Eicosapentaenoic acid and arachidonic acid differentially regulate adipogenesis, acquisition of a brite phenotype and mitochondrial function in primary human adipocytes.

SCOPE: n-3 and n-6 PUFAs have several opposing biological effects and influence white adipose tissue (WAT) function. The recent discovery of thermogenic UCP1-expressing brite adipocytes within WAT raised the question whether n-3 and n-6 PUFAs exert differential effects on brite adipocyte formation and mitochondrial function. METHODS AND RESULTS: Primary human preadipocytes were treated with n-3 PUFAs (eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) or n-6 PUFA (arachidonic acid, ARA) during differentiation, and adipogenesis, white and brite gene expression markers, mitochondrial content and function were analyzed at day 12 of differentiation. Adipogenesis was equally increased by n-3 and n-6 PUFAs. The n-6 PUFA ARA increased lipid droplet size and expression of the white-specific marker TCF21 while decreased mitochondrial protein expression and respiratory function. In contrast, EPA increased expression of the brown adipocyte-related genes UCP1 and CPT1B, and improved mitochondrial function of adipocytes. The opposing effects of EPA and ARA on gene expression and mitochondrial function were also observed in cells treated from day 8 to 12 of adipocyte differentiation. CONCLUSION: EPA promotes brite adipogenesis and improves parameters of mitochondrial function, such as increased expression of CPTB1, citrate synthase activity and higher maximal respiratory capacity, while ARA reduced mitochondrial spare respiratory capacity in vitro.

Nutritional ingredients modulate adipokine secretion and inflammation in human primary adipocytes.

Nutritional factors such as casein hydrolysates and long chain polyunsaturated fatty acids have been proposed to exert beneficial metabolic effects. We aimed to investigate how a casein hydrolysate (eCH) and long chain polyunsaturated fatty acids could affect human primary adipocyte function in vitro. Incubation conditions with the different nutritional factors were validated by assessing cell vitality with lactate dehydrogenase (LDH) release and neutral red incorporation. Intracellular triglyceride content was assessed with Oil Red O staining. The effect of eCH, a non-peptidic amino acid mixture (AA), and long-chain polyunsaturated fatty acids (LC-PUFAs) on adiponectin and leptin secretion was determined by enzyme-linked immunosorbent assay (ELISA). Intracellular adiponectin expression and nuclear factor-κB (NF-κB) activation were analyzed by Western blot, while monocyte chemoattractant protein-1 (MCP-1) release was explored by ELISA. The eCH concentration dependently increased adiponectin secretion in human primary adipocytes through its intrinsic peptide bioactivity, since the non-peptidic mixture, AA, could not mimic eCH's effects on adiponectin secretion. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and DHA combined with arachidonic acid (ARA) upregulated adiponectin secretion. However, only DHA and DHA/ARA exerted a potentanti-inflammatory effect reflected by prevention of tumor necrosis factor-α (TNF-α) induced NF-κB activation and MCP-1 secretion in human adipocytes. eCH and DHA alone or in combination with ARA, may hold the key for nutritional programming through their anti-inflammatory action to prevent diseases with low-grade chronic inflammation such as obesity or diabetes.

Browning of white fat: does irisin play a role in humans?

The discovery of irisin as an exercise-regulated myokine inducing browning of WAT has gained interest as a potential new strategy to combat obesity and its associated disorders, such as type 2 diabetes. However, there are inconsistencies regarding the relevance of irisin in humans. The regulation of FNDC5 mRNA expression by exercise and contraction could not be reproduced by a number of human studies using several exercise protocols and in vitro approaches. Furthermore, the nature of FNDC5 fragments and the presence of irisin in humans are questionable and probably contribute to conflicting data obtained with commercially available ELISA kits. Most importantly, the information regarding the concentration of circulating irisin in humans is not clear, as different studies using different kits measure irisin levels in a wide range. Data about the role of irisin in states of human obesity and metabolic diseases are conflicting and, in some cases, changes in irisin levels have been observed; they were only moderate in 10-20%. Independent of the presence and regulation of FNDC5/irisin in humans, the application of recombinant irisin could still represent a therapeutic strategy to fight obesity. However, the current data obtained from human cell models reveal that FNDC5/irisin has no effect on browning of the major WAT depots in humans and is likely to selectively target a small subpopulation of adipocytes, which are located in classical BAT regions, such as the supraclavicular adipose tissue. Thus, other candidates, such as BMP7 or CNPs, seem to be more prominent candidates as inducers of browning in humans.

Lactate regulates myogenesis in C2C12 myoblasts in vitro.

Satellite cells (SCs) are the resident stem cells of skeletal muscle tissue which play a major role in muscle adaptation, e.g. as a response to physical training. The aim of this study was to examine the effects of an intermittent lactate (La) treatment on the proliferation and differentiation of C2C12 myoblasts, simulating a microcycle of high intensity endurance training. Furthermore, the involvement of reactive oxygen species (ROS) in this context was examined. C2C12 myoblasts were therefore repeatedly incubated for 2 h each day with 10 mM or 20 mM La differentiation medium (DM) and in some cases 20 mM La DM plus different antioxidative substances for up to 5 days. La free (0 mM) DM served as a control. Immunocytochemical staining, Western blot analysis and colorimetric assays were used to assess oxidative stress, proliferation, and differentiation. Results show that La induces oxidative stress, enhances cell-cycle withdrawal, and initiates early differentiation but delays late differentiation in a timely and dose-dependent manner. These effects can be reversed by the addition of antioxidants to the La DM. We therefore conclude that La has a regulatory role in C2C12 myogenesis via a ROS-sensitive mechanism which elicits implications for reassessing some aspects of training and the use of nutritional supplements.

Chitinase-3-like protein 1 protects skeletal muscle from TNFα-induced inflammation and insulin resistance.

CHI3L1 (chitinase-3-like protein 1) is a glycoprotein consisting of 383 amino acids with a molecular mass of 40 kDa, and its serum level is elevated in inflammatory diseases. Although CHI3L1 is described as a biomarker of inflammation, the function of this protein is not completely understood. In the present study, we examined the regulation of CHI3L1 in primary human skeletal muscle cells. Moreover, we analysed potential autocrine effects of CHI3L1. We show that myotubes express CHI3L1 in a differentiation-dependent manner. Furthermore, pro-inflammatory cytokines up-regulate CHI3L1 expression (6-fold) and release (3-fold). Importantly, CHI3L1 treatment blocked TNFα (tumour necrosis factor α)-induced inflammation by inhibiting NF-κB (nuclear factor κB) activation in skeletal muscle cells. We show that this effect is mediated via PAR2 (protease-activated receptor 2). In addition, CHI3L1 treatment diminished the TNFα-induced expression and secretion of IL (interleukin)-8, MCP1 (monocyte chemoattractant protein 1) and IL-6. In addition, impaired insulin action at the level of Akt and GSK3α/ß (glycogen synthase kinase 3α/ß) phosphoryl-ation and insulin-stimulated glucose uptake was normalized by CHI3L1. In conclusion, the novel myokine CHI3L1, which is induced by pro-inflammatory cytokines, can counteract TNFα-mediated inflammation and insulin resistance in human skeletal muscle cells, potentially involving an auto- and/or para-crine mechanism.

BMP4 and BMP7 induce the white-to-brown transition of primary human adipose stem cells.

While white adipose tissue (AT) is an energy storage depot, brown AT is specialized in energy dissipation. Uncoupling protein 1 (UCP1)-expressing adipocytes with a different origin than classical brown adipocytes have been found in white AT. These "brite" (brown-in-white) adipocytes may represent a therapeutic target to counteract obesity. Bone morphogenetic proteins (BMPs) play a role in the regulation of adipogenesis. Based on studies with murine cells, BMP4 is assumed to induce stem cell commitment to the white adipocyte lineage, whereas BMP7 promotes brown adipogenesis. There is evidence for discrepancies between mouse and human AT. Therefore, we compared the effect of BMP4 and BMP7 on white-to-brown transition in primary human adipose stem cells (hASCs) from subcutaneous AT. Long-term exposure of hASCs to recombinant BMP4 or BMP7 during differentiation increased adipogenesis, as determined by lipid accumulation and peroxisome proliferator-activated receptor-γ (PPARγ) expression. Not only BMP7, but also BMP4, increased UCP1 expression in hASCs and decreased expression of the white-specific marker TCF21. The ability of hASCs to induce UCP1 in response to BMP4 and BMP7 markedly differed between donors and could be related to the expression of the brite marker CD137. However, mitochondrial content and oxygen consumption were not increased in hASCs challenged with BMP4 and BMP7. In conclusion, we showed for the first time that BMP4 has similar effects on white-to-brown transition as BMP7 in our human cell model. Thus the roles of BMP4 and BMP7 in adipogenesis cannot always be extrapolated from murine to human cell models.

Evidence against a beneficial effect of irisin in humans.

Brown adipose tissue has gained interest as a potential target to treat obesity and metabolic diseases. Irisin is a newly identified hormone secreted from skeletal muscle enhancing browning of white fat cells, which improves systemic metabolism by increasing energy expenditure in mice. The discovery of irisin raised expectations of its therapeutic potential to treat metabolic diseases. However, the effect of irisin in humans is unclear. Analyses of genomic DNA, mRNA and expressed sequence tags revealed that FNDC5, the gene encoding the precursor of irisin, is present in rodents and most primates, but shows in humans a mutation in the conserved start codon ATG to ATA. HEK293 cells transfected with a human FNDC5 construct with ATA as start codon resulted in only 1% full-length protein compared to human FNDC5 with ATG. Additionally, in vitro contraction of primary human myotubes by electrical pulse stimulation induced a significant increase in PGC1α mRNA expression. However, FNDC5 mRNA level was not altered. FNDC5 mRNA expression in muscle biopsies from two different human exercise studies was not changed by endurance or strength training. Preadipocytes isolated from human subcutaneous adipose tissue exhibited differentiation to brite human adipocytes when incubated with bone morphogenetic protein (BMP) 7, but neither recombinant FNDC5 nor irisin were effective. In conclusion, our findings suggest that it is rather unlikely that the beneficial effect of irisin observed in mice can be translated to humans.

Functional annotation of the human fat cell secretome.

CONTEXT: Recent secretome analyses suggest that human fat cells secrete hundreds of proteins (adipokines). OBJECTIVE: We made an overall analysis of their potential functional importance. MATERIALS AND METHODS: A secretome of 347 adipokines was evaluated by in silico analysis of their expression during adipocyte differentiation, regulation by obesity and adipose region. The gene expression in human adipose tissue was investigated in microarray studies using samples from different adipose depots from lean or obese patients. RESULTS: 60% of the adipokines were regulated by obesity and 50% between visceral and subcutaneous adipose region. Eight adipokines, all novel, scored particularly high in the in silico analysis. Among those, four were both regulated by obesity and adipose region, namely WNT1-inducible-signaling pathway protein 2, transmembrane glycoprotein NMB, inter-alpha-trypsin inhibitor heavy chain H5, and complement C4-A. Furthermore, many adipokines were extracellular matrix proteins. CONCLUSION: Several novel adipokines have potential important functional features warranting in depth analysis.