Extracellular Matrix Expression in Human Pancreatic Fat Cells of Patients with Normal Glucose Regulation, Prediabetes and Type 2 Diabetes.

Previously, we found that human pancreatic preadipocytes (PPAs) and islets influence each other and that the crosstalk with the fatty liver via the hepatokine fetuin-A/palmitate induces inflammatory responses. Here, we examined whether the mRNA-expression of pancreatic extracellular matrix (ECM)-forming and -degrading components differ in PPAs from individuals with normal glucose regulation (PPAs-NGR), prediabetes (PPAs-PD), and type 2 diabetes (PPAs-T2D), and whether fetuin-A/palmitate impacts ECM-formation/degradation and associated monocyte invasion. Human pancreatic resections were analyzed (immuno)histologically. PPAs were studied for mRNA expression by real-time PCR and protein secretion by Luminex analysis. Furthermore, co-cultures with human islets and monocyte migration assays in Transwell plates were conducted. We found that in comparison with NGR-PPAs, TIMP-2 mRNA levels were lower in PPAs-PD, and TGF-ß1 mRNA levels were higher in PPAs-T2D. Fetuin-A/palmitate reduced fibronectin, decorin, TIMP-1/-2 and TGF-ß1 mRNA levels. Only fibronectin was strongly downregulated by fetuin-A/palmitate independently of the glycemic status. Co-culturing of PPAs with islets increased TIMP-1 mRNA expression in islets. Fetuin-A/palmitate increased MMP-1, usherin and dermatopontin mRNA-levels in co-cultured islets. A transmigration assay showed increased monocyte migration towards PPAs, which was enhanced by fetuin-A/palmitate. This was more pronounced in PPAs-T2D. The expression of distinct ECM components differs in PPAs-PD and PPAs-T2D compared to PPAs-NGR, suggesting that ECM alterations can occur even in mild hyperglycemia. Fetuin-A/palmitate impacts on ECM formation/degradation in PPAs and co-cultured islets. Fetuin-A/palmitate also enhances monocyte migration, a process which might impact on matrix turnover.

Fetuin-A and risk of diabetes-related vascular complications: a prospective study.

BACKGROUND: Fetuin-A is a hepatokine which has the capacity to prevent vascular calcification. Moreover, it is linked to the induction of metabolic dysfunction, insulin resistance and associated with increased risk of diabetes. It has not been clarified whether fetuin-A associates with risk of vascular, specifically microvascular, complications in patients with diabetes. We aimed to investigate whether pre-diagnostic plasma fetuin-A is associated with risk of complications once diabetes develops. METHODS: Participants with incident type 2 diabetes and free of micro- and macrovascular disease from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort (n = 587) were followed for microvascular and macrovascular complications (n = 203 and n = 60, respectively, median follow-up: 13 years). Plasma fetuin-A was measured approximately 4 years prior to diabetes diagnosis. Prospective associations between baseline fetuin-A and risk of complications were assessed with Cox regression. RESULTS: In multivariable models, fetuin-A was linearly inversely associated with incident total and microvascular complications, hazard ratio (HR, 95% CI) per standard deviation (SD) increase: 0.86 (0.74; 0.99) for total, 0.84 (0.71; 0.98) for microvascular and 0.92 (0.68; 1.24) for macrovascular complications. After additional adjustment for cardiometabolic plasma biomarkers, including triglycerides and high-density lipoprotein, the associations were slightly attenuated: 0.88 (0.75; 1.02) for total, 0.85 (0.72; 1.01) for microvascular and 0.95 (0.67; 1.34) for macrovascular complications. No interaction by sex could be observed (p > 0.10 for all endpoints). CONCLUSIONS: Our data show that lower plasma fetuin-A levels measured prior to the diagnosis of diabetes may be etiologically implicated in the development of diabetes-associated microvascular disease.

Liver-targeting drugs and their effect on blood glucose and hepatic lipids.

The global epidemic of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) and the high prevalence among individuals with type 2 diabetes has attracted the attention of clinicians specialising in liver disorders. Many drugs are in the pipeline for the treatment of NAFLD/NASH, and several glucose-lowering drugs are now being tested specifically for the treatment of liver disease. Among these are nuclear hormone receptor agonists (e.g. peroxisome proliferator-activated receptor agonists, farnesoid X receptor agonists and liver X receptor agonists), fibroblast growth factor-19 and -21, single, dual or triple incretins, sodium-glucose cotransporter inhibitors, drugs that modulate lipid or other metabolic pathways (e.g. inhibitors of fatty acid synthase, diacylglycerol acyltransferase-1, acetyl-CoA carboxylase and 11ß-hydroxysteroid dehydrogenase type-1) or drugs that target the mitochondrial pyruvate carrier. We have reviewed the metabolic effects of these drugs in relation to improvement of diabetic hyperglycaemia and fatty liver disease, as well as peripheral metabolism and insulin resistance.

The hepatokine fetuin-A disrupts functional maturation of pancreatic beta cells.

AIMS/HYPOTHESIS: Neonatal beta cells carry out a programme of postnatal functional maturation to achieve full glucose responsiveness. A partial loss of the mature phenotype of adult beta cells may contribute to a reduction of functional beta cell mass and accelerate the onset of type 2 diabetes. We previously found that fetuin-A, a hepatokine increasingly secreted by the fatty liver and a determinant of type 2 diabetes, inhibits glucose-stimulated insulin secretion (GSIS) of human islets. Since fetuin-A is a ubiquitous fetal glycoprotein that declines peripartum, we examined here whether fetuin-A interferes with the functional maturity of beta cells. METHODS: The effects of fetuin-A were assessed during in vitro maturation of porcine neonatal islet cell clusters (NICCs) and in adult human islets. Expression alterations were examined via microarray, RNA sequencing and reverse transcription quantitative real-time PCR (qRT-PCR), proteins were analysed by western blotting and immunostaining, and insulin secretion was quantified in static incubations. RESULTS: NICC maturation was accompanied by the gain of glucose-responsive insulin secretion (twofold stimulation), backed up by mRNA upregulation of genes governing beta cell identity and function, such as NEUROD1, UCN3, ABCC8 and CASR (Log2 fold change [Log2FC] > 1.6). An active TGFß receptor (TGFBR)-SMAD2/3 pathway facilitates NICC maturation, since the TGFBR inhibitor SB431542 counteracted the upregulation of aforementioned genes and de-repressed ALDOB, a gene disallowed in mature beta cells. In fetuin-A-treated NICCs, upregulation of beta cell markers and the onset of glucose responsiveness were suppressed. Concomitantly, SMAD2/3 phosphorylation was inhibited. Transcriptome analysis confirmed inhibitory effects of fetuin-A and SB431542 on TGFß-1- and SMAD2/3-regulated transcription. However, contrary to SB431542 and regardless of cMYC upregulation, fetuin-A inhibited beta cell proliferation (0.27 ± 0.08% vs 1.0 ± 0.1% Ki67-positive cells in control NICCs). This effect was sustained by reduced expression (Log2FC ≤ -2.4) of FOXM1, CENPA, CDK1 or TOP2A. In agreement, the number of insulin-positive cells was lower in fetuin-A-treated NICCs than in control NICCs (14.4 ± 1.2% and 22.3 ± 1.1%, respectively). In adult human islets fetuin-A abolished glucose responsiveness, i.e. 1.7- and 1.1-fold change over 2.8 mmol/l glucose in control- and fetuin-A-cultured islets, respectively. In addition, fetuin-A reduced SMAD2/3 phosphorylation and suppressed expression of proliferative genes. Of note, in non-diabetic humans, plasma fetuin-A was negatively correlated (p = 0.013) with islet beta cell area. CONCLUSIONS/INTERPRETATION: Our results suggest that the perinatal decline of fetuin-A relieves TGFBR signalling in islets, a process that facilitates functional maturation of neonatal beta cells. Functional maturity remains revocable in later life, and the occurrence of a metabolically unhealthy milieu, such as liver steatosis and elevated plasma fetuin-A, can impair both function and adaptive proliferation of beta cells. DATA AVAILABILITY: The RNAseq datasets and computer code produced in this study are available in the Gene Expression Omnibus (GEO): GSE144950; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144950.

[New pharmacological treatment options for nonalcoholic fatty liver disease]. / Neue arzneitherapeutische Optionen bei nichtalkoholischer Fettlebererkrankung.

The prevalence of nonalcoholic fatty liver disease (NAFLD) continues to increase worldwide, presently affecting 25% of the adult population, and is associated with an elevated risk of total and liver-specific mortality. NAFLD is a chronic disease and results from a combination of genetic, environmental and predominantly lifestyle-related causes. Therefore, NAFLD, similarly to cardiovascular disease, type 2 diabetes and many different types of cancer, can be considered a noncommunicable disease. Consequently, lifestyle intervention, particularly if associated with a large amount of weight loss, is considered highly effective and safe to treat NAFLD. For patients with advanced-stage NAFLD or that cannot lose weight, metabolically-based pharmacotherapy is effective to improve liver histology and cardiometabolic risk profile. If a moderate or advanced stage of liver fibrosis is present, additional antifibrotic therapy is necessary to halt the progression of the disease.

Associations of short stature and components of height with incidence of type 2 diabetes: mediating effects of cardiometabolic risk factors.

AIMS/HYPOTHESIS: This study aimed to evaluate associations of height as well as components of height (sitting height and leg length) with risk of type 2 diabetes and to explore to what extent associations are explainable by liver fat and cardiometabolic risk markers. METHODS: A case-cohort study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study comprising 26,437 participants who provided blood samples was designed. We randomly selected a subcohort of 2500 individuals (2029 diabetes-free at baseline and with anamnestic, anthropometrical and metabolic data for analysis). Of the 820 incident diabetes cases identified in the full cohort during 7 years of follow-up, 698 remained for analyses after similar exclusions. RESULTS: After adjustment for age, potential lifestyle confounders, education and waist circumference, greater height was related to lower diabetes risk (HR per 10 cm, men 0.59 [95% CI 0.47, 0.75] and women 0.67 [0.51, 0.88], respectively). Leg length was related to lower risk among men and women, but only among men if adjusted for total height. Adjustment for liver fat and triacylglycerols, adiponectin and C-reactive protein substantially attenuated associations between height and diabetes risk, particularly among women. CONCLUSIONS/INTERPRETATION: We observed inverse associations between height and risk of type 2 diabetes, which was largely related to leg length among men. The inverse associations may be partly driven by lower liver fat content and a more favourable cardiometabolic profile.

Effect of SGLT2 inhibitors on body composition, fluid status and renin-angiotensin-aldosterone system in type 2 diabetes: a prospective study using bioimpedance spectroscopy.

BACKGROUND: SGLT2-inhibitors are potent antihyperglycemic drugs for patients with type 2 diabetes and have been shown to reduce body weight. However, it is unclear which body compartments are reduced and to what extent. METHODS: In this longitudinal observational study, we analyzed the body composition of 27 outpatients with type 2 diabetes mellitus during the first week and up to 6 months after initiation of treatment with SGLT2-inhibitors (n = 18 empagliflozin, n = 9 dapagliflozin) using bioimpedance spectroscopy (BCM, Fresenius). Fluid status of hypertensive patients taking medication with hydrochlorothiazide (n = 14) and healthy persons (n = 16) were analyzed for comparison. RESULTS: At 6 months, HbA1c decreased by 0.8% (IQR 2.3; 0.4), body weight and BMI by 2.6 kg (1.5; 9.3) and 0.9 kg/m2 (0.4; 3.3), respectively. Bioimpedance spectroscopy revealed significant decrease in adipose tissue mass and fat tissue index while lean tissue parameters remained stable. Overhydration (OH) and extracellular water (ECW) decreased by - 0.5 L/1.73 m2 (- 0.1; - 0.9) and - 0.4 L/1.73 m2 (- 0.1; - 0.8) at day 3, respectively, and returned to the initial value after 3 and 6 months. Plasma renin activity increased by 2.1-fold (0.5; 3.6) at 1 month and returned to the initial level at month 3 and 6. Fluid status of patients with SGLT2 inhibitors after 6 months showed no difference from that of hypertensive patients taking hydrochlorothiazide or healthy persons. CONCLUSIONS: Body weight reduction under the treatment with SGLT2-inhibitors is caused by reduction of adipose tissue mass and transient loss of extracellular fluid, which is accompanied by upregulation of renin-angiotensin-aldosterone system (RAAS). Permanent loss of extracellular water does not occur under SGLT2 inhibition.

The hepatokines fetuin-A and fetuin-B are upregulated in the state of hepatic steatosis and may differently impact on glucose homeostasis in humans.

The liver is a central regulator of whole body glucose, and lipid homeostasis and hepatokines, like fetuin-A, have been identified as markers and mediators of fatty liver-induced cardiometabolic risk. The closely related protein fetuin-B was shown to be upregulated in the fatty liver and to impact on glucose homeostasis in mice. In the present study we aimed to test the relevance of these findings in humans. In 55 subjects, hepatic mRNA expression of both hepatokines, fetuin-A and fetuin-B, associated positively with liver triglyceride content, whereas only fetuin-A expression associated with the homeostatic model assessment of insulin resistance. In 220 subjects who underwent precise metabolic phenotyping, circulating fetuin-A, but not fetuin-B, associated positively with liver fat content, and negatively with insulin sensitivity, measured during the oral glucose tolerance test (OGTT) and during the euglycemic, hyperinsulinemic clamp. Both circulating fetuin-A and fetuin-B correlated positively with the glucose area under the curve during the OGTT, but after additional adjustment for insulin sensitivity this relationship remained significant only for fetuin-B. In conclusion, despite the fact that the two hepatokines, fetuin-A and fetuin-B, are upregulated in the state of hepatic steatosis in humans, it appears that they differently impact on glucose homeostasis. Our data are in agreement with observations that fetuin-A can alter insulin signaling and that fetuin-B may regulate glucose homeostasis via so far unknown effects, possibly on glucose effectiveness.

Effects of resveratrol supplementation on liver fat content in overweight and insulin-resistant subjects: A randomized, double-blind, placebo-controlled clinical trial.

We performed the largest randomized, placebo-controlled clinical trial to date (N = 112, 12-week intervention) to investigate the effects and safety of resveratrol supplementation on liver fat content and cardiometabolic risk parameters in overweight and obese and insulin-resistant subjects. At baseline the variability in liver fat content was very large, ranging from 0.09% to 37.55% (median, 7.12%; interquartile range, 3.85%-12.94%). Mean (SD) liver fat content was 9.22 (6.85) % in the placebo group and 9.91 (7.76) % in the resveratrol group. During the study liver fat content decreased in the placebo group (-0.7%) but not in the resveratrol group (-0.03%) (differences between groups: P = .018 for the intention-to-treat [ITT] population; N = 54, resveratrol, N = 54, placebo and P = .0077 for the per protocol [PP] population). No effects of resveratrol supplementation on cardiometabolic risk parameters were observed. Resveratrol supplementation was well tolerated and safe. In conclusion, these data suggest that resveratrol supplementation is safe and that it does not considerably impact liver fat content or cardiometabolic risk parameters in humans.

Non-alcoholic fatty liver disease and impaired proinsulin conversion as newly identified predictors of the long-term non-response to a lifestyle intervention for diabetes prevention: results from the TULIP study.

AIMS/HYPOTHESIS: Lifestyle intervention is effective to prevent type 2 diabetes. However, a considerable long-term non-response occurs to a standard lifestyle intervention. We investigated which risk phenotypes at baseline and their changes during the lifestyle intervention predict long-term glycaemic non-response to the intervention. METHODS: Of 300 participants at high risk for type 2 diabetes who participated in a 24 month lifestyle intervention with diet modification and increased physical activity, 190 participants could be re-examined after 8.7 ± 1.6 years. All individuals underwent a five-point 75 g OGTT and measurements of body fat compartments and liver fat content with MRI and spectroscopy at baseline, 9 and 24 months during the lifestyle intervention, and at long-term follow-up. Fasting proinsulin to insulin conversion (PI/I ratio) and insulin sensitivity and secretion were calculated from the OGTT. Non-response to lifestyle intervention was defined as no decrease in glycaemia, i.e. no decrease in AUC for glucose at 0-120 min during OGTT (AUCglucose0-120 min). RESULTS: Before the lifestyle intervention, 56% of participants had normal glucose regulation and 44% individuals had impaired fasting glucose and/or impaired glucose tolerance. At long-term follow-up, 11% had developed diabetes. Multivariable regression analysis with adjustment for age, sex, BMI and change in BMI during the lifestyle intervention revealed that baseline insulin secretion and insulin sensitivity, as well as change in insulin sensitivity during the lifestyle intervention, predicted long-term glycaemic control after 9 years. In addition, increased hepatic lipid content as well as impaired fasting proinsulin conversion at baseline were newly detected phenotypes that independently predicted long-term glycaemic control. CONCLUSIONS/INTERPRETATION: Increased hepatic lipid content and impaired proinsulin conversion are new predictors, independent of change in body weight, for non-response to lifestyle intervention in addition to the confirmed factors, impaired insulin secretion and insulin sensitivity.

Metabolic crosstalk between fatty pancreas and fatty liver: effects on local inflammation and insulin secretion.

AIMS/HYPOTHESIS: Obesity-linked ectopic fat accumulation is associated with the development of type 2 diabetes. Whether pancreatic and liver steatosis impairs insulin secretion is controversial. We examined the crosstalk of human pancreatic fat cells with islets and the role of diabetogenic factors, i.e. palmitate and fetuin-A, a hepatokine released from fatty liver. METHODS: Human pancreatic resections were immunohistochemically stained for insulin, glucagon, somatostatin and the macrophage/monocyte marker CD68. Pancreatic adipocytes were identified by Oil Red O and adiponectin staining. Primary pancreatic pre-adipocytes and differentiated adipocytes were co-cultured with human islets isolated from organ donors and the metabolic crosstalk between fatty liver and fatty pancreas was mimicked by the addition of palmitate and fetuin-A. Insulin secretion was evaluated by ELISA and RIA. Cytokine expression and secretion were assessed by RT-PCR and multiplex assay, respectively. Subcellular distribution of proteins was examined by confocal microscopy and protein phosphorylation by western blotting. RESULTS: In human pancreatic parenchyma, highly differentiated adipocytes were detected in the proximity of islets with normal architecture and hormone distribution. Infiltration of adipocytes was associated with an increased number of CD68-positive cells within islets. In isolated primary pancreatic pre-adipocytes and differentiated adipocytes, palmitate and fetuin-A induced IL6, CXCL8 and CCL2 mRNA expression. Cytokine production was toll-like receptor 4 (TLR4)-dependent and further accentuated in pre-adipocytes when co-cultured with islets. In islets, IL6 and CXCL8 mRNA levels were also increased by fetuin-A and palmitate. Only in macrophages within the isolated islets, palmitate and fetuin-A stimulated the production of the cytotoxic cytokine IL-1ß. Palmitate, but not fetuin-A, exerted pro-apoptotic effects in islet cells. Instead, fetuin-A impaired glucose-induced insulin secretion in a TLR4-independent, but c-Jun N-terminal kinase- and Ca2+-dependent, manner. CONCLUSIONS/INTERPRETATION: These results provide the first evidence that fetuin-A-mediated metabolic crosstalk of fatty liver with islets may contribute to obesity-linked glucose blindness of beta cells, while fatty pancreas may exacerbate local inflammation.

Intra- and interindividual variability of fatty acid unsaturation in six different human adipose tissue compartments assessed by 1 H-MRS in vivo at 3 T.

It is generally accepted that the amount and distribution of adipose tissue (AT) in the human body play an important role in the pathogenesis of metabolic diseases. In addition, metabolic effects of released saturated fatty acids (FAs) in blood are known to be more critical than those of unsaturated FAs. However, little is known about the variability in unsaturation of FAs in various AT compartments. The aim of this prospective study was the assessment of mono- and polyunsaturated FAs in various AT compartments by localized 1 H-MRS in order to obtain insight into the intra- and interindividual variability. Associations of FA unsaturation with intrahepatic lipids (IHLs), insulin sensitivity and related AT volumes were analyzed. Fifty healthy Caucasians (36 male, 14 female) participated in this study. Spectroscopic examinations were performed in subcutaneous adipose tissue in the neck (SCATneck ), abdominal deep subcutaneous adipose tissue (DSCAT), abdominal superficial subcutaneous adipose tissue (SSCAT), visceral adipose tissue (VAT), tibial bone marrow (BM) and subcutaneous adipose tissue of the lower leg (SCATcalf ) at 3 T. Unsaturated index (UI) was calculated by the ratio of olefinic and methyl resonances, polyunsaturated index (PUI) by the ratio of diallylic and methyl resonances. Volumes of AT compartments (by T1 -weighted MRI) and IHL (single-voxel STEAM) were assessed at 1.5 T, insulin sensitivity by an oral glucose tolerance test. UI was highest for SCATcalf (0.622) and lowest for BM (0.527). Highest PUI was observed for SSCAT (0.108), lowest for BM (0.093). Significant intraindividual differences between UIs-but not PUIs-are present for most compartments. There is a non-significant trend for higher UI in males but otherwise no correlation to anthropometric data (age, BMI). A significant negative correlation between UI and AT volume was observed for VAT but for none of the other compartments. Neither UIs nor PUIs show a relation with IHL; insulin sensitivity is significantly correlated to UI in BM (p < 0.01). Unsaturation indices in several distinct AT compartments are location dependent. Our cohort showed only moderate gender-related differences, with a trend towards less unsaturated FAs (mainly PUI) in females. In BM, insulin resistant subjects are characterized by a higher UI compared with the insulin sensitive ones. Further studies in larger cohorts are necessary to gain further insight into unsaturation of AT.

Obesity and renal disease: not all fat is created equal and not all obesity is harmful to the kidneys.

The prevalence of obesity is increasing worldwide and contributes to many health problems, including kidney disease. Unexpectedly, 10-30% of obese individuals are apparently not at increased risk of metabolic diseases, e.g. type 2 diabetes, cardiovascular disease and risk of renal disease. Their phenotype is labeled 'metabolically healthy obesity'. In the search for mechanisms explaining this unexpected condition, a favourable type of body fat distribution with low insulin resistance and with low subclinical inflammation has been identified. Furthermore, signalling pathways have been found that distinguish between metabolically benign and malignant obesity. In addition, the important roles of fatty acids, adipokines and hepatokines were identified. These factors regulate insulin resistance and subclinical inflammation. Onset and evolution of chronic kidney disease (CKD) are affected by obesity. CKD also increases the risk of insulin resistance and subclinical inflammation, two pathways that play an important role in the pathogenesis of renal malfunction. This brief review summarizes novel insights, specifically how distinct body fat compartments (including perivascular and even renal sinus fat) may have an impact on progression of CKD.

A high-risk phenotype associates with reduced improvement in glycaemia during a lifestyle intervention in prediabetes.

AIMS/HYPOTHESIS: Lack of reversal of prediabetes (impaired glucose tolerance and/or impaired fasting glucose) to normal glucose regulation (NGR) during a lifestyle intervention is strongly associated with a higher incidence of diabetes later in life. In the Tübingen Lifestyle Intervention Program (TULIP) we hypothesised that an at-risk phenotype may exist at baseline that associates with this nonresponse to the intervention. METHODS: A total of 120 participants of TULIP with prediabetes at baseline were studied. Participants underwent 9 months of lifestyle intervention and had measurements of insulin secretion and insulin sensitivity during a 75 g OGTT, and measurements of liver fat content by proton magnetic resonance spectroscopy. RESULTS: During the lifestyle intervention, 55% of the participants did not revert to NGR. Even among participants with the largest body fat loss (upper quartile: -6.9 ± 3.3%, mean ± SD), 40% did not revert to NGR. In this regard, we identified at baseline a high-risk phenotype (n = 72) consisting of low disposition index or low insulin sensitivity + nonalcoholic fatty liver disease (NAFLD) and a low-risk phenotype (n = 48, all other traits). While the adjusted decrease in body fat was almost identical between these phenotypes (-5.7 ± 15.3% vs -7.7 ± 15.2%, p = 0.49), the high-risk phenotype had a smaller decrease in adjusted 2 h blood glucose levels (-3.7 ± 20.3% vs -18.5 ± 20.0%, p = 0.0009). In addition, only 31% of the participants with the high-risk phenotype, but 67% with the low-risk phenotype, reverted to NGR (p < 0.0001). The odds ratio for reaching the status NGR was 4.54 (95% CI 2.08, 9.94) for participants having the low-risk phenotype. CONCLUSIONS/INTERPRETATION: Stratification of individuals with prediabetes at baseline into a high-risk and a low-risk phenotype, based on corrected insulin secretion and insulin-resistant NAFLD, may help to determine the effectiveness of a lifestyle intervention to revert individuals to NGR.

Solutions for low and high accuracy mass spectrometric data matching: a data-driven annotation strategy in nontargeted metabolomics.

Ultra high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) has become a widespread analytical technique in metabolomics investigations, however the benefit of high-performance chromatographic separation is often blunted due to insufficient mass spectrometric accuracy. A strategy that allows for the matching of UHPLC-MS data to highly accurate direct infusion electrospray ionization (DI-ESI) Fourier transform ion cyclotron resonance/mass spectrometry (FTICR/MS) data is developed in this manuscript. Mass difference network (MDiN) based annotation of FTICR/MS data and matching to unique UHPLC-MS peaks enables the consecutive annotation of the chromatographic data set. A direct comparison of experimental m/z values provided no basis for the matching of both platforms. The matching of annotation-based exact neutral masses finally enabled the integration of platform specific multivariate statistical evaluations, minimizing the danger to compare artifacts generated on either platform. The approach was developed on a non-alcoholic fatty liver disease (NAFLD) data set.

PNPLA3 variant I148M is associated with altered hepatic lipid composition in humans.

AIMS/HYPOTHESIS: The common sequence variant I148M of the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) is associated with increased hepatic triacylglycerol (TAG) content, but not with insulin resistance, in humans. The PNPLA3 (I148M) variant was previously reported to alter the specificity of the encoded enzyme and subsequently affect lipid composition. METHODS: We analysed the fatty acid composition of five lipid fractions from liver tissue samples from 52 individuals, including 19 carriers of the minor PNPLA3 (I148M) variant. RESULTS: PNPLA3 (I148M) was associated with a strong increase (1.75-fold) in liver TAGs, but with no change in other lipid fractions. PNPLA3 (I148M) minor allele carriers had an increased n-3 polyunsaturated fatty acid (PUFA) α-linolenic acid content and reductions in several n-6 PUFAs in the liver TAG fraction. Furthermore, there was a strong inverse correlation between n-6 PUFA and TAG content independent of PNPLA3 genotype. In a multivariate model including liver fat content, PNPLA3 genotype and fatty acid composition, two significant differences could be exclusively attributed to the PNPLA3 (I148M) minor allele: reduced stearic acid and increased α-linolenic acid content in the hepatic TAG fraction. CONCLUSIONS: These changes therefore suggest a mechanism to explain the PNPLA3 (I148M)-dependent increase in liver fat content without causing insulin resistance. Stearic acid can induce insulin resistance, whereas α-linolenic acid may protect against it.

Fetuin-A influences vascular cell growth and production of proinflammatory and angiogenic proteins by human perivascular fat cells.

AIMS/HYPOTHESIS: Fetuin-A (alpha2-Heremans-Schmid glycoprotein), a liver-derived circulating glycoprotein, contributes to lipid disorders, diabetes and cardiovascular diseases. In a previous study we found that perivascular fat cells (PVFCs) have a higher angiogenic potential than other fat cell types. The aim was to examine whether fetuin-A influences PVFC and vascular cell growth and the expression and secretion of proinflammatory and angiogenic proteins, and whether TLR4-independent pathways are involved. METHODS: Mono- and co-cultures of human PVFCs and endothelial cells were treated with fetuin-A and/or palmitate for 6-72 h. Proteins were quantified by ELISA and Luminex, mRNA expression by real-time PCR, and cell growth by BrDU-ELISA. Some PVFCs were preincubated with a nuclear factor κB NFκBp65 inhibitor, or the toll-like receptor 4 (TLR4) inhibitor CLI-095, or phosphoinositide 3-kinase (PI3K)/Akt inhibitors and/or stimulated with insulin. Intracellular forkhead box protein O1 (FoxO1), NFκBp65 and inhibitor of κB kinase ß (IKKß) localisation was visualised by immunostaining. RESULTS: PVFCs expressed and secreted IL-6, IL-8, plasminogen activator inhibitor 1 (PAI-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), placental growth factor (PLGF) and hepatocyte growth factor (HGF). Fetuin-A upregulated IL-6 and IL-8, and this was potentiated by palmitate and blocked by CLI-095. Immunostaining and electrophoretic mobility shift assay (EMSA) showed partial NFκBp65 activation. MCP-1 was upregulated and blocked by CLI-095, but not by palmitate. However, HGF was downregulated, which was slightly potentiated by palmitate. This effect persisted after TLR4 pathway blockade. Stimulation of insulin-PI3K-Akt signalling by insulin resulted in nuclear FoxO1 extrusion and HGF upregulation. Fetuin-A counteracted these insulin effects. CONCLUSIONS/INTERPRETATION: Fetuin-A and/or palmitate influence the expression of proinflammatory and angiogenic proteins only partially via TLR4 signalling. HGF downregulation seems to be mediated by interference with the insulin-dependent receptor tyrosine kinase pathway. Fetuin-A may also influence angiogenic and proinflammatory proteins involved in atherosclerosis.