Insulin Resistance In Adipocytes Research Articles

A Fructose-Rich Diet Decreases Insulin-Stimulated Glucose Incorporation into Lipids but Not Glucose Transport in Adipocytes of Normal and Diabetic Rats

To study the cellular mechanisms underlying fructose-induced insulin resistance in rats, the effects of fructose feeding on insulin-stimulated glucose transport, oxidation and incorporation into lipids in epididymal adipocytes were evaluated in 27 normal and 27 noninsulin-dependent diabetic male Sprague-Dawley rats. Diabetes was induced by streptozotocin injection 2 d after birth. At 5 wk of age, both normal and diabetic rats were fed a diet containing 62% carbohydrate as fructose, dextrose or cornstarch. Fructose feeding for 6 wk induced glucose intolerance in normal rats (P < 0.05) and aggravated that of diabetic rats (P < 0.05). Plasma triacylglycerol concentration was higher in fructose-fed than in starch-fed or dextrose-fed rats (P < 0.05). Adipocytes of fructose-fed rats had significantly lower maximum insulin-stimulated glucose incorporation into total lipids than those of rats fed starch, and tended (P = 0.22) to have lower production of CO2 from glucose than adipocytes of the other dietary groups. Glucose transport in adipocytes of dextrose-, starch- and fructose-fed rats did not differ. We conclude that in both normal and diabetic rats, a chronic fructose-rich diet induced hypertriacylglycerolemia, glucose intolerance and insulin resistance of adipocytes.

In Vitro Hypoglycemic Diterpenoids from the Roots of Croton yunnanensis.

Fifteen compounds including nine new diterpenes were isolated from the roots of Croton yunnanensis. By HRESIMS, NMR, ECD data, and X-ray diffraction analysis, the new compounds were characterized as eight neo-clerodane diterpenes (compounds 1-8) and one 15,16-dinor-ent-pimarane diterpene (9). All diterpenes were assayed for their hypoglycemic activities. Compounds 1-4, 6, 7, and 10 promoted glucose uptake activity in insulin-resistant 3T3-L1 adipocytes. Compounds 1 and 6 showed insulin sensitizing activity, potentiating conspicuously their glucose uptake activity at a concentration of 20 μM when treated synergistically with low-concentration insulin at 1 nM.

Improvement of obesity by Liupao tea is through the IRS-1/PI3K/AKT/GLUT4 signaling pathway according to network pharmacology and experimental verification

BackgroundObesity is a state of accumulating excessive body fat, charactering by a high blood lipid and associating with various metabolic diseases. As a kind of dark tea, many studies revealed that long-term drinking Liupao tea (LT) can reduce weight (Liu et al., 2014). However, the anti-obesity mechanism and active ingredients of LT are not known. MethodsLiquid chromatography-mass spectrometry (LC-MS) combined with network pharmacology was used to screen the active components and related targets of Liupao tea water extract (LTWE). The key anti-obesity targets and pathways of LTWE were predicted by protein-protein interaction (PPI) networks, and enrichment analyses using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. Then, the active components selected by high-performance liquid chromatography (HPLC) fingerprinting were used together with LTWE in an adipogenic model and insulin resistance (IR) model in vitro. ResultsMost of the compounds identified from LTWE were flavonofids, esters, and amides. Key targets such as RAC-alpha serine/threonine-protein kinase, insulin, and tumor necrosis factor (TNF) were involved in the phosphatidylinositol-3-kinase-protein kinase B (PI3K-AKT) signaling pathway, pathways in cancer, and other pathways. Four active components were screened by network pharmacology combined with HPLC fingerprinting. The in vitro experiment of LTWE and its four active components showed that in insulin-resistant 3T3-L1 cells, LTWE, (−)-epigallocatechin gallate (EGCG) and gallic acid (GA) inhibited adipocyte differentiation. Three factors could inhibit the differentiation of 3T3-L1 cells by decreasing gene expression of peroxisome proliferators-activated receptor γ (PPARγ), fatty acid synthase (FAS), CCAAT/enhancer binding proteins-α (C/EBPα) and interleukin-6 (IL-6). Caffeine and ellagic acid (EA) showed opposite results, but their effects on promoting adipose differentiation diminished with increasing concentrations of drug. In dexamethasone-induced insulin-resistant 3T3-L1 cells, the fluorescence intensity of 2-Deoxy-2-[(7-nitro-2,1,3-Benzoxadiazol-4-yl)amino]-d-glucose revealed that LTWE, GA, EGCG, caffeine, and EA significantly promoted glucose consumption. LTWE, GA, and EA improved insulin resistance in adipocytes by upregulating gene expression of insulin receptor substrate-1 (IRS-1), PI3K, AKT, and glucose transporter 4 (GLUT4). ConclusionLC-MS combined with network pharmacology preliminarianized that LTWE acts mainly on the PI3K-AKT signaling pathway. Cell experiments revealed that the anti-obesity effect of LTWE is the result of multi-component action, which inhibits the proliferation and differentiation of preadipocytes by regulating gene expression of adipogenic transcription factors and proinflammatory factors, and improves IR by activating the IRS-1/PI3K/AKT/GLUT4 pathway.

Statin-Induced Geranylgeranyl Pyrophosphate Depletion Promotes PCSK9–Dependent Adipose Insulin Resistance

Statin treatment is accepted to prevent adverse cardiovascular events. However, statin therapy has been reported to be dose-dependently associated with increased risk for new-onset type 2 diabetes mellitus (T2DM). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed in adipose tissue and is positively correlated with lipid metabolism. It is, however, unknown if PCSK9 participates in adipocyte insulin resistance occurring as a result of statin use. Our goal was to use an in vitro adipose tissue explant approach to support the hypothesis that PCSK9 regulates statin-induced new-onset T2DM. Studies were performed using Pcsk-/- and C57Bl/6J control mice. Pcsk9-/- and control mice were fed a high-fat diet to affect a state of chronically altered lipid metabolism and increased PCSK9. Epididymal fat was excised and incubated with atorvastatin (1 µmol/L) in the absence and presence of insulin or geranylgeranyl pyrophosphate (GGPP). PCSK9 mRNA was evaluated using quantitative rtPCR. We further examined the effects of atorvastatin on insulin-mediated AKT signaling in adipose tissue explants by immunoblotting. Atorvastatin was found to upregulate PCSK9 gene expression in adipose tissue. The metabolic intermediate GGPP is required to downregulate PCSK9 expression. PCSK9 deficiency protects against statin-induced impairments in insulin signaling. Moreover, supplementation with GGPP reversed atorvastatin-induced suppression of insulin signaling. Furthermore, the basal and atorvastatin-stimulated release of free fatty acids was observed in adipose tissue from wild-type mice but not PCSK9 deficient mice. Collectively, we describe a novel mechanism for PCSK9 expression in adipose tissue that could mediate statin-impaired adipose insulin resistance.

MiR-210-3p Promotes Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Targeting SOCS1-Mediated NF-κB Pathway.

Under the condition of chronic obesity, an increased level of free fatty acids along with low oxygen tension in the adipose tissue creates a pathophysiological adipose tissue microenvironment (ATenv), leading to the impairment of adipocyte function and insulin resistance. Here, we found the synergistic effect of hypoxia and lipid (H + L) surge in fostering adipose tissue macrophage (ATM) inflammation and polarization. ATenv significantly increased miR-210-3p expression in ATMs which promotes NF-κB activation-dependent proinflammatory cytokine expression along with the downregulation of anti-inflammatory cytokine expression. Interestingly, delivery of miR-210-3p mimic significantly increased macrophage inflammation in the absence of H + L co-stimulation, while miR-210-3p inhibitor notably compromised H + L-induced macrophage inflammation through increased production of suppressor of cytokine signaling 1 (SOCS1), a negative regulator of the NF-κB inflammatory signaling pathway. Mechanistically, miR-210 directly binds to the 3'-UTR of SOCS1 mRNA and silences its expression, thus preventing proteasomal degradation of NF-κB p65. Direct delivery of anti-miR-210-3p LNA in the ATenv markedly rescued mice from obesity-induced adipose tissue inflammation and insulin resistance. Thus, miR-210-3p inhibition in ATMs could serve as a novel therapeutic strategy for managing obesity-induced type 2 diabetes.

Never-in-Mitosis A-Related Kinase 8 (NEK8) Regulates Adipogenesis, Glucose Homeostasis, and Obesity.

Adipogenesis is a complex biological process and the leading main cause of obesity. We evaluated the role of never-in-mitosis A-related kinase 8 (NEK8) in adipocyte development and insulin sensitivity in the present study. NEK8 expression was manipulated using a specific shRNA or the NEK8-full-length expressing recombinant plasmids. The interaction between NEK8 and Tafazzin (TAZ, an oncogenic transcriptional regulator) was examined by Co-immunoprecipitation (Co-IP) and confocal immunofluorescence staining. Western blot assay was performed to determine the protein expression. The in vivo role of NEK8 was explored in a mouse model of high-fat diet- (HFD-) induced insulin resistance. During adipogenesis, the expression of NEK8 was elevated while TAZ was downregulated. Overexpression of NEK8 promoted lipid accumulation and expression of markers for adipocyte differentiation. Mechanically, NEK8 interacted with TAZ and suppressed its expression in adipocytes. Functionally, lentiviral-mediated NEK8 inhibition ameliorates HFD-induced insulin resistance in adipocytes. These findings suggest that NEK8 plays a critical role in adipocyte proliferation, providing novel insight into the link between NEK8 and type 2 diabetes- (T2DM-) related obesity.

Impact and mechanism of CHL1 in insulin resistant adipocytes and insulin resistant mouse model induced by high glucose and high fat

Objective: To investigate the role and mechanism of cell adhesion molecule L1 like (CHL1) in insulin resistant adipocytes and insulin resistant mouse model induced by high glucose and high fat. Methods: The 3T3-L1 preadipocytes were randomly divided into control group (transfected with empty vector) and CHL1 overexpression group (transfected with CHL1 vector), cells were then induced to mature adipocytes by insulin, and insulin resistance was then induced by high sugar and high fat. The glucose content was measured to determine the glucose consumption of cells from the two groups. Protein expression levels of CHL1 and glucose transporter 4 (GLUT4), serine/threonine protein kinase (AKT) phosphorylation levels were detected by Western blot (WB), the mRNA expression levels of TNF-α and IL-6 were detected by real-time quantitative PCR (RT-qPCR). 24 C57BL/6 adult male mouse were randomly divided into conventional diet group (regular group), high-fat diet group (high-fat group), empty vector overexpression+high-fat group and CHL1 overexpression+high-fat group (n=6 each group). CHL1 overexpression was induced by tail vein injection of lentivirus. Four months later, mice were sacrificed, body weight was determined, and the epididymal white adipose tissue was collect. Hematoxylin-eosin staining (HE) was used to observe the pathology of mouse epididymal white adipose tissue, the expression of CHL1 was evaluated by immunohistochemical staining(IHC), RT-qPCR was used to detect the mRNA expression levels of CHL1, TNF-α and IL-6 in mouse epididymal white adipose tissue. Results: In vitro, glucose consumption was significantly higher in the CHL1 overexpression group than in the control group (P<0.05), and the protein expressions of CHL1 and GLUT4 were higher in the CHL1 overexpression group than those in the control group (P<0.01), and the mRNA expressions levels of TNF-α and IL-6 were lower in the CHL1 overexpression group than those in the control group (P<0.01). In vivo, the body weight and epididymal white adipose tissue of mouse were higher in the high-fat group and the empty vector overexpression+high-fat group than those in the conventional group (P<0.01), which were lower in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group (P<0.01). HE results showed that the volume of epididymal white adipocytes was larger in the high-fat group and the overexpression control+high-fat group than that in the conventional group, which was smaller in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group (P<0.01). The mRNA expression levels of IL-6 and TNF-α in epididymal white adipose tissue of mice were higher in the high-fat group and the empty vector overexpression+high-fat group than those in the conventional group (P<0.01), which were lower in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group (P<0.05). IHC results showed that protein expression of CHL1 in epididymal white adipose tissue was lower in the high-fat group and the empty vector overexpression+high-fat group than in regular group, which was upregulated in the CHL1 overexpression+high fat group than in the empty vector overexpression+high-fat group (P<0.01). RT-qPCR results showed that mRNA expression of CHL1 in epididymal white adipose tissue was lower in the high-fat group and the empty vector overexpression+high-fat group than in regular group (P<0.01), which was higher in the CHL1 overexpression+high fat group than in the empty vector overexpression+high fat group (P<0.01). Conclusion: Overexpression of CHL1 can improve insulin resistance in adipocytes and mouse insulin resistance model induced by high glucose and high fat, and the beneficial effects might be mediated by the inhibition of AKT activation and the reduction of related inflammatory responses.

ODP011 Single Nuclei RNA-Sequencing of Frozen Human Adipose Tissue and PET/CT Imaging Coupled With Deep-Learning Reveals Single-Cell Landscape and Transcriptional Dynamics Related to Adipocyte Hypertrophy and in Vivo Dysmetabolism

Abstract Background Adipocyte hypertrophy is associated with glucose and lipid dysmetabolism, ectopic fat accumulation, and metabolic complications independently of the level of obesity [1]. In this study, we aimed to explore potential mechanisms driving the obesity-independent, hypertrophy-associated dysmetabolism at a single-cell resolution and to assess the applicability of single nuclei RNA-sequencing of flash-frozen human subcutaneous adipose tissue (AT) to probe for these metabolic differences. Methods In 40 healthy participants, peri-umbilical subcutaneous adipose tissue biopsies were collected by needle aspiration to determine adipocyte size. Tissue uptake of dietary fat was quantified via oral 18-fluoro-thiaheptadecanoic acid administration with positron emission tomography (PET) [2]. Rates of fatty acid oxidation and esterification were measured using intravenous [11C]-palmitate with PET and multi-compartmental modeling. PET/CT segmentation using deep convolutional neural networks was done with DeepImageTranslator [3,4]. Single nuclei RNA-sequencing was performed in two individuals with AT hypertrophy vs. hyperplasia but matched for the same sex, ethnicity, glucose-tolerance status, BMI, total and percent body fat, and waist circumference. Nuclei were extracted from frozen AT and purified by flow cytometry following a protocol that we have developed and validated. Capture of single nuclei and preparation of RNA libraries were done with 10X Genomics technology. Results Unsupervised clustering identified a number of cell-type markers identical to those previously reported in murine epididymal AT and human AT. We found reduced quantities of fibroadipogenic cells per volume of AT with fat cell hypertrophy. Furthermore, the fraction of DPP4+ fibroblasts is increased with AT hypertrophy. Analyses of RNA velocity shows decreased velocity of adipocyte transformation from a transcriptionally active (TADA) phenotype to mature adipocytes in AT hypertrophy. Furthermore, in AT hypertrophy, adipocytes from the early TADA stage are destined to become insulin-resistant adipocytes, whereas those in AT hyperplasia are developing into an insulin-sensitive phenotype. Metabolic imaging results show that hypertrophied subcutaneous AT is associated with lower subcutaneous and higher visceral AT volume and dietary fatty acid storage capacity. Conclusion We have documented, for the first time, the applicability and consistency of single-nuclei RNA-sequencing of frozen human subcutaneous AT. We uncovered changes in cell clusters and subclusters of adipocytes, fibroblasts, pericytes, endothelial and immune cells as well as differences in transcriptional dynamics and developmental trajectory. [1] Ye RZ, et al. Endocrine reviews. 2022 Feb;43(1): 35-60. [2] Montastier É, Ye RZ, et al. American Journal of Physiology-Endocrinology and Metabolism. 2021 Jun 1;320(6): E1093-106. [3] R. Z. Ye, et al., DeepImageTranslator: A free, user-friendly graphical interface for image translation using deep-learning and its applications in 3D CT image analysis, SLAS Technology, https://doi.org/10.1016/j.slast.2021.10. 014, [4] Ye EZ, Ye EH, Ye RZ. DeepImageTranslator V2: analysis of multimodal medical images using semantic segmentation maps generated through deep learning. bioRxiv. 2021 Jan 1. Presentation: No date and time listed

Tumor necrosis factor-α promotes lipolysis and reduces insulin sensitivity by activating nuclear factor kappa B and c-Jun N-terminal kinase in primary bovine adipocytes.

Sustained lipolysis and insulin resistance increase the risk of metabolic dysfunction in dairy cows during the transition period. Proinflammatory cytokines are key regulators of adipose tissue metabolism in nonruminants, but biological functions of these molecules in ruminants are not well known. Thus, the objective of this study was to investigate whether tumor necrosis factor-α (TNF-α) could affect insulin sensitivity and lipolysis in bovine adipocytes as well as the underlying mechanisms. Bovine adipocytes (obtained from the omental and mesenteric adipose depots) isolated from 5 Holstein female calves (1 d old) with similar body weight (median: 36.9 kg, range: 35.5-41.2 kg) were differentiated and used for (1) treatment with different concentrations of TNF-α (0, 0.1, 1, or 10 ng/mL) for 12 h; (2) pretreatment with 10 μM lipolytic agonist isoproterenol (ISO) for 3 h, followed by treatment with or without 10 ng/mL TNF-α for 12 h; and (3) pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 μM for 2 h) and nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082 (10 μM for 1 h) followed by treatment with or without 10 ng/mL TNF-α for 12 h. The TNF-α increased glycerol content in supernatant, decreased triglyceride content and insulin-stimulated phosphorylation of protein kinase B suggesting activation of lipolysis and impairment of insulin sensitivity. The TNF-α reduced cell viability, upregulated mRNA abundance of Caspase 3 (CASP3), an apoptosis marker, and increased activity of Caspase 3. In addition, increased phosphorylation of NF-κB and JNK, upregulation of mRNA abundance of interleukin-6 (IL-6), TNFA, and suppressor of cytokine signaling 3 (SOCS3) suggested that TNF-α activated NF-κB and JNK signaling pathways. Furthermore, ISO plus TNF-α-activated NF-κB and JNK signaling pathway to a greater extent than TNF-α alone. Combining TNF-α and ISO aggravated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. In the absence of TNF-α, inhibition of NF-κB and JNK did not alter glycerol content in supernatant, triglyceride content or insulin-stimulated phosphorylation of protein kinase B. In the presence of TNF-α, inhibition of NF-κB and JNK alleviated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. Overall, TNF-α impairs insulin sensitivity and induces lipolysis and apoptosis in bovine adipocytes, which may be partly mediated by activation of NF-κB and JNK. Thus, the data suggested that NF-κB and JNK are potential therapeutic targets for alleviating lipolysis dysregulation and insulin resistance in adipocytes.

Disruption of adipocyte YAP improves glucose homeostasis in mice and decreases adipose tissue fibrosis

ObjectiveAdipose tissue is a very dynamic metabolic organ that plays an essential role in regulating whole-body glucose homeostasis. Dysfunctional adipose tissue hypertrophy with obesity is associated with fibrosis and type 2 diabetes. Yes-associated protein 1 (YAP) is a transcription cofactor important in the Hippo signaling pathway. However, the role of YAP in adipose tissue and glucose homeostasis is unknown. MethodsTo study the role of YAP with metabolic stress, we assessed how increased weight and insulin resistance impact YAP in humans and mouse models. To further investigate the in vivo role of YAP specifically in adipose tissue and glucose homeostasis, we developed adipose tissue-specific YAP knockout mice and placed them on either chow or high fat diet (HFD) for 12–14 weeks. To further study the direct role of YAP in adipocytes we used 3T3-L1 cells. ResultsWe found that YAP protein levels increase in adipose tissue from humans with type 2 diabetes and mouse models of diet-induced obesity and insulin resistance. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. On an HFD, adipose tissue YAP knockout mice had improved glucose tolerance compared to littermate controls. Perigonadal fat pad weight was also decreased in knockout animals, with smaller adipocyte size. Adipose tissue fibrosis and gene expression associated with fibrosis was decreased in vivo and in vitro in 3T3-L1 cells treated with a YAP inhibitor or siRNA. ConclusionsWe show that YAP is increased in adipose tissue with weight gain and insulin resistance. Disruption of YAP in adipocytes prevents glucose intolerance and adipose tissue fibrosis, suggesting that YAP plays an important role in regulating adipose tissue and glucose homeostasis with metabolic stress.

Astragaloside IV Regulates Insulin Resistance and Inflammatory Response of Adipocytes via Modulating CTRP3 and PI3K/AKT Signaling.

Emerging evidence showed that adipocytes are important regulators in controlling insulin resistance in type 2 diabetes mellitus (T2DM). So far, compounds isolated from natural plants have been widely studied for their roles in alleviating T2DM-associated complications. This work evaluated the actions of astragaloside IV (AS-IV) on insulin resistance and inflammatory biomarker expression in adipocytes and explored the potential mechanisms. Glucose consumption of the adipocytes was determined by a glucose assay kit; the mRNA expression levels of glucose transporter type 4 (GLUT-4), interleukin-6 (IL-6), TNF-α and C1q tumor necrosis factor-related protein 3 (CTRP3) were measured by quantitative real-time PCR (qRT-PCR); the protein levels were determined by western blot assay and enzyme-linked immunosorbent assay. AS-IV concentration-dependently increased glucose consumption in the insulin resistance adipocytes. Further qRT-PCR results showed that AS-IV concentration-dependently reduced adipocyte IL-6 and TNF-α expression. Moreover, GLUT-4 expression in adipocytes was also significantly upregulated by AS-IV. Furthermore, we found that AS-IV concentration-dependently increased CTRP3 expression in adipocytes. CTRP3 silence decreased glucose consumption, upregulated IL-6 and TNF-α expression and downregulated GLUT-4 mRNA expression in 200µM AS-IV-treated adipocytes. Moreover, AS-IV treatment enhanced the activity of phosphoinositide 3-kinase (PI3K)/AKT signaling in adipocytes, which was markedly attenuated by CTRP3 silencing. Importantly, inhibition of PI3K/AKT signaling also attenuated AS-IV induced an increase in glucose consumption and GLUT-4 expression and a decrease in IL-6 and TNF-α expression of adipocytes. Collectively, our data indicated that AS-IV attenuated insulin resistance and inflammation in adipocytes via targeting CTRP3/PI3K/Akt signaling.

Betulinic acid improves TNF-α-induced insulin resistance by inhibiting negative regulator of insulin signalling and inflammation-activated protein kinase in 3T3-L1 adipocytes

Context Obesity is related to insulin resistance, and adipose tissue-secreted TNF-α may play a role in inducing obesity. TNF-α activates inflammatory protein kinase and impairs insulin signalling. Objectives We investigated the effect of betulinic acid on insulin resistance caused by TNF-α treatment in 3T3-L1 adipocytes. Material and methods 3T3-L1 was exposed to TNF-α in the presence and absence of betulinic acid. Various parameters such as glucose uptake assay, cell viability, expression of proteins involved in insulin resistance were studied. Results Betulinic acid increased glucose uptake in TNF-α pre-treated cells and inhibited the activation of PTP1B and JNK and reduced IκBα degradation. Tyrosine phosphorylation was increased, and serine phosphorylation was decreased in IRS-1. Discussion Betulinic acid restored TNF-α impaired insulin signalling and increased PI3K activation and phosphorylation of Akt and increased plasma membrane expression of GLUT 4, which stimulated glucose uptake concentration-dependently. Conclusion These results suggest that betulinic acid is effective at improving TNF-α-induced insulin resistance in adipocytes via inhibiting the activation of negative regulator of insulin signalling and inflammation-activated protein kinase and may potentially improve insulin resistance.

Prenylated flavonoids from Morus nigra and their insulin sensitizing activity

Six undescribed prenylated flavonoids, nigragenons H–M, and four known compounds, were isolated from Morus nigra L. Their structures were elucidated through extensive analysis of spectroscopic data, and their absolute configurations were established by time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculation. The insulin sensitizing activities of all compounds were investigated using insulin-resistant 3T3-L1 adipocytes. At a high concentration (30 μM), all compounds except nigragenon I enhanced insulin-stimulated glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, nigragenons J–L and the promoted adiponectin secretion in the model cells. Among them, nigragenon L showed the most potent effect at a low concentration of 10 μM, which was comparable to that of rosiglitazone at a concentration of 1 μM. Furthermore, using the Lantha Screen™ TR-FRET assay, nigragenon L was confirmed to be the ligand of PPARγ, showing potent binding affinity toward PPARγ with an IC50 value of 2.8 μM.

Effects of two triterpenoids from Nigella sativa seeds on insulin resistance of 3T3-L1 adipocytes.

Insulin resistance (IR) is a physiological abnormality that occurs when insulin fails to activate the signal transduction pathway in target organs. It was found that supplementation of Nigella sativa seeds with oral antidiabetic medicines helps improve blood glucose control by enhanced β cells activity and alleviation of IR. However, the activities and related mechanisms of phytochemicals from N. sativa seeds have not been thoroughly explored. In this study, the effects of two triterpenoids, 3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]-28-O-[α-L-rhamnose-(1→4)-β-D-glucopyranose-L-(1→6)-β-D-glucopyranose]-hederagenin (Hxrarg) and 3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]-hederagenin (Hxra), on IR were studied by 3T3-L1 adipocytes model. The results demonstrated that Hxrarg and Hxra inhibited maturation of 3T3-L1 preadipocytes, dramatically stimulated glucose uptake of IR-3T3-L1 adipocytes, promoted transcription of IRS, AKT, PI-3K, and GLUT4 mRNA. Western Blot results suggested that Hxrarg and Hxra were able to markedly up-regulate expression of p-IRS, p-AKT, PI-3K, and GLUT4 proteins. These findings could provide a basic foundation for the continued development and application of N. sativa in medicine and functional foods.

The Stress-Responsive microRNA-34a Alters Insulin Signaling and Actions in Adipocytes through Induction of the Tyrosine Phosphatase PTP1B.

Metabolic stresses alter the signaling and actions of insulin in adipocytes during obesity, but the molecular links remain incompletely understood. Members of the microRNA-34 (miR-34 family play a pivotal role in stress response, and previous studies showed an upregulation of miR-34a in adipose tissue during obesity. Here, we identified miR-34a as a new mediator of adipocyte insulin resistance. We confirmed the upregulation of miR-34a in adipose tissues of obese mice, which was observed in the adipocyte fraction exclusively. Overexpression of miR-34a in 3T3-L1 adipocytes or in fat pads of lean mice markedly reduced Akt activation by insulin and the insulin-induced glucose transport. This was accompanied by a decreased expression of VAMP2, a target of miR-34a, and an increased expression of the tyrosine phosphatase PTP1B. Importantly, PTP1B silencing prevented the inhibitory effect of miR-34a on insulin signaling. Mechanistically, miR-34a decreased the NAD+ level through inhibition of Naprt and Nampt, resulting in an inhibition of Sirtuin-1, which promoted an upregulation of PTP1B. Furthermore, the mRNA expression of Nampt and Naprt was decreased in adipose tissue of obese mice. Collectively, our results identify miR-34a as a new inhibitor of insulin signaling in adipocytes, providing a potential pathway to target to fight insulin resistance.

Cytokines secreted from adipose tissues mediate tumor proliferation and metastasis in triple negative breast cancer

BackgroundObesity is a high-risk factor for development and poor prognosis of triple-negative breast cancer (TNBC), which was considered as a high malignant and poor clinical outcome breast cancer subtype. TNBC proliferation and migration regulated by obesity is complex. Here, we studied effects of cytokines secreted from adipose tissue on development of TNBC.MethodsForty postmenopausal cases by Yuebei People’s Hospital of Shaoguan with stage I/IIA TNBC were enrolled. Cytokine concentrations were examined using ELISA analysis. Proliferation and migration of TNBC cell lines were performed using CCK8 assays and Transwell tests. The Log-rank (Mantel-Cox) test, two-tailed Mann-Whitney U test and two-tailed unpaired t test were performed using GraphPad Prism 8.4.2.ResultsSurvival analysis indicated that obese patients with TNBC had worse disease free survival (DFS) as compared with normal weight group (Hazard Ratio 4.393, 95% confidence interval (CI) of ratio 1.071–18.02, p < 0.05). Obese patients with TNBC had severe insulin resistance and high plasma triglycerides. However, plasma adiponectin concentration was decreased and interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) concentration was increased in obese TNBC patients as compared with the nonobese group. The similar results were found in the cytokine secretion from adipose tissues and insulin-resistant adipocytes. The secretion of adipose tissue from obese TNBC patients could promote proliferation and migration of TNBC cell lines, including MDA-MB-157, MDA-MB-231, MDA-MB-453 and HCC38 cells. These TNBC cell lines co-incubated with insulin-resistant 3T3-L1 adipocytes or supplementing these cytokines medium also exhibited increase of proliferative and migratory capacity.ConclusionTNBC patients with obesity had worse prognosis compared with the normal weight groups. Alteration of cytokines secreted from adipose tissues mediated proliferation and migration of TNBC, leading to tumor progression in TNBC patients with obesity.

Modulating effects of steviol and steviol glycosides on adipogenesis, lipogenesis, glucose uptake and insulin resistance in 3T3-L1 adipocyte model

• Stevia-derived compounds modulate adipogenic and lipogenic gene expression. • Steviol decreases lipid accumulation in mature 3T3-L1 adipocytes. • Steviol and stevioside improve glucose uptake and mitigate insulin resistance. Stevia rebaudiana Bertoni is famous for sweetness due to the content of steviol glycosides (SG). This study aimed to determine the effect of SG (stevioside, rebaudioside A) and steviol on adipogenesis and lipogenesis in the 3T3-L1 model. Test compounds were also analysed for their ability to affect glucose uptake in hypertrophied insulin-resistant 3T3-L1 adipocytes. The most active compound in modulating adipogenesis, lipogenesis and insulin resistance was steviol, SG metabolism product. Steviol (10 μM, 100 μM) significantly down-regulated the expression of adipogenic transcription factors (PPARγ, C/EBPα, SREBP1) and lipogenic genes (FAS, aP2, LPL), which caused decreased lipid accumulation and triglyceride content in adipocytes. Treatment of insulin-resistant adipocytes with steviol (1 μM) and stevioside (1 μM, 10 μM) increased GLUT-4 transcript level and improved glucose uptake. Steviol also lowered resistin gene expression, which may mitigate insulin resistance in hypertrophied adipocytes. This study promotes SG as potential health-promoting ingredients in low-calorie functional foods.

Inhibition of Adipose Tissue Beiging by HIV Integrase Inhibitors, Dolutegravir and Bictegravir, Is Associated with Adipocyte Hypertrophy, Hypoxia, Elevated Fibrosis, and Insulin Resistance in Simian Adipose Tissue and Human Adipocytes.

For people living with HIV, treatment with integrase-strand-transfer-inhibitors (INSTIs) can promote adipose tissue (AT) gain. We previously demonstrated that INSTIs can induce hypertrophy and fibrosis in AT of macaques and humans. By promoting energy expenditure, the emergence of beige adipocytes in white AT (beiging) could play an important role by limiting excess lipid storage and associated adipocyte dysfunction. We hypothesized that INSTIs could alter AT via beiging inhibition. Fibrosis and gene expression were measured in subcutaneous (SCAT) and visceral AT (VAT) from SIV-infected, dolutegravir-treated (SIVART) macaques. Beiging capacity was assessed in human adipose stromal cells (ASCs) undergoing differentiation and being exposed to dolutegravir, bictegravir, or raltegravir. Expression of beige markers, such as positive-regulatory-domain-containing-16 (PRDM16), were lower in AT of SIVART as compared to control macaques, whereas fibrosis-related genes were higher. Dolutegravir and bictegravir inhibited beige differentiation in ASCs, as shown by lower expression of beige markers and lower cell respiration. INSTIs also induced a hypertrophic insulin-resistant state associated with a pro-fibrotic phenotype. Our results indicate that adipocyte hypertrophy induced by INSTIs is involved via hypoxia (revealed by a greater hypoxia-inducible-factor-1-alpha gene expression) in fat fibrosis, beiging inhibition, and thus (via positive feedback), probably, further hypertrophy and associated insulin resistance.

Microbiota and adipocyte mitochondrial damage in type 2 diabetes are linked by Mmp12+ macrophages.

Microbiota contribute to the induction of type 2 diabetes by high-fat/high-sugar (HFHS) diet, but which organs/pathways are impacted by microbiota remain unknown. Using multiorgan network and transkingdom analyses, we found that microbiota-dependent impairment of OXPHOS/mitochondria in white adipose tissue (WAT) plays a primary role in regulating systemic glucose metabolism. The follow-up analysis established that Mmp12+ macrophages link microbiota-dependent inflammation and OXPHOS damage in WAT. Moreover, the molecular signature of Mmp12+ macrophages in WAT was associated with insulin resistance in obese patients. Next, we tested the functional effects of MMP12 and found that Mmp12 genetic deficiency or MMP12 inhibition improved glucose metabolism in conventional, but not in germ-free mice. MMP12 treatment induced insulin resistance in adipocytes. TLR2-ligands present in Oscillibacter valericigenes bacteria, which are expanded by HFHS, induce Mmp12 in WAT macrophages in a MYD88-ATF3-dependent manner. Thus, HFHS induces Mmp12+ macrophages and MMP12, representing a microbiota-dependent bridge between inflammation and mitochondrial damage in WAT and causing insulin resistance.

227-OR: Disruption of Adipocyte YAP Improves Glucose Homeostasis in Mice and Decreases Adipose Tissue Fibrosis

Adipose tissue is a dynamic metabolic organ that is essential for the regulation of whole-body glucose homeostasis. Dysfunctional adipose tissue hypertrophy with obesity is associated with glucose intolerance and insulin resistance underlying type 2 diabetes. Yes-associated protein 1 (YAP) is a transcription cofactor important in the Hippo signaling pathway. However, the role of YAP in adipose tissue and glucose homeostasis is unknown. To understand the role of YAP in vivo under metabolic stress conditions, we assessed how increased weight and insulin resistance impact YAP protein levels. We found that YAP protein levels increase in adipose tissue in humans with type 2 diabetes and mouse models. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. To further investigate the role of YAP in adipose tissue and glucose homeostasis in vivo, we developed adipose tissue-specific YAP knockout (YAP-KO) mice and placed them on either chow or high fat diet (HFD) for 12-14 weeks. On an HFD, adipose tissue YAP-KO mice show improved glucose tolerance compared to controls. Perigonadal fat pad weight was also decreased in knockout animals, with smaller adipocyte size. Adipose tissue fibrosis and gene expression associated with fibrosis was decreased in vivo and in 3T3-L1 cells treated with a YAP inhibitor. Together, these data indicate that YAP is increased in adipose tissue with weight gain and insulin resistance. Disruption of YAP in adipocytes improves glucose tolerance and adipose tissue fibrosis; suggesting that YAP plays an important role in adipose tissue and modulating glucose homeostasis under metabolic stress conditions. Disclosure D.Han: None. R.Aslam: None. T.Ojha: n/a. D.Yuen: Consultant; Fibrocor Therapeutics. C.T.Luk: None. Funding NSERC Discovery Grant (RGPIN-2018-05671) BBDC Gales Family Charitable Foundation Pilot and Feasibility GrantCanadian Institutes of Health Research Project Grant (PJT 168996)