Selective Modulator of Nuclear Receptor PPARy with Reduced Adipogenic Potential Ameliorates Experimental Nephrotic Syndrome

Background: Glomerular disease, often manifesting as nephrotic syndrome (NS) with high proteinuria, can be refractory to standard treatment and is typically associated with hypoalbuminemia, hypercholesterolemia and hypercoagulopathy. We hypothesized that the nuclear receptor PPARy can be selectively modulated using a novel partial agonist, GQ-16, to gain therapeutic advantage over traditional PPARy agonists (e.g. thiazolidinediones) for the treatment of glomerular disease. Methods: Nephropathy was induced with puromycin amino-nucleoside (PAN) in Wistar rats and treated with Pioglitazone (Pio) or GQ-16. Plasma, serum, and urine chemistries were performed, and kidneys, glomeruli, liver, and white adipose tissue (WAT) were harvested. Lipid accumulation and adipogenic gene expression were measured in adipocytes. Results: PAN-induced proteinuria was significantly reduced with Pio to 64% of PAN-value. It was reduced robustly with GQ-16 to 81% of PAN, which was comparable to controls. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct restored glomerular genes downstream of Pio and GQ-16. Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Pio induced more lipid accumulation than GQ-16 in differentiated adipocytes. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees. Conclusions: Selective modulation of PPARy by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria and NS associated co-morbidities, while reducing the adipogenic side-effects conferred by traditional PPARy full agonists.

Abstract P186: Chemical And Mechanical Activation Of The Mechanosensor Piezo1 Alters Adipogenesis In Pvat Preadipocytes

During hypertension, vascular remodeling allows the blood vessel to withstand high blood pressure (BP). This process is well characterized in the media and intima layers of the vessel. In the perivascular adipose tissue (PVAT) there is evidence for fibrosis development during hypertension; but PVAT remodeling is poorly understood. In stem cells (i.e., adipocyte progenitors) from non-PVAT depots, mechanical forces affect adipogenesis' commitment and lipogenic stages. The mechanism involves PIEZO1, a mechanosensor that boosts the differentiation of preadipocytes towards osteogenic and fibroblastic lineages. However, PVAT's particular anatomical location continuously exposes it to forces generated by blood flow that could affect adipogenesis during normotensive and hypertensive states. Our objective was to evaluate PIEZO1's role in the adipogenic potential of preadipocytes. We hypothesize that activation of PIEZO1 reduces Adipogenesis in PVAT preadipocytes. Aortic (APVAT) was collected from male SD rats at 10 weeks of age (n=15) to harvest preadipocytes by Liberase™ digestion. Nonselective cationic channel PIEZO1 activity was evaluated with Ca 2+ indicator Fluo-4AM. Piezo1 was reduced with siRNA. Preadipocytes were differentiated for 4 d in adipogenic media containing PIEZO1 agonist Yoda1 (CON=0; YODA1=10μM). Mechanical strain (MS) was applied with FlexCell System at 12%, half-sine at 1 Hz for 4 d (MS+; MS-). Adipogenesis was evaluated by quantification of adipogenic gene network expression using PCR; lipid accumulation using lipophilic stains (Bodipy, siRNA experiments) or Oil Red O (FlexCell experiments). Adipogenesis efficiency is reported as Adipocyte/Total cells as measured in the IncuCyte Live-Cell ® system. Yoda1 reduced adipogenesis by 33% compared with CON and as expected, increased cytoplasmic Ca 2+ . In si Piezo1 cells, the anti-adipogenic effect of Yoda1 was reversed. MS+ reduced adipogenesis efficiency (0.15±0.06) compared with MS- (0.22±0.1). These data demonstrate that Piezo1 activation in PVAT may be an adaptive or pathogenic mechanism by which adipocyte populations are reduced, thus minimizing their secretion of vasoactive adipokines, and enhancing the deleterious impact of hypertension on PVAT function.

Effect of Temperature and Selection for Growth on Intracellular Lipid Accumulation and Adipogenic Gene Expression in Turkey Pectoralis Major Muscle Satellite Cells

As multipotential stem cells, satellite cells (SCs) have the potential to express adipogenic genes resulting in lipid synthesis with thermal stress. The present study determined the effect of temperature on intracellular lipid synthesis and adipogenic gene expression in SCs isolated from the pectoralis major (p. major) muscle of 7-day-old fast-growing modern commercial (NC) turkeys compared to SCs from unselected slower-growing turkeys [Randombred Control Line 2 (RBC2)]. Since proliferating and differentiating SCs have different responses to thermal stress, three incubation strategies were used: (1) SCs proliferated at the control temperature of 38°C and differentiated at 43° or 33°C; (2) SCs proliferated at 43° or 33°C and differentiated at 38°C; or (3) SCs both proliferated and differentiated at 43°, 38°, or 33°C. During proliferation, lipid accumulation increased at 43°C and decreased at 33°C with the NC line showing greater variation than the RBC2 line. During proliferation at 43°C, peroxisome proliferator-activated receptor-γ (PPARγ) and neuropeptide-Y (NPY) expression was reduced to a greater extent in the NC line than the RBC2 line. At 33°C, expression of PPARγ, NPY, and CCAAT/enhancer-binding protein-β (C/EBPβ) was upregulated, but only in the RBC2 line. During differentiation, both lines showed greater changes in lipid accumulation and in C/EBPβ and NPY expression if the thermal challenge was initiated during proliferation. These data suggest that adipogenic gene expression is more responsive to thermal challenge in proliferating SCs than in differentiating SCs, and that growth-selection has increased temperature sensitivity of SCs, which may significantly affect breast muscle structure and composition.

Novel adipokine asprosin modulates browning and adipogenesis in white adipose tissue

Obesity is an increasingly serious epidemic worldwide characterized by an increase in the number and size of adipocytes. Adipose tissue maintains the balance between lipid storage and energy utilization. Therefore, adipose metabolism is of great significance for the prevention, treatment and intervention of obesity. Asprosin, a novel adipokine, is a circulating hormone mainly secreted by white adipose tissue. Previous studies have shown that asprosin plays a role in fasting-induced homeostasis, insulin resistance, and glucose tolerance. However, whether it can regulate the metabolism of adipose tissue itself has not been studied. This study intended to examine the roles and potential mechanisms of asprosin in adipose regulation. We first demonstrated that the expression level of asprosin was significantly downregulated in subcutaneous white adipose tissue (scWAT) of high-fat diet (HFD)-fed or cold-stimulated mice. Overexpression of asprosin in scWAT reduced heat production, decreased expression of the browning marker uncoupling protein 1 (UCP1) and other browning-related genes, along with upregulation of adipogenic gene expression. Mechanistically, we found that Nrf2 was activated upon cold exposure, but this activation was suppressed after asprosin overexpression. In primary cultured adipocytes, adenovirus mediated asprosin overexpression inhibited adipose browning and aggravated lipid deposition, while Nrf2 agonist oltipraz could reverse these changes. Our findings suggest that novel adipokine asprosin negatively regulated browning and elevate lipid deposition in adipose tissue via a Nrf2-mediated mechanism. Asprosin may be a promising target for the prevention and treatment of obesity and other metabolic diseases.

Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

Ciglitazone Increases Adiponectin and Adipogenic Gene Expression in Bovine Skeletal Muscle Satellite Cells

Background: Ciglitazone is a member of the thiazolidinedione (TZD) family, and specifically binds to peroxisome proliferator-activated receptor-γ (PPARγ) , thereby promoting adipocyte differentiation. We hypothesized that ciglitazone as a PPARγ ligand in the absence of an adipocyte differentiation cocktail would increase adiponectin and adipogenic gene expression in bovine satellite cells (BSC).Methods: Muscle-derived bovine satellite cells were isolated from six, 18-month-old Yanbian Yellow Cattle. The BSC were cultured for 96 h in differentiation medium containing 5 µM ciglitazone (CL), 10 µM ciglitazone (CM), or 20 µM ciglitazone (CH). Control (CON) BSC were cultured only in differentiation medium (containing 2% horse serum).Results: The presence of myogenin, desmin, and paired box 7 (Pax7) proteins were confirmed in the BSC by immunofluorescence staining. The CL, CM, and CH treatments produced higher concentrations of triacylglycerol and lipid droplet accumulation in myotubes than those of the CON treatment. Ciglitazone treatments significantly increased the relative expression of PPARγ , CCAAT/enhancer-binding protein alpha (C/EBPα), C/EBPβ, fatty acid synthase stearoyl-CoA desaturase (SCD) and perilipin 2. Ciglitazone treatments increased gene expression of paired box 3 (Pax3) and Pax7 and decreased expression of myogenic differentiation-1, myogenin, myogenic regulatory factor-5 (MRF5), and MYF4 (P < 0.01). Adiponectin concentration caused by ciglitazone treatments was significantly greater than CON (P < 0.01). RNA sequencing showed that 281 differentially expressed genes (DEGs) were found in the treatments of ciglitazone. DEGs gene ontology (GO) analysis showed that the top 10 GO enrichment significantly changed the biological processes such as protein trimerization, negative regulation of cell proliferation, adipocytes differentiation, and cellular response to external stimulus. KEGG pathway analysis showed that DEGs were involved into p53 signaling pathway, PPAR signaling pathway, biosynthesis of amino acids, TNF signaling pathway, non-alcoholic fatty liver disease (NAFLD), PI3K-Akt signaling pathway, and Wnt signaling pathway.Conclusion: These results indicate that ciglitazone acts as PPARγ agonist, effectively increasing the adiponectin concentration and adipogenic gene expression, and stimulating the conversion of BSC to adipocyte-like cells in the absence of adipocyte differentiation cocktail.

217 Young Scholar Presentation: The use of diverse cattle breeds to understand marbling development and growth

Wagyu cattle have been genetically selected to have superior marbling deposition compared with many other cattle breeds, including Angus. It was hypothesized that greater marbling deposition by Wagyu influenced cattle may be due to an additional wave of adipogenic gene expression compared with Angus cattle. The present study compared the expression of lipid metabolism genes within the longissimus muscle (LM) of Wagyu and Angus sired cattle at similar harvest endpoints: days on feed and age, or body weight (613 kg). Angus sired steers were sired by an Angus bull selected for marbling (T1), while Wagyu sired steers were sired by either a Wagyu bull selected for growth (T2 & T4) or marbling (T3 & T5), at the respective age or body weight endpoints. Cattle were weaned at 7 months of age, entered the feedlot 54 d later, and consumed the same growing (119 d) and finishing ration. Muscle biopsies were collected from the LM posterior to the 13th rib at approximately 7, 9, 11, 14, and 17 months of age (harvest) for T1, T2, and T3 cattle. Muscle biopsies for T4 and T5 cattle were collected when their body weight was similar to T1 cattle at the time of biopsy, approximately 241, 276, 400, 531, and 613 kg (harvest). The high marbling sired Wagyu steers (T3 &T5) had greater marbling scores and percent LM lipid compared with high growth Wagyu (T2 & T4) and Angus sired steers. Gene expression results from high marbling sired Wagyu steers (T3 &T5) demonstrated additional peaks of PPARδ mRNA expression, greater and earlier PPARγ mRNA expression, and greater or additional peaks of GPAM1, LPIN1, and HSL mRNA expression compared with high growth Wagyu (T2 & T4) and Angus sired steers.

β-actin contributes to open chromatin for activation of the adipogenic pioneer factor CEBPA during transcriptional reprograming

We demonstrate that nuclear actin regulates heterochromatin during adipogenesis by mediating Brg1 deposition at specific adipogenic gene loci to ensure binding of the transcription factor CEBPB. This mechanism contributes to CEBPA gene expression during adipogenic transcriptional reprograming.