Dysregulation of macrophage PEPD in obesity determines adipose tissue fibro-inflammation and insulin resistance.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes.

Decreased lipid metabolism but increased FA biosynthesis are coupled with changes in liver microRNAs in obese subjects with NAFLD.

BACKGROUND/OBJECTIVE: Many controversies regarding the association of liver miRNAs with obesity and nonalcoholic fatty liver diseases (NAFLD) call for additional validations. This study sought to investigate variations in genes and hepatic miRNAs in a sample of obese patients with or without NAFLD and human hepatocytes (HH). SUBJECTS/METHODS: A total of 60 non-consecutive obese women following bariatric surgery were recruited. Subjects were classified as NAFLD (n=17), borderline (n=24) and controls (n=19) with normal enzymatic profile, liver histology and ultrasound assessments. Profiling of 744 miRNAs was performed in 8 obese women with no sign of hepatic disease and 11 NAFLD patients. Additional validation and expression of genes related to de novo fatty acid (FA) biosynthesis, uptake, transport and ß-oxidation; glucose metabolism, and inflammation was tested in the extended sample. Induction of NAFLD-related genes and miRNAs was examined in HepG2 cells and primary HH treated with palmitic acid (PA), a combination of palmitate and oleic acid, or high glucose, and insulin (HG) mimicking insulin resistance in NAFLD. RESULTS: In the discovery sample, 14 miRNAs were associated with NAFLD. Analyses in the extended sample confirmed decreased miR-139-5p, miR-30b-5p, miR-122-5p and miR-422a, and increased miR-146b-5p in obese subjects with NAFLD. Multiple linear regression analyses disclosed that NAFLD contributed independently to explain miR-139-5p (P=0.005), miR-30b-5p (P=0.005), miR-122-5p (P=0.021), miR-422a (P=0.007) and miR-146a (P=0.033) expression variance after controlling for confounders. Decreased miR-122-5p in liver was associated with impaired FA usage. Expression of inflammatory and macrophage-related genes was opposite to decreased miR-30b-5p, miR-139-5p and miR-422a, whereas increased miR-146b-5p was associated with FABP4 and decreased glucose metabolism and FA mobilization. In partial agreement, PA (but not HG) led to decreased miR-139-5p, miR-30b-5p, miR-422a and miR-146a in vitro, in parallel with increased lipogenesis and FA transport, decreased glucose metabolism and diminished FA oxidation. CONCLUSION: This study confirms decreased liver glucose and lipid metabolism but increased FA biosynthesis coupled with changes in five unique miRNAs in obese patients with NAFLD.

CIDEC/FSP27 and PLIN1 gene expression run in parallel to mitochondrial genes in human adipose tissue, both increasing after weight loss.

OBJECTIVE: FSP27 KO mice showed enhanced expression of mitochondrial genes, increased mitochondrial activity and smaller lipid droplets. Here, we aimed to investigate lipid droplet protein (CIDEC/FSP27 and perilipinA (PLIN1)) gene expression in human adipose tissue in association with obesity, insulin resistance and mitochondrial gene expression. DESIGN AND SUBJECTS: In cohort 1, CIDEC/FSP27, PLIN1, adipogenic (FASN, ACACA, PPARG, GLUT4) and mitochondrial (PPARGC1A, PPARGC1B, TFAM, MT-CO3) gene expression were analyzed in 171 adipose tissue samples (88 visceral adipose tissue (VAT) and 83 subcutaneous adipose tissue (SAT) depots) and in a time course experiment in human subcutaneous and visceral preadipocytes using real-time PCR. In cohort 2, the effects of bariatric surgery-induced weight loss were also evaluated in six caucasian morbidly obese women. Additionally, in cohort 2 FSP27 and PLIN1 protein levels were measured using western blotting. RESULTS: CIDEC/FSP27 (1.03±0.52 vs 0.49±0.23 relative gene expression unit (R.U.), P<0.0001) and PLIN1 (1.32±0.82 vs 0.63±0.42 R.U., P<0.0001) gene were significantly more expressed in SAT than in VAT. In VAT, CIDEC/FSP27 and PLIN1 gene expression decreased with body mass index, percent fat mass, fasting glucose, fasting insulin, HOMA and were positively associated with adipogenic (PPARG, GLUT4, FASN and ACACA) and mitochondrial biogenesis (PPARGC1A, PPARGC1B, TFAM and MT-CO3)-related genes. Mitochondrial gene expression increased during adipocyte differentiation in parallel to FSP27 and PLIN1 and other adipogenic genes. After bariatric surgery-induced weight loss, PLIN1 and CIDEC/FSP27 gene and protein expression in SAT increased significantly in parallel to adipogenic and mitochondrial genes. CONCLUSION: These findings suggest a positive functional interaction between CIDEC/FSP27, PLIN1 and mitochondrial biogenesis-related genes in human adipose tissue.

Inflammation and insulin resistance exert dual effects on adipose tissue tumor protein 53 expression.

OBJECTIVE: The purpose of this study was to investigate the expression of human adipose tissue protein 53 (p53) in subjects who varied widely in terms of obesity and insulin resistance. We also analyzed different in vivo and in vitro models to try to comprehend the associations found in humans. METHODS: p53 was analyzed in human adipose and isolated adipocytes, in high fat-fed and GLP-1R KO mice, during in vitro adipogenesis, and in adipocytes after high glucose, rosiglitazone and inflammatory conditions. The effects of surgery-induced weight loss and ex vivo metformin were also evaluated. RESULTS: Omental (OM) p53 gene expression (+27%, P=0.001) and protein (+11%, P=0.04) were increased in obese subjects and high fat diet-induced obese mice (+86%, P=0.018). Although the obesity-associated inflammatory milieu was associated with increased OM p53, this was negatively related to insulin resistance and glycated hemoglobin, and positively with biomarkers for insulin sensitivity. Multiple linear regression analyses revealed that glycated hemoglobin (P<0.0001) and body mass index (P=0.048) contributed independently to explain 13.7% (P<0.0001) of the OM p53 variance. Accordingly, the improvement of insulin sensitivity with surgery-induced weight loss (+51%, P=0.01) and metformin (+42%, P=0.02) led to increased adipose p53. While the glucose-intolerant GLP-1R KO mice showed decreased mesenteric p53 (-45.4%, P=0.017), high glucose led to decreased p53 in pre-adipocytes (-27%, P<0.0001). Inflammatory treatments led to increased p53 (+35%, P<0.0001), while Rs downregulated this expression (-40%, P=0.005) in mature adipocytes. CONCLUSION: Inflammation and insulin resistance exert dual effects on adipose p53, which seems to be the final result of these opposing forces.

Liver, but not adipose tissue PEDF gene expression is associated with insulin resistance.

OBJECTIVE: Recent studies linked circulating pigment epithelium-derived factor (PEDF) to obesity-associated insulin resistance, but the main source of circulating PEDF is unknown. We aimed to investigate liver and adipose tissue PEDF gene expression in association with obesity and insulin resistance. DESIGN, SUBJECTS AND METHODS: Three (two cross-sectional and one longitudinal) independent cohorts have been studied, for adipose tissue (n=80 and n=30) and liver gene expression (n=32 and n=14). Effects of high glucose and cytokines on HepG2 cell line were also investigated. PEDF gene expression and circulating PEDF were analyzed using real-time PCR and ELISA, respectively. RESULTS: In a first cohort of subjects, PEDF relative gene expression was higher in subcutaneous (SC) than in omental (OM) adipose tissue (P<0.0001) being also higher in mature adipocytes compared with stromo-vascular cells (P<0.0001). However, OM PEDF relative gene expression was decreased in morbidly obese subjects (P=0.01). Both OM PEDF and OM PEDF receptor (PEDFR) correlated positively with lipogenic and lipolytic genes, and with genes implicated in the lipid vacuole formation. Circulating PEDF levels were not associated with fat PEDF gene expression. In the second cohort, SC PEDF was decreased in subjects with type 2 diabetes and did not change significantly after weight loss. We next explored circulating PEDF in association with markers of liver-related insulin resistance injury (alanine aminotransferase, r=0.59, P=0.001). Interestingly, liver PEDF gene expression increased with obesity and insulin resistance in men, being significantly associated with fasting glucose and glycated hemoglobin in two independent cohorts. In fact, high glucose led to increased PEDF in HepG2 cells, while inflammatory stimuli present in the adipose tissue environment downregulated PEDF. CONCLUSION: Liver, but not adipose tissue, might be the source of increased circulating PEDF linked to insulin resistance.

The postprandial inflammatory response after ingestion of heated oils in obese persons is reduced by the presence of phenol compounds.

SCOPE: Heating during the process of cooking alters the chemical properties of foods and may affect subsequent postprandial inflammation. We tested the effects of four meals rich in different oils subjected to heating on the postprandial inflammatory metabolism of peripheral blood mononuclear cells (PBMCs). METHODS AND RESULTS: Twenty obese participants received four breakfasts following a randomized crossover design, consisting of milk and muffins made with different oils (virgin olive oil (VOO), sunflower oil (SFO), and a mixture of seeds oil (SFO/canola oil) with added either dimethylpolysiloxane (SOD), or natural antioxidants from olive mill wastewater alperujo (phenols; SOP)), previously subjected to 20 heating cycles. Postprandial inflammatory status in PBMCs was assessed by the activation of nuclear NF-κB, the concentration in cytoplasm of the NF-κB inhibitor (IκB-α), the mRNA levels of NF-κB subunits and activators (p65, IKKß, and IKKα) and other inflammatory molecules (TNF-α, IL-1ß, IL-6, MIF, and JNK), and lipopolysaccharide (LPS) levels. VOO and SOP breakfasts reduced NF-κB activation, increased IκB-α, and decreased LPS plasma concentration. SFO increased IKKα, IKKß, p65, IL-1b, IL-6, MIF, and JNK mRNA levels, and plasma LPS. CONCLUSION: Oils rich in phenols, whether natural (VOO) or artificially added (SOP), reduce postprandial inflammation, compared with seed oil (sunflower).

Type I iodothyronine 5'-deiodinase mRNA and activity is increased in adipose tissue of obese subjects.

Differentiation and metabolism of adipose tissue are modulated by thyroid hormones (THs), but relatively little is known about the metabolism of THs in this tissue. Expression of the genes for type I iodothyronine 5'-deiodinase (D1), leptin (LEP) and stearoyl-CoA desaturase 1 (SCD-1) was evaluated in omental (OM) and subcutaneous (SC) fat using a cohort of 70 humans. Activities of iodothyronine deiodinases (D1, D2 and D3) were assessed in a randomly selected subpopulation of 19 subjects. D1 expression was upregulated in both OM (P=0.011) and SC (P=0.003) fat of obese subjects. Concomitantly, OM (P=0.002) and SC (P=0.028) LEP expression were increased in obesity, associated with both D1 mRNA (r=0.315, P=0.014) and activity (r=0.647, P=0.023) and inversely related to SCD-1 (r=-0.266, P=0.034) expression in SC fat. Also D1 (but not D2 and D3) activity was increased in OM (∼fourfold, P=0.010) and SC (∼eightfold, P=0.004) fat of obese when compared with non-obese subjects and correlated in both OM (r=0.528, P=0.036) and SC (r=0.749, P=0.005) fat with body mass index. Our results document increased D1 gene expression and activity in adipose tissue of obese humans and suggest a role of 3,5,3'-triiodo-L-thyronine formed by D1 in response to leptin in the modulation of adipose tissue metabolism.

Telomere length of subcutaneous adipose tissue cells is shorter in obese and formerly obese subjects.

Obesity and increased fat mass are associated with increased adipocyte proliferation. Telomere length can serve as a biomarker of a cell's biological (vs chronological) age. To gain insight in the physiology of adipose tissue, we aimed to investigate telomere length in subcutaneous adipose tissue in relation to age and obesity. Telomere length was measured in 72 subcutaneous adipose tissue samples from 21 nonobese and 51 obese subjects. Telomere length of subcutaneous adipose tissue cells was negatively associated with body mass index (BMI), systolic blood pressure and fasting triglycerides. After controlling for age, fasting glucose, triglycerides and smoking status, BMI (P=0.009) contributed independently to 16% of telomere length variance. Interestingly, formerly obese patients (n=10) had shorter telomere length than never-obese subjects (n=12) of similar age, sex and BMI (7.1+/-1.3 vs 9.08+/-1.8 kb, P=0.01). In summary, adipose tissue cells from obese subjects show a shorter telomere length. The shorter telomere length of formerly obese subjects suggests that this is an established, irreversible feature of obesity that could contribute to its comorbidities.

Thyroid hormone responsive Spot 14 increases during differentiation of human adipocytes and its expression is down-regulated in obese subjects.

CONTEXT: Very limited information is available regarding the function of human thyroid hormone responsive Spot 14 (human S14, hS14) in adipogenesis and human adiposity. OBJECTIVE: To evaluate hS14 levels during differentiation of human pre-adipocytes, in human fat depots and isolated fat cells. DESIGN: This was a cross-sectional study. SUBJECTS: A total of 161 omental (OM) and 87 subcutaneous (SC) adipose tissue samples obtained during elective surgical procedures from a population who varied widely in terms of obesity. MEASUREMENTS: hS14 gene expression and protein levels during adipogenesis were assessed by RT-PCR, western blot, and using an automated confocal imaging approach. RESULTS: hS14 gene expression levels were decreased in OM adipose tissue from overweight (-42.0%) and obese subjects (-56.5%) compared with lean subjects (P<0.05 and P<0.0001, respectively). hS14 mRNA (but not hS14-related) was inversely associated with obesity measures such as body mass index (P=0.001), percent fat mass (P=0.001), waist-to-hip ratio (P=0.020), and systolic blood pressure (P=0.031). hS14 gene expression and protein levels were up-regulated at the early stages of differentiation of human pre-adipocytes as well as for 3T3-L1 cells. That observation was most prominent in those individual cells exhibiting the more marked differentiation features. hS14 gene expression levels increased by approximately 45 000-fold in mature adipocytes. Increased hS14 levels were also found in stromal-vascular cells/pre-adipocytes (3.8-fold, P<0.05) and in adipose tissue samples (1.9-fold, P<0.0001) from SC compared with OM fat depots. CONCLUSIONS: These results suggest that hS14 is involved in human adipogenesis, but inversely related to obesity and OM fat accumulation.

Metabolic endotoxemia and saturated fat contribute to circulating NGAL concentrations in subjects with insulin resistance.

OBJECTIVE: Lipocalin-2 (neutrophil gelatinase-associated lipocalin, NGAL) is an innate immune system protein that has been linked to insulin resistance and obesity, but the mechanisms behind these associations are poorly known. We hypothesized that endotoxin (lipopolysaccharide, LPS) and fat intake were in the background of these associations. DESIGN: We studied four cohorts: (1) a cross-sectional study in 194 subjects; (2) the changes in NGAL concentration induced by diet and weight loss in 36 obese women (with circadian rhythm in 8 of them); (3) the effects of acute fat intake on circulating NGAL concentration in 42 morbidly obese subjects; and (4) LPS-induced NGAL secretion ex vivo (whole blood and adipose tissue explants). RESULTS: Serum NGAL concentration was significantly associated with fasting triglycerides and LPS-binding protein in patients with type 2 diabetes. In obese subjects, the intake of saturated fatty acids was the factor that best explained the variance of NGAL changes after weight loss (contributing independently to 14% of NGAL variance). In fact, weight loss significantly changed the circadian rhythm of NGAL. The acute increase in circulating NGAL after fat overload was significantly associated with fasting insulin (r=0.52, P<0.001), homeostasis model assessment of insulin resistance (HOMA-IR) (r=0.36, P=0.02) and post-load triglyceride concentrations (r=0.38, P=0.018). LPS-induced NGAL secretion from adipose tissue explants did not change significantly, but LPS led to a significant increase in NGAL concentration in the whole blood obtained from patients with type 2 diabetes. CONCLUSION: Metabolic endotoxemia and saturated fat might contribute to circulating NGAL concentration in patients with insulin resistance.

LIGHT is associated with hypertriglyceridemia in obese subjects and increased cytokine secretion from cultured human adipocytes.

BACKGROUND: LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells) is a member of the tumor necrosis factor (TNF) family, primarily expressed in lymphocytes, which was associated with the induction of pro-inflammatory cytokines and alterations of lipid homeostasis in animal models. We aimed to analyze whether LIGHT has a role in the human obesity-associated inflammatory status. METHODS: The association between circulating LIGHT concentrations and clinical variables was studied in 190 subjects with different degrees of obesity and glucose tolerance. The expression and release of 21 different cytokines, and the expression of genes involved in lipid metabolism were also evaluated after stimulation with LIGHT in cultured human differentiated adipocytes. RESULTS: Serum LIGHT concentrations positively associated with body mass index (BMI), fat mass, glycated hemoglobin and fasting triglycerides, and negatively with high-density lipoprotein cholesterol. Circulating LIGHT concentrations were significantly increased in morbidly obese subjects and in patients with type 2 diabetes. LIGHT induced the secretion of several cytokines and upregulated the expression and secretion of interleukin-6 (IL-6), IL-8, Growth Regulated Oncogene (GRO) and monocyte chemotactic protein-1 (MCP-1). These observations were concomitant with the activation of nuclear factor (NF)-kappaB signalling in human differentiated adipocytes. LIGHT also upregulated the expression and synthesis of its own receptor (herpesvirus entry mediator (HVEM)) and decreased the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and fatty acid synthase. CONCLUSION: These data suggest that LIGHT may have a role in mediating chronic inflammation and alterations of lipid metabolism in obese subjects.

Lactoferrin increases (172Thr)AMPK phosphorylation and insulin-induced (p473Ser)AKT while impairing adipocyte differentiation.

OBJECTIVE: Lactoferrin is a pleiotropic glycoprotein of the innate immune system with known effects on immunomodulation and cell differentiation. To gain an insight into the interaction among obesity, inflammation and insulin action, we aimed to examine the effects of lactoferrin on adipogenesis and the response to insulin in human hepatocarcinoma (HepG2) and 3T3-L1 cell lines. DESIGN: The cells were cultured with increasing lactoferrin concentration under non-inflammatory, inflammatory and standard conditions. The response to insulin was evaluated through (473Ser)AKT phosphorylation. The effects of lactoferrin on adipogenesis were studied through the expression of different lipogenic markers, AMP-activated protein kinase (AMPK) activation, retinoblastoma (Rb) activity and Oil Red O staining in 3T3-L1 cells. RESULTS: Lactoferrin increased dose-dependent insulin-induced (473Ser)AKT phosphorylation in both cell lines. Inflammation-induced decreased (473Ser)AKT phosphorylation was also rescued by lactoferrin. In addition, lactoferrin led to increased (p172Thr)AMPK during 3T3-L1 differentiation and to decreased adipogenesis (as shown by decreased expression of fatty acid synthase, acetyl-coenzyme A carboxylase-alpha and peroxisome proliferator-activated receptor-gamma in parallel with decreased formation of lipid droplets). Lactoferrin also increased dose-dependent Rb activity (expression and hypophosphorylation) during 3T3-L1 differentiation. CONCLUSION: Lactoferrin administration increased insulin-induced (473Ser)AKT phosphorylation, even in those conditions wherein the response to insulin was downregulated, and led to blunted adipogenesis in the context of increased (p172Thr)AMPK and Rb activity.

Decreased circulating lactoferrin in insulin resistance and altered glucose tolerance as a possible marker of neutrophil dysfunction in type 2 diabetes.

CONTEXT: Lactoferrin is an innate immune system protein with multiple beneficial health activities. OBJECTIVE: To gain insight in the interaction between innate immune system and metabolic disturbances (obesity and insulin resistance), we investigated the relationship between circulating lactoferrin and chronic inflammation-associated insulin resistance according glucose tolerance status in Caucasian population. DESIGN, SETTING, PARTICIPANTS, AND MAIN OUTCOME MEASURES: Circulating nonstressed lactoferrin (ELISA), metabolic variables, and inflammatory markers were measured in 229 men, 94 with normal (NGT) and 135 with altered glucose tolerance (AGT). Lactoferrin secretion by neutrophil was investigated in whole-blood culture (four young NGT subjects, four older NGT subjects, and four patients with type 2 diabetes) under microbial lipopolysaccharide (LPS) with IL-6 and rosiglitazone treatment. We also tested the lactoferrin action in THP-1 cells under LPS stimulus. RESULTS: Circulating lactoferrin was significantly decreased in patients with AGT (431.5 +/- 187.5 vs. 493.5 +/- 238.9 ng/ml, P = 0.02). In addition, circulating lactoferrin was negatively associated with hyperglycemia and obesity measures and positively with insulin sensitivity. Lactoferrin was negatively related to inflammatory markers, especially in AGT subjects. In ex vivo experiments, we found a significant decrease in LPS-induced lactoferrin release from neutrophils in subjects with type 2 diabetes. IL-6 coincubation decreased LPS-induced lactoferrin release in NGT subjects (P < 0.001). Finally, rosiglitazone treatment led to increased lactoferrin secretion (398 +/- 193 vs. 280.1 +/- 104.9 ng/ml, P < 0.0001). Lactoferrin decreased nuclear factor-kappabeta activation and IL-6, IL-8, and macrophage chemoattractant protein-1 expression under LPS challenge. CONCLUSIONS: Decreased circulating lactoferrin levels may play a role in chronic low level inflammation-associated insulin resistance.