
identified.A recent prospective, multicenter study aimed at investigating the efficacy and safety of the insulin-sensitizer pioglitazone, 15 mg QD, added to
the pegylated IFN-α2a, 180 μg QW/ribavirin, 1 000–1 200 mg QD
combination therapy in chronic hepatitis C patients who were previously nonresponders
to a pegylated IFN-α/ribavirin
combination without the insulin sensitizer [34]. All patients had a baseline
HOMA > 2 as additional inclusion criterion, because this was the threshold
discriminating responders from nonresponders in previous works [16, 21]. Diabetic
patients were however excluded. Unfortunately, none of the first five patients enrolled
into the trial had a satisfactory virological response after 12 weeks of
retreatment, despite the fact that in at least three of them the insulin
resistance score improved, and thus the study was prematurely terminated [34].Additional
data on this topic have been presented at the 2008 annual meeting of the
American Association for the Study of Liver Diseases. In an interim analysis of
a clinical trial, 30 mg QD of pioglitazone were given for four weeks as
monotherapy and then added for the first four weeks of a standard therapy of
treatment-naïve, nondiabetic chronic hepatitis C patients. The authors showed
that the triple regimen containing pioglitazone increased significantly the
rate of virological response after 4 weeks of therapy compared to pegylated
IFN-α/ribavirin
combination [35]. Long-term data are eagerly awaited. However, in another
randomized, double-blind, placebo-controlled study, adding pioglitazone 30 mg QD simultaneously to the standard of care (i.e., without a preceding
administration as monotherapy) clearly increased the on-treatment virological
response, but failed to increase the sustained virological response after the
end of treatment [36]. Additional studies evaluating different schedules are
clearly warranted. This approach, however, should also take into consideration
the known effects of PPAR agonists on serum lipid profile and their potential
consequences on the HCV life cycle. HCV circulates bound to lipoproteins in
complexes known as lipoviroparticles [37]. As a result, HCV entry into
hepatocytes appears to be mediated and facilitated, among others, by the LDL
receptor [38]. In keeping with this, at least two recent studies have suggested
that baseline LDL-associated cholesterol levels may affect response to
antiviral therapy [39, 40]. In fact, higher levels of cholesterol- and
ApoB-rich lipoproteins could facilitate viral clearance by impeding HCV
interaction with cell surface receptors. Thus, drugs like thiazolidinediones that
modify the circulating lipoprotein profile may have unexpected—and potentially unwanted—effects on the HCV life cycle. On the other
hand, in addition to lipid-lowering effects, PPARα agonists have been shown to decrease also the
expression of the LDL receptor in experimental models [41]. This may offset the
untoward effect on lipoproteins by impairing HCV entry into target cells.
Although highly speculative, these hypotheses deserve being appropriately
evaluated in clinical trials.Modulating
the levels and/or the activity of intracellular factors involved in HCV-induced
insulin resistance may not only reverse hepatic insulin resistance but also
help establishing the IFN-α-induced
antiviral state at the very site of HCV replication.However, specific
inhibitors of SOCS family members and of the protein phosphatase 2A are either
not suitable for in vivo
administration or toxic. Alternatively, increasing insulin sensitivity may be
achieved by modulating serum levels of specific cytokines, such as TNF-α, associated with insulin resistance [24, 25], but the
administration of anti-TNF-α antibodies to chronic
hepatitis C patients may be risky [42]. Insulin sensitizers may also inhibit
HCV replication by decreasing serum free fatty acids flow to hepatocytes; saturated
and monounsaturated free fatty acids have indeed been shown to stimulate HCV replication
in an in vitro model [43].Thus, although increasing insulin sensitivity may be a rational option in chronic
hepatitis C patients not responding to current combination therapy, more work
is warranted to identify the appropriate treatment schedule. It is not clear
whether the best approach would be using a thiazolidindiones, activating PPARs,
or a biguanide such as metformin, whose mechanism of action is specifically
directed on the hepatic AMP-activated protein kinase [44]. Another major issue
concerns the treatment schedule. It is unclear whether one should start the
antiviral retreatment together with the insulin sensitizer or only once the
HOMA-IR score has decreased to a level predicting a sufficient SVR rate [16].
As an alternative, higher dosages of thiazolidindiones (as compared to the
above quoted study) or metformin may be used. Finally, insulin sensitizing
therapy might need to be tailored according to HCV genotype, and PPARs agonists
should probably be considered only in insulin-resistant patients with HCV
genotype 3a [23]. Finally, interactions of insulin sensitizing agents with
other drugs, most notably those taken for psychiatric comorbidities, should be
considered. In the quoted study, a paradoxical increase of the HOMA score was observed
in two patients during therapy, and both patients were taking drugs for their
psychiatric comorbidities that may also alter insulin sensitivity, that is, zopiclone
and olanzapine. Thus, the beneficial effect of pioglitazone may be nullified in
patients taking drugs for psychiatric indications. In addition to the potential
effects of these and other drugs, also the acute administration of IFN-α may induce some degree of insulin resistance in both
healthy subjects [45] and chronic hepatitis C patients [46]. The interactions
among all these drugs are, however, speculative, but require further studies,
in view of the frequent use of antidepressants in chronic hepatitis C patients.
Thus, further clinical trials aiming at reducing the insulin resistance in
chronic hepatitis C via
different pharmacological interventions are warranted.



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