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Natural bone is a complex composite, consisting predominantly of collagen and hydroxyapatite (HA), which form a highly organized, hierarchical structure from the nano- to the macroscale. Because of its biphasic, anisotropic, ultrafine structural design, bone tissue possesses excellent mechanical properties. Herein, inspired by the composition and microstructure of natural bone, a biphasic composite consisting of highly aligned strontium/copper-doped one-dimensional hydroxyapatite (Sr/Cu-doped 1D HA) and poly(d,l-lactide) (PDLA) was developed. The presence and alignment of Sr/Cu-doped 1D HA crystals resulted in mechanical reinforcement of the polymer matrix, including compressive and tensile strength and modulus, fracture toughness, swelling resistance, and long-term structural stability. The compressive strength, tensile strength, and Young's modulus of the biomimetic composite were comparable to that of cortical bone. Biologically, the biomimetic composite showed a sustained release of the incorporated Sr and Cu ions, facilitated mineral deposition from simulated body fluid, and supported attachment, proliferation, and alkaline phosphatase activity of human mesenchymal stromal cells (hMSCs). Moreover, the highly aligned Sr/Cu-doped 1D HA crystals in the 3D porous scaffolds induced the alignment of hMSCs and secretion of an anisotropic collagen fiber matrix in 3D. The biomimetic Sr/Cu-doped 1D HA/PDLA composite presented here contributes to the current efforts aiming at the design and development of load-bearing bioactive synthetic bone graft substitutes. Moreover, the biomimetic composite may serve as a 3D platform for studying cell-extracellular matrix interactions in bone tissue.

Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, various dynamic hydrogel architectures are created, conferring a controlled and biomimetic environment for organoid encapsulation. How hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers are investigated. The authors observe that stiff hydrogel encapsulation leads to an absence of certain renal cell types and signs of an epithelial-mesenchymal transition (EMT), whereas encapsulation in soft, stress-relaxing hydrogels leads to all major renal segments, fewer fibrosis or EMT associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. The findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure-property-function relationships can enable the rational design of materials, bringing us closer to functional engraftments and disease-modeling applications.

Surface topography is a tool to endow biomaterials with bioactive properties. However, the large number of possible designs makes it challenging to find the optimal surface structure to induce a specific cell response. The TopoChip platform is currently the largest collection of topographies with 2176 in silico designed microtopographies. Still, it is exploring only a small part of the design space due to design algorithm limitations and the surface engineering strategy. Inspired by the diversity of natural surfaces, it is assessed as to what extent the topographical design space and consequently the resulting cellular responses can be expanded using natural surfaces. To this end, 26 plant and insect surfaces are replicated in polystyrene and their surface properties are quantified using white light interferometry. Through machine-learning algorithms, it is demonstrated that natural surfaces extend the design space of the TopoChip, which coincides with distinct morphological and focal adhesion profiles in mesenchymal stem cells (MSCs) and Pseudomonas aeruginosa colonization. Furthermore, differentiation experiments reveal the strong potential of the holy lotus to improve osteogenesis in MSCs. In the future, the design algorithms will be trained with the results obtained by natural surface imprint experiments to explore the bioactive properties of novel surface topographies.

Context Long-term weight loss (WL) maintenance is the biggest challenge for overweight and obesity because of the almost unavoidable phenomenon of partial or even total weight regain (WR) after WL. Objective In the present study we investigated the relations of (the changes of) adipocyte size and other risk biomarkers with WR during the follow-up of the Yoyo dietary intervention. Methods In this randomized controlled study, 48 overweight/obese participants underwent a very-low-calorie diet to lose weight, followed by a weight-stable period of 4 weeks and a follow-up period of 9 months. Anthropometric measurements, adipocyte volume of abdominal subcutaneous adipose tissue, and plasma metabolic parameters (free fatty acids [FFAs], triglycerides [TGs], total cholesterol, glucose, insulin, homeostasis model assessment of insulin resistance [HOMA-IR], interleukin 6 [IL-6], angiotensin-converting enzyme [ACE] activity, retinol binding protein 4 [RBP4]) at the beginning and the end of follow-up were analyzed. Results Our results show that changes of TGs, IL-6, HOMA-IR, and ACE are significantly positively correlated with WR. Multiple linear regression analysis shows that only TG and IL-6 changes remained significantly correlated with WR and increased body fat mass. Moreover, the change in HOMA-IR was tightly correlated with the change in TGs. Surprisingly, change in adipocyte volume during follow-up was not correlated with WR nor with other factors, but positive correlations between adipocyte volume and HOMA-IR were found at the beginning and end of the follow-up. Conclusion These results suggest that TGs and IL-6 are independently linked to WR via separate mechanisms, and that HOMA-IR and adipocyte volume may indirectly link to WR through the change of plasma TGs.

Based on several randomized clinical trials, it has been suggested that baseline glucose homeostasis interacts with the influence of diet composition on weight loss and weight loss maintenance. In this secondary analysis of the YoYo study, a study investigating predictors of weight loss maintenance, we tested the hypothesis that (self-selected) dietary carbohydrate and/or fibre intake interact with the glucose homeostasis parameters for weight loss maintenance. Sixty-one overweight or obese individuals lost around 10 kg of body weight on an energy-restricted diet and were then followed for 9 months. During this period, participants were advised to maintain their body weight and eat a healthy diet without further recommendations on calorie intake or diet composition. Contrary to our hypothesis, carbohydrate intake showed no positive association with weight regain after weight loss, and no interaction with baseline fasting glucose concentration was found. There was a non-significant negative association between fibre intake and weight regain (B = −0.274, standard error (SE) 0.158, p = 0.090), but again, no interaction with fasting plasma glucose was found. In conclusion, the data from the YoYo study do not support a role for baseline glucose homeostasis in determining the association between self-reported carbohydrate and/or fibre intake and weight regain after weight loss.

Long-term weight loss maintenance is a problem of overweight and obesity. Changes of gene expression during weight loss (WL) by calorie restriction (CR) are linked to the risk of weight regain (WR). However, detailed information on genes/proteins involved in the mechanism is still lacking. Therefore, we developed an in-vitro model system for glucose restriction (GR) and refeeding (RF) to uncover proteome differences between GR with RF vs normal feeding, of which we explored the relation with WR after WL. Human Simpson-Golabi-Behmel Syndrome cells were subjected to changing levels of glucose to mimic the condition of CR and RF. Proteome profiling was performed by liquid chromatography tandem mass spectrometry. This in-vitro model revealed 44 proteins differentially expressed after GR and RF versus feeding including proteins of the focal adhesions. Four proteins showed a persistent up- or down-regulation: liver carboxylesterase (CES1), mitochondrial superoxide dismutase [Mn] (SOD2), alpha-crystallin B-chain (CRYAB), alpha-enolase (ENO1). In-vivo weight loss-induced RNA expression changes linked CES1, CRYAB and ENO1 to WR. Moreover, of these 44 proteins, CES1 and glucosidase II alpha subunit (GANAB) during follow up correlated with WR. Correlation clustering of in-vivo protein expression data indicated an interaction of these proteins with structural components of the focal adhesions and cytoplasmic filaments in the adipocytes.

Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. Statement of Significance: The challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering.

We previously found that surface topographies induce the expression of the Scxa gene, encoding Scleraxis in tenocytes. Because Scxa is a TGF-beta responsive gene, we investigated the link between mechanotransduction and TGF-beta signaling. We discovered that mesenchymal stem cells exposed to both micro-topographies and TGF-beta 2 display synergistic induction of SMAD phosphorylation and transcription of the TGF-beta target genes SCX, alpha-SMA, and SOX9. Pharmacological perturbations revealed that Rho/ROCK/SRF signaling is required for this synergistic response. We further found an activation of the early response genes SRF and EGR1 during the early adaptation phase on micro-topographies, which coincided with higher expression of the TGF-beta type II receptor gene. Of interest, PKC activators Prostratin and Ingenol-3, known for inducing actin reorganization and activation of serum response elements, were able to mimic the topography-induced TGF-beta response. These findings provide novel insights into the convergence of mechanobiology and TGF-beta signaling, which can lead to improved culture protocols and therapeutic applications.

Surface topography guides cell behavior and is a tool to endow biomaterials with bioactive properties. The large number of possible designs makes it challenging to find the optimal surface structure to induce a specific cell response. The TopoChip platform is currently the largest collection of topographies with 2176 in silico designed micro-topographies. Still, it is exploring only a small part of the design space due to the boundary conditions of the design algorithm and the surface engineering strategy. Inspired by the diversity of natural surfaces, we assessed to what extend we could expand the topographical design space and consequently the resulting cellular responses using natural surfaces. To this end, we replicated twenty-six plant and insect surfaces in polystyrene and quantified their surface properties using white light interferometry, image analysis and principle component analysis. Next, we quantified mesenchymal stem cell morphology and the pattern of Pseudomonas aeruginosa colonization and compared it to previous data from TopoChip screens. Our data show that natural surfaces extended the TopoChip design space. Moreover, the natural surfaces induced MSC morphologies and bacterial attachment patterns not previously observed on the TopoChip. In the future, we will train our design algorithms with the results obtained by natural surface imprint experiments to further explore the design space and bio-active properties of surface topography.

Learning rules by which cell shape impacts cell function would enable control of cell physiology and fate in medical applications, particularly, on the interface of cells and material of the implants. We defined the phenotypic response of human bone marrow-derived mesenchymal stem cells (hMSCs) to 2176 randomly generated surface topographies by probing basic functions such as migration, proliferation, protein synthesis, apoptosis, and differentiation using quantitative image analysis. Clustering the surfaces into 28 archetypical cell shapes, we found a very strict correlation between cell shape and physiological response and selected seven cell shapes to describe the molecular mechanism leading to phenotypic diversity. Transcriptomics analysis revealed a tight link between cell shape, molecular signatures, and phenotype. For instance, proliferation is strongly reduced in cells with limited spreading, resulting in down-regulation of genes involved in the G2/M cycle and subsequent quiescence, whereas cells with large filopodia are related to activation of early response genes and inhibition of the osteogenic process. In this paper we were aiming to identify a universal set of genes that regulate the material-induced phenotypical response of human mesenchymal stem cells. This will allow designing implants that can actively regulate cellular, molecular signalling through cell shape. Here we are proposing an approach to tackle this question.

Fibroblast growth factor 21 (FGF21) is an important regulator of energy metabolism. FGF21 is inactivated by fibroblast activation protein (FAP). We investigated whether FGF21 and/or FAP are secreted from human white adipose tissue of individuals with obesity by measuring total FGF21, active FGF21, and FAP concentrations in arterialized blood and venous blood draining the subcutaneous abdominal adipose tissue (scAT). Measurements were performed under fasting conditions and after a high fat meal before and after diet-induced weight loss in 16 adults with BMI 27–35 kg/m2. FGF21 was not released from scAT, neither before nor after weight loss in agreement with an undetectable gene expression of FGF21 in this tissue. Although scAT showed significant gene expression of FAP, no release of FAP from the tissue could be detected. The high fat meal increased postprandial circulating FGF21 but not FAP. Circulating levels of FAP but not FGF21 were significantly reduced after weight loss. On the other hand, FAP expression in scAT was increased. In conclusion, release from scAT does not appear to contribute to circulating concentrations of FGF21 and FAP and their responses to ingestion of a high fat meal or weight loss, respectively, in individuals with obesity.

We previously found that surface topographies induce the expression of theScxagene, encoding Scleraxis in tenocytes. BecauseScxais a TGF-β responsive gene, we investigated the link between mechanotransduction and TGF-β signaling. We discovered that mesenchymal stem cells exposed to both micro-topographies and TGF-β2 display synergistic induction of SMAD phosphorylation and transcription of the TGF-β target genesSCXA,a-SMA, andSOX9. Pharmacological perturbations revealed that Rho/ROCK/SRF signaling is required for this synergistic response. We further found an activation of the early response genesSRFandEGR1during the early adaptation phase on micro-topographies, which coincided with higher expression of the TGF-β type-II receptor gene. Of interest, PKC activators Prostratin and Ingenol-3, known for inducing actin reorganization and activation of serum response elements, were able to mimic the topography-induced TGF-β response. These findings provide novel insights into the convergence of mechanobiology and TGF-β signaling, which can lead to improved culture protocols and therapeutic applications.

Objective During weight loss, the volume of adipocytes decreases, leading to stress because of the misfit between the cell contents and the surrounding extracellular matrix (ECM). This stress can be resolved by remodeling the ECM or the restorage of triglycerides within the adipocytes. The objective of this study was to investigate the existence of a connection between stress-related and ECM-related genes that is associated with weight regain. Methods Thirty-one participants with overweight or obesity followed a 5-week very-low-calorie diet (500 kcal/d) with a subsequent 4-week weight-stable diet (WS), and then an uncontrolled 9-month follow-up. Adipose tissue biopsies were collected for microarray analysis. A correlation and interaction analysis was performed with the weight regain percentage (WR%) ([weight after follow-up - weight after WS] ÷ weight after WS × 100%) by using two gene sets that were previously defined as "stress-related" (n = 107) and "ECM-related" genes (n = 277). Results During WS, a coexpression network of 8 stress-related genes and 15 ECM-related genes correlating with WR% could be constructed, with links to multiple biological processes. Interaction analysis between stress- and ECM-related genes revealed that several gene combinations were highly related to weight regain. Conclusions Our findings underscore the importance of the connection between stress- and ECM-related genes in the risk for weight regain.

Objective This study investigated whether diet-induced weight loss alters indices of in vivo postprandial fat uptake in adipose tissue (AT) and whether these changes are associated with weight regain in adults with overweight and obesity. Methods In this randomized controlled trial, 16 (6 male) individuals (BMI: 28-35 kg/m2) were randomized to either a low-calorie diet (1,250 kcal/d) for 12 weeks or a very-low-calorie diet (500 kcal/d) for 5 weeks (weight loss [WL] period) followed by a 4-week weight-stable (WS) period (together, the dietary intervention [DI] period) and a 9-month follow-up period. Arteriovenous difference measurements combined with stable isotope labeling ([U-13C] palmitate) of a mixed meal were used to determine postprandial fatty acid uptake in AT. Results Body weight was significantly reduced during the WL period (−8.2 ± 0.6 kg, P

Background: Weight loss (WL) is often followed by weight regain after an energy-restricted dietary intervention (DI). When people are following a diet, the volume of an adipocyte decreases by loss of triglycerides, which creates stress between the cell contents and the surrounding extracellular matrix (ECM). Previously, we observed that genetic variations in ECM genes are associated with an increased risk of weight regain. Objective: We investigated the relation between the expression of ECM genes during WL and a period of weight stabilization (WS) and the risk of weight regain. Design: In this randomized controlled trial, 61 healthy overweight or obese participants followed either a 5-wk very-low-calorie diet (VLCD; 500 kcal/d) or a 12-wk low-calorie diet (1250 kcal/d) (WL period) with a subsequent 4-wk WS period and a 9-mo follow-up. The WL and WS periods combined were considered the DI. Abdominal subcutaneous adipose tissue biopsy samples were collected for microarray analysis. Gene expression changes for a broad set of ECM-related genes were correlated with the weight-regain percentage (WR%). Results: A total of 26 of the 277 genes were significantly correlated with WR% during WL, WS, or the DI periods. Most correlations were observed in the VLCD group during the WS period. Four genes code for leukocyte-specific receptors. These and other genes belong to a group of 26 genes, among which the expression changes were highly correlated (r ≥ 0.7, P ≤ 0.001). This group could be divided into 3 subclusters linking to 2 biological processes-leukocyte integrin gene activity and ECM remodeling-and a link to insulin sensitivity was also apparent. Conclusions: Our present findings indicate the importance of adipose tissue leukocytes for the risk of weight regain. ECM modification also seems to be involved, and we observed a link to insulin sensitivity. This trial was registered at clinicaltrials.gov as NCT01559415.

BACKGROUND/OBJECTIVES: Although adipose tissue (AT) hypoxia is present in rodent models of obesity, evidence for this in humans is limited. Here, we investigated the effects of diet-induced weight loss (WL) on abdominal subcutaneous AT oxygen tension (pO(2)), AT blood flow (ATBF), AT capillary density, AT morphology and transcriptome, systemic inflammatory markers and insulin sensitivity in humans.SUBJECTS/METHODS: Fifteen overweight and obese individuals underwent a dietary intervention (DI), consisting of a 5-week verylow-calorie diet (VLCD, 500 kcal day(-1); WL), and a subsequent 4-week weight stable diet (WS). Body composition, AT pO(2) (optochemical monitoring), ATBF (Xe-133 washout), and whole-body insulin sensitivity were determined, and AT biopsies were collected at baseline, end of WL (week 5) and end of WS (week 9).RESULTS: Body weight, body fat percentage and adipocyte size decreased significantly during the DI period. The DI markedly decreased AT pO2 and improved insulin sensitivity, but did not alter ATBF. Finally, the DI increased AT gene expression of pathways related to mitochondrial biogenesis and non-mitochondrial oxygen consumption.CONCLUSIONS: VLCD-induced WL markedly decreases abdominal subcutaneous AT pO(2), which is paralleled by a reduction in adipocyte size, increased AT gene expression of mitochondrial biogenesis markers and non-mitochondrial oxygen consumption pathways, and improved whole-body insulin sensitivity in humans.

BACKGROUND/OBJECTIVE: Weight loss is often followed by weight regain after the dietary intervention (DI). Cellular stress is increased in adipose tissue of obese individuals. However, the relation between cellular stress and weight regain is unclear. Previously, we observed increased adipose tissue cellular stress of participants regaining weight compared with participants maintaining weight loss. In the current study, we further investigated the relation between weight regain and changes in the expression of stress-related genes and stress protein levels to determine possible predictors of weight regain.PARTICIPANTS/METHODS: In this randomized controlled trial, sixty-one healthy overweight/obese participants followed a DI of either a 5-week very-low-calorie diet (500 kcal per day) or a 12-week low-calorie diet (1250 kcal per day; WL period) with a subsequent 4-week weight stable diet (WS period), and a 9-month follow-up. The WL and WS period taken together was named the DI. Abdominal subcutaneous adipose tissue biopsies were collected in 53 participants for microarray and liquid chromatography-mass spectrometry analysis. RNA and protein levels for a broad set of stress-related genes were correlated to the weight regain percentage.RESULTS: Different gene sets correlated to weight regain percentage during WS and DI. Bioinformatics clustering suggests that during the WS phase-defined genes for actin filament dynamics, glucose handling and nutrient sensing are related to weight regain. HIF-1 (hypoxia-inducible factor-1) is indicated as an important regulator. With regard to DI, clustering of correlated genes indicate that LGALS1, ENO1 and ATF2 are important nodes for conferring risk for weight regain.CONCLUSIONS: Our present findings indicate that the risk for weight regain is related to expression changes of distinct sets of stress-related genes during the first 4 weeks after returning to energy balance, and during the DI. Further research is required to investigate the mechanistic significance of these findings and find targets for preventing weight regain.

Given the association of disturbances in non-esterified fatty acid (NEFA) metabolism with the development of Type 2 Diabetes and Non-Alcoholic Fatty Liver Disease, computational models of glucose-insulin dynamics have been extended to account for the interplay with NEFA. In this study, we use arteriovenous measurement across the subcutaneous adipose tissue during a mixed meal challenge test to evaluate the performance and underlying assumptions of three existing models of adipose tissue metabolism and construct a new, refined model of adipose tissue metabolism. Our model introduces new terms, explicitly accounting for the conversion of glucose to glyceraldehye-3-phosphate, the postprandial influx of glycerol into the adipose tissue, and several physiologically relevant delays in insulin signalling in order to better describe the measured adipose tissues fluxes. We then applied our refined model to human adipose tissue flux data collected before and after a diet intervention as part of the Yoyo study, to quantify the effects of caloric restriction on postprandial adipose tissue metabolism. Significant increases were observed in the model parameters describing the rate of uptake and release of both glycerol and NEFA. Additionally, decreases in the model’s delay in insulin signalling parameters indicates there is an improvement in adipose tissue insulin sensitivity following caloric restriction., Author summary The adipose tissue is no longer considered a metabolically quiescent tissue. Disturbances in non-esterified fatty acid (NEFA) metabolism leading to ectopic fat deposition have been associated with the development of insulin resistance. In recent years, the use of stable isotope tracers coupled with arteriovenous sampling across tissue depots has greatly improved our knowledge of postprandial NEFA dynamics. In this study, we make use of arteriovenous measurements collected across the abdominal subcutaneous adipose tissue in humans during a high fat mixed meal to evaluate three existing computational models of adipose tissue metabolism. As the three models included in this study were not capable of fully describing the measured adipose tissue fluxes, we present a new model of human in vivo adipose tissue metabolism, introducing novel terms such as the postprandial uptake of glycerol. We then utilised our refined model to quantify the effect of caloric restriction on adipose tissue metabolism by fitting the model to mixed meal challenge test data collected before and after a weight-loss intervention. Parameter estimates indicate an increase in the rates of glycerol and NEFA release coupled with a decrease in the delay of insulin signalling for all reactions, suggesting improved insulin sensitivity following caloric restriction.

Human mesenchymal stem (hMSCs) are defined as multi-potent colony-forming cells expressing a specific subset of plasma membrane markers when grown on flat tissue culture polystyrene. However, as soon as hMSCs are used for transplantation, they are exposed to a 3D environment, which can strongly impact cell physiology and influence proliferation, differentiation and metabolism. Strategies to control in vivo hMSC behavior, for instance in stem cell transplantation or cancer treatment, are skewed by the un-physiological flatness of the standard well plates. Even though it is common knowledge that cells behave differently in vitro compared to in vivo, only little is known about the underlying adaptation processes. Here, we used micrometer-scale defined surface topographies as a model to describe the phenotype of hMSCs during this adaptation to their new environment. We used well established techniques to compare hMSCs cultured on flat and topographically enhanced polystyreneand observed dramatically changed cell morphologies accompanied by shrinkage of cytoplasm and nucleus, a decreased overall cellular metabolism, and slower cell cycle progression resulting in a lower proliferation rate in cells exposed to surface topographies. We hypothesized that this reduction in proliferation rate effects their sensitivity to certain cancer drugs, which was confirmed by higher survival rate of hMSCs cultured on topographies exposed to paclitaxel. Thus, micro-topographies can be used as a model system to mimic the natural cell micro-environment, and be a powerful tool to optimize cell treatment in vitro.

Primary cilia are organelles that are present on many different cell types, either transiently or permanently. They play a crucial role in receiving signals from the environment and passing these signals to other parts of the cell. In that way, they are involved in diverse processes such as adipocyte differentiation and olfactory sensation. Mutations in genes coding for ciliary proteins often have pleiotropic effects and lead to clinical conditions, ciliopathies, with multiple symptoms. In this study, we reviewed observations from ciliopathies with obesity as one of the symptoms. It shows that variation in cilia-related genes is itself not a major cause of obesity in the population but may be a part of the multifactorial aetiology of this complex condition. Both common polymorphisms and rare deleterious variants may contribute to the obesity risk. Genotype–phenotype relationships have been noticed. Among the ciliary genes, obesity differs with regard to severity and age of onset, which may relate to the influence of each gene on the balance between pro- and anti-adipogenic processes. Analysis of the function and location of the proteins encoded by these ciliary genes suggests that obesity is more linked to activities at the basal area of the cilium, including initiation of the intraflagellar transport, but less to the intraflagellar transport itself. Regarding the role of cilia, three possible mechanistic processes underlying obesity are described: adipogenesis, neuronal food intake regulation and food odour perception.

Initial successful weight loss is often followed by weight regain after the dietary intervention. Compared with lean people, cellular stress in adipose tissue is increased in obese subjects. However, the relation between cellular stress and the risk for weight regain after weight loss is unclear. Therefore, we determined the expression levels of stress proteins during weight loss and weight maintenance in relation to weight regain. In vivo findings were compared with results from in vitro cultured human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes. In total, eighteen healthy subjects underwent an 8-week diet programme with a 10-month follow-up. Participants were categorised as weight maintainers or weight regainers (WR) depending on their weight changes during the intervention. Abdominal subcutaneous adipose tissue biopsies were obtained before and after the diet and after the follow-up. In vitro differentiated SGBS adipocytes were starved for 96 h with low (0·55 mm) glucose. Levels of stress proteins were determined by Western blotting. WR showed increased expressions of β-actin, calnexin, heat shock protein (HSP) 27, HSP60 and HSP70. Changes of β-actin, HSP27 and HSP70 are linked to HSP60, a proposed key factor in weight regain after weight loss. SGBS adipocytes showed increased levels of β-actin and HSP60 after 96 h of glucose restriction. The increased level of cellular stress proteins in the adipose tissue of WR probably resides in the adipocytes as shown by in vitro experiments. Cellular stress accumulated in adipose tissue during weight loss may be a risk factor for weight regain.

Objective To investigate the effect of rate of weight loss, with similar total weight loss, on weight regain in individuals with overweight and obesity. Methods Fifty-seven participants (BMI: 28-35 kg/m2) underwent a dietary intervention (DI). They were randomized to a low-calorie diet (LCD; 1250 kcal/day) for 12 weeks (slow weight loss) or a very-low-calorie diet (VLCD; 500 kcal/day) for 5 weeks (rapid weight loss) (weight loss (WL) period) followed by a 4-week weight-stable (WS) period and 9 months follow-up. Body weight and body composition (BodPod) were determined at study start and after each period. Results Weight change was similar in both groups after WL (LCD: −8.2 kg and VLCD: −9.0 kg, P = 0.24). Weight regain after follow-up was not significantly different between groups (LCD: 4.2 kg and VLCD: 4.5 kg, P = 0.73). Percentage fat-free mass loss (%FFML) was higher in the VLCD-group compared to the LCD-group after DI (8.8% and 1.3%, respectively, P = 0.034) and was associated with weight regain during follow-up in the whole group (r = 0.325, P = 0.018). Conclusions The present study showed that, with similar total weight loss, rate of weight loss did not affect weight regain. However, %FFML after DI was associated with weight regain.

Stress during pregnancy increases the risk to develop psychological disorders such as depression during pregnancy or in the postpartum period. According to the neurotrophin hypothesis of depression, the pathophysiology of depression is caused by reduced neurotrophic activity in the brain. However, most studies only focus on the molecular changes happening to the offspring upon gestational stress. To gain insight into the potential molecular changes happening in the stressed dams, C57B16/J mice were stressed during their first week of gestation. At 28 days postpartum, the hippocampus and nucleus accumbens core of the dams, two brain regions heavily implicated in depression, were evaluated using immunohistochemistry to detect changes in the neurotrophin system. Gestational stress decreased the weight of the dams, increased the chance for spontaneous abortion and increased the weight of offspring. Litter size, survival rates and sex distribution were not altered as a consequence of gestational stress. Hippocampal brain-derived neurotrophic factor (BDNF) decreased following exposure to stress during pregnancy. Hippocampal protein levels of p75(NTR), a low-affinity receptor for BDNF which can induce apoptosis, were increased following exposure to stress. Protein levels of p11, of which the expression is regulated by BDNF, were decreased in the hippocampus. No changes were found for TrkB immunostaining or apoptosis. Taken together, this shows that stress during pregnancy negatively affects the neurotrophin system in the hippocampus of the dams, thereby reducing hippocampal plasticity. These data confirm that gestational stress has a negative impact on pregnancy. Open Access funded by VSNU

Adipose tissue autophagy (AT) is associated with human obesity and increased metabolic risk. Recent findings establish a role for autophagy in lipid metabolism. Here, we compared the expression of autophagy-related and lipolysis genes in human abdominal subcutaneous AT (SCAT) in overweight/obese subjects (n = 17) with or without impaired glucose tolerance in comparison with lean normal glucose tolerant individuals (n = 9), and investigated the association between AT autophagy and lipolysis. Human multipotent adipose-derived stem cells (hMADS) were used to investigate the effect of pharmacological HSL inhibition on changes in the autophagic flux. The expression of autophagy-related genes (ATG) 5, 7 and 12 in SCAT was significantly higher (p = 0.043, p = 0.015, p = 0.004, respectively) in overweight/obese compared to lean men, while expression of the classical lipases HSL (p = 0.092) and ATGL (p = 0.084) tended to be lower. ATG12 mRNA was positively correlated with BMI (r = 0.407, p = 0.039). ATG7 mRNA correlated positively with waist/hip ratio (WHR) (r = 0.420, p = 0.041), 2 h glucose concentration (r = 0.488, p = 0.011) and insulin (r = 0.419, p = 0.033). Multiple linear regressions revealed that ATG7 gene expression was positively related to 2 h glucose, independent of BMI, WHR and insulin. Gene expression interaction analysis showed that ATG7 mRNA negatively correlated with HSL (p

Weight regain after successful weight loss is a big problem in obesity management. This study aimed to investigate whether weight regain after a weight loss period is correlated with the macronutrient composition and/or the inflammatory index of the diet during that period. Sixty one overweight and obese adults participated in this experimental study. Subjects lost approximately 10% of their initial weight by means of very low-calorie diet for five weeks, or a low calorie diet for 12 weeks. After that, subjects in both groups followed a strict weight maintenance diet based on individual needs for four weeks, which was followed by a nine-month weight maintenance period without dietary counseling. Anthropometrics and dietary intake data were recorded before weight loss (baseline) and during the weight maintenance period. On average, participants regained approximately half of their lost weight. We found no evidence that macronutrient composition during the weight maintenance period was associated with weight regain. The dietary inflammatory index (r = 0.304, p = 0.032) was positively correlated with weight regain and remained significant after correction for physical activity (r = 0.287, p = 0.045). Our data suggest that the inflammatory properties of diet play a role in weight regain after weight loss in overweight and obese adults.

During weight loss, the volume of adipocytes decreases, leading to stress because of the misfit between the cell contents and the surrounding extracellular matrix (ECM). This stress can be resolved by remodeling the ECM or the restorage of triglycerides within the adipocytes. The objective of this study was to investigate the existence of a connection between stress-related and ECM-related genes that is associated with weight regain.Thirty-one participants with overweight or obesity followed a 5-week very-low-calorie diet (500 kcal/d) with a subsequent 4-week weight-stable diet (WS), and then an uncontrolled 9-month follow-up. Adipose tissue biopsies were collected for microarray analysis. A correlation and interaction analysis was performed with the weight regain percentage (WR%) ([weight after follow-up - weight after WS] ÷ weight after WS × 100%) by using two gene sets that were previously defined as "stress-related" (n = 107) and "ECM-related" genes (n = 277).During WS, a coexpression network of 8 stress-related genes and 15 ECM-related genes correlating with WR% could be constructed, with links to multiple biological processes. Interaction analysis between stress- and ECM-related genes revealed that several gene combinations were highly related to weight regain.Our findings underscore the importance of the connection between stress- and ECM-related genes in the risk for weight regain.

It is so frustrating to finally lose some weight only to gain it back almost immediately. Research conducted at Maastricht University provides new insight into the way this yo-yo effect works. When we lose weight, our fat cells lose some of the fat they have stored. As a result, the cytoskeleton does not match the cell volume anymore. That is why the fat cells will develop stress. A fat cell can lose stress by hormonally stimulating the host to eat more and store fat in the fat cells. This will lead to weight gain. In addition to stress in fat cells, the immune system plays a role in this process, too.

Obesity is characterized by dysfunctional white adipose tissue (WAT) that ultimately may lead to metabolic diseases. Calorie restriction (CR) reduces the risk for age and obesity-associated complications. The impact of CR on obesity has been examined with human intervention studies, which showed alterations in circulating adipokines. However, a direct effect of CR on the human adipocyte secretome remains elusive. Therefore, the effect of a 96h low glucose CR on the secretion profile of in vitro cultured mature human SGBS adipocytes was investigated by using proteomics technology. Low-glucose CR decreased the adipocyte triglyceride contents and resulted in an altered secretion profile. Changes in the secretome indicated an improved inflammatory phenotype. In addition, several adipocyte-secreted proteins related to insulin resistance showed a reversed expression after low-glucose CR. Furthermore, 6 novel CR-regulated adipocyte-secreted proteins were identified. Since resveratrol (RSV) mimics CR we compared results from this study with data from our previous RSV study on the SGBS adipocyte secretome. The CR and RSV adipocyte secretomes partly differed from each other, although both treatment strategies lead to secretome changes indicating a less inflammatory phenotype. Furthermore, both treatments induced SIRT1 expression and resulted in a reversed expression of detrimental adipokines associated with metabolic complications.

Motivation Microscopy imaging is an essential tool for medical diagnosis and molecular biology. It is particularly useful for extracting information about disease states, tissue heterogeneity and cell specific parameters such as cell type or cell size from biological specimens. However, the information obtained from the images is likely to be subjected to sampling and observational bias with respect to the underlying cell size/type distributions. Results We present an algorithm, Estimate Tissue Cell Size/Type Distribution (EstiTiCS), for the adjustment of the underestimation of the number of small cells and the size of measured cells while accounting for the section thickness independent of the tissue type. We introduce the sources of bias under different tissue distributions and their effect on the measured values with simulation experiments. Furthermore, we demonstrate our method on histological sections of paraffin-embedded adipose tissue sample images from 57 people from a dietary intervention study. This data consists of measured cell size and its distribution over the dietary intervention period at four time points. Adjusting for the bias with EstiTiCS results in a closer fit to the true/expected adipocyte size distribution with earlier studies. Therefore, we conclude that our method is suitable as the final step in estimating the tissue wide cell type/size distribution from microscopy imaging pipeline. Availability and Implementation Source code and its documentation are available at https://github.com/michaelLenz/EstiTiCS. The whole pipeline of our method is implemented in R and makes use of the ‘nloptr’ package. Adipose tissue data used for this study are available on request. Contact Michael.Lenz@Maastrichtuniversity.nl, Gokhan.Ertaylan@Maastrichtuniversity.nl

Obesity is a global epidemic identified as a major risk factor for multiple chronic diseases and, consequently, diet-induced weight loss is used to counter obesity. The adipose tissue is the primary tissue affected in diet-induced weight loss, yet the underlying molecular mechanisms and changes are not completely deciphered. In this study, we present a network biology analysis workflow which enables the profiling of the cellular processes affected by weight loss in the subcutaneous adipose tissue. Time series gene expression data from a dietary intervention dataset with two diets was analysed. Differentially expressed genes were used to generate co-expression networks using a method that capitalises on the repeat measurements in the data and finds correlations between gene expression changes over time. Using the network analysis tool Cytoscape, an overlap network of conserved components in the co-expression networks was constructed, clustered on topology to find densely correlated genes, and analysed using Gene Ontology enrichment analysis. We found five clusters involved in key metabolic processes, but also adipose tissue development and tissue remodelling processes were enriched. In conclusion, we present a flexible network biology workflow for finding important processes and relevant genes associated with weight loss, using a time series co-expression network approach that is robust towards the high inter-individual variation in humans.

Angiopoietin‐like protein 4 (ANGPTL4) plays a role in lipid partitioning by inhibiting lipoprotein lipase (LPL)‐dependent plasma clearance of triacylglycerol in adipose tissue. We investigated the effects of diet‐induced weight loss on plasma ANGPTL4 concentrations in relation to in vivo adipose tissue LPL activity and lipolysis and adipose tissue ANGPTL4 release in overweight/obese participants. Sixteen individuals (BMI: 28–35 kg/m2; 10 women) were randomized to a dietary intervention composed of either a low‐calorie diet (1250 kcal/day) for 12 weeks (n = 9) or a very low‐calorie diet (500 kcal/day) for 5 weeks, followed by a 4‐week weight stable period. Before and after the intervention, we measured arteriovenous concentration differences in combination with adipose tissue blood flow before and after intake of a high‐fat mixed meal with [U‐13C]‐palmitate to assess in vivo adipose tissue LPL activity and lipolysis. The intervention significantly reduced body weight (−8.6 ± 0.6 kg, P

BACKGROUND: Energy restriction causes adaptations in energy expenditure (total-,TEE; resting-,REE; activity induced-,AEE). OBJECTIVE: To determine if changes in the levels of proteins involved in adipocyte glucose and fatty acid metabolism as indicators for energy deficiency are related to adaptations in energy expenditure during weight loss. METHODS: Forty-eight healthy subjects (18 men, 30 women), mean +/- SD age 42 +/- 8 y and BMI 31.4 +/- 2.8 kg/m(2), followed a very low energy diet for 8 wk. Protein levels of fatty acid binding protein 4 (FABP4), fructose-bisphosphate aldolase C (AldoC) and short chain 3-hydroxyacyl-CoA dehydrogenase (HADHsc) (adipose tissue biopsy, western blot), TEE (doubly labeled water), REE (ventilated hood), and AEE were assessed before and after the 8-wk diet. RESULTS: There was a positive correlation between the decrease in AldoC and the decrease in TEE (R = 0.438, P < 0.01) and the decrease change in AEE (R = 0.439, P < 0.01). Furthermore, there was a negative correlation between the increases in HADHsc and the decrease in REE (R = 0.343, P < 0.05). CONCLUSION: The decrease in AldoC correlated with the decrease in AEE, which may be explained by a decreased glycolytic flux. Additionally, the change in HADHsc, a crucial enzyme for a step in beta-oxidation, correlated with the adaptation in REE. TRIAL REGISTRATION: CLINICAL TRIAL REGISTRATION NUMBER: NCT01015508 at clinicaltrials.gov.

To investigate the effect of rate of weight loss, with similar total weight loss, on weight regain in individuals with overweight and obesity.Fifty-seven participants (BMI: 28-35 kg/m(2) ) underwent a dietary intervention (DI). They were randomized to a low-calorie diet (LCD; 1250 kcal/day) for 12 weeks (slow weight loss) or a very-low-calorie diet (VLCD; 500 kcal/day) for 5 weeks (rapid weight loss) (weight loss (WL) period) followed by a 4-week weight-stable (WS) period and 9 months follow-up. Body weight and body composition (BodPod) were determined at study start and after each period.Weight change was similar in both groups after WL (LCD: -8.2 kg and VLCD: -9.0 kg, P = 0.24). Weight regain after follow-up was not significantly different between groups (LCD: 4.2 kg and VLCD: 4.5 kg, P = 0.73). Percentage fat-free mass loss (%FFML) was higher in the VLCD-group compared to the LCD-group after DI (8.8% and 1.3%, respectively, P = 0.034) and was associated with weight regain during follow-up in the whole group (r = 0.325, P = 0.018).The present study showed that, with similar total weight loss, rate of weight loss did not affect weight regain. However, %FFML after DI was associated with weight regain.

Adipokines and other biomarkers were previously identified with roles in energy expenditure, appetite, satiety, and adiposity. Therefore, we investigated whether dietary weight loss‐induced changes in adipokines and other biomarkers known to play a role in weight regulation or energy expenditure could predict weight regain in people with overweight and obesity. In this randomized controlled trial 26 males and 30 females (BMI: 28–35 kg/m 2 ) followed either a low‐calorie diet (LCD; 1250 kcal/day) for 12 weeks or a very‐low‐calorie diet (VLCD; 500 kcal/day) for 5 weeks followed by a weight stable period of 4 weeks (dietary intervention (DI) period) and a 9‐month follow‐up period. Blood samples were taken before and after each period to measure FFA, TAG, total cholesterol, insulin, glucose, angiotensin‐converting enzyme (ACE) activity, IL‐6, RBP4, apelin, leptin, adiponectin, vaspin, and nesfatin‐1 concentrations. Weight loss was similar between groups (LCD: −8.2 ± 0.5 kg; VLCD: −8.9 ± 0.4 kg, P = 0.30). Only changes in ACE activity, FFA and RBP4 concentrations during DI were correlated with weight regain in the whole group ( r = −0.299, P = 0.030, r = −0.274, P = 0.047, and r = 0.357, P = 0.008, respectively). Together they explained 28% ( r = 0.532) of weight regain variation. Dietary weight loss‐induced changes in ACE activity, FFA and RBP4 independently contribute to weight regain prediction.

BACKGROUND/OBJECTIVES: Moderate weight loss (WL) can ameliorate adverse health effects associated with obesity, reflected by an improved adipose tissue (AT) gene expression profile. However, the effect of rate of WL on the AT transcriptome is unknown. We investigated the global AT gene expression profile before and after two different rates of WL that resulted in similar total WL, and after a subsequent weight stabilization period.SUBJECTS/METHODS: In this randomized controlled trial, 25 male and 28 female individuals (body mass index (BMI): 28-35 kg m(-)(2)) followed either a low-calorie diet (LCD; 1250 kcal day(-1)) for 12 weeks or a very-low-calorie diet (VLCD; 500 kcal day(-1)) for 5 weeks (WL period) and a subsequent weight stable (WS) period of 4 weeks. The WL period and WS period together is termed dietary intervention (DI) period. Abdominal subcutaneous AT biopsies were collected for microarray analysis and gene expression changes were calculated for all three periods in the LCD group, VLCD group and between diets (ΔVLCD-ΔLCD).RESULTS: WL was similar between groups during the WL period (LCD: -8.1±0.5 kg, VLCD: -8.9±0.4 kg, difference P=0.25). Overall, more genes were significantly regulated and changes in gene expression appeared more pronounced in the VLCD group compared with the LCD group. Gene sets related to mitochondrial function, adipogenesis and immunity/inflammation were more strongly upregulated on a VLCD compared with a LCD during the DI period (positive ΔVLCD-ΔLCD). Neuronal and olfactory-related gene sets were decreased during the WL period and DI period in the VLCD group.CONCLUSIONS: The rate of WL (LCD vs VLCD), with similar total WL, strongly regulates AT gene expression. Increased mitochondrial function, angiogenesis and adipogenesis on a VLCD compared with a LCD reflect potential beneficial diet-induced changes in AT, whereas differential neuronal and olfactory regulation suggest functions of these genes beyond the current paradigm.International Journal of Obesity advance online publication, 7 December 2016; doi:10.1038/ijo.2016.201.

Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.

The extracellular matrix (ECM) of adipocytes is important for body weight regulation. Here, we investigated whether genetic variation in ECM-related genes is associated with weight regain among participants of the European DiOGenes study. Overweight and obese subjects (n = 469, 310 females, 159 males) were on an 8-week low-calorie diet with a 6-month follow-up. Body weight was measured before and after the diet, and after follow-up. Weight maintenance scores (WMS, regained weight as percentage of lost weight) were calculated based on the weight data. Genotype data were retrieved for 2903 SNPs corresponding to 124 ECM-related genes. Regression analyses provided us with six significant SNPs associated with the WMS in males: 3 SNPs in the POSTN gene and a SNP in the LAMB1, COL23A1, and FBLN5 genes. For females, 1 SNP was found in the FN1 gene. The risk of weight regain was increased by: the C/C genotype for POSTN in a co-dominant model (OR 8.25, 95 % CI 2.85–23.88) and the T/C–C/C genotype in a dominant model (OR 4.88, 95 % CI 2.35–10.16); the A/A genotype for LAMB1 both in a co-dominant model (OR 18.43, 95 % CI 2.35–144.63) and in a recessive model (OR 16.36, 95 % CI 2.14–124.9); the G/A genotype for COL23A1 in a co-dominant model (OR 3.94, 95 % CI 1.28–12.10), or the A-allele in a dominant model (OR 2.86, 95 % CI 1.10–7.49); the A/A genotype for FBLN5 in a co-dominant model (OR 13.00, 95 % CI 1.61–104.81); and the A/A genotype for FN1 in a recessive model (OR 2.81, 95 % CI 1.40–5.63). Concluding, variants of ECM genes are associated with weight regain after weight loss in a sex-specific manner. Electronic supplementary material The online version of this article (doi:10.1007/s12263-015-0506-y) contains supplementary material, which is available to authorized users.