Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome

The relationship between stress and obesity remains elusive. In response to stress, some people lose weight, whereas others gain. Here we report that stress exaggerates diet-induced obesity through a peripheral mechanism in the abdominal white adipose tissue that is mediated by neuropeptide Y (NPY). Stressors such as exposure to cold or aggression lead to the release of NPY from sympathetic nerves, which in turn upregulates NPY and its Y2 receptors (NPY2R) in a glucocorticoid-dependent manner in the abdominal fat. This positive feedback response by NPY leads to the growth of abdominal fat. Release of NPY and activation of NPY2R stimulates fat angiogenesis, macrophage infiltration, and the proliferation and differentiation of new adipocytes, resulting in abdominal obesity and a metabolic syndrome-like condition. NPY, like stress, stimulates mouse and human fat growth, whereas pharmacological inhibition or fat-targeted knockdown of NPY2R is anti-angiogenic and anti-adipogenic, while reducing abdominal obesity and metabolic abnormalities. Thus, manipulations of NPY2R activity within fat tissue offer new ways to remodel fat and treat obesity and metabolic syndrome.     

Dipeptidyl-peptidase-IV by cleaving neuropeptide Y induces lipid accumulation and PPAR-γ expression

We evaluated the effects of dipeptidyl peptidase-IV (DPPIV), and its inhibitor, vildagliptin, on adipogenesis and lipolysis in a pre-adipocyte murine cell line (3T3-L1). The exogenous rDPPIV increased lipid accumulation and PPAR-γ expression, whereas an inhibitor of DPPIV, the anti-diabetic drug vildagliptin, suppresses the stimulatory role of DPPIV on adipogenesis and lipid accumulation, but had no effect on lipolysis. NPY immunoneutralization or NPY Y(2) receptor blockage inhibited DPPIV stimulatory effects on lipid accumulation, collectively, indicating that DPPIV has an adipogenic effect through NPY cleavage and subsequent NPY Y(2) activation. Vildagliptin inhibits PPAR-γ expression and lipid accumulation without changing lipolysis, suggesting that this does not impair the ability of adipose tissue to store triglycerides inside lipid droplets. These data indicate that DPPIV and NPY interact on lipid metabolism to promote adipose tissue depot.     

Histone deacetylase 9 is a negative regulator of adipogenic differentiation

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.     

Effects of macrophages and CXCR2 on adipogenic differentiation of bone marrow mesenchymal stem cells

Macrophages and many chemokines are closely associated with the adipogenic differentiation of bone marrow mesenchymal stem cells (MSCs), but their roles in adipogenesis and the underlying mechanisms are not fully understood. Here, we first investigated the influence of macrophages on the differentiation of MSCs in vitro. We found that RAW246.7 macrophages cocultured with MSCs strongly blocked the differentiation progress and inhibited the expression of C-X-C motif chemokine ligand 1 (CXCL1) during adipogenesis. Coculture with MSCs mainly induced macrophages toward M2 polarization. In addition, the expression of CXCL1 and its receptor, C-X-C chemokine receptor type 2, CXCR2 are high during adipogenic differentiation of MSCs and not in mature adipocytes. Although CXCL1 had no effect on adipogenesis, treatment with a specific CXCR2 inhibitor, SB225002, hampered the adipogenic differentiation of MSCs. Blocking CXCR2 decreased p38 and Elk1 phosphorylation but increased the extracellular signal–regulated kinase (ERK) phosphorylation at the initial stage of adipogenesis, which suppressed the phosphorylation of p38/ERK-Elk1 at the late stage. Inhibition of ERK had similar effects on adipogenesis and Elk1 phosphorylation. Our data suggest that MSCs interact with macrophages during adipogenic differentiation. CXCR2 regulates the adipogenic differentiation of MSCs by altering the activation of the p38/ERK-Elk1 signaling pathway.     

Selenium promotes adipogenic determination and differentiation of chicken embryonic fibroblasts with regulation of genes involved in fatty acid uptake,triacylglycerol synthesis and lipolysis

Selenium (Se) has been utilized in the differentiation of primary pig and rat preadipocytes, indicating that it may have proadipogenic potential; however, some studies have also demonstrated that Se has antiadipogenic activity. In this study, chicken embryonic fibroblasts (CEFs) were used to investigate the role of Se in adipogenesis in vitro and in ovo. Se supplementation increased lipid droplet accumulation and inhibited proliferation of cultured CEFs isolated from 6-day-old embryos dose-dependently. This suggests that Se may play a role in cell cycle inhibition, thereby promoting the differentiation of fibroblasts to adipocytes. Se did not stimulate adipogenic differentiation of CEFs isolated from 9- to 12-day-old embryos, implying a permissive stage of adipogenic determination by Se at earlier embryonic ages. Microarray analysis comparing control and Se treatments on CEFs from 6-day-old embryos and confirmatory analysis by quantitative real-time polymerase chain reaction revealed that genes involved in adipocyte determination and differentiation, fatty acid uptake and triacylglycerol synthesis were up-regulated. In addition, up-regulation of an anti-lipolytic G0/G1 switch gene 2 and down-regulation of a prolipolytic monoglyceride lipase may lead to inhibition of lipolysis by Se. Both osteogenic and myogenic genes were down-regulated, and several genes related to oxidative stress response during adipogenesis were up-regulated. In ovo injection of Se at embryonic day 8 increased adipose tissue mass by 30% and caused adipocyte hypertrophy in 17-day-old chicken embryos, further supporting the proadipogenic role of Se during the embryonic development of chickens. These results suggest that Se plays a significant role in several mechanisms related to adipogenesis.     

miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ.     

Heparin-binding epidermal growth factor-like growth factor inhibits adipocyte differentiation at commitment and early induction stages

Abstract Adipocytokines, bioactive molecules secreted from adipose tissues, play important roles in physiology, development, and disease. Recently, heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an adipocytokine whose expression correlates with obesity. However, the biological role of fat-secreted HB-EGF is still unclear. In this study, we investigated the effects of HB-EGF on the adipocyte differentiation of C3H10T1/2 pluripotent mesenchymal cells. Upon adipogenic conversion of C3H10T1/2 cells, HB-EGF displayed dynamic changes in expression where an initial decrease was followed by increased levels of expression at later stages. HB-EGF treatment during adipogenic induction inhibited lipid accumulation and decreased the expression of adipocyte molecular markers (fatty acid-binding protein, peroxisome proliferator-activated receptor γ, and CAAT enhancer-binding protein α) and lipogenic genes (glucose transporter, fatty acid synthetase, and lipoprotein lipase). Therefore, HB-EGF has an inhibitory effect on adipocyte differentiation. Administration of HB-EGF at various intervals during adipocyte differentiation revealed that HB-EGF acts during the early stages of adipocyte differentiation, but not at the later stages of differentiation. Furthermore, HB-EGF was able to block the commitment of pluripotent mesenchymal cells to the adipocyte lineage triggered by bone morphogenic protein 4 treatment. These data suggest that HB-EGF acts as a negative regulator of adipogenesis by inhibiting the commitment and early differentiation of the adipose lineage. The inhibitory role of HB-EGF on adipocyte differentiation of pluripotent mesenchymal cells sheds light on potential mechanisms that control adipose tissue homeostasis.     

Long-term neuropeptide Y administration in the periphery induces abnormal baroreflex sensitivity and obesity in rats

Neuropeptide Y (NPY) is an important neuronal element involved in cardiovascular regulation. Since elevated plasma levels of NPY have been observed in numerous pathological situations, this study aimed to determine whether long-term elevated plasma concentrations of NPY could result in aberrant baroreflex sensitivity. Mini-osmotic pump containing NPY (85 μg per 30 days) was subcutaneously implanted between scapulae in male rats for 4 months. The rats treated with NPY showed the following characters compared with control group: (1) attenuated heart rate responding to the increases in mean arterial blood pressure (MABP) induced by phenylephrine, but enhanced heart rate responding to the decreases in MABP induced by sodium nitroprusside; (2) decreased protein levels of substance P (SP) and GluR2, while increased the expression of γ-aminobutyric acid A receptor (GABA(A)R) in brainstem; (3) abdominal obesity indicated by increased body weight and accumulated fat mass in peritoneal cavity; (4) significant increases in total cholesterol, triglycerides, and low density lipoprotein levels in the periphery. These findings indicate that long-term NPY administration in the periphery leads to abnormal baroreflex sensitivity due, at least in part, to the down-regulated expression of SP/GluR2 and elevated expression of GABA(A)R in both protein and RNA levels, which indicate the alternations in glutamate function and GABA action in the nucleus tractus solitarii in NPY-treated rats. Furthermore, long-term NPY administration results in abdominal obesity and dyslipidemia.     

Human bone marrow-derived stromal cells show highly efficient stress-resistant adipogenesis on denatured collagen IV matrix but not on its native counterpart: Implications for obesity

Collagen IV is the major matrix component associated with differentiating adipocytes in adipose tissues, and the understanding of its contribution in adipogenic differentiation could be important for elucidation of mechanisms and processes driving the obesity. Therefore, in the light of our previous findings of differential effects of structural conformation of collagen I matrix on differentiation of bone marrow stromal cells, we investigated whether similar phenomenon occurs on collagen IV matrix in native and denatured structural states. The results of the present study show that native collagen IV is unsupportive of adipogenic differentiation and very little if any adipogenesis occurs on this matrix in the presence of adipogenic stimuli. In sharp contrast to native collagen IV, the same matrix in denatured structural state drives highly efficient adipogenic differentiation suggesting that it might be the major driver of adipogenesis in adipose tissues and that the ratio of native to denatured matrix might regulate the intensity of adipogenesis and possibly underlies the obesity. In contrast to observations that adipogenesis on denatured collagen I (collagen I is the major matrix component in musculoskeletal tissues) is suppressed by stress, adipogenesis on denatured collagen IV appears to be stress-resistant suggesting an explanation for the observed ineffectiveness of physical exercise, i.e. mechanical stress, in the reduction of adipose tissues. The obesity was shown to be associated with overproduction of MMPs and decline in levels of TIMPs. Such a shift in MMP/TIMP balance was considered a consequence of the pathology. In the light of the present study, however, this shift might constitute the primary source of the decease. The findings of the present study suggest strategies for the treatment of obesity, raise significant questions and indicate directions for further experimentation.     

Protein tyrosine phosphatase profiling studies during brown adipogenic differentiation of mouse primary brown preadipocytes

There is a correlation between obesity and the amount of brown adipose tissue; however, the molecular mechanism of brown adipogenic differentiation has not been as extensively studied. In this study, we performed a protein tyrosine phosphatase (PTP) profiling analysis during the brown adipogenic differentiation of mouse primary brown preadipocytes. Several PTPs, including PTPRF, PTPRZ, and DUSP12 showing differential expression patterns were identified. In the case of DUSP12, the expression level is dramatically downregulated during brown adipogenesis. The ectopic expression of DUSP12 using a retroviral expression system induces the suppression of adipogenic differentiation, whereas a catalytic inactive DUSP12 mutant showed no effect on differentiation. These results suggest that DUSP12 is involved in brown adipogenic differentiation and may be used as a target protein for the treatment or prevention of obesity by the regulation of brown adipogenic differentiation. [BMB Reports 2013; 46(11): 539-543]     

Wnt5a regulates the cell proliferation and adipogenesis via MAPK‐independent pathway in early stage of obesity

The early stage of obesity is an important stage in the development of obesity. However, there are few studies which explored the property or changes in obesity at early stage especially involving Wnt5a. The associated gene expression of Wnt5a on cell regeneration and the effect of Wnt5a on rat adipose‐derived stem cell (rASC) proliferation and adipogenesis need additional study. Here, we investigated the changes in obesity at early stage and how Wnt5a regulates rASC regeneration, proliferation, and adipogenesis. Our data revealed that obesity at early stage measured by Lee index presented a state with impaired adipogenesis and more infiltrated inflammatory cells but without significant changes in adipocyte sizes and inflammatory factors. The process might be associated with anti‐canonical Wnt pathway and a reciprocal Wnt5a/JNK pathway. Besides the gene expression of Wnt5a decreased from cell passage 1 to passage 3. The cell proliferation was regulated by increasing dose of Wnt5a with the maximal effect at 50 ng/mL and 50 ng/mL Wnt5a suppressed adipogenic differentiation at middle‐late stage of adipogenesis via anti‐β‐catenin and a mitogen‐activated protein kinase (MAPK) signaling‐independent manner. Accordingly, the research helps to gain further insights into the early stage of obesity and its associated changes on a cellular and molecular level.     

GRK5 deficiency decreases diet-induced obesity and adipogenesis

Identification of the protein factors that regulate the adipogenesis and lipid metabolism of adipose tissue is critical for the understanding of the physiology and pathology of obesity and energy homeostasis. In this study, we found that G protein coupled receptor (GPCR) kinase 5 (GRK5) was expressed at a relatively high level in the white adipose tissue. When fed on a high-fat diet, GRK5(-/-) mice gained significantly less weight and had decreased WAT mass than their wild type littermates, which could not be attributed to alterations in food consumption or energy expenditure. However, GRK5(-/-) mice showed a 30-70% decreased expression of lipid metabolism and adipogenic genes in WAT. Moreover, GRK5(-/-) embryonic fibroblasts and preadipocytes exhibited 40-70% decreased expression of adipogenic genes and impaired adipocyte differentiation when induced in vitro. Taken together, these results suggest that GRK5 is an important regulator of adipogenesis and is crucial for the development of diet-induced obesity.     

Cytoglobin: a potential marker for adipogenic differentiation in preadipocytes in vitro

Obesity, mainly characterized by the excess fat storage, is a global health problem resulting in serious morbidity and mortality. Identification of molecular mechanisms in adipogenic differentiation pathway might lead to development of new strategies for diagnosis, prevention and therapy of obesity and associated diseases. Discovery of new genes and proteins in the differentiation pathway could help to understand the key specific regulators of the adipogenesis. Cytoglobin (Cygb), identified as a new globin family member protein, is expressed in various tissues. Although its interaction with oxygen and nitric oxide indicates the potential role in antioxidant pathways, the exact role remains unclear. In the current study, expression level of Cygb was determined in proliferating and differentiating 3T3-F442A cells by gene expression and protein expression analysis. Results revealed that Cygb expression up-regulated in differentiated cells in parallel with adipogenic differentiation markers; PPARγ, CEBPα and FABP4 expressions. Besides, Cygb overexpression in preadipocytes contributed to the adipogenic differentiation as verified by detection of higher lipid droplets and increased PPARγ, CEBPα and FABP4 expressions with respect to control cells. These findings will shed light on the unknown roles of Cygb in adipogenesis and obesity.     

MicroRNA-425 controls lipogenesis and lipolysis in adipocytes

An increasing number of studies have demonstrated that some microRNAs participate in the regulation of growth and development of adipocytes. The present study shows that microRNA-425-5p (miR-425) is a novel strong regulator of adipogenesis and adipolysis in adipocytes. Forced expression of miR-425 in mice promoted body fat accumulation and the development of obesity due to high-fat intake, whereas silencing miR-425 prevented mice from being obese. Mechanically, the expression of miR-425 is controlled by PPARγ during the adipogenesis process in adipocytes. MiR-425 overexpression resulted in a reduction in the proliferation of 3t3-L1 pre-adipocytes but significantly accelerated cellular adipogenic differentiation. Mapk14, a negative regulator of adipogenesis, was predicted and confirmed as a real target gene of miR-425. Moreover, knocking down miR-425 remarkably intensified intracellular lipolysis and promoted lipid oxidation, which is related to the activation of AMPK, a monitor for intracellular energy balance. MiR-425 activated AMPK not only by decreasing cellular ATP concentrations but also by targeting the gene of Cab39, which is an upstream co-activator of AMPK. The findings of the present study suggest that miR-425 could control adipogenesis and adipolysis in adipocytes by simultaneously triggering multidirectional targets.     

Role of Wnt10b and C/EBPalpha in spontaneous adipogenesis of 243 cells

This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPalpha and PPARgamma. The 243 cells express C/EBPalpha early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPalpha is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPalpha, results in induction of PPARgamma and spontaneous adipogenesis of 243 cells.