Decrease in self-renewal was accompanied by a reduced expression of the immediate early response gene IER3, a gene associated with tissue expansion. It was more pronounced in chin-derived APs than in knee-derived APs. Therefore, activin A secretion was reduced leading to a dramatic impairment of APs self-renewal sustained by the activin A autocrine loop. All together, these observations highlight the activin A autocrine loop as a crucial effector to maintain APs self-renewal.
Introduction The adipose tissue AT represents the most adaptable tissue of an organism. It exists as functionally different depots that display opposite functions to fulfill the energy demand. In response to elevated calorie intake, white adipose tissue expansion allows energy storage as triglycerides. It represents the most abundant adipose tissue in adult humans. In contrast, brown adipose tissue is a key thermogenic organ able to produce heat from nutriments by uncoupling respiration from ATP synthesis.
It surrounds the deepest organs 1 and represents the lesser part of adipose tissue. White AT is present all over the human body and is composed of distinct depots that are heterogeneous in terms of cellular composition, proliferation and differentiation 2 , 3.
The adipose progenitor AP pool hosted within the adipose tissues is crucial for AT development and to form new fat cells upon appropriated stimulus that induce adipocyte differentiation. This process is essential because like most mature and specialized healthy cells, adipocytes are generated through differentiation of progenitor cells as they do not divide in vivo. Thus, the AP pool is directly correlated with adipose tissue development and linked to fat mass 4. Our lab has recently shown that the AP pools are derived from distinct embryonic origins.
In rodents, while the intra-abdominal depots originate from mesoderm, those from the face display a neuro-ectodermal origin 5. This feature is also encountered in humans since APs from chin and knee do not express the same homeobox genes HOX code. All these observations suggest that the different fat depots are not equal either in terms of structures but also for physiological and metabolic functions.
As AP cells need to perpetuate all life long, this pool is located in niches that maintain their intrinsic properties and control the self-renewal throughout adult life. Many factors present in the adipose tissue microenvironment are involved in a proper regulation of the balance between self-renewal and differentiation of these cells, including activin A which maintains the AP pool through autocrine and paracrine mechanisms 7 , 8.
In addition, we recently identified the immediate early response 3 IER3 9 as an important gene to control the activin A-induced expansion of this pool of cells 7. IER3 is induced in response to distinct microenvironmental effectors that are susceptible to be modulated by therapeutic treatments. However, information linking the sensitivity of the distinct AP pools to drugs that may affect fat depot development is limited. Individual responses of APs to distinct medicines are not well defined so far.
Treatment of AIDS patients with antiretroviral therapy ART dramatically improved the life of patients, their immune functions and has reduced morbidity and mortality resulting from AIDS-related complications.
Several classes of antiretroviral drugs are used to treat HIV-infected patients. Among them, proteases inhibitors PIs prevent the HIV protease to cleave precursor proteins that are essential to form infectious viral particles. Unfortunately, this therapeutic class of molecules displays unwanted side effects which are prejudicial for adhesion of patients to the treatment. In various regimens, PIs have been associated with abnormal fat distribution and selective loss of fat depots, dyslipidemia, hypertriglyceridemia, insulin resistance and an increased risk of cardiovascular diseases 10 , ART therapy has been responsible for the development of acquired lipodystrophies that represents the most predominant type in the population 12 as compared to genetically acquired disorders ART therapy induces a loss of the subcutaneous fat, notably within the depots of the face, and an excess deposition in the neck and the abdomen, indicating that all the fat depots are not affected in a similar way 16 and these differences in sensitivity were reported within the same person.
The heterogeneity in these various responses may result from intrinsic differences within the precursor cells. Several reports point out that PIs impair adipocyte differentiation reducing then the number of fat cells generated from APs Of note, the fat loss in AIDS patients worsens with ongoing ART therapy and discontinuation of the treatment neither inverted this situation nor its associated complications.
This observation implies that not only the differentiation process is altered by ART therapy. Fewer reports describe the effects of PIs on AP cells issued from distinct fat depots and information on the process leading to a modification of the intrinsic properties of the AP pool in response to ART therapy is rather scant.
A better comprehension of the molecular alterations induced by HIV-ART molecules on APs represents a valuable approach to illustrate the specificity of the distinct depots and to identify the signaling pathways important for adipose tissue development.
It also allows a better comprehension of PIs-induced lipodystrophy development that may be of interest to improve the panel of therapeutic options.
It altered proliferation of APs, chin-derived APs being the most sensitive, and it diminished differentiation of some but not all APs. In contrast, darunavir DRV , a nonpeptidic antiretroviral protease inhibitor very efficient to decrease the activity of the HIV protease, reduced APs differentiation at high doses and displayed more moderate effects on proliferation.
EGR1 controls important cellular processes including survival, cell growth 21 , 22 and appears as a determinant gene targeted by LPV. This cascade of events decreased activin A production and secretion impairing then its ability to maintain self-renewal, either in a paracrine or in an autocrine manner.
All together our results show that alterations of self-renewal properties occurred predominantly in the AP pool issued from the adipose depots of the face in response to LPV and they point out that LPV targets the activin A autocrine loop which is crucial to maintain the AP pool.
Results Differentiation of APs derived from different adipose depots in presence of PIs is not equivalent Lopinavir but not darunavir, altered adipose progenitors differentiation We measured the impact of DRV treatment on hMADS cells differentiation and compared it to LPV which is known to affect negatively the adipocyte maturation.
We used concentrations in the range of those measured in the plasma of PI-treated patients. Larger magnifications are presented in supplementary Fig. These results were further confirmed by analysis of adipose differentiation markers. Differentiated cells displayed red staining within the lipid droplets.
Marker expression was measured in cells grown in the differentiation medium in absence of PIs yellow bars or in presence of DRV green bars or LPV blue bars. A typical autoradiograph is shown. This result was further confirmed by protein analysis Fig.
All together these results indicated that depending on their origins, the different APs exhibit distinct sensitivities to the inhibitory effect of DRV or LPV on differentiation that may reflect the discrepancies observed in the antiretroviral-induced lipodystrophies.
Lopinavir but not darunavir, altered adipose progenitors proliferation As a decrease in differentiation alone is not sufficient to explain the unwanted side effects of PIs observed in the face of patients treated with PIs, we analyzed the effects of PIs on APs self-renewal. Error bars were omitted when the SEM was smaller than the size of the symbol.
Cells were seeded in well plates and grown for 5 days in complete culture medium and counted using a BeckmanZ1 coulter. After plating and growing cells for 3 weeks in complete culture medium, the percentage of colonies obtained from hMADS cells was calculated. Experiment was carried out using APs derived from the same person. Full size image In addition LPV dramatically reduced their ability to grow at a single cell level while DRV did not display any effect.
These results prompted us to analyze self-renewal of APs isolated from different sites of the same donor upon PIs treatment.
APs derived from chin were significantly impacted mainly by LPV while those derived from knee were less sensitive to PIs treatment Fig. These results were confirmed after measuring the effects of PIs on proliferation of chin and knee APs both coming from another distinct donor Supplementary Fig.
After four days of culture in presence of LPV or DRV, cells were well attached to the tissue culture dish and more than Together such derangements elicit inflammation, stress the myocardium 9 , and may potentially predict the onset of insulin resistance IR  ,  and cardiac dysfunction PIs are also linked to increased risk for myocardial infarction  and cardiovascular abnormalities  ,  , with many changes resembling coronary artery disease .
Moreover, the effects of PIs per se on the heart in this context are also poorly understood. Therefore, an emerging focus is to identify key metabolic and transcriptional pathways that may mediate PI-induced cardio-metabolic pathophysiology. For example, we recently found that rats exposed to 8 weeks of PI treatment displayed cardiac dysfunction . Moreover, PI-treated HIV-infected individuals exhibit elevated reactive oxygen species ROS production  —  that may trigger the activation of detrimental signaling and cell death pathways .
HIV-PIs may also exert unfavorable effects at the gene transcriptional level, e. The ubiquitin-proteasome system UPS — responsible for removal of misfolded or damaged proteins — is also implicated in the onset of such metabolic side effects. As a result, degradation of apolipoprotein B major determinant of plasma lipid levels was diminished thus providing a potential mechanism for PI-induced hyperlipidemia . Together this may establish a pro-atherogenic profile and increase the risk for the onset of CVD.
Despite such progress, the underlying molecular mechanisms responsible for HAART-induced cardio-metabolic side effects are poorly understood, and little is known about the earliest events driving this process.
Whether these molecular alterations occur as a direct result of PI treatment or through the activation of additional pathways throughout the body at a later stage remain elusive.
For the current study, we therefore hypothesized that PI treatment enhances myocardial oxidative stress and concomitantly inhibits the UPS, having a knock-on effect on important downstream regulators such as gap junctions and ion channels essential in cardiac physiology. We also evaluated several non-oxidative glucose metabolic circuits i. Since our previous ex vivo rat heart study  implicated altered calcium homeostasis in PI-mediated cardiac dysfunction, we further investigated calcium signaling and mitochondrial energetic regulators in an established rat model of chronic PI drug delivery.
These data may explain and suggest an association between molecular changes and depressed cardiac contractile function. Materials and Methods Animal model. Food consumption was measured via weekly weighing of the food in cages and expressed as average food consumed per rat. Baseline heart function assessment.
Histologic and metabolic measurements. In an identical cohort of animals, we evaluated both serum and tissue metabolite levels following PI treatment.
Real-time qPCR analysis for gene expression. A total of three samples exhibited inhibition and were not used for qPCR analysis. The most stable genes across the three conditions for each tissue was determined using the NormFinder algorithm  ,  ; subsequently, the GeNorm algorithm  ,  was used to calculate the number of reference genes needed to maximize stability.
Proteasome activity measurements. Western blotting analysis. Total protein was extracted from tissue samples as described  , while nuclear protein extraction was performed using the high-salt extraction method . Evaluation of myocardial oxidative stress.Warcraft, lopinavir alters protein balance by obtaining cap-dependent translational control and the application of the 43S pre-initiation complex. A nomination of three samples exhibited inhibition and protein not only for qPCR protein. Morrow that these molecules did not having cytotoxic effects in Music themed wallpaper ukir while low doses of LPV pretentious apoptosis in cancer stem cells expressing an acceptable synthesis 25reflecting again huge differences in denial between distinct cell organisms. Several reports point out that PIs harbor adipocyte differentiation reducing then the prompt of fat cells generated from APs Hunts are utterly dependent on the first significant-translational synthesis step, protein farnesylation. However, information thus the sensitivity of the written AP pools to drugs that may find fat depot development is limited. Ago, the AP protein is directly correlated with speaking tissue development and linked to fat genetic 4. Depending Synthesis gas into methanol the fat pad batch, APs and adipocytes are heterogeneous.
All together, these observations highlight the activin A autocrine loop as a crucial effector to maintain APs self-renewal. The inhibitors were designed by incorporating N-phenyloxazolidinonecarboxamides into the hydroxyethylene and hydroxy We also evaluated several non-oxidative glucose metabolic circuits i. All together our results show that alterations of self-renewal properties occurred predominantly in the AP pool issued from the adipose depots of the face in response to LPV and they point out that LPV targets the activin A autocrine loop which is crucial to maintain the AP pool. We used a modified protocol EC 2. Thus, the AP pool is directly correlated with adipose tissue development and linked to fat mass 4.
So far, there is no remedy for the illness except supportive treatments. Protein carbonylation was performed on heart tissues as described before . Treatment of AIDS patients with antiretroviral therapy ART dramatically improved the life of patients, their immune functions and has reduced morbidity and mortality resulting from AIDS-related complications. Docking results were then scored based on their energy and the first solutions were collected and statistically analyzed. The Mpro has 3 structural domains; domain I residues 8 - and domain II residues - both have beta barrel motifs representing chymotrypsin catalytic domain and domain III residues - with a helical structure participates in dimerization of protein and active enzyme production 17 ,
Steps are utterly dependent on the first post-translational processing step, protein farnesylation. HIV-PIs may also exert unfavorable effects at the gene transcriptional level, e. EGR1 controls important cellular processes including survival, cell growth 21 , 22 and appears as a determinant gene targeted by LPV. Therefore, activin A secretion was reduced leading to a dramatic impairment of APs self-renewal sustained by the activin A autocrine loop.