Matthias Laudes

Expression of the thermogenic nuclear hormone receptor coactivator PGC-1α is reduced in the adipose tissue of morbidly obese subjects

Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) is an accessory protein which can potentiate the transcriptional activation function of many nuclear hormone receptors. Its tissue distribution and physiological studies suggest that its principal in vivo roles are to promote cold-induced thermogenesis, mitochondrial biogenesis, hepatic gluconeogenesis, and fatty acid β-oxidation. It is expressed in the white adipose tissue of both humans and rodents, and in rodents it has been suggested to mediate in part the leptin-induced conversion of white adipocytes from fat storing to fat oxidising cells. In this study, quantitative real-time PCR has been used in human tissue to demonstrate that (1) PGC1α mRNA levels in subcutaneous fat are three-fold lower in morbidly obese than in slim subjects; (2) there are no differences in PGC1α mRNA between omental and subcutaneous mature adipocytes; (3) there is a robust induction of PGC1α expression during subcutaneous human preadipocyte differentiation ex vivo. Whether low PGC1α expression is a prelude to the development of obesity, or a consequence of that obesity, attempts to upregulate endogenous white adipose tissue expression may prove a valuable new avenue to explore in obesity therapy.

The Wnt antagonist Dickkopf-1 and its receptors are coordinately regulated during early human adipogenesis

Secretion of Wnts by adipose cells has an important role in the control of murine adipogenesis. We present the first evidence that a Wnt antagonist, Dickkopf 1 (Dkk1), is secreted by human preadipocytes and promotes adipogenesis. DKK1 mRNA increases six hours after onset of human adipogenesis and this is followed by an increase in Dkk1 protein. With further differentiation, the mRNA and protein levels progressively decline such that they are undetectable in mature adipocytes. The transient induction in DKK1 correlates with downregulation of cytoplasmic and nuclear β-catenin levels, this being a surrogate marker of canonical Wnt signalling, and Wnt/β-catenin transcriptional activity. In addition, constitutive expression of Dkk1 in 3T3-L1 preadipocytes promotes their differentiation, further supporting the functional significance of increased Dkk1 levels during human adipogenesis. Concomitant downregulation of the Dkk1 receptors LRP5 and LRP6 is likely to potentiate the ability of Dkk1 to inhibit Wnt signalling and promote differentiation. Notably, Dkk1 is not expressed in primary murine preadipocytes or cell lines. The involvement of Dkk1 in human but not murine adipogenesis indicates that inter-species differences exist in the molecular control of this process. Given the public health importance of disorders of adipose mass, further knowledge of the pathways involved specifically in human adipocyte differentiation might ultimately be of clinical relevance.

Role of WNT signalling in the determination of human mesenchymal stem cells into preadipocytes.

The development of obesity is characterised not only by increased storage of lipids in existing fat cells but also by the generation of new adipocytes from progenitor cells. This process, called adipogenesis, can be divided into two related steps. First, during determination, multipotent mesenchymal stem cells commit to preadipocytes. These cells exhibit similar morphology compared with stem cells; however, they are committed to the adipogenic lineage and are not longer able to transform into osteoblasts, myocytes or chondrocytes. Secondly, during differentiation, preadipocytes become mature fat cells. As in other developmental processes, adipogenesis is tightly regulated at a molecular level by several transcription factors. Within the last decade, it has also become clear how the activity of these transcription factors is coordinated by extracellular signals. In this respect, secreted WNT signalling molecules are particularly important. Several members of the WNT family have been shown to inhibit early steps of adipogenesis. Conversely, endogenous inhibitors of WNT signalling were found to promote generation of adipocytes, indicating a fundamental role of these bioactive peptides in adipogenesis. From a pathophysiological point of view, it is of interest that polymorphisms in genes of the WNT signalling system have been associated with the development of obesity and type 2 diabetes in humans. Moreover, recent findings indicate that certain WNT molecules are involved in the so-called low-grade inflammation of adipose tissue, which is crucial in the development of obesity-associated insulin resistance. These important findings in nutritional and metabolic medicine will be summarised in the present review.

Role of Wnt-5a in the Determination of Human Mesenchymal Stem Cells into Preadipocytes

Increasing adipocyte size as well as numbers is important in the development of obesity and type 2 diabetes, with adipocytes being generated from mesenchymal precursor cells. This process includes the determination of mesenchymal stem cells (MSC) into preadipocytes (PA) and the differentiation of PA into mature fat cells. Although the process of differentiation has been highly investigated, the determination in humans is poorly understood. In this study, we compared human MSC and human committed PA on a cellular and molecular level to gain further insights into the regulatory mechanisms in the determination process. Both cell types showed similar morphology and expression patterns of common mesenchymal and hematopoietic surface markers. However, although MSC were able to differentiate into adipocytes and osteocytes, PA were only able to undergo adipogenesis, indicating that PA lost their multipotency during determination. WNT-5a expression showed significantly higher levels in MSC compared with PA suggesting that WNT-5a down-regulation might be important in the determination process. Indeed, incubation of human MSC in medium containing neutralizing WNT-5a antibodies abolished their ability to undergo osteogenesis, although adipogenesis was still possible. An opposite effect was achieved using recombinant WNT-5a protein. On a molecular level, WNT-5a was found to promote c-Jun N-terminal kinase-dependent intracellular signaling in MSC. Activation of this noncanonical pathway resulted in the induction of osteopontin expression further indicating pro-osteogenic effects of WNT-5a. Our data suggest that WNT-5a is necessary to maintain osteogenic potential of MSC and that inhibition of WNT-5a signaling therefore plays a role in their determination into PA in humans.

Transcript and metabolite analysis of the effects of tamoxifen in rat liver reveals inhibition of fatty acid synthesis in the presence of hepatic steatosis

Nonalcoholic steatohepatitis (NASH) is a common feature of the metabolic syndrome and toxic reactions to pharmacological drugs. Tamoxifen, (TMX) a widely used anti‐breast cancer drug, can induce NASH and changes in plasma cholesterol levels through mechanisms that are unclear. We studied primary actions of TMX using a short‐term treatment (5 days) that induces microvesicular hepatic steatosis and marked hypercholesterolemia in male rats. Using a combined approach of gene expression profiling and NMR‐based metabolite analysis, we found that TMX‐treated livers have increased saturated fatty acid content despite changes in gene expression, indicating decreased de novo lipogenesis and increased fatty acid oxidation. Our results show that TMX predominantly down‐regulates FAS expression and activity as indicated by the accumulation of malonyl‐CoA, a known inhibitor of mitochondrial β‐oxidation. In the face of a continued supply of exogenous free fatty acids, the blockade of fatty acid oxidation produced by elevated malonyl‐CoA is likely to be the major factor leading to steatosis. Use of a combination of metabolomic and transcriptomic analysis has allowed us to identify mechanisms underlying important metabolic side effects of a widely prescribed drug. Given the broader importance of hepatic steatosis, the novel molecular mechanism revealed in this study should be examined in other forms of steatosis and nonalcoholic steatohepatitis. Lelliott C. J., López M., Curtis R. K., Parker N., Laudes M., Yeo G., Jimenez‐Linan M., Grosse J., Saha A. K., Wiggins D., Hauton D., Brand M. D. ORahilly S., Griffin J. L., Gibbons G. F., Vidal‐Puig A. Transcript and metabolite analysis of the effects of tamoxifen in rat liver reveals inhibition of fatty acid synthesis in the presence of hepatic steatosis. FASEB J. 19, 1–12 (2005)

ETO/MTG8 Is an Inhibitor of C/EBPβ Activity and a Regulator of Early Adipogenesis

ABSTRACT The putative transcriptional corepressor ETO/MTG8 has been extensively studied due to its involvement in a chromosomal translocation causing the t(8;21) form of acute myeloid leukemia. Despite this, the role of ETO in normal physiology has remained obscure. Here we show that ETO is highly expressed in preadipocytes and acts as an inhibitor of C/EBPβ during early adipogenesis, contributing to its characteristically delayed activation. ETO prevents both the transcriptional activation of the C/EBPα promoter by C/EBPβ and its concurrent accumulation in centromeric sites during early adipogenesis. ETO expression rapidly reduces after the initiation of adipogenesis, and this is essential to the normal induction of adipogenic gene expression. These findings define, for the first time, a molecular role for ETO in normal physiology as an inhibitor of C/EBPβ and a novel regulator of early adipogenesis.

Adipose tissue macrophages inhibit adipogenesis of mesenchymal precursor cells via wnt-5a in humans

In patients with obesity and type 2 diabetes, adipose tissue is infiltrated by macrophages known to alter adipogenesis of mesenchymal precursor cells via secretion of proinflammatory cytokines. Recently, it has been shown that under certain conditions, immune cells can also express wnt-5a, a factor known to inhibit adipogenesis in humans. Therefore, in this study we aimed to investigate whether macrophages affect adipogenesis of mesenchymal precursor cells via wnt-5a. Wnt-5a was found to be expressed in adipose tissue macrophages in obese and type 2 diabetic human subjects in vivo by immunohistochemistry of adipose tissue biopsies. Furthermore, wnt-5a was detectable in circulating CD14+ blood monocytes of human subjects with obesity and type 2 diabetes on RNA level by real-time PCR. Besides expression analysis in vivo, we also performed functional studies to explore the role of wnt-5a in low-grade inflammation of adipose tissue. In a cell culture experiment, macrophage-conditioned differentiation medium inhibited adipogenesis of 3T3-L1 cells. This inhibitory effect was restored by adding neutralising anti-wnt-5a antibodies. In conclusion, our data indicate that macrophages alter adipogenesis of 3T3-L1 cells not only via classical proinflammatory cytokines, but also via wnt signalling molecules.

Secreted frizzled-related protein 1 regulates adipose tissue expansion and is dysregulated in severe obesity.

Aim:The Wnt/β-catenin signaling network offers potential targets to diagnose and uncouple obesity from its metabolic complications. In this study, we investigate the role of the Wnt antagonist, secreted frizzled-related protein 1 (SFRP1), in promoting adipogenesis in vitro and adipose tissue expansion in vivo.Methods:We use a combination of human and murine, in vivo and in vitro models of adipogenesis, adipose tissue expansion and obesity-related metabolic syndrome to profile the involvement of SFRP1.Results:SFRP1 is expressed in both murine and human mature adipocytes. The expression of SFRP1 is induced during in vitro adipogenesis, and SFRP1 is preferentially expressed in mature adipocytes in human adipose tissue. Constitutive ectopic expression of SFRP1 is proadipogenic and inhibits the Wnt/β-catenin signaling pathway. In vivo endogenous levels of adipose SFRP1 are regulated in line with proadipogenic states. However, in longitudinal studies of high-fat-diet-fed mice, we observed a dynamic temporal but biphasic regulation of endogenous SFRP1. In agreement with this profile, we observed that SFRP1 expression in human tissues peaks in patients with mild obesity and gradually falls in morbidly obese subjects.Conclusions:Our results suggest that SFRP1 is an endogenous modulator of Wnt/β-catenin signaling and participates in the paracrine regulation of human adipogenesis. The reduced adipose expression of SFRP1 in morbid obesity and its knock-on effect to prevent further adipose tissue expansion may contribute to the development of metabolic complications in these individuals.

Identification of a region in the human IRS2 promoter essential for stress induced transcription depending on SP1, NFI binding and ERK activation in HepG2 cells

Recent studies have discovered changes in the insulin-/IGF1 signaling affecting glucose metabolism and the molecular pathogenesis of human hepatocellular cancer. Insulin/IGF1 receptor mediates its intracellular effects by recruitment of one out of the four different insulin receptor substrates (IRS). To investigate mechanisms of IRS2 expression, we analyzed transcriptional regulation of IRS2 in human HepG2 cells. We identified a region 688 bp upstream of the translation start codon responsible for approximately 90% of basal human IRS2 promoter activity in HepG2 cells, and confirmed binding of specificity protein 1 (also called Sp1 transcription factor, SP1) and nuclear factor 1 (NFI) in this region. Mutation of both SP1 and NFI binding sites or inhibition of extracellular signal regulated kinase (ERK) suppressed IRS2 promoter activity almost completely, revealing a major role of MAP kinases (MAPK) for IRS2 transcription. Activating this cascade with oxidative stress increased IRS2 promoter activity and endogenous IRS2 expression substantially. IRS2 promoter activity rose even more after additional inhibition of p38MAPK indicating an inhibitory effect of p38MAPK on ERK mediated IRS2 transcription. Activation of the MAPK pathway using interleukin 1, beta (IL1B) increased IRS2 promoter activity similar to oxidative stress. In contrast IL1B decreases and inhibition of the MAPK pathway increases IRS1 promoter activity revealing opposed effects of IL1B and ERK on the expression of different IRS proteins. In conclusion we discovered a specific region (-688 to -611 bp) in the IRS2 promoter essential for basal promoter activity and oxidative stress induced transcription depending on ERK activation and SP1 and NFI binding in human hepatocytes.

Hypoadiponectinemia in Extremely Low Gestational Age Newborns with Severe Hyperglycemia - A Matched- Paired Analysis

Background Hyperglycemia is commonly observed in extremely low gestational age newborns (ELGANs) and is associated with both increased morbidity and mortality. The objective of this study was to examine the relationship between neonatal hyperglycemia and adiponectin levels in ELGANs. Methodology/Principal Findings Ten preterm infants between 22+6/7 and 27+3/7 weeks’ gestation with neonatal hyperglycemia (defined as pre-feeding blood glucose levels above 200mg/dl on two consecutive measurements with a maximum parenteral glucose infusion of 4mg/kg*min−1) formed the case cohort of this study. To every single patient of this case cohort a patient with normal fasting (u200a=u200apre-feeding) blood glucose levels was matched in terms of gestational age and gender. Adiponectin ELISAs were performed both at onset of hyperglycemia and at term-equivalent age. In the case cohort 9/10 patients had to be treated with insulin for 1–26 days (range 0.01–0.4 IU/kg*h−1). Compared to matched-paired controls, significant hypoadiponectinemia was observed at onset of hyperglycemia in these affected patients (6.9µg/ml versus 15.1µg/ml, pu200a=u200a0.009). At term equivalent age, normoglycemia without any insulin treatment was found in both groups. Moreover, adiponectin levels at that time were no longer significantly different (12.3µg/ml versus 20.0µg/ml; pu200a=u200a0.051) possibly indicating a mechanistic relevance of this adipokine in regulating insulin sensitivity in ELGANs. Conclusions/Significance Decreased circulating adiponectin levels are correlated with hyperglycemia in ELGANs and may contribute to the pathogenesis of impaired glucose homeostasis in these infants. These findings suggest that adiponectin might be a potential future drug target for the potentially save treatment of hyperglycemia in pre-term infants.