CD10 marks non-canonical PPARγ-independent adipocyte maturation and browning potential of adipose-derived stem cells.

BACKGROUND: Effective stem cell therapy is dependent on the stem cell quality that is determined by their differentiation potential, impairment of which leads to poor engraftment and survival into the target cells. However, limitations in our understanding and the lack of reliable markers that can predict their maturation efficacies have hindered the development of stem cells as an effective therapeutic strategy. Our previous study identified CD10, a pro-adipogenic, depot-specific prospective cell surface marker of human adipose-derived stem cells (ASCs). Here, we aim to determine if CD10 can be used as a prospective marker to predict mature adipocyte quality and play a direct role in adipocyte maturation. METHODS: We first generated 14 primary human subject-derived ASCs and stable immortalized CD10 knockdown and overexpression lines for 4 subjects by the lentiviral transduction system. To evaluate the role of CD10 in adipogenesis, the adipogenic potential of the human subject samples were scored against their respective CD10 transcript levels. Assessment of UCP1 expression levels was performed to correlate CD10 levels to the browning potential of mature ASCs. Quantitative polymerase chain reaction (qPCR) and Western blot analysis were performed to determine CD10-dependent regulation of various targets. Seahorse analysis of oxidative metabolism and lipolysis assay were studied. Lastly, as a proof-of-concept study, we used CD10 as a prospective marker for screening nuclear receptor ligands library. RESULTS: We identified intrinsic CD10 levels as a positive determinant of adipocyte maturation as well as browning potential of ASCs. Interestingly, CD10 regulates ASC's adipogenic maturation non-canonically by modulating endogenous lipolysis without affecting the classical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent adipogenic pathways. Furthermore, our CD10-mediated screening analysis identified dexamethasone and retinoic acid as stimulator and inhibitor of adipogenesis, respectively, indicating CD10 as a useful biomarker for pro-adipogenic drug screening. CONCLUSION: Overall, we establish CD10 as a functionally relevant ASC biomarker, which may be a prerequisite to identify high-quality cell populations for improving metabolic diseases.

Heredity of type 2 diabetes confers increased susceptibility to oxidative stress and inflammation.

INTRODUCTION AND OBJECTIVE: Heredity of type 2 diabetes mellitus (T2DM) is associated with greater risk for developing T2DM. Thus, individuals who have a first-degree relative with T2DM (FDRT) provide a natural model to study factors of susceptibility towards development of T2DM, which are poorly understood. Emerging key players in T2DM pathophysiology such as adverse oxidative stress and inflammatory responses could be among possible mechanisms that predispose FDRTs to develop T2DM. Here, we aimed to examine the role of oxidative stress and inflammatory responses as mediators of this excess risk by studying dynamic postprandial responses in FDRTs. RESEARCH DESIGN AND METHODS: In this open-label case-control study, we recruited normoglycemic men with (n=9) or without (n=9) a family history of T2DM. We assessed plasma glucose, insulin, lipid profile, cytokines and F2-isoprostanes, expression levels of oxidative and inflammatory genes/proteins in circulating mononuclear cells (MNC), myotubes and adipocytes at baseline (fasting state), and after consumption of a carbohydrate-rich liquid meal or insulin stimulation. RESULTS: Postprandial glucose and insulin responses were not different between groups. Expression of oxidant transcription factor NRF2 protein (p<0.05 for myotubes) and gene (pgroup=0.002, ptime×group=0.016), along with its target genes TXNRD1 (pgroup=0.004, ptime×group=0.007), GPX3 (pgroup=0.011, ptime×group=0.019) and SOD-1 (pgroup=0.046 and ptime×group=0.191) was upregulated in FDRT-derived MNC after meal ingestion or insulin stimulation. Synergistically, expression of target genes of inflammatory transcription factor nuclear factor kappa B such as tumor necrosis factor alpha (pgroup=0.001, ptime×group=0.007) was greater in FDRT-derived MNC than in non-FDRT-derived MNC after meal ingestion or insulin stimulation. CONCLUSIONS: Our findings shed light on how heredity of T2DM confers increased susceptibility to oxidative stress and inflammation. This could provide early insights into the underlying mechanisms and future risk of FDRTs for developing T2DM and its associated complications.

Oxidative stress mediates depot-specific functional differences of human adipose-derived stem cells.

BACKGROUND: Visceral (VS) fat depot is known to have defective adipogenic functions compared to subcutaneous (SC) fat, but its mechanism of origin is unclear. OBJECTIVE: We tested our hypothesis that the degree of oxidative stress in adipose-derived stem cells (ASCs) from these depots may account for this difference. METHODS: ASCs were isolated from VS (omental region) and SC (abdominal region) fat depots of human subjects undergoing bariatric surgery. ASCs from VS and SC fat were investigated for their cellular characteristics in reactive oxygen species (ROS), metabolism, gene expression, proliferation, senescence, migration, and adipocyte differentiation. ASCs were also treated with antioxidant ascorbic acid (vitamin C). RESULTS: We found that human VS-derived ASCs exhibit excessive oxidative stress characterized by high reactive oxygen species (ROS), compared to SC-derived ASCs. Gene expression analyses indicate that the VS-ASCs exhibit higher levels of genes involved in pro-oxidant and pro-inflammatory pathways and lower levels of genes in antioxidant and anti-inflammatory pathways. VS-ASCs have impaired cellular functions compared to SC-ASCs, such as slower proliferation, early senescence, less migratory activity, and poor adipogenic capability in vitro. Treatment with ascorbic acid decreased ROS levels drastically in VS-ASCs. Ascorbic acid treatment substantially improved proliferation, senescence, migration, and adipogenic capacities of compromised ASCs caused by high ROS. CONCLUSIONS: This finding suggests the fat depot-specific differences of cellular defects originating from stem cell population. Considering clinical potentials of human ASCs for cell therapies, this also offers a possible strategy for improving their therapeutic qualities through antioxidants.

Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches.

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1 flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.

Right-Site Care Programme with a community-based family medicine clinic in Singapore: secondary data analysis of its impact on mortality and healthcare utilisation.

OBJECTIVE: Stable patients with chronic conditions could be appropriately cared for at family medicine clinics (FMC) and discharged from hospital specialist outpatient clinics (SOCs). The Right-Site Care Programme with Frontier FMC emphasised care organised around patients in community rather than hospital-based providers, with one identifiable primary provider. This study evaluated impact of this programme on mortality and healthcare utilisation. DESIGN: A retrospective study without randomisation using secondary data analysis of patients enrolled in the intervention matched 1:1 with unenrolled patients as controls. SETTING: Programme was supported by the Ministry of Health in Singapore, a city-state nation in Southeast Asia with 5.6 million population. PARTICIPANTS: Intervention group comprises patients enrolled from January to December 2014 (n=684) and control patients (n=684) with at least one SOC and no FMC attendance during same period. INTERVENTIONS: Family physician in Frontier FMC managed patients in consultation with relevant specialist physicians or fully managed patients independently. Care teams in SOCs and FMC used a common electronic medical records system to facilitate care coordination and conducted regular multidisciplinary case conferences. PRIMARY OUTCOME MEASURES: Deidentified linked healthcare administrative data for time period of January 2011 to December 2017 were extracted. Three-year postenrolment mortality rates and utilisation frequencies and charges for SOC, public primary care centres (polyclinic), emergency department attendances and emergency, non-day surgery inpatient and all-cause admissions were compared. RESULTS: Intervention patients had lower mortality rate (HR=0.37, p<0.01). Among those with potential of postenrolment polyclinic attendance, intervention patients had lower frequencies (incidence rate ratio (IRR)=0.60, p<0.01) and charges (mean ratio (MR)=0.51, p<0.01). Among those with potential of postenrolment SOC attendance, intervention patients had higher frequencies (IRR=2.06, p<0.01) and charges (MR=1.86, p<0.01). CONCLUSIONS: Intervention patients had better survival, probably because their chronic conditions were better managed with close monitoring, contributing to higher total outpatient attendance frequencies and charges.

TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin.

Transient receptor potential (TRP) channels are polymodal cell sensors responding to diverse stimuli and widely implicated in the developmental programs of numerous tissues. The evidence for an involvement of TRP family members in adipogenesis, however, is scant. We present the first comprehensive expression profile of all known 27 human TRP genes in mesenchymal progenitors cells during white or brown adipogenesis. Using positive trilineage differentiation as an exclusion criterion, TRP polycystic (P)3, and TPR melastatin (M)8 were found to be uniquely adipospecific. Knockdown of TRPP3 repressed the expression of the brown fat signature genes uncoupling protein (UCP)-1 and peroxisome proliferator-activated receptor γ coactivator (PGC)-1α as well as attenuated forskolin-stimulated uncoupled respiration. However, indices of generalized adipogenesis, such as lipid droplet morphology and fatty acid binding protein (FAPB)-4 expression, were not affected, indicating a principal mitochondrial role of TRPP3. Conversely, activating TRPM8 with menthol up-regulated UCP-1 expression and augmented uncoupled respiration predominantly in white adipocytes (browning), whereas streptomycin antagonized TRPM8-mediated calcium entry, downregulated UCP-1 expression, and mitigated uncoupled respiration; menthol was less capable of augmenting uncoupled respiration (thermogenesis) in brown adipocytes. TRPP3 and TRPM8 hence appear to be involved in the priming of mitochondria to perform uncoupled respiration downstream of adenylate cyclase. Our results also underscore the developmental caveats of using antibiotics in adipogenic studies.-Goralczyk, A., van Vijven, M., Koch, M., Badowski, C., Yassin, M. S., Toh, S.-A., Shabbir, A., Franco-Obregón, A., Raghunath, M. TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin.

Meal rich in carbohydrate, but not protein or fat, reveals adverse immunometabolic responses associated with obesity.

BACKGROUND: Obesity-related insulin resistance is linked to inflammation. Immunometabolic function differs between lean and obese subjects, but whether macronutrient composition of ingested meals affects these responses is not well known. We examined the effects of a single meal rich in fat, protein, or carbohydrate on immunometabolic responses. METHODS: Nine lean insulin sensitive (LIS) men and 9 obese insulin resistant (OIR) men ingested high-carbohydrate (HC), high-fat (HF) or high-protein (HP) mixed meals in random order. We assessed plasma glucose, insulin, and cytokine responses and cytokine gene expression in circulating mononuclear cells (MNC) at fasting and postprandial states (up to 6-h). RESULTS: Expression of NF-κB and TNFα genes were greater; whereas that of TGFß and IL-6 genes were lower, in the OIR compared to the LIS individuals. The differences were significantly greater after the HC meal, but not after the HP or HF meal. Similar results were obtained for plasma concentrations of TNFα and IL-6. CONCLUSIONS: Our findings indicate that a single HC meal has a distinct adverse effect on immunometabolic responses in the OIR individuals. The cumulative effect of such adverse responses to meals rich in carbohydrate may predispose the OIR individuals to a higher risk of cardiovascular disease.

Retinoic Acid Mediates Visceral-Specific Adipogenic Defects of Human Adipose-Derived Stem Cells.

Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots.

ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs.

Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced 'browning' in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow.

Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots.

Adipose-derived stem/stromal cells (ASCs) from the anatomically distinct subcutaneous and visceral depots of white adipose tissue (WAT) differ in their inherent properties. However, little is known about the molecular identity and definitive markers of ASCs from these depots. In this study, ASCs from subcutaneous fat (SC-ASCs) and visceral fat (VS-ASCs) of omental region were isolated and studied. High-content image screening of over 240 cell-surface markers identified several potential depot-specific markers of ASCs. Subsequent studies revealed consistent predominant expression of CD10 in SC-ASCs and CD200 in VS-ASCs across 12 human subjects and in mice. CD10-high-expressing cells sorted from SC-ASCs differentiated better than their CD10-low-expressing counterparts, whereas CD200-low VS-ASCs differentiated better than CD200-high VS-ASCs. The expression of CD10 and CD200 is thus depot-dependent and associates with adipogenic capacities. These markers will offer a valuable tool for tracking and screening of depot-specific stem cell populations.

Selective peroxisome proliferator-activated receptor-γ modulation to reduce cardiovascular risk in patients with insulin resistance.

The thiazolidinediones (TZDs) rosiglitazone and pioglitazone improve glucose homeostasis through activation of peroxisome proliferator-activated receptor (PPAR)-γ. Their use, however, has been limited due to adverse effects that include body weight gain and edema leading to congestive heart failure. Selective PPAR-γ modulators (SPPARMs) are second generation PPAR-γ ligands designed to improve insulin sensitivity with minimal undesirable effects associated with first generation PPAR-γ agonists. INT131 is one of the first SPPARMs to reach human trials. Early phase human studies with INT131 look promising with changes in plasma lipids and glucose being equal or better than what is seen with rosiglitazone and pioglitazone treatment but without evidence of edema. This profile of improved glucose homeostasis, improved plasma lipids, and reduced inflammation in the absence of edema would be expected to reduce cardiovascular risk in patients with Type 2 diabetes mellitus. Recent patents of novel approaches for the use of PPAR-γ related compounds with the potential for this improved risk-benefit ratio are discussed.

PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI.

PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/(3)H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20 mg/kg/day) did not change (3)H-tracer plasma appearance, but surprisingly decreased fecal (3)H-free sterol excretion by 43% (P<0.01) over 48h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with (3)H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived (3)H-CE uptake by adipose tissue (P<0.005) with concomitant 22% decrease in HDL derived (3)H-CE uptake by the liver (P<0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived (3)H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.

Trib1 is a lipid- and myocardial infarction-associated gene that regulates hepatic lipogenesis and VLDL production in mice.

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic-specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans.

Overexpression of apolipoprotein F reduces HDL cholesterol levels in vivo.

OBJECTIVE: Apolipoprotein F (ApoF) is a protein component of several lipoprotein classes including HDL. It is also known as lipid transfer inhibitor protein (LTIP) based on its ability to inhibit lipid transfer between lipoproteins ex vivo. We sought to investigate the role of ApoF in HDL metabolism. METHODS AND RESULTS: Adeno-associated viruses (AAV) based on serotype 8, were used to overexpress either murine or human ApoF in mice. Overexpression of murine ApoF significantly reduced total cholesterol levels by 28% (P<0.001), HDL by 27% (P<0.001), and phospholipid levels by 19% (P<0.001). Overexpression of human ApoF had similar effects. Human ApoF was nearly exclusively HDL-associated in mice. In agreement with this finding, greater than 90% of the ApoF in human plasma was found on HDL(3), with only a small amount on LDL. Overexpression of mouse ApoF accelerated the plasma clearance of [(3)H]-cholesteryl ether labeled HDL. Plasma from mice overexpressing ApoF showed improved macrophage cholesterol efflux on a per HDL-C basis. CONCLUSIONS: ApoF overexpression reduces HDL cholesterol levels in mice by increasing clearance of HDL-CE. ApoF may be an important determinant of HDL metabolism and reverse cholesterol transport.

Modulating angiogenesis: the yin and the yang in ginseng.

BACKGROUND: Ginseng is a commonly used nutraceutical. Intriguingly, existing literature reports both wound-healing and antitumor effects of ginseng extract through opposing activities on the vascular system. To elucidate this perplexity, we merged a chemical fingerprinting approach with a deconstructional study of the effects of pure molecules from ginseng extract on angiogenesis. METHODS AND RESULTS: A mass spectrometric compositional analysis of American, Chinese and Korean, and Sanqi ginseng revealed distinct "sterol ginsenoside" fingerprints, especially in the ratio between a triol, Rg1, and a diol, Rb1, the 2 most prevalent constituents. Using a Matrigel implant model and reconstituting the extracts using distinct ratios of the 2 ginsenosides, we demonstrate that the dominance of Rg1 leads to angiogenesis, whereas Rb1 exerts an opposing effect. Rg1 also promoted functional neovascularization into a polymer scaffold in vivo and the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro, an effect mediated through the expression of nitric oxide synthase and the phosphatidylinositol-3 kinase-->Akt pathway. In contrast, Rb1 inhibited the earliest step in angiogenesis, the chemoinvasion of endothelial cells. CONCLUSIONS: The present study explains, for the first time, the ambiguity about the effects of ginseng in vascular pathophysiology based on the existence of opposing active principles in the extract. We also unraveled a speciogeographic variation impinging on the compositional fingerprint that may modulate the final phenotype. This emphasizes the need for regulations standardizing herbal therapy, currently under the Dietary Supplement and Health Education Act. Furthermore, we propose that Rg1 could be a prototype for a novel group of nonpeptide molecules that can induce therapeutic angiogenesis, such as in wound healing.