Stromal-vascular Research Articles

EFFECT OF HIGH FAT AND CAGED CHICKEN DIET ON OVARIAN HISTOMORPHOLOGY IN FEMALE ALBINO RATS

Background: Consumption of high fat diet, caged chicken meat and sedentary lifestyle have seriously increased the risks of hyperlipidema that leads to increased ovarian weight as well as ovarian stromal changes associated with difficulties in reproduction. This study is designed to compare the effects of high fat and caged chicken diet on histomorphology of ovaries of female albino rats. Methods: This study was conducted in collaboration with National Institute of Health and Anatomy Department of Islamic International Medical College, Islamabad after approval from Ethics Review Committee. The study duration was 12 months from Sep 2021 to Sep 2022, and was performed on 30 albino rats. Control group A was given standard rat diet. Experimental group B was given High Fat Diet (HFD) of 60% fat. Experimental group C was given cubes of caged chicken meat in the raw form. At the end of experiment, animals’ ovaries were collected and weighed. The samples were processed, sectioned in 5 ?m thickness, stained with H&E and observed under light microscope for stromal vascular changes and congestion. Results: The ovarian weight and stromal vascularity significantly increased in caged chicken diet group C compared to control group A and experimental group B. Conclusion: Caged chicken diet is more harmful than HFD in causing ovarian weight gain and ovarian stromal hypervascularity. Pak J Physiol 2023;19(4):23-5

Bone morphogenic protein 9 is a novel thermogenic hepatokine secreted in response to cold exposure

ObjectiveMaintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. MethodsTo investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. ResultsHepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. ConclusionsBMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.

Adipocytes and Stromal Cells Regulate Brown Adipogenesis Through Secretory Factors During the Postnatal White-to-Brown Conversion of Adipose Tissue in Syrian Hamsters.

Brown adipose tissue (BAT) is a specialized tissue that regulates non-shivering thermogenesis. In Syrian hamsters, interscapular adipose tissue is composed primarily of white adipocytes at birth, which is converted to BAT through the proliferation and differentiation of brown adipocyte progenitors and the simultaneous disappearance of white adipocytes. In this study, we investigated the regulatory mechanism of brown adipogenesis during postnatal BAT formation in hamsters. Interscapular adipose tissue of a 10-day-old hamster, which primarily consists of brown adipocyte progenitors and white adipocytes, was digested with collagenase and fractioned into stromal–vascular (SV) cells and white adipocytes. SV cells spontaneously differentiated into brown adipocytes that contained multilocular lipid droplets and expressed uncoupling protein 1 (Ucp1), a marker of brown adipocytes, without treatment of adipogenic cocktail such as dexamethasone and insulin. The spontaneous differentiation of SV cells was suppressed by co-culture with adipocytes or by the addition of white adipocyte-conditioned medium. Conversely, the addition of SV cell-conditioned medium increased the expression of Ucp1. These results indicate that adipocytes secrete factors that suppress brown adipogenesis, whereas SV cells secrete factors that promote brown adipogenesis. Transcriptome analysis was conducted; however, no candidate suppressing factors secreted from adipocytes were identified. In contrast, 19 genes that encode secretory factors, including bone morphogenetic protein (BMP) family members, BMP3B, BMP5, and BMP7, were highly expressed in SV cells compared with adipocytes. Furthermore, the SMAD and MAPK signaling pathways, which represent the major BMP signaling pathways, were activated in SV cells, suggesting that BMPs secreted from SV cells induce brown adipogenesis in an autocrine manner through the SMAD/MAPK signaling pathways. Treatment of 5-day-old hamsters with type I BMP receptor inhibitor, LDN-193189, for 5 days reduced p38 MAPK phosphorylation and drastically suppressed BAT formation of interscapular adipose tissue. In conclusion, adipocytes and stromal cells regulate brown adipogenesis through secretory factors during the postnatal white-to-brown conversion of adipose tissue in Syrian hamsters.

Unraveling Secretory Mechanisms that Control Pentraxin 3 Secretion in Adipocytes During Inflammation

As a soluble pattern recognition receptor, Pentraxin 3 (PTX3) plays an important role in innate immunity and obesity-associated metabolic inflammation. PTX3 is abundantly expressed and secreted in adipocytes in response to lipopolysaccharide (LPS) stimulation. Appropriate regulation of PTX3 secretion is critical for maintaining inflammatory homeostasis. This study aims to unravel the mechanisms that control PTX3 secretion in adipocytes during LPS-induced inflammation. Upon 6h treatment of LPS, PTX3 expression and secretion were significantly induced in 3T3-L1 and stromal-vascular (SV) differentiated adipocytes, but to a lesser extent in SV cells or 3T3-L1 fibroblasts. However, LPSdoes not significantly stimulate PTX3 expression and secretion in macrophages. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms for controlling LPS-stimulated PTX3 secretion. 3T3-L1 adipocytes were treated with LPS for 6h in the presence or absence of various inhibitors blocking protein secretion from the Golgi complex (Monensin and Brefeldin A), mitochondrial oxidation (carbonyl cyanide 3-chlorophenylhydrazone [CCCP]), autophagy-lysosome (chloroquine and 3-methyladenine) and inflammasome (Bay 11–7082 and wedelolactone) activation, or exosome synthesis and trafficking (GW4869, manumycin A, calpeptin, and Y-27632). There were no significant effects of all inhibitors except for Monensin, Brefeldin A, and CCCP on intracellular and secreted levels of PTX3 in adipocytes. We found that Monensin and Brefeldin A significantly blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. Disrupting mitochondrial membrane potential by CCCP caused the reduction in PTX3 secretion from adipocytes. Additionally, we detected PTX3 in exosomes isolated from LPS-treated adipocytes. Inhibiting exosome synthesis by Manumycin A attenuated LPS-stimulated PTX3 secretion in both adipocyte culture media and isolated exosomes but not in the non-exosomal fraction of media, suggesting the involvement of the exosomal pathway in PTX3 secretion. However, the levels of exosomal PTX3 were significantly lower than that of the non-exosomal PTX3, and only 4.3% of secreted PTX3 was detected in the exosomal fraction of cultural media. Inhibiting the Golgi complex pathway blocked both the exosomal and non-exosomal secretion of PTX3 in adipocytes. After further fractionation of isolated crude exosomes by the iodixanol density gradient centrifugation, we showed that the majority of PTX3 was found in the non-extracellular vesicular (EV) fractions; only a small portion of secreted PTX3 overlapped with the exosomal marker CD63 in the small EV fractions. We conclude that PTX3 is secreted mainly through the conventional protein secretion pathway and minimally through the exosomal or EV pathway in response to LPS stimulation.

MiR-340-5p inhibits sheep adipocyte differentiation by targeting ATF7.

Several microRNAs (miRNAs) have been identified to play roles in adipocyte differentiation. However, little is known about their involvement in the differentiation of ovine intramuscular adipocytes. Here, the role of one such miRNA, miR-340-5p, in ovine adipocyte differentiation was investigated. Stromal vascular (SV) cells were isolated from skeletal muscle tissues of 1-month-old lambs and induced to differentiate into mature adipocytes. miRNA mimics and inhibitors were used for miR-340-5p overexpression and knockdown assays. For overexpression and knockdown of activating transcription factor 7 (ATF7), lentivirus infection was performed. Luciferase reporter assay was performed to determine the relationship between miR-340-5p and ATF7. The expression of adipogenesis marker genes, PPARγ, C/EBPα, FABP4, ADIPOQ, and ACC, and formation of lipid droplets were detected after the overexpression and inhibition of miR-340-5p, or upon overexpression or knockdown of ATF7. miR-340-5p inhibited the expression of the marker genes and the formation of lipid droplets. ATF7 positively regulated the expression of the marker genes and the formation of lipids. Thus, ATF7 is the target of miR-340-5p in sheep. Overall, these findings indicate that miR-340-5p acts as an inhibitor of the differentiation of intramuscular adipocytes by targeting ATF7. Our study provides a new theoretical basis for improving sheep meat quality.

ROCK2 inhibition enhances the thermogenic program in white and brown fat tissue in mice.

The RhoA/ROCK-mediated actin cytoskeleton dynamics have been implicated in adipogenesis. The two ROCK isoforms, ROCK1 and ROCK2, are highly homologous. The contribution of ROCK2 to adipogenesis in vivo has not been elucidated. The present study aimed at the in vivo and in vitro roles of ROCK2 in the regulation of adipogenesis and the development of obesity. We performed molecular, histological, and metabolic analyses in ROCK2+/- and ROCK2+/KD mouse models, the latter harboring an allele with a kinase-dead (KD) mutation. Both ROCK2+/- and ROCK2+/KD mouse models showed a lean body mass phenotype during aging, associated with increased amounts of beige cells in subcutaneous white adipose tissue (sWAT) and increased thermogenic gene expression in all fat depots. ROCK2+/- mice on a high-fat diet showed increased energy expenditure accompanying by reduced obesity, and improved insulin sensitivity. In vitro differentiated ROCK2+/- stromal-vascular (SV) cells revealed increased beige adipogenesis associated with increased thermogenic gene expressions. Treatment with a selective ROCK2 inhibitor, KD025, to inhibit ROCK2 activity in differentiated SV cells reproduced the pro-beige phenotype of ROCK2+/- SV cells. In conclusion, ROCK2 activity-mediated actin cytoskeleton dynamics contribute to the inhibition of beige adipogenesis in WAT, and also promotes age-related and diet-induced fat mass gain and insulin resistance.

PTX3 Regulates miR-21 Expression and Secretion in Brown Adipocytes During Lipopolysaccharide-induced Inflammation (P21-072-19)

ObjectivesMetabolic endotoxemia is known to induce systemic inflammation and adipose tissue dysfunction during obesity. Pentraxin 3 (PTX3), a member of the pentraxin family can be induced by inflammatory stimuli in adipocytes. However, how PTX3 regulates inflammation in adipocytes is not fully understood. MicroRNAs (miRNAs) are key regulators of inflammatory gene expression. Specifically, miR-21 plays a role in the resolution of inflammation through inhibiting Toll-like receptor 4 (TLR4) signaling pathway. This study aimed to investigate how PTX3 regulates inflammatory homeostasis involving miR-21 during lipopolysaccharide (LPS) stimulation in brown adipocytes. MethodsUsing PTX3 knockout (KO) mouse and primary stromal-vascular (SV) cell culture models, we determined the effect of PTX3 deficiency on miR-21 expression, secretion, and inflammatory response in brown adipocytes, as well as the rescue effect of recombinant PTX3 on LPS-induced inflammation. ResultsBrown adipose tissue (BAT) had significantly higher levels of Dicer protein expression than inguinal and epididymal adipose depots; fully differentiated brown adipocytes expressed higher levels of Dicer than SV cells. These results suggest that BAT is the major site of miRNA production, and brown adipocyte is the main cell type that produces miRNAs. Moreover, we found brown adipocytes but not SV cells are the LPS responsive cells in miR-21 expression and secretion. In WT brown adipocytes, LPS stimulation significantly up-regulated cellular levels of miR-21. Interestingly, PTX3 deficiency led to a significant increase in the basal levels of both cellular and secreted miR21 compared to WT cells. Unlike in WT cells, LPS treatment suppressed cellular expression as well as secretion of miR-21 in PTX3 KO brown adipocytes. Treatment of recombinant PTX3 slightly up-regulated cellular expression of miR-21, but significantly increased the secretion of miR-21 in PTX3 KO brown adipocytes. Moreover, Treatment of recombinant PTX3 significantly attenuated LPS-stimulated increase in the expression of TNFα and MCP1 genes in PTX3 KO adipocytes. ConclusionsWe conclude that PTX3 plays an anti-inflammatory role in part via regulating the expression and secretion of miR-21. Funding SourcesAmerican Diabetes Association.

Vascular-Associated Muc4/Vwf Co-Localization in Human Conjunctival Malignant Melanoma Specimens—Tumor Metastasis by Migration?

ABSTRACTPurpose: To investigate whether vascular differentiation marker von Willebrand factor (vWf) and proliferation marker KI67 expression correlate with MUC4 localization around stromal tumor vascularization in human conjunctival malignant melanoma (CMM).Materials and methods: For the purposes of this study, we analyzed samples from human CMMs (n = 4), conjunctival compound nevi (n = 7), and samples from healthy conjunctiva (n = 7) for MUC1, 4, and 16 by immunohistochemistry. To test CMM vessel association of MUC4, we investigated the co-localization of MUC4 with vWf or KI67 in human CMM specimens (n = 10) by immunohistochemistry. Also, we investigated the MUC4 localization around vessels of healthy conjunctiva (n = 10).Results: The immunohistochemical analysis demonstrated membrane-associated mucin expression in epithelia of CMM, nevi and healthy conjunctiva, whereas only MUC4 was localized perivascular in CMM tissue in this preliminary analysis. Co-staining analysis with vWf and KI67 demonstrated MUC4 localization around stromal vessels in human CMM specimens. In contrast, no MUC4 localization has been seen around healthy conjunctiva stroma vessels.Conclusions: MUC4 was detected around vWf/KI67-positive CMM stromal vascular tissue, but not around healthy conjunctival stroma vessels. Therefore, we assume that MUC4 might play a role in tumor cell migration toward vessels inducing metastasis.

Cold Exposure Induces Proliferation of Mature Brown Adipocyte in a ß3-Adrenergic Receptor-Mediated Pathway.

Hyperplasia of brown adipose tissue (BAT) is a fundamental mechanism for adaptation to survive in the cold environment in rodents. To determine which cell types comprising BAT contribute to tissue hyperplasia, immunohistochemical analysis using a proliferative marker Ki67 was performed on the BAT from 6-week-old C57BL/6J mice housed at 23°C (control) or 10°C (cold) for 5 days. Interestingly, in the control group, the cell proliferative marker Ki67 was detected in the nuclei of uncoupling protein 1-positive mature brown adipocytes (7.2% ± 0.4% of brown adipocyte), as well as in the non-adipocyte stromal-vascular (SV) cells (19.6% ± 2.3% of SV cells), which include preadiopocytes. The percentage of Ki67-positive brown adipocytes increased to 25.6% ± 1.8% at Day 1 after cold exposure and was significantly higher than the non-cold acclimated control until Day 5 (21.8% ± 1.7%). On the other hand, the percentage of Ki67-positive SV cells gradually increased by a cold exposure and peaked to 42.1% ± 8.3% at Day 5. Injection of a ß3-adrenergic receptor (ß3-AR) agonist for continuous 5 days increased the number of Ki67-positive brown adipocytes even at Day 1 but not that of SV cells. In addition, the ß3-AR antagonist, but not ß1-AR antagonist, attenuated the cold exposure-induced increase in the number of Ki67-positive brown adipocytes. These results suggest that mature brown adipocytes proliferate immediately after cold exposure in a ß3-AR-mediated pathway. Thus, proliferation of mature brown adipocytes as well as preadipocytes in SV cells may contribute to cold exposure-induced BAT hyperplasia.

Loss of DRO1/CCDC80 results in obesity and promotes adipocyte differentiation

To investigate the role of DRO1 in obesity and adipogenesis in vivo, we generated a constitutive Dro1 knockout mouse model and analyzed the effect of DRO1 loss on body growth under standard and high fat diet feeding conditions. Loss of DRO1 resulted in a significant increase in body weight which was accompanied by a substantial expansion of white adipose tissue depots. The obese phenotype could be further enhanced by a high fat dietary challenge which also resulted in impaired glucose metabolism and the development of hepatosteatosis in Dro1 knockout mice. To study the role of DRO1 in adipocyte differentiation, primary stromal-vascular (SV) cells were isolated from inguinal white fat pads of knockout and control mice. In primary SV cells, depletion of DRO1 significantly promoted adipogenesis with upregulation of markers for adipogenesis (Cebpa, Pparg, Adipoq) and lipid metabolism (Dgat1, Dgat2). Our results demonstrate that DRO1 is a crucial regulator of energy homeostasis in vivo and functions as an inhibitor of adipogenesis in primary cells.

Abstract 270: IκB Kinase β Signaling in Adipose Progenitor Cells Promotes Obesity and Metabolic Disorders

Objective: Obesity is associated with a state of chronic low-grade inflammation that is a major contributor to diabetes and cardiovascular diseases. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been implicated in the pathogenesis of obesity-associated metabolic disorders. However, the role of IKKβ in adipose tissue development and metabolism remains elusive. We have recently revealed a potential role of IKKβ in the regulation of adipocyte differentiation. This study aims to investigate the impact of adipose progenitor cell IKKβ deficiency in diet-induced obesity and metabolic disorders. Approach and Results: To determine the role of IKKβ in adipose tissue development, we selectively deleted IKKβ in the white adipose lineage. This was attained by using a PDGFRβ-Cre/IKKβ-loxP system. Cell lineage analysis confirmed that PDGFRβ-Cre is active in primary adipose stromal vascular (SV) cells and PDGFRβ-positive SV cells had higher adipogenic potential than did PDGFRβ-negative cells. PDGFRβ-Cre-mediated IKKβ deletion diminished the ability of SV cells to differentiate into adipocytes. By analyzing mice that selectively lacked IKKβ in the white adipose lineage, we found that deficiency of IKKβ protected mice from high-fat diet-induced obesity. Moreover, IKKβ deficient mice had decreased plasma proinflammatory cytokine levels and enhanced insulin sensitivity. Mechanistically, IKKβ deficiency inhibited proteasome-mediated β-catenin ubiquitination and degradation in adipose SV cells, causing an accumulation of nuclear β-catenin protein levels and impaired adipocyte differentiation. Consistently, CRISPR-mediated IKKβ deletion in murine 3T3-L1 preadipocytes also increased nuclear β-catenin protein levels and blocked 3T3-L1 cell differentiation. Conclusions: Our results establish IKKβ as an important regulator of adipogenesis and adipose tissue development. Overnutrition-mediated IKKβ activation may serve as an initial signal that triggers adipose progenitor cell differentiation in response to consumption of a high-fat diet. Inhibition of IKKβ may present as a novel therapeutic approach to combat obesity and diabetes.

BMP7 drives human adipogenic stem cells into metabolically active beige adipocytes.

Adult humans have a substantial amount of inducible-brown (or beige) fat, which is associated with increased energy expenditure and reduced weight gain via thermogenesis. Despite the identification of key regulators of beige adipogenesis, impacts of dietary factors on adaptive thermogenesis are largely unknown, partly due to a lack of validated human cell models. Bone morphogenetic protein 7 (BMP7) is known to promote brown adipogenesis in rodent and human progenitor cells. However, controversy still surrounds the cellular identity in BMP7-mediated transition of white to brown adipocytes. The aim of this study was to confirm BMP7-derived human adipocytes as a relevant in vitro model of human beige adipocyte by verifying the cellular lineage and metabolic activity. In this study, we hypothesized that pre-exposure of the stromal vascular (SV) fraction of primary human adipogenic precursor cells (hASC) to BMP7 would convert metabolically active brown adipocytes. Our results showed that exposure of hASC to human BMP7 was associated with significant escalation of (1) UCP1 gene expression, a signature gene of brown adipocytes, (2) beige specific marker gene expression (i.e., CD137 and TMEM26), (3) glucose and fatty acid uptake, and (4) basal and cAMP-stimulated oxygen consumption rate compared to white adipocyte control. Taken together, we demonstrated that BMP7 mediates conversion of hASC into metabolically active beige adipocytes. By confirming the cellular identity and metabolic activity, this BMP7-induced human beige adipocytes from hASC should aid in the discovery and assessment of bioactive molecules to promote adaptive thermogenesis.

Nutritional milieu of isolated stromal vascular cells determines their proliferative, adipogenic, and lipogenic capacity in vitro

The objective was to determine the effect of nutritional milieu of isolated stromal vascular (SV) cells on proliferative capacity of preadipocytes, and adipogenic and lipogenic capacity in adipocytes in vitro. Proliferation of the preadipocytes increased over time with 48 and 72 h being greater than 24 h; however, preadipocytes from steers supplemented with corn (LC) had lower proliferation rates compared with those without corn grain supplementation (L) at 72 h. Adipocyte cultures isolated from LC group had higher mean diameter on d 4 and 6, and higher mean volume on d 0, 4, 6, and 12 of culture. Adipocytes from steers supplemented with corn grain (LC) had lower expression of key adipogenic genes during extended days in culture. The results show that prior nutritional treatment of the donor animal used to isolate SV cultures alters their proliferative, adipogenic, and lipogenic capacity in culture. These differences may be related to lower induction/expression of AP2 gene in the adipose cultures from corn supplemented group. Corn grain supplementation to steers grazing legumes could have stimulated more active adipogenic progenitor cells to differentiate, which would leave fewer behind in the SV pool for subsequent isolation.

Eicosapentaenoic acid promotes thermogenic and fatty acid storage capacity in mouse subcutaneous adipocytes

In this study, we determined if eicosapentaenoic acid (EPA) promotes beneficial metabolic activities of subcutaneous adipocytes. Stromal-vascular (SV) cells were isolated from inguinal adipose tissue of C57BL/6 mice and induced to differentiate into adipocytes. EPA effect on thermogenic and mitochondrial gene expression and oxidative metabolism were assessed in inguinal adipocytes. When added to SV cell cultures during 8day differentiation, EPA significantly increased the expression of thermogenic genes UCP1-3, CIDEA and VEGFα. Moreover, EPA increased mitochondrial DNA content and the expression of genes involved in mitochondrial biogenesis including PGC1α, Nrf1 and COXiv. However, this effect was not perceived when EPA was added to mature inguinal adipocytes for 24h, suggesting that EPA exerts its browning effect via recruiting brite adipocytes. Consistently, long-term EPA treatment also upregulated AMPKα phosphorylation and CPT1 expression and increased glucose uptake and GLUT4 mRNA expression, suggesting improved mitochondrial oxidation. Additionally, EPA-treated adipocytes had enlarged lipid droplets and increased expression of triglyceride synthesis genes GPAT1 and GPAT3, while significantly decreased glycerol release and down-regulation of HSL and ATGL gene expression. We conclude that EPA enhances energy dissipation capacity by recruiting brite adipocytes to stimulate oxidative metabolism and reduces fatty acid release by facilitating fatty acid storage in subcutaneous adipocytes.

Preadipocyte and adipose tissue differentiation in meat animals: influence of species and anatomical location.

Early in porcine adipose tissue development, the stromal-vascular (SV) elements control and dictate the extent of adipogenesis in a depot-dependent manner. The vasculature and collagen matrix differentiate before overt adipocyte differentiation. In the fetal pig, subcutaneous (SQ) layer development is predictive of adipocyte development, as the outer, middle, and inner layers of dorsal SQ adipose tissue develop and maintain layered morphology throughout postnatal growth of SQ adipose tissue. Bovine and ovine fetuses contain brown adipose tissue but SQ white adipose tissue is poorly developed structurally. Fetal adipose tissue differentiation is associated with the precocious expression of several genes encoding secreted factors and key transcription factors like peroxisome proliferator activated receptor (PPAR)γ and CCAAT/-enhancer-binding protein. Identification of adipocyte-associated genes differentially expressed by age, depot, and species in vivo and in vitro has been achieved using single-gene analysis, microarrays, suppressive subtraction hybridization, and next-generation sequencing applications. Gene polymorphisms in PPARγ, cathepsins, and uncoupling protein 3 have been associated with back fat accumulation. Genome scans have mapped several quantitative trait loci (QTL) predictive of adipose tissue-deposition phenotypes in cattle and pigs.

EPA promotes fatty acid storage and thermogenic capacity in primary differentiated mouse subcutaneous adipocytes

Adipose tissue dysfunction has been associated with lipid dysregulation and insulin resistance. Compared with visceral fat, subcutaneous adipose tissue has been recently emerged as a lipid storage and thermogenic depot, playing a beneficial role in lipid homeostasis and insulin resistance. N‐3 PUFAs are known to have a role against hyperlipidemia and insulin resistance. Herein, we determined if EPA promotes beneficial metabolic activities of subcutaneous adipocytes. Primary stromal‐vascular (SV) cells isolated from mouse inguinal adipose tissue were cultured and induced to differentiate into adipocytes. EPA was added to SV cell culture during differentiation. On day 8 of differentiation, glucose metabolism, fatty acid storage, and thermogenic capacity were assessed. When compared to controls, EPA‐treated subcutaneous adipocytes had increased levels of basal glucose uptake and GLUT4 mRNA expression, but decreased glucose and lactate levels in the cultured media, suggesting decreased glycolysis. To assess fatty acid storage capacity, lipolysis, fatty acid uptake, and TAG synthesis were determined. We observed that EPA‐treated adipocytes had significantly decreased glycerol release and downregulation of HSL and ATGL gene expression, while markedly increased expression of genes involved in fatty acid uptake, CD36 and TAG synthesis, GPAT1 and GPAT3. Moreover, EPA treatment led to a significant increase in the expression of thermogenic genes including PGC1α, COXiv, UCP1, UCP2 and UCP3 as well as adiponectin expression. We conclude that EPA enhances fatty acid storage and energy dissipation capacity in subcutaneous adipocytes, contributing in part to its metabolic benefits.Grant Funding Source: NIH

Stromal Vascular Cells and Adipogenesis: Cells within Adipose Depots Regulate Adipogenesis.

A collection of investigations indicate the importance of adipose tissue stromal/stem cells to vasculogenesis and angiogenesis during adipogenesis. Early in development the stromal-vascular (S-V) elements control and dictate the extent of adipogenesis. For instance, the vasculature and connective tissue collagen matrix develops before overt adipocyte differentiation. Definitive studies of human adipose tissue stem cells (ADSC) provided an understanding of stem cell identity and function. In this regard, a novel vascular stem cell theory proposes that ADSC are a mixed population of vascular stem cells (VSC) with differential potential proportional to the angiogenic potential of the vasculature. The differential potential of VSC can range considerably in a continuous fashion and can include vascular smooth cells, endothelial cells (EC) and adipocytes. These observations are consistent with fetal adipose tissue studies that show location-dependent angiogenic potential ranging from more to less in regards to a predominant presence of EC and developing arterioles before overt adipogenesis.

Acquisition of pig intramuscular preadipocytes through dedifferentiation of mature adipocytes and establishment of optimal induction conditions

Intramuscular fat deposition is a major contributing factor to variations in pork quality. Understanding the mechanisms driving the differentiation and metabolism of muscle-derived adipocytes is important for regulating the fat deposition in muscle. Studies on intramuscular adipocytes commonly involve stromal-vascular (SV) cell cultures, which contain preadipocytes but also several other types of primordial cells. Hence, it is crucial to obtain pure intramuscular preadipocytes for investigating adipocyte differentiation and metabolism in muscle tissue. In this study, we established cultures of pure intramuscular preadipocytes that were derived from mature adipocytes of newborn pigs. Pure mature adipocytes were isolated from the longissimus dorsi (LD) muscle and allowed to dedifferentiate into fibroblast-like cells in ceiling culture. These fibroblast-like cells turned out to be preadipocytes; they exhibited the ability to redifferentiate into mature adipocytes when adipogenically induced in vitro. The redifferentiation process was confirmed by lipid accumulation in the cytoplasm and expression patterns of peroxisome proliferator-activated receptor gamma 2 (PPARγ2), CCAAT/enhancer binding protein alpha (C/EBPα), lipoprotein lipase (LPL), and adiponectin genes, which were all similar to those observed in previous preadipocyte studies. We optimized the induction conditions for intramuscular preadipocytes by adding 0.25 nM dexamethasone (DEX), 5 μg/mL insulin (INS), and 0.1 mM 3-isobutyl-1-methylxanthine (IBMX). Therefore, this study provides a new model for studying the mechanisms of intramuscular preadipocyte differentiation and metabolism.

Alternative splicing and developmental and hormonal regulation of porcine comparative gene identification-58 (CGI-58) mRNA1

The process of lipolysis is essential for regulating the catabolism of cellular fat stores. Therefore, knowledge of lipolysis contributes to improving porcine production, such as reducing back fat, enhancing lean meat, and controlling marbling. Comparative gene identification-58 (CGI-58) plays an important role in the multi-enzyme-mediated process of lipolysis. It was identified as the co-activator of adipose triglyceride lipase (ATGL), which performs the first step in breaking down triacylglycerol and generating diacylglycerol and NEFA. We cloned and sequenced the CGI-58 cDNA and deduced the AA sequences in 3 breeds of swine (Duroc, Berkshire, and Landrace). Homologies were found with the human, mouse, and chicken for the lipid droplet binding domain, the α/β hydrolase domain, and the lysophosphatidic acid acyltransferase (LPAAT) domain, which demonstrates conservation of CGI-58 across species. An alternatively spliced isoform with an exon 3 deletion was identified. Interestingly, this unique isoform contains the lipid droplet-binding domain but lacks the LPAAT domain due to an open reading frame (ORF) shift that creates a premature stop codon. Furthermore, porcine CGI-58 is expressed in multiple organs and tissues but is most predominant in adipose tissue. Porcine adipose and stromal-vascular (SV) cell fractionation reveals that CGI-58 and ATGL are highly expressed (P < 0.01) in mature adipocytes. The expressions of both CGI-58 and ATGL mRNA were found to increase (P < 0.05) at d 6 of SV cell culture, confirming their upregulation during adipogenesis and differentiation. Also, the results from in vitro cell culture showed that insulin decreased (P < 0.05) the expressions of both CGI-58 and ATGL in a dose-dependent manner. Overall, these results report the cDNA and AA sequences of porcine CGI-58 with identification of its unique alternatively spliced variant. The results of the study also reveal the developmental and hormonal regulation of porcine CGI-58 gene, which contributes to the understanding of the role of CGI-58 in lipid metabolism. These findings suggest that CGI-58 may be a new target for enhancing the quality of pork products as well as offering the potential of CGI-58 for human obesity treatment.

The Effect of dietary fatty acids on the recruitment of brown‐like adipocytes in inguinal white adipose tissue

The physiological significance of brown adipose tissue (BAT) in energy metabolism in adult humans has been recently re‐evaluated. The evidence from recent studies indicates a potential of converting white to brown adipocytes in white adipose tissue (WAT). Herein, we explored the regulatory effect of dietary fatty acids on this conversion in WAT. We found that BAT‐specific UCP1 was expressed at a significantly higher level in inguinal adipose tissue (ING) compared to epididymal adipose tissue in mice. High fat diet (HFD) modulated UCP1 expression in BAT and ING in a different manner. After one week of HFD, UCP1 was increased in BAT, but not in ING of mice. However, UCP1 was dramatically decreased after chronic HFD feeding in both tissues. Additionally, we examined the effects of saturated and polyunsaturated fatty acids on the differentiation of brown‐like adipocytes in primary culture of stromal‐vascular (SV) cells from ING of normal mice. Fatty acids were added to SV cell culture either during 8 days of differentiation or after differentiation for 24 hours. In differentiated adipocytes, 24h treatment with palmitate significantly reduced UCP1, while n‐3 fatty acid (EPA) treatment did not change UCP1 expression. However, UCP1 and PGC1α expression was significantly increased in SV cell cultures after 8‐ day EPA treatment. Our data suggests that EPA is capable of inducing the differentiation of inguinal SV cells into brown‐like adipocytes, but failed to induce UCP1 expression in mature white adipocytes. We conclude that HFD or saturated FA reduces UCP1, while EPA has a potential to increase the recruitment of brown‐like adipocytes in inguinal WAT.Grant Funding Source : General Mills Foundation, MN Obesity Consortium