Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes.

AIMS/HYPOTHESIS: In mammals, the evolutionary conserved family of Mg(2+)-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. METHODS: The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. RESULTS: Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. CONCLUSIONS/INTERPRETATION: Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.

Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells.

Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

The stress-activated protein kinase Hog1 develops a critical role after resting state.

Quiescence is an essential process in eukaryotes. Control of cell cycle progression by stress-activated protein kinases (SAPK) is critical for cell adaptation to extracellular stimuli. In yeast, activation of the HOG MAPK signalling pathway results in the control of cell cycle at several phases. In this manuscript, we describe the role of Hog1p modulating re-entry into cell cycle from a resting state. Cells deficient in Hog1p activation show a delay in entering the mitotic cell cycle from the stationary phase. Furthermore, a repressible Hog1p allele (Hog1AS) presents a comparable behaviour at this phase to the deleted strain. In addition, the role of Hog1p at the stationary phase exit is not related to loss of cell viability. Moreover, when cells enter the mitotic cell cycle after being in the stationary phase, Hog1p is rapidly activated and concentrates in the nucleus where it modifies the expression of several genes. Similar results are obtained in higher eukaryotic cells by activation of p38. Thus, these results reveal a novel role of the SAPK Hog1p in the control of cell cycle progression as cells leave a resting state.

Resveratrol induces antioxidant defence via transcription factor Yap1p.

Resveratrol is a polyphenol suggested to play a protective role against ageing and age-related diseases. We demonstrate that administering low-doses of resveratrol causes ROS accumulation and transcriptional changes in yeast cells and human adipocytes. These changes in gene expression depend on the oxidative transcription factor Yap1p. In particular, resveratrol induces expression of Yap1p gene targets, such as TRX2, TRR1 or AHP1, in a Yap1p-dependent mode. Under resveratrol treatment, Yap1p is phosphorylated and accumulated in the nucleus. Yap1p knockout causes resveratrol sensitivity, which totally depends on the presence of the C-terminal region of Yap1p. Thus, resveratrol may enhance cellular lifespan by hormetic ROS accumulation, which leads to strengthening the cells' antioxidant capacity.

Zinc alpha-2 glycoprotein is implicated in dyslipidaemia in HIV-1-infected patients treated with antiretroviral drugs.

OBJECTIVES: Treated HIV-1-infected patients with lipodystrophy often develop insulin resistance and proatherogenic dyslipidaemia. Zinc alpha-2 glycoprotein (ZAG) is a recently characterized adipokine which has been shown to be involved in the development of obesity and metabolic syndrome in uninfected subjects. We assessed the relationship between circulating ZAG levels and metabolic derangements in HIV-1-infected patients receiving antiretroviral drugs. METHODS: Plasma ZAG levels were assessed in 222 individuals: 166 HIV-1-infected patients treated with antiretroviral drugs (77 with lipodystrophy and 89 without lipodystrophy) and 56 uninfected controls. Plasma ZAG levels were assessed by enzyme-linked immunosorbent assay (ELISA) and were correlated with fat distribution abnormalities and metabolic parameters. RESULTS: HIV-1-infected patients had lower plasma ZAG levels compared with uninfected controls (P < 0.001). No differences were found in ZAG plasma levels according to the presence of lipodystrophy, components of the metabolic syndrome or type of antiretroviral treatment regimen. Circulating ZAG levels were strongly determined by high-density lipoprotein cholesterol (HDLc) in men (B = 0.644; P < 0.001) and showed a positive correlation with total cholesterol (r = 0.312; P < 0.001) and HDLc (r = 0.216; P = 0.005). CONCLUSIONS: HIV-1-infected patients have lower plasma ZAG levels than uninfected controls. In infected patients, plasma ZAG levels are in close relationship with total cholesterol and HDLc.

Lpin1 in human visceral and subcutaneous adipose tissue: similar levels but different associations with lipogenic and lipolytic genes.

LPIN1 is a gene with important effects on lipidic and metabolic homeostasis. Human subcutaneous LPIN1 expression levels in adipose tissue are related with a better metabolic profile, including insulin sensitivity markers. However, there are few data on the regulation of LPIN1 in visceral adipose tissue (VAT). Our aim was to perform a cross-sectional analysis of VAT compared with subcutaneous (SAT) LPIN1 expression in a well-characterized obese cohort, its relation with the expression of genes involved in lipid metabolism, and the in vitro response to lipogenic and lipolytic stimuli. A downregulation of total LPIN1 mRNA expression in subjects with obesity was found in VAT similarly to that in SAT. Despite similar total LPIN1 mRNA levels in SAT and VAT, a close relationship with clinical parameters and with many lipogenic and lipolytic genes was observed primarily in SAT depot. As shown in the in vitro analysis, the low-grade proinflammatory environment and the insulin resistance associated with obesity may contribute to downregulate LPIN1 in adipose tissue, leading to a worse metabolic profile.

Human subcutaneous adipose tissue LPIN1 expression in obesity, type 2 diabetes mellitus, and human immunodeficiency virus--associated lipodystrophy syndrome.

The aim of this study was to analyze LPIN1 adipose tissue gene expression levels in 3 clinical insulin-resistant conditions-obesity, type 2 diabetes mellitus, and human immunodeficiency virus (HIV)-associated lipodystrophy-and its relationship with adipogenic and inflammatory markers. Subcutaneous adipose tissue samples were obtained from 2 cohorts: 98 subjects with different degrees of adiposity and with or without the presence of type 2 diabetes mellitus and 37 HIV-infected patients. Real-time polymerase chain reaction was used to measure gene expression of LPIN1 and adipogenic (PPARgamma, SREBP1c) and inflammatory markers (IL6, TNFalpha, TNFR1, and TNFR2). LPIN1 messenger RNA expression levels were significantly lower in the obese group (P = .002), were similar in type 2 diabetes mellitus patients and control subjects (P = .211), and were significantly higher in HIV-infected patients (P < .001). LPIN1 messenger RNA levels positively correlated with insulin sensitivity in all subjects. Moreover, an inverse correlation with proinflammatory cytokines was observed.

FABP4 dynamics in obesity: discrepancies in adipose tissue and liver expression regarding circulating plasma levels.

BACKGROUND: FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile. OBJECTIVE: In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed. METHODS: The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot. RESULTS: In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group. CONCLUSION: The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.

Lipodystrophy and insulin resistance in combination antiretroviral treated HIV-1-infected patients: implication of resistin.

BACKGROUND: Little information is available with respect to the involvement of resistin in lipodystrophy and metabolic disturbances in HIV-1-infected patients treated with combination antiretroviral therapy (cART). We determined whether the resistin (rest) -420C>G single-nucleotide polymorphism and plasma resistin are associated with the development of lipodystrophy and metabolic disturbances in HIV-1-infected patients treated with cART. METHODS: The study group comprised 299 HIV-1-infected patients treated with a stable cART for at least 1 year (143 with lipodystrophy and 156 without) and 175 uninfected controls. Anthropometric, clinical, and metabolic variables were determined. Homeostasis model assessment for insulin resistance was used to evaluate insulin resistance. Plasma resistin levels were determined by enzyme-linked immunosorbent assay. The rest -420C>G was assessed using restriction fragment length polymorphism. Student t test, 1-way and 2-way analysis of variance, χ2 test, and Pearson and Spearman correlations were performed for statistical analysis. RESULTS: Genotypes containing the rest -420G variant allele were significantly more common in HIV-1-infected patients without lipodystrophy compared with those with lipodystrophy (P = 0.037). Infected patients had significantly greater plasma resistin levels than uninfected controls (P < 0.001). Among infected patients, plasma resistin levels were significantly lower in patients with lipodystrophy with respect to those without (P = 0.034). In infected patients, plasma resistin levels had a significant positive correlation with insulin and homeostasis model assessment for insulin resistance: P < 0.001 and P = 0.002 in the lipodystrophy subset and P = 0.002 and P = 0.03 in the nonlipodystrophy subset, respectively. CONCLUSIONS: In our cohort of white Spaniards, the rest -420C>G single-nucleotide polymorphism may be associated with cART-related lipodystrophy. Plasma resistin correlates with insulin resistance in infected patients with and without lipodystrophy.

PP2A regulatory subunit PP2A-B' counteracts S6K phosphorylation.

The insulin/TOR signaling pathway plays a crucial role in animal homeostasis, sensing nutrient status to regulate organismal growth and metabolism. We identify here the Drosophila B' regulatory subunit of PP2A (PP2A-B') as a novel, conserved component of the insulin pathway that specifically targets the PP2A holoenzyme to dephosphorylate S6K. PP2A-B' knockout flies have elevated S6K phosphorylation and exhibit phenotypes typical of elevated insulin signaling such as reduced total body triglycerides and reduced longevity. We show that PP2A-B' interacts with S6K both physically and genetically. The human homolog of PP2A-B', PPP2R5C, also counteracts S6K1 phosphorylation, indicating a conserved mechanism in mammals. Since S6K affects development of cancer and metabolic disease, our data identify PPP2R5C as a novel factor of potential medical relevance.

Gene expression of paired abdominal adipose AQP7 and liver AQP9 in patients with morbid obesity: relationship with glucose abnormalities.

The trafficking of glycerol from adipose and hepatic tissue is mainly mediated by 2 aquaporin channel proteins: AQP7 and AQP9, respectively. In rodents, both aquaporins were found to act in a coordinated manner. The aim was to study the relationship between adipose AQP7 and hepatic AQP9 messenger RNA expression and the presence of glucose abnormalities simultaneously in morbid obesity. Adipose tissue (subcutaneous [SAT] and visceral [VAT]) and liver biopsies from the same patient were obtained during bariatric surgery in 30 (21 male and 9 female) morbidly obese subjects. Real-time quantification of AQP7 in SAT and VAT and hepatic AQP9 gene expression were performed. A 75-g oral glucose tolerance test was performed in all subjects. The homeostasis model assessment of insulin resistance and lipidic profile were also determined. Visceral adipose tissue AQP7 expression levels were significantly higher than SAT AQP7 (P = .009). Subcutaneous adipose tissue AQP7 positively correlated with both VAT AQP7 and hepatic AQP9 messenger RNA expression (r = 0.44, P = .013 and r = 0.45, P = .012, respectively). The correlation between SAT AQP7 and liver AQP9 was stronger in intolerant and type 2 diabetes mellitus subjects (r = 0.602, P = .011). We have found no differences in compartmental AQP7 adipose tissue distribution or AQP9 hepatic gene expression according to glucose tolerance classification. The present study provides, for the first time, evidence of coordinated regulation between adipose aquaglyceroporins, with a greater expression found in visceral fat, and between subcutaneous adipose AQP7 and hepatic AQP9 gene expression within the context of human morbid obesity.

PPARgamma Pro12Ala polymorphism in HIV-1-infected patients with HAART-related lipodystrophy.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in obesity and in some components of the metabolic syndrome in unselected population. To determine whether PPARgamma genetic variants are associated with the risk of developing lipodystrophy and its associated metabolic disturbances in HIV-1-infected patients treated with HAART and to assess PPARgamma mRNA expression in subcutaneous adipose tissue (SAT). The study group comprised 278 patients infected with HIV-1 and treated with antiretroviral drugs (139 with lipodystrophy and 139 without) and 105 uninfected controls (UC). The PPARgamma Pro12Ala (C%>G) single nucleotide polymorphism (SNP) was assessed using PCR-RFLPs on white cell DNA. PPARgamma mRNA expression in SAT was assessed in 38 patients (25 with lipodystrophy and 13 without) and in 21 UC by real-time PCR. Statistical analysis was based on Student's T tests, Chi(2) tests, Spearman's correlations tests and logistic regression tests. PPARgamma Pro12Ala genotype distribution and allele frequencies were non-significantly different between both HIV-1-infected categories, lipodystrophy vs non-lipodystrophy (p=0.9 and p=0.87, respectively). Lipodystrophic patients harbouring the rare X/Ala genotype (Ala/Ala plus Pro/Ala) had significantly greater plasma total and LDL cholesterol levels compared with carriers of the common Pro/Pro genotype (p=0.029 and p=0.016, respectively) at univariate analyses. At multivariate analyses these associations were no longer significant. There was a near-significant decreased SAT PPARgamma mRNA expression in patients with lipodystrophy compared to UC (p=0.054). PPARgamma Pro12Ala SNP has no effect on the risk of developing lipodystrophy in HIV-1-infected patients treated with HAART. PPARgamma mRNA SAT expression appears decreased in lipodystrophy.

LMNA mRNA expression is altered in human obesity and type 2 diabetes.

OBJECTIVE: The aim of this study was to analyze lamin A/C mRNA levels in abdominal subcutaneous adipose tissue in two conditions-obesity and type 2 diabetes-that share common inflammatory and metabolic features, and to assess their relationship with selected inflammatory and adipogenic genes. METHODS AND PROCEDURES: This is a cross-sectional study involving 52 nondiabetic and 54 type 2 diabetes patients. Anthropometrical and analytical measurements (glycemic, lipidic, and inflammatory profiles) were performed, and mRNA expression was determined using real-time PCR. RESULTS: Lamin A and C isoforms are expressed differentially. Lamin A/C mRNA levels were increased in obese and in type 2 diabetes patients. We also observed a strong relationship between both isoforms (B = 2.218, P < 0.001) and among lamin C mRNA expression and adipogenic (sterol-responsive element binding protein-1 (SREBP1c)) and inflammatory (interleukin-6 (IL-6)) markers (B = 0.854, P = 0.001, and B = 0.557, P < 0.001, respectively). DISCUSSION: These data suggest that lamin A/C may be involved in the adipocyte gene profile observed in obesity and type 2 diabetes.

The IL-6 system in HIV-1-infection and in HAART-related fat redistribution syndromes.

We determined the IL-6 -174 G>C single nucleotide polymorphism, IL-6 mRNA expression in subcutaneous adipose tissue (SAT) and IL-6 plasma levels in HIV-1-infected patients with and without lipodystrophy and uninfected controls. HIV-1-infected patients had a greater prevalence of the IL-6 -174 C/C genotype and the C allele, higher SAT IL-6 mRNA expression and plasma IL-6 levels than controls. The IL-6 -174 G>C genotype distribution and allele frequencies, SAT IL-6 mRNA expression and IL-6 plasma levels were non-significantly different between HIV-1-infected patients with and without lipodystrophy.

LMNA messenger RNA expression in highly active antiretroviral therapy-treated HIV-positive patients.

The LMNA gene encodes for lamins A and C as major products, which are involved in nuclear stability, chromatin structure, and gene expression. Several LMNA mutations cause an insulin-resistant lipodystrophy that shares features with HIV-related lipodystrophy. Some HIV-treatment agents alter lamin A/C maturation, organization, and stability in 3T3-L1. We aimed to test the hypothesis that human adipose tissue LMNA expression can be altered in highly active antiretroviral therapy (HAART)-treated HIV-positive patients with lipodystrophy. We have also analyzed both isoforms and explored if their expression is associated with insulin resistance or inflammation in these patients. A cross-sectional study that analyzed abdominal subcutaneous adipose tissue from 39 treated HIV-positive patients (25 of whom had lipodystrophy) and 21 uninfected control subjects was performed. We have observed lower levels of lamin A isoform but normal levels of lamin C isoform in all HIV-infected patients, irrespective of the presence or absence of lipodystrophy, which reinforces the idea that an altered lamin A/C ratio could reflect a pathogenic condition. We have also found a correlation between LMNA adipose expression and several cytokine and adipogenic gene markers in HIV-positive patients, regardless of the presence or absence of lipodystrophy. Hence, in the present study, the lower lamin A expression observed in HIV-positive patients is related to HIV itself or to treatments rather than to the presence of lipodystrophy.

Monocyte chemoattractant protein-1 in obesity and type 2 diabetes. Insulin sensitivity study.

OBJECTIVE: Our goal was to test any association between human plasma circulating levels of monocyte chemoattractant protein-1 (cMCP-1) and insulin resistance and to compare monocyte chemoattractant protein-1 (MCP-1) adipose tissue gene expression and cMCP-1 in relation with inflammatory markers. RESEARCH METHODS AND PROCEDURES: cMCP-1 was measured in n = 116 consecutive control male subjects to whom an insulin sensitivity (S(i)) test was performed. Circulating levels of soluble CD14, soluble tumor necrosis factor receptor type 2 (sTNFR2), soluble interleukin-6 (sIL-6), and adiponectin also were measured. Subcutaneous adipose tissue samples were obtained from n = 107 non-diabetic and type 2 diabetic subjects with different degrees of obesity. Real-time polymerase chain reaction was used to measure gene expression of MCP-1, CD68, tumor necrosis factor-alpha (TNF-alpha), and its receptor TNFR2. RESULTS: In the S(i) study, no independent effect of cMCP-1 levels on insulin sensitivity was observed. In the expression study, in non-diabetic subjects, MCP-1 mRNA had a positive correlation with BMI (r = 0.407, p = 0.003), TNF-alpha mRNA (r = 0.419, p = 0.002), and TNFR2 mRNA (r = 0.410, p = 0.003). In these subjects, cMCP-1 was found to correlate with waist-to-hip ratio (r = 0.322, p = 0.048). In patients with type 2 diabetes, MCP-1 mRNA was up-regulated compared with non-diabetic subjects. TNF-alpha mRNA was found to independently contribute to MCP-1 mRNA expression. In this group, CD68 mRNA was found to correlate with BMI (r = 0.455, p = 0.001). DISCUSSION: cMCP-1 is not associated with insulin sensitivity in apparently healthy men. TNF-alpha is the inflammatory cytokine associated with MCP-1 expression in subcutaneous adipose tissue.