Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells.

Characterization of DLK1(PREF1)+/CD34+ cells in vascular stroma of human white adipose tissue.

Sorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34-, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1-/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34- cells. Permeabilized cs-DLK1-/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31-). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34- and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34- perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31- cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity.

Bariatric surgery and diet-induced long-term caloric restriction protect subcutaneous adipose-derived stromal/progenitor cells and prolong their life span in formerly obese humans.

A key effect of prolonged reducing diets and bariatric surgeries in formerly obese people is long-term caloric restriction (CR). The analysis of the impact of these interventions on specific tissues will contribute to a better understanding of their mechanisms of action. The physiological functions of subcutaneous white adipose tissues are mainly fulfilled by adipocytes arising out of adipose-derived stromal/progenitor cells (ASCs), which are crucial for adipose tissue homeostasis. In the present study we analyzed ASC from age-matched long-term calorically restricted formerly obese (CRD), obese (OD) and normal weight donors (NWDs). We demonstrate that ASC derived from CRD has a significant longer replicative lifespan than ASC isolated from OD and NWD. This correlated with strongly reduced DNA-damage and improved survival of the CRD ASC, both are hallmarks of CR. The adipogenic capacity was significantly lower in ASC derived from CRD than that from OD, as shown by reduced expression of the adipogenic key regulator PPARγ2 and the differentiation marker FABP4. The adipogenic capacity of ASCs from CRD and NWD differed only slightly. In conclusion, we provide evidence that bariatric surgery and diet-induced long-term CR substantially reprogram ASCs in formerly obese humans, comprising reduced DNA-damage, improved viability, extended replicative lifespan and reduced adipogenic differentiation potential.

Adipogenic differentiation is impaired in replicative senescent human subcutaneous adipose-derived stromal/progenitor cells.

We demonstrate that adipose-derived stromal/progenitor cells isolated from abdominal subcutaneous fat pads of adult donors successively enter replicative senescence after long-term cultivation. This is characterized by enlarged cell size, flattened morphology, and upregulated senescence-associated ß-galactosidase activity. Moreover, the senescence- associated cyclin-dependent kinase inhibitors p16(Ink4A) and p21(Cip1) were induced correlating with activation of the G1/S cell cycle inhibitor retinoblastoma protein and terminal proliferation arrest. The number of cells in the adipose-derived stromal/progenitor cell population with high adipogenic capacity declined inversely with the increase of senescent cells. Adipogenic hormone cocktail induced expression of the adipogenic key regulators peroxisome proliferator-activated receptor-γ2 and CCAAT/enhancer-binding protein α was significantly reduced in senescent adipose-derived stromal/ progenitor cells. Furthermore, the expression of the adipogenic differentiation genes fatty acid binding protein-4, adiponectin, and leptin and the formation of fat droplets were impaired. We conclude cellular senescence contributes to dysfunctions in adipose-derived stromal/progenitor cell replication, adipogenesis, triglyceride storage, and adipokine secretion.

Mechanisms of resveratrol-induced inhibition of clonal expansion and terminal adipogenic differentiation in 3T3-L1 preadipocytes.

We show that resveratrol prevents clonal expansion and terminal adipogenesis in 3T3-L1 preadipocytes. An early resveratrol effect was the inhibition of AKT and mitogen-activated protein kinase signaling, accompanied by down regulation of cyclin D1 expression, abrogation of retinoblastoma protein hyperphosphorylation, and subsequent inhibition of cell cycle reentry and clonal expansion, as indicated by cyclin A2 repression. Resveratrol inhibited terminal adipogenesis at the level of peroxisome proliferator-activated receptor-γ2 expression and activity. This was independent from the preceding inhibition of clonal expansion. Peroxisome proliferator-activated receptor-γ2 overexpression and activation partially restored fatty acid-binding protein 4 induction in resveratrol-treated 3T3-L1. Resveratrol activated AMP-activated protein kinase (AMPK) but did not induce PPAR-γ co-activator 1α (PGC1α) and mitochondrial biogenesis in 3T3-L1. Treatment with the Sirt1 inhibitor splitomicin augmented downregulation of peroxisome proliferator-activated receptor-γ2 and fatty acid-binding protein 4 expressions in resveratrol-treated 3T3-L1 and did not prevent the inhibition of terminal adipogenesis. Moreover, splitomicin could not obviate resveratrol-induced cyclin D1 repression, retinoblastoma protein hypophosphorylation, and inhibition of clonal expansion. Our data suggest that resveratrol inhibits clonal expansion and terminal adipogenesis in 3T3-L1 by several mechanisms.

Role of C/EBPß-LAP and C/EBPß-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

We investigated the role of the major isoforms of CCAAT enhancer binding protein ß (C/EBPß), C/EBPß-LAP and C/EBPß-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPß gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPß mRNA and protein levels declined. The C/EBPß-LIP protein steady-state level decreased considerably stronger than the C/EBPß-LAP level and the C/EBPß-LIP half-life was significantly shorter than the C/EBPß-LAP half-life. The turn-over of both C/EBPß-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPß-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPß-LIP had antiadipogenic activity in human ASC. C/EBPß-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPß-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPß-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPß gene expression and C/EBPß-LIP and C/EBPß-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.

DLK1(PREF1) is a negative regulator of adipogenesis in CD105⁺/CD90⁺/CD34⁺/CD31⁻/FABP4⁻ adipose-derived stromal cells from subcutaneous abdominal fat pats of adult women.

The main physiological function of adipose-derived stromal/progenitor cells (ASC) is to differentiate into adipocytes. ASC are most likely localized at perivascular sites in adipose tissues and retain the capacity to differentiate into multiple cell types. Although cell surface markers for ASC have been described, there is no complete consensus on the antigen expression pattern that will precisely define these cells. DLK1(PREF1) is an established marker for mouse adipocyte progenitors which inhibits adipogenesis. This suggests that DLK1(PREF1) could be a useful marker to characterize human ASC. The DLK1(PREF1) status of human ASC is however unknown. In the present study we isolated ASC from the heterogeneous stromal vascular fraction of subcutaneous abdominal fat pats of adult women. These cells were selected by their plastic adherence and expanded to passage 5. The ASC were characterized as relatively homogenous cell population with the capacity to differentiate in vitro into adipocytes, chondrocytes, and osteoblasts and the immunophenotype CD105⁺/CD90⁺/CD34⁺/CD31⁻/FABP4⁻. The ASC were positive for DLK1(PREF1) which was well expressed in proliferating and density arrested cells but downregulated in the course of adipogenic differentiation. To investigate whether DLK1(PREF1) plays a role in the regulation of adipogenesis in these cells RNAi-mediated knockdown experiments were conducted. Knockdown of DLK1(PREF1) in differentiating ASC resulted in a significant increase of the expression of the adipogenic key regulator PPARγ2 and of the terminal adipogenic differentiation marker FABP4. We conclude that DLK1(PREF1) is well expressed in human ASC and acts as a negative regulator of adipogenesis. Moreover, DLK1(PREF1) could be a functional marker contributing to the characterization of human ASC.

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2.

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO(2) have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression.

The human papillomavirus type 16 E7 oncoprotein targets Myc-interacting zinc-finger protein-1.

We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21(Cip1) and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21(Cip1) and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21(Cip1) expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21(Cip1) expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21(Cip1) core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21(Cip1) promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21(Cip1) expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21(Cip1) expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21(Cip1) gene expression.

Reduced insulin-like growth factor-I serum levels in formerly obese women subjected to laparoscopic-adjustable gastric banding or diet-induced long-term caloric restriction.

Life-span extension in laboratory rodents induced by long-term caloric restriction correlates with decreased serum insulin-like growth factor-I (IGF-I) levels. Reduced activity of the growth hormone/IGF-I signaling system slows aging and increases longevity in mutant mouse models. In the present study, we show that long-term caloric restriction achieved by two different interventions for 4 years, either laparoscopic-adjustable gastric banding or reducing diet, leads to reduced IGF-I serum levels in formerly obese women relative to normal-weight women eating ad libitum. Moreover, we present evidence that the long-term caloric restriction interventions reduce fasting growth hormone serum levels. The present study indicates that the activity of the growth hormone/IGF-I axis is reduced in long-term calorically restricted formerly obese humans. Furthermore, our findings suggest that the duration and severity of the caloric restriction intervention are important for the outcome on the growth hormone/IGF-I axis in humans.

Adipokine profile and insulin sensitivity in formerly obese women subjected to bariatric surgery or diet-induced long-term caloric restriction.

To better understand the contribution of the fat mass to the effects of long-term caloric restriction in humans, we compared adipokine profile and insulin sensitivity in long-term calorically restricted formerly obese women (CRW) subjected to different interventions, bariatric surgery, or reducing diet, with age- and BMI-matched obese (OW) and normal-weight women (NW) eating ad libitum. Our key findings are that despite a considerably stronger weight loss induced by bariatric surgery, both long-term caloric restriction interventions improved insulin sensitivity to the same degree and led to significantly lower retinol-binding protein-4 and interleukin-6 serum levels than in OW, suggesting that lowering of these two adipokines contributes to the improved insulin sensitivity. Moreover, serum leptin was considerably lower in CRW than in OW as well as in NW, suggesting that CRW develop hypoleptinemia.

Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply.

The glycolytic key regulator pyruvate kinase M2 (M2-PK or PKM2) can switch between a highly active tetrameric and an inactive dimeric form. The transition between the two conformations regulates the glycolytic flux in tumor cells. We developed specific M2-PK-binding peptide aptamers which inhibit M2-PK, but not the 96% homologous M1-PK isoenzyme. In this study we demonstrate that, at normal blood glucose concentrations, peptide aptamer-mediated inhibition of M2-PK induces a significant decrease of the population doubling (PDL rate) and cell proliferation rate as well as an increase in cell size, whereas under glucose restriction an increase in PDL and cell proliferation rates but a decrease in cell size was observed. Moreover, M2-PK inhibition rescues cells from glucose starvation-induced apoptotic cell death by increasing the metabolic activity. These findings suggest that M2-PK is a metabolic sensor which regulates cell proliferation, cell growth and apoptotic cell death in a glucose supply-dependent manner.