Pathophysiology of Adipocyte Defects and Dyslipidemia in HIV Lipodystrophy: New Evidence from Metabolic and Molecular Studies

Despite a burgeoning mass of descriptive information regarding the epidemiology, clinical features, body composition changes, hormonal alterations and dyslipidemic patterns in patients with HIV lipodystrophy syndrome (HLS), the specific biochemical pathways that are dysregulated in the condition and the molecular mechanisms that lead to their dysfunction, remain relatively unexplored. In this paper, we review studies that detail the metabolic basis of the dyslipidemia specifically, the hypertriglyceridemia that is the serologic hallmark of HLS and present new data relevant to mechanisms of dyslipidemia in the postprandial state. We also describe preliminary experiments showing that in addition to the well-known effects of highly-active antiretroviral drugs, the functional disruption of adipocytes and preadipocytes by factors intrinsic to HIV-infected immunocytes may play a role in the pathogenesis of HLS.


INTRODUCTION
Characteristics of dyslipidemia and its relationship to lipodystrophy: A characteristic dyslipidemic pattern observed in the majority of patients with HLS is moderate to severe hypertriglyceridemia and reduced serum HDL cholesterol, with serum LDL cholesterol that is slightly elevated or altered in composition to small, dense particles [1][2][3][4][5] . This resembles the typical dyslipidemia of the Metabolic Syndrome, albeit in an accelerated and exaggerated form. Indeed, insulin resistance [6][7][8][9] and increased cardiovascular risk [10][11][12][13][14][15][16] have been associated with HLS in several studies. A subset of these patients also manifest generalized lipoatrophy or truncal fat accumulation associated with peripheral lipoatrophy. We have previously proposed, on the basis of whole body lipid kinetic data, a pathophysiologic scheme whereby fundamental defects in adipocyte function lead to a chain of metabolic consequences that explain hypertriglyceridemia, insulin resistance and increased cardiovascular risk in HLS [17,18] . The mechanisms associated with depotspecific adipose alterations, i.e., atrophy in some regions and hypertrophy in others, are not easy to explain on the basis of whole-body metabolic measurements and they require investigations of lipid turnover in specific body regions, which are currently under way.

Metabolic basis of HLS -studies in the fasting and fed state:
Whole body kinetic studies have demonstrated defects in specific lipid metabolic pathways in HLS patients in both the fasting [19][20][21] and fed (25) states. Studies in the fasted state uniformly reveal accelerated whole body lipolysis in patients with different phenotypic forms of HLS. Sekhar et al. studied patients with the "mixed" form (i.e., with peripheral atrophy and central obesity) of HLS using infusions of 13 C 1 -palmitate, 2 H 2 -glycerol and indirect calorimetry [19] and showed that, compared to age-, gender-and BMI-matched non-HIV healthy controls, these patients had markedly increased rates of both total and net lipolysis. Despite a modest concomitant increase in the rate of intra-adipocyte reesterification, there was a net excess release of free fatty acids (FFA's) into the plasma pool. The HLS patients lacked the ability to increase fat oxidation proportionately, resulting in a net increase in hepatic flux of FFA's resulting in increased availability of FFAs for hepatic reesterification and increased release of VLDLtriglyceride into the circulation. Reeds et al. confirmed the latter finding by direct measurement of the rate of VLDL-triglyceride synthesis in HLS patients [20] .
Another fate of un-oxidized plasma FFA's derived from excessive lipolysis would be increased deposition of fatty acids in myocytes and hepatocytes. Indeed, excessive intramyocellular fat has been noted in patients with HLS [7] and this is strongly associated with the development of insulin resistance in skeletal muscle [7,22] . An indirect correlate of this effect is that the FFA's that form the substrate for fat oxidation in HLS patients are derived mainly from non-plasma sources [19] such as intramyocellular lipid depots. Finally, elevated plasma FFA's in HLS patients would also exacerbate an atherogenic plasma lipid profile by increasing the activity of cholesteryl ester transfer protein, which catalyzes transfer of triglycerides of VLDL to HDL and LDL [23,24] . The resulting TG-rich HDL and LDL would be good substrates for hepatic lipase, generating smaller, more atherogenic (small, dense LDL) and less atheroprotective (HDL3) lipoprotein particles. We have proposed the term "systemic steatosis" for the whole body disruption of lipid metabolism that results from a primary adipocyte defect in HLS, to emphasize that this unusual pathogenic cascade causes not only dyslipidemia but also excess lipid deposition in other tissues (muscle, liver, beta cells), leading to lipotoxic insulin resistance [17] .
These findings in HLS patients in the fasted state do not completely account for the degree of hypertriglyceridemia in HLS patients. Recently, Sekhar et al. have demonstrated profound defects in adipocyte function and lipase action in the postprandial state that result in significant hypertriglyceridemia in the plasma chylomicron fraction [25] . HLS patients were fed a meal mixed with two grams of a triglyceride labeled in the terminal carbon position on all three fatty acids ( 13 C 3tripalmitin). At baseline, in the fasted state, chylomicron-TG concentrations were markedly higher in the HLS patients than in normal non-HIV controls. Blood and breath sampling for 8 hours following the meal showed that the ability of HLS patients to dispose of the labeled triglyceride from the CM pool was impaired five-fold compared to that of non-HIV healthy persons. Furthermore, tracking the labeled free palmitic acid revealed that the HLS patients also had a marked inability to store diet-derived fatty acids within adipocytes in the postprandial period (10% of the fatty acid label within the chylomicron pool was stored 8 hours after the meal in HLS patients, compared to 90% in the controls). These data imply that there is a profound defect in the postprandial function or regulation of adipocyte lipoprotein lipase in HLS patients (Fig. 2). Since the elevated lipolytic rates in the fasted state point to dysregulation of hormone sensitive lipase (HSL), the lipid kinetic data in toto indicate that two key insulin-regulated lipases -HSL and LPL -are severely dysfunctional in HLS and that these defects can account for most of the cascade of metabolic abnormalities that form the basis of the dyslipidemia in HLS. The extent to which these defects arise from specific inhibition of the enzymes as opposed to a general disruption of adipocyte function, remains an open issue, as is the question of the underlying mechanisms (HAART drugs, viral factors, immune responses).
"Rational" treatments aimed at the metabolic defects: Treatment of the dyslipidemia of HLS has proven to be difficult and in some patients, an intractable problem. There is inadequate response to many standard lipid lowering agents, a high frequency of adverse effects and interference by some agents of the metabolism of antiretroviral drugs. Metformin, a drug that enhances insulin sensitivity, has produced modest benefit, suggesting an interconnection between the mechanisms of dyslipidemia and insulin resistance [26,27] . There is also evidence that the dietary habits and physical activity of patients with HLS tend to be quite suboptimal [28] . In this context, the specific metabolic defects uncovered by the lipid kinetic studies offer specific targets at which to direct rational treatment to improve dyslipidemia in HLS (Fig 2). Agents directed at inhibiting lipolysis (such as niacin or acipimox), increasing fat oxidation (such as fibrates and other PPAR-alpha agonists, or leptin), attenuating the redistribution of fat (such as growth hormone) or enhancing adipocyte development and function (such as thiazolidinediones or other PPAR-gamma agonists) would appear to be attractive therapeutic candidates, alone or in combination. Lifestyle interventions would also be a key aspect of rational therapy: a low fat diet (rather than the usual low-carbohydrate diet recommended for hypertriglyceridemia) might help attenuate post-prandial hypertriglyceridemia and regular exercise would diminish plasma and tissue free fatty acids by enhancing their oxidative disposal. Each of these interventions is currently under study in different clinical trials. One multiple arm, blinded, placebo-controlled trial sponsored by the NIH, termed "Heart Positive", is investigating the effects of stepwise addition of sustained-release niacin, fenofibrate, or both agents against the background of a carefully supervised program of aerobic and resistance exercise together with a regulated diet designed for treatment of the Metabolic Syndrome (the National Cholesterol Education Program Adult Treatment Panel-III diet). The effects of these interventions on the primary endpoint (plasma triglyceride lowering) will not be available for three years; however, it is interesting to note that some of the most significant improvements in hypertriglyceridemia in HLS patients have been observed in small trials of fibrates or niacin [4,[29][30][31] .
We have utilized a mechanistically-driven approach that complements standard clinical trials for assessing rational therapy for dyslipidemia in patients with HLS. Because the stable isotope protocols have been optimized to define the specific lipid kinetic defects in a quantitative manner in HLS patients, targeted interventions can be applied for brief periods of time to assess their effects on the defective kinetic  rates and metabolic endpoints. In this manner, the effects of growth hormone replacement therapy on lipid kinetics in HLS have been assessed and those of treatment with leptin and rosiglitazone are under way. The rationale for GH treatment is that patients with HLS are frequently partially GH-deficient (in relative proportion to the degree of visceral adiposity) [32] and that a putative metabolic benefit of GH treatment is to enhance fat oxidation. GH replacement in HLS patients with unequivocal GH deficiency appears to have the salutary though somewhat paradoxical effect of decreasing the rates of lipolysis without increasing fat oxidation (33). These preliminary results suggest a mechanistic basis for the recently reported clinical effects of restoring physiologic plasma levels of GH on improving body composition and fat redistribution in patients with HLS [33] .

Molecular basis of HLS -role of immunocyteadipocyte interactions:
What is the molecular basis of the adipocyte defects that lead to the metabolic disorders described above? A great deal of in vitro investigation has been focused on the role of antiretroviral drugs on adipocyte function [34,35] and on the ability of preadipocytes to differentiate into mature adipocytes [36] . As in most metabolic illnesses, the pathophysiology is likely to be multifactorial and complex and there is clearly evidence that hypertriglyceridemia was observed in HIV-infected patients even prior to the era of treatment with effective antiretroviral drugs. We are currently investigating several novel hypotheses regarding the molecular pathogenesis of HLS. One hypothesis is that interactions between specific, HIV-infected or activated immune cells and adipocytes or preadipocytes lead to defects in function or differentiation of the adipocytes. It is based on the observation that adipocyte depots are anatomically associated with specific lymph node aggregates -hence it is possible that there are distinct functional interactions between activated lymphocytes and the surrounding adipocytes and pre-adipocytes (37]. We have investigated these potential interactions in the context of in vitro HIV-1 infection, using an assay system in which primary human pre-adipocytes (stromal vascular cells) or 3T3-L1 mouse preadipocytes are incubated with uninfected or HIVinfected lymphocytes for varying periods of time, with or without direct contact between the two cell types and each cell type is assayed for cell cycle distributions, apoptosis and biochemical function. Exposure of acutely HIV-infected T lymphocytes to human preadipocytes leads to a marked increase in HIV-1 production in the lymphocytes. In chronically HIVinfected lymphocytes, exposure to the pre-adipocytes leads to a cell cycle block and increased apoptosis in the lymphocytes. These effects are clearly mediated by a soluble factor secreted by the preadipocytes in response to the cell-cell interaction. Conversely, the pre-adipocytes undergo a differentiation block following exposure to the lymphocytes, as demonstrated by a marked reduction in both lipid staining by Oil Red O and quantitative gene expression of key adipocyte developmental transcription factors. These effects are observed in both human and murine pre-adipocytes, suggesting that they are not due to direct HIV-1 infection of the pre-adipocytes. The specificity of these interactive effects is underscored by the fact that these results can be replicated using three different human T lymphocyte lines, but not using two different human macrophage cell lines.
These data indicate that there are significant interactions between pre-adipocytes and HIV-infected lymphocytes, which result on the side of the lymphocytes in increased HIV-1 production, cell cycle block and apoptosis and on the side of the preadipocytes in a marked block to differentiation. The interactions are mediated by soluble factors secreted by the pre-adipocytes (? adipokines), lymphocytes (? cytokines) or secreted components of HIV-1 itself. Identifying these factors and specifying their mechanism could be important for understanding the complex pathophysiology of HIV lipodystrophy.