Alexander Sorisky

Adipocytes Produce Aldosterone Through Calcineurin-Dependent Signaling Pathways: Implications in Diabetes Mellitus–Associated Obesity and Vascular Dysfunction

We reported aldosterone as a novel adipocyte-derived factor that regulates vascular function. We aimed to investigate molecular mechanisms, signaling pathways, and functional significance of adipocyte-derived aldosterone and to test whether adipocyte-derived aldosterone is increased in diabetes mellitus–associated obesity, which contributes to vascular dysfunction. Studies were performed in the 3T3-L1 adipocyte cell line and mature adipocytes isolated from human and mouse (C57BL/6J) adipose tissue. Mesenteric arteries with and without perivascular fat and mature adipocytes were obtained from obese diabetic db/db and control db/+ mice. Aldosterone synthase (CYP11B2; mRNA and protein) was detected in 3T3-L1 and mature adipocytes, which secrete aldosterone basally and in response to angiotensin II (Ang II). In 3T3-L1 adipocytes, Ang II stimulation increased aldosterone secretion and CYP11B2 expression. Ang II effects were blunted by an Ang II type 1 receptor antagonist (candesartan) and inhibitors of calcineurin (cyclosporine A and FK506) and nuclear factor of activated T-cells (VIVIT). FAD286 (aldosterone synthase inhibitor) blunted adipocyte differentiation. In candesartan-treated db/db mice (1 mg/kg per day, 4 weeks) increased plasma aldosterone, CYP11B2 expression, and aldosterone secretion were reduced. Acetylcholine-induced relaxation in db/db mesenteric arteries containing perivascular fat was improved by eplerenone (mineralocorticoid receptor antagonist) without effect in db/+ mice. Adipocytes possess aldosterone synthase and produce aldosterone in an Ang II/Ang II type 1 receptor/calcineurin/nuclear factor of activated T-cells–dependent manner. Functionally adipocyte-derived aldosterone regulates adipocyte differentiation and vascular function in an autocrine and paracrine manner, respectively. These novel findings identify adipocytes as a putative link between aldosterone and vascular dysfunction in diabetes mellitus–associated obesity.

From Preadipocyte to Adipocyte: Differentiation-Directed Signals of Insulin from the Cell Surface to the Nucleus

An alarming rise in obesity, and the accompanying threat of type 2 diabetes mellitus and cardiovascular disease, have attracted worldwide attention. The pathogenic mechanism(s) underlying obesity remains obscure. However, new cellular and molecular insights about the development of adipose tissue, with respect to adipocyte number (hyperplasia) and size (hypertrophy), are occurring at a rapid pace. Specialized fibroblasts (preadipocytes) committed to the adipocyte lineage are present throughout life. Primary cell culture systems and immortalized cell line models of preadipocytes have advanced the study of adipocyte differentiation (adipogenesis). Differentiation-inducing cues are able to trigger a complex network of intracellular signaling pathways in the preadipocyte, allowing signals from cell-surface receptors to reach nuclear transcription factors that regulate the genetic program of adipocyte differentiation. The extracellular matrix environment of the preadipocyte, known to modulate adipogenesis, may act by altering some of these signaling events.

Macrophage-conditioned medium inhibits the differentiation of 3T3-L1 and human abdominal preadipocytes

Aims/hypothesisIn obesity, a limited adipogenic capacity may promote adipocyte hypertrophy and increase the risk of insulin resistance and type 2 diabetes. Recent data indicate that macrophages reside within adipose tissue in obese rodents and humans. We hypothesised that secreted macrophage factors may inhibit adipogenesis.Materials and methodsConditioned media from cultured murine J774 or human THP-1 macrophages were collected, and added to either murine 3T3-L1 preadipocytes or human abdominal stromal preadipocytes from subcutaneous or omental fat depots.ResultsMacrophage-conditioned medium (MacCM) strongly inhibited 3T3-L1 adipogenesis. Dose–response studies with J774-MacCM revealed that 80 and 100% of J774-MacCM completely suppressed triacylglycerol accumulation as well as the induction of fatty acid synthase, peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and adiponectin. Similar inhibitory effects on 3T3-L1 preadipocytes were observed with THP-1-MacCM. Differentiation of human abdominal subcutaneous stromal preadipocytes was moderately reduced (subcutaneous>omental) by J744-MacCM. In contrast, the differentiation of both subcutaneous and omental stromal preadipocytes was completely inhibited by THP-1-MacCM, as determined on the basis of morphology and triacylglycerol accumulation, as well as fatty acid synthase and adiponectin protein expression.Conclusions/interpretationSecreted macrophage products inhibit the differentiation of 3T3-L1 preadipocytes as well as human abdominal stromal preadipocytes.

Ritonavir increases the level of active ADD-1/SREBP-1 protein during adipogenesis.

ObjectiveA novel lipodystrophy syndrome characterized by truncal adiposity, peripheral fat atrophy, type 2 diabetes mellitus, and dyslipidemia occurs in HIV-infected individuals, and may be aggravated by HIV-1 protease inhibitors. The increase in truncal fat could be due to enhanced preadipocyte differentiation. Using the 3T3-L1 preadipocyte model, we reported that ritonavir enhances adipocyte differentiation in culture. The goal of this study was to characterize the molecular mechanism of ritonavir on preadipocyte differentiation. Designs and methodsTime course studies of 3T3-L1 preadipocytes placed in standard differentiation medium (insulin, dexamethasone, and isobutylmethylxanthine) were performed. Glycerol phosphate dehydrogenase (GPDH) was assayed enzymatically, and triacylglycerol (TG) mass was quantified. The adipogenic transcription factors adipocyte determination and differentiation-dependent factor 1 (ADD-1)/sterol regulatory element binding protein 1 (SREBP-1), CCAAT/enhancer-binding protein-α (CEBPα), and peroxisome proliferator activated receptor-γ (PPARγ), were measured by Western analysis. ResultsRitonavir (10 μg/ml) enhanced 3T3-L1 preadipocyte differentiation (30% increase in TG mass; 50% increase in GPDH activity), and transiently raised levels of the 68 kDa active mature form of ADD-1/SREBP-1 during adipogenesis by threefold, compared with standard differentiation. In contrast, ritonavir attenuated the differentiation-induced increase in CEBPα and PPARγ. ConclusionsOur data suggest that ritonavir enhances 3T3-L1 adipogenesis by increasing the level of active mature ADD-1/SREBP-1. This effect may be due to reduced proteolysis of ADD-1/SREBP-1, as ritonavir inhibits an N-acetyl-leucyl-leucyl-norleucinal (ALLN)-sensitive proteosomal degradation pathway in lymphocytes, and ALLN itself inhibits the breakdown of mature ADD-1/SREBP-1. As mature ADD-1/SREBP-1 regulates several lipogenic enzymes, higher levels may explain the effect of ritonavir on TG accumulation and GPDH activity. Studying ADD-1/SREBP-1 may lead to better understanding and prevention of the lipodystrophy syndrome.

Rapamycin-sensitive phase of 3T3-L1 preadipocyte differentiation after clonal expansion

Inhibition of insulin‐induced 3T3‐L1 preadipocyte differentiation by rapamycin has been attributed to a blockade of the early critical clonal expansion phase of the adipogenic program. Rapamycin binds to, and inhibits, mTOR (mammalian target of rapamycin), leading to diminution of p70 S6 kinase activity and eukaryotic initiation factor 4E binding protein 1 (eIF4E‐BP1) function. Our objective was to determine if rapamycin‐sensitive pathways exist subsequent to the clonal expansion phase. We determined that the mitotic clonal expansion was complete by day 4 of the differentiation protocol, based on the response to Ara‐C (cytosine β‐d‐arabinofuranoside), which only inhibits differentiation when administered during this phase. Treatment of differentiating 3T3‐L1 cells with rapamycin, starting on day 4, exerted potent negative effects on glycerol phosphate dehydrogenase activity, and triacylglycerol accumulation, as well as on the protein expression of adipogenic transcription factors, C/EBPα and PPARγ. Insulin‐stimulated p70 S6 kinase activity, and its inhibition by rapamycin, were comparable in preadipocytes at day 0 vs. day 4 post‐differentiation. We conclude that a component of the adipogenic program, operating after the completion of clonal expansion, is inhibited by rapamycin, suggesting an ongoing need for mTOR function in this process.

Efficacy and Safety of a New Hydroxymethylglutaryl-Coenzyme A Reductase Inhibitor, Atorvastatin, in Patients with Combined Hyperlipidemia: Comparison with Fenofibrate

This 24-week, randomized, open-label multicenter study evaluated the efficacy and safety of atorvastatin compared with fenofibrate in the treatment of patients with combined hyperlipidemia (CHL). Following a 6-week baseline period, 84 patients with CHL were randomly assigned to either atorvastatin treatment, 10 mg QD for 12 weeks increasing to 20 mg QD for 12 weeks, or fenofibrate treatment, 100 mg TID for 24 weeks. Changes from baseline in lipid parameters were evaluated at weeks 12 and 24. At both 10- and 20-mg doses, atorvastatin treatment resulted in significantly greater reductions in LDL cholesterol, apolipoprotein (apo) B, total cholesterol, LDL-apoB, and lipoprotein-B compared to 300-mg fenofibrate treatment (P < .05). While atorvastatin also resulted in clinically significant reductions in triglyceride, VLDL cholesterol, apoB in VLDL, triglyceride in VLDL, and apoC-III and significant increases in HDL cholesterol and apoA-I levels, fenofibrate was more effective than atorvastatin in altering all these parameters. However, by significantly affecting both the cholesterol-rich and triglyceride-rich particles, atorvastatin holds promise as a lipid-regulator able to adequately treat a broad range of patients that includes those with CHL.

Anti-adipogenic effect of PDGF is reversed by PKC inhibition.

Healthy adipose tissue function depends on adipogenesis. The capacity to form new adipocytes prevents the emergence of insulin‐resistant hypertrophied adipocytes, as well as the deleterious lipid deposition in muscle, liver, and pancreas. It is therefore important to understand how adipogenesis is modulated. Platelet‐derived growth factor (PDGF) is anti‐adipogenic, but the stage of differentiation that it targets, and the signaling pathways that it triggers, are not defined. We have studied the inhibitory effect of PDGF on murine 3T3‐L1 preadipocyte and human preadipocyte differentiation. There was a significant attenuation in the protein expression of the adipogenic transcription factors, PPARγ and C/EBPα, as well as in the levels of later differentiation markers, including adiponectin, aP2, and fatty acid synthase. PDGF treatment resulted in the persistence of PDGF receptor and PKCα expression, in contrast to the expected downregulation of both proteins that occurs during differentiation. Inactivation of conventional PKC isoforms, by bisindolylmaleimide I or PKC pseudosubstrate M20–28, partially reversed the inhibition of 3T3‐L1 and human preadipocyte differentiation by PDGF, as assessed by fatty acid synthase expression and morphological appearance.

Apoptosis of human abdominal preadipocytes before and after differentiation into adipocytes in culture

Differentiation of murine 3T3-L1 preadipocytes into adipocytes is associated with the acquisition of apoptotic resistance accompanied by the upregulation of cell survival genes. We have now examined the effect of adipogenesis on apoptotic susceptibility of human abdominal preadipocytes in primary culture. To induce apoptosis, human preadipocytes, or their differentiated counterparts, were serum-deprived for 24 or 48 hours. When indicated, ceramide was also used as an apoptotic trigger. Cell death was assessed by enumeration of adherent viable cells, and its apoptotic nature was verified by Hoechst staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After 48 hours of serum withdrawal, cell death was 26% +/- 4% in preadipocytes and was increased to 41% +/- 4% in differentiated adipocytes (mean +/- SE; n = 7 patients; P <.002). Under serum-free conditions for 24 hours, ceramide-induced cell death was 40% +/- 6% in preadipocytes and increased to 68% +/- 8% in adipocytes (mean +/- SE; P <.01; n = 8 patients). Neuronal apoptosis inhibitor protein (NAIP), an antiapoptotic protein cell survival that increases upon 3T3-L1 adipogenesis, was reduced in human preadipocytes undergoing differentiation (n = 6 patients). Preadipocytes derived from omental versus subcutaneous abdominal fat were more susceptible to apoptosis induced by serum deprivation, 16% +/- 4% versus 31% +/- 3% cell death, respectively (mean +/- SE; P <.02; n = 7 patients). Although the murine 3T3-L1 preadipocyte cell line is a useful model that approximates primary preadipocyte cell biology, our data derived from human preadipocyte studies suggest important differences with respect to the regulation of apoptosis.

Macrophage-Induced Adipose Tissue Dysfunction and the Preadipocyte: Should I Stay (and Differentiate) or Should I Go?

Adipose tissue can be regarded as a multidepot organ responsible for metabolic homeostasis by managing sophisticated energy transactions as well as by producing bioactive molecules that regulate insulin sensitivity and immune and vascular responses. Chronic nutrient excess expands adipose tissue, and concomitant variations in its cellular and matrix remodeling can affect the extent of the metabolic dysfunction that is associated with obesity. Preadipocytes, also termed adipose progenitor cells, play a pivotal role in determining whether a dysfunctional hypertrophic state arises as opposed to a hyperplastic process in which mature adipocytes remain relatively responsive. Obesity is associated with infiltration of macrophages, and these immune cells have been shown to communicate with preadipocytes to influence how they differentiate, survive, and proliferate. Understanding macrophage-preadipocyte interactions and their effect on adipose remodeling mechanisms may identify potential therapeutic molecular targets to improve adipose tissue function, even in the face of obesity.

Phosphatidylinositol-3,4,5-Trisphosphate Is Required for Insulin-Like Growth Factor 1-Mediated Survival of 3T3-L1 Preadipocytes1

Adipocyte number, a determinant of adipose tissue mass, reflects the balance between the rates of proliferation/differentiation vs. apoptosis of preadipocytes. The percentage of 3T3-L1 preadipocytes undergoing cell death following serum deprivation was reduced by 10 nM insulin-like growth factor (IGF)-1 (from 50.0 +/- 0.7% for control starved cells to 27.5 +/- 3.1%). TUNEL staining confirmed the apoptotic nature of the cell death. The protective effect of IGF-1 was blocked by phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin, and LY294002, but was unaffected by rapamycin, PD98059, or SB203580, which inhibit mammalian target of rapamycin (mTOR), ERK kinase (MEK1), and p38 MAPK respectively. Exogenous PI(3,4,5)P3 (10 microM), the principal product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, had a modest survival effect on its own, reducing cell death from 47.9 +/- 3.4% to 35.6 +/- 3.5%. When added to the combination of IGF-1 and LY294002, PI(3,4,5)P3 reversed most of the inhibitory effect of LY294002 on IGF-1-dependent cell survival, protein kinase B/Akt phosphorylation, and caspase-3 activity. Taken together, these results implicate PI(3,4,5)P3 as a necessary signal for the anti-apoptotic action of IGF-1 on 3T3-L1 preadipocytes.