Insulin Resistance In 3T3-L1 Adipocytes Research Articles

A Neutral Polysaccharide from Medicago Sativa L.: Structural Properties and Hypoglycemic Activity In Vitro and In Vivo.

Medicago sativa polysaccharides (MSPs) are beneficial compounds extracted from Medicago sativa L. that exhibit multiple medicinal activities. However, little is known about their hypoglycemic effects. In this study, MSP-II-a, a neutral polysaccharide with an Mw of 4.3×104 Da, was isolated and purified from M. sativa L. Monosaccharide composition analysis determined that MSP-II-a was composed of arabinose, glucose, galactose, mannose, rhamnose, and xylose in a molar ratio of 2.1 : 4.0 : 1.1:0.4 : 1.4 : 1.1. Structural characterization of MSP-II was performed using a combination of methylation analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The results showed that MSP-II-a was mainly comprised of 1,4-p-Glc, 1,3,4-Rha, and 1,3-p-Gal glycosidic linkages, revealing a mesh-like texture with irregular blade shapes. In vitro assays demonstrated that MSP-II-a, at concentrations of 200 and 400 μg/mL, promoted glucose uptake in insulin-resistant 3T3-L1 adipocytes. In vivo studies have shown that MSP-II-a significantly alleviates insulin resistance by reducing fasting blood glucose levels and increasing hepatic glycogen synthesis in HFD/STZ-induced diabetic mice. These findings revealed that MSP-II-a is a promising source of bioactive polysaccharides with potential hypoglycemic activity.

Effect of Alpha-Phellandrene on Glucose Uptake and Adipogenesis in Insulin Resistant 3T3-L1 Adipocytes: an in vitro and in silico Approach

Effect of Alpha-Phellandrene on Glucose Uptake and Adipogenesis in Insulin Resistant 3T3-L1 Adipocytes: an in vitro and in silico Approach

Lactobacillus amylovorus KU4 induces adipose browning in obese mice by regulating PP4C.

We previously reported that Lactobacillus amylovorus KU4 (LKU4) promotes adipocyte browning in mice fed a high-fat diet (HFD mice) in part by remodeling the PPARγ transcription complex. However, the mechanism through which LKU4 enables PPARγ to drive adipocyte browning remains elusive. Here, we report that LKU4 inhibits the expression of PP4C in inguinal white adipose tissue of HFD mice and in insulin-resistant 3T3-L1 adipocytes, which promotes SIRT1-dependent PPARγ deacetylation by activating AMPK, leading to the browning of adipocytes. Consistently, the silencing of PP4C further enhances this pathway. Furthermore, we observed that lactate, a key LKU4 metabolite, reduces insulin-induced PP4C expression and suppresses PP4C inhibition of PPARγ deacetylation and transcriptional activity via AMPK-SIRT1, thereby facilitating the browning of adipocytes. Together, these data demonstrate that LKU4 promotes the AMPK-SIRT1-PPARγ pathway by inhibiting PP4C, thereby facilitating adipocyte browning in HFD mice.

The SWGEDWGEIW from Soybean Peptides Reduces Insulin Resistance in 3T3-L1 Adipocytes by Activating p-Akt/GLUT4 Signaling Pathway.

Diabetes mellitus, a group of metabolic disorders characterized by persistent hyperglycemia, affects millions of people worldwide and is on the rise. Dietary proteins, from a wide range of food sources, are rich in bioactive peptides with anti-diabetic properties. Notably, the protective mechanism of the single peptide SWGEDWGEIW (TSP) from soybean peptides (SBPs) on insulin resistance of adipocytes in an inflammatory state was investigated by detecting the lipolysis and glucose absorption and utilization of adipocytes. The results showed that different concentrations of TSP (5, 10, 20 µg/mL) intervention can reduce 3T3-L1 adipocytes' insulin resistance induced by inflammatory factors in a dose-dependent manner and increase glucose utilization by 34.2 ± 4.6%, 74.5 ± 5.2%, and 86.7 ± 6.1%, respectively. Thus, TSP can significantly alleviate the lipolysis of adipocytes caused by inflammatory factors. Further mechanism analysis found that inflammatory factors significantly reduced the phosphorylation (p-Akt) of Akt, two critical proteins of glucose metabolism in adipocytes, and the expression of GLUT4 protein downstream, resulting in impaired glucose utilization, while TSP intervention significantly increased the expression of these two proteins. After pretreatment of adipocytes with PI3K inhibitor (LY294002), TSP failed to reduce the inhibition of p-Akt and GLUT4 expression in adipocytes. Meanwhile, the corresponding significant decrease in glucose absorption and the increase in the fat decomposition of adipocytes indicated that TSP reduced 3T3-L1 adipocytes' insulin resistance by specifically activating the p-Akt/GLUT4 signal pathway. Therefore, TSP has the potential to prevent obesity-induced adipose inflammation and insulin resistance.

Phenols from Potentilla anserina L. Improve Insulin Sensitivity and Inhibit Differentiation in 3T3-L1 Adipocytes in Vitro.

Potentilla anserina L., a well-known perennial herb, is widely used in traditional Tibetan medicine and used as a delicious food in humans. The present investigation reports on the activity of P. anserina phenols (PAP) in regulating glycolipid metabolism in 3T3-L1 adipocytes. Insulin sensitivity tests showed that PAP improved insulin-stimulated glucose uptake by promoting the phosphorylation of serine/threonine kinase Akt. Moreover, an assay involving the differentiation of 3T3-L1 preadipocytes demonstrated that PAP also decreased the accumulation of lipid droplets by suppressing the expression of adipokines during the differentiation process. In addition, the underlying mechanism from the aspects of energy metabolism and oxidative stress is also discussed. The improvement in energy metabolism was supported by an increase in mitochondrial membrane potential (MMP) and intracellular ATP. Amelioration of oxidative stress was supported by decreased levels of intracellular reactive oxygen species (ROS). In summary, our findings suggest that PAP can ameliorate the disorder of glycolipid metabolism in insulin resistant 3T3-L1 adipocytes by improving energy metabolism and oxidative stress and might be an attractive candidate for the treatment of diabetes.

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.

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.

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.

HM-chromanone attenuates TNF-α-mediated inflammation and insulin resistance by controlling JNK activation and NF-κB pathway in 3T3-L1 adipocytes

Obesity is a major public health problem worldwide and causes inflammation and insulin resistance in adipose tissue. We investigated the ability of (E)-5-hydroxy-7-methoxy-3-(2′-hydroxybenzyl)-4-chromanone (HM-chromanone) isolated from Portulaca oleracea to attenuate the activation of inflammatory cytokines and signaling pathways associated with tumor necrosis factor (TNF)-α-mediated inflammation and insulin resistance in 3T3-L1 adipocytes. TNF-α triggers the release of inflammatory cytokines and activation of the mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathways. In this study, HM-chromanone inhibited the production of inflammatory cytokines and chemokines [TNF-α, interleukin (IL)-6, IL-1β, and monocyte chemoattractant protein 1] involved in inflammation and insulin resistance. Furthermore, TNF-α treatment increased c-Jun-NH2 terminal kinase (JNK) phosphorylation, whereas HM-chromanone significantly decreased JNK phosphorylation in a dose-dependent manner. TNF-α treatment increased the activation of inhibitor kappa B (IκB) kinase (IKK), IκBα, and NF-κBp65 compared with that of the control. However, HM-chromanone significantly blocked IKK, IκBα, and NF-κBp65 activation. Upon adipocyte stimulation with TNF-α, phosphorylated insulin receptor substrate (pIRS)-1 serine 307 levels increased and pIRS-1 tyrosine 612 levels decreased compared with those of the control. Upon treatment with HM-chromanone, serine 307 phosphorylation of IRS-1 was inhibited and tyrosine 612 phosphorylation of IRS-1 was increased. Thus, HM-chromanone improved TNF-α-mediated inflammation and insulin resistance by regulating JNK activation and the NF-κB pathway, thereby reducing inflammatory cytokine secretion and inhibiting serine phosphorylation of IRS-1 in the insulin signaling pathway. These results suggest the potential of HM-chromanone to improve inflammatory conditions and insulin resistance in adipocytes.

MiRNA-27a mediates insulin resistance in 3T3-L1 cells through the PPARγ.

The biological actions of insulin have been originated by activation of membrane receptors, which trigger a diversity of signaling pathways in facilitating their biological activities. Insulin homeostasis functions in promoting metabolism balance and promotes cell growth and proliferation. If these mechanisms are reformed, this could lead to insulin resistance as a result of defective insulin signaling triggered by mutations in receptors or effector molecules located downstream or by abnormal posttranslational modifications. The purpose of this is to preliminarily investigate the mechanism of miRNA-27a-mediating insulin resistance in 3T3-L1 cells. Insulin resistance in 3T3-L1 adipocytes as a cell model was induced by tumor necrosis factor-alpha (TNF-α) and the miRNA-27a expression in 3T3-L1 adipocytes had been experiential. The regulation of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA by miRNA-27a had been studied by reverse transcription receptor polymerase chain reaction (RT-PCR). MiRNA-27a was up-regulated in 3T3-L1 cells, miRNA-27a mimics reserved expression of PPARγ mRNA, and miRNA-27a inhibitors up-regulated the expression of PPARγ mRNA. The insulin resistance in 3T3-L1 cells mediated by miRNA-27a may be achieved by targeting PPARγ.

Carnosic Acid Attenuates the Free Fatty Acid-Induced Insulin Resistance in Muscle Cells and Adipocytes

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine kinases including JNK, mTOR, and p70 S6K cause serine phosphorylation of the insulin receptor substrate (IRS) and have been implicated in insulin resistance. Activation of AMP-activated protein kinase (AMPK) increases glucose uptake, and in recent years, AMPK has been viewed as an important target to counteract insulin resistance. We reported previously that carnosic acid (CA) found in rosemary extract (RE) and RE increased glucose uptake and activated AMPK in muscle cells. In the present study, we examined the effects of CA on palmitate-induced insulin-resistant L6 myotubes and 3T3L1 adipocytes. Exposure of cells to palmitate reduced the insulin-stimulated glucose uptake, GLUT4 transporter levels on the plasma membrane, and Akt activation. Importantly, CA attenuated the deleterious effect of palmitate and restored the insulin-stimulated glucose uptake, the activation of Akt, and GLUT4 levels. Additionally, CA markedly attenuated the palmitate-induced phosphorylation/activation of JNK, mTOR, and p70S6K and activated AMPK. Our data indicate that CA has the potential to counteract the palmitate-induced muscle and fat cell insulin resistance.

Adrenomedullin ameliorates palmitic acid-induced insulin resistance through PI3K/Akt pathway in adipocytes.

White adipose tissue (WAT) dysfunction has been associated with adipose tissue low-grade inflammation and oxidative stress leading to insulin resistance (IR). Adrenomedullin (ADM), an endogenous active peptide considered as an adipokine, is associated with adipocytes function. We evaluated the protective effects of ADM against IR in 3T3-L1 adipocytes treated by palmitic acid (PA) and in visceral white adipose tissue (vWAT) of obese rats fed with high-fat diet. We found that endogenous protein expressions of ADM and its receptor in PA-treated adipocytes were markedly increased. PA significantly induced impaired insulin signaling by affecting phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) axis and glucose transporter-4 (GLUT-4) levels, whereas ADM pretreatment enhanced insulin signaling PI3K/Akt and GLUT-4 membrane protein levels, decreased pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) and IL-6 levels, and improved oxidative stress accompanied with reduced reactive oxygen species (ROS) levels and increased anti-oxidant enzymes manganese superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx1) and catalase (CAT) protein expressions. Furthermore, ADM treatment not only improved IR in obese rats, but also effectively restored insulin signaling, and reduced inflammation and oxidative stress in vWAT of obese rats. This study demonstrates a prevention potential of ADM against obesity-related metabolic disorders, due to its protective effects against IR, inflammation and oxidative stress in adipocytes.

Total flavonoids from Lagerstroemia speciosa (L.) Pers inhibits TNF-α-induced insulin resistance and inflammatory response in 3T3-L1 adipocytes via MAPK and NF-кB signaling pathways

Adipocyte tissue inflammation orchestrates the occurrence and development of diseases induced by obesity. Here, the effects of Lagerstroemia speciosa (L.) Pers total flavonoids (LTF) on TNF-α-induced insulin resistance (IR) and inflammatory response in 3T3-L1 adipocytes and underlying mechanisms were studied. Cell glucose uptake test confirmed that TNF-α could induce IR in 3T3-L1 adipocytes. The sensitivity of 3T3-L1 adipocytes to insulin was increased by LTF treatment. The phosphorylation of IRS1 and Akt in TNF-α-treated 3T3-L1 adipocytes was markedly restored by LTF treatment. The results revealed that interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) mRNA expression was elevated in TNF-α-administered 3T3-L1 adipocytes, whereas the mRNA expression of adiponectin (ADPN) was considerably decreased. When treated with 50 μg/mL LTF, the IL-6 and MCP-1 mRNA expression levels were drastically reduced, and the ADPN mRNA expression level was significantly raised. Moreover, the concentration of IL-6 in the supernatant of the medium was decreased after LTF treatment compared with the model group. Western blotting results showed that the phosphorylation of p38, JNK, and ERK of mitogen-activated protein kinase (MAPK) signaling pathways and IкB of NF-кB signaling pathway were inhibited. It suggested that LTF improves TNF-α-induced IR and inflammatory response in 3T3-L1 adipocytes via inhibiting MAPK and NF-кB signaling pathways.

꾸지뽕 열매 발효식초의 3T3-L1 세포에서 항염증 및 인슐린 저항성 개선 효과

Obesity is associated with chronic low-grade adipose tissue inflammation, leading to insulin resistance. Inflammation associated with insulin resistance is an interesting area of biomedical research and is expected to affect insulin signaling pathways via the downregulation of glucose transporters. In the present study, we investigated the inhibitory effects of Maclura tricuspidata fruit vinegar (MFV) against TNF-α-induced inflammation and insulin resistance in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were pretreated with MFV at various concentrations and then cultured with TNF-α to induce insulin resistance. Then, lipid droplets, inflammatory cytokine and glucose uptake, PPARγ and GLUT4 expression, and IRS-1 phosphorylation were analyzed. MFV attenuated the TNF-α-induced decrease in lipid droplets and glucose uptake and inhibited TNF-α-induced inflammatory cytokine, IL-6, and MCP-1 production. MFV reversed the decrease in adiponectin produced by TNF-α. Furthermore, MFV upregulated the TNF-α-induced suppression of PPARγ and GLUT4 protein expression and reduced the TNF-α-induced phosphorylation of IRS-1. These findings suggest that MFV effectively ameliorates TNF-α-induced insulin resistance; accordingly, we recommend the use of MFV for the control and management of insulin resistance and its associated complications.

Comparing the effect of cinnamaldehyde and metformin on expression of MiR320 and MiR26-b in insulin resistant 3T3L1 adipocytes

BackgroundSome micro-RNAs are linked with insulin resistance and their related signals. They can act as a predicting factor for respond to anti-diabetic medications. Cinnamon extract is considered as an herbal drug in Type 2 Diabetes. PurposeThis study was designed to evaluate the effect of Cinnamon extract on miR320 and miR26-b expression in insulin-resistant adipocytes in comparison to Metformin. Methods3T3L1 pre-adipocytes were cultured in DMEM medium and differentiated into adipocytes. Then, the cells were induced to insulin resistance with high glucose and high insulin medium, and treated with 100µg/ml Cinnamaldehyde, 10mM Metformin and combination of both. The expression of miRs was determined by quantitative real-time PCR method. GLUT4 protein expression was determined by western blotting method. ResultsMiR320 was significantly over-expressed and conversely miR26-b expression was significantly decreased in insulin resistant 3T3L1 adipocytes compared to non-insulin resistant adipocytes (P-value<0.01). Cinnamaldehyde treatment, similar to Metformin decreased the content of miR320 and increased the content of miR26-b after 2h (P-value<0.01). Drug treatment increased GLUT4 protein expression (P-value<0.05). ConclusionThese findings suggest Cinnamaldehyde similar to Metformin can change miRs expression related to insulin resistance toward pre-insulin resistance conditions.

Lipocalin-type Prostaglandin D2 Synthase appears to function as a Novel Adipokine Preventing Adipose Dysfunction in response to a High Fat Diet

Adipose dysfunction is the primary defect in obesity that contributes to the development of dyslipidemia, insulin resistance, cardiovascular diseases, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and some cancers. Previously, we demonstrated the development of NAFLD in lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice regardless of diet. In the present study, we examined the role of L-PGDS in adipose in response to a high fat diet. We observed decreased expression of L-PGDS in adipose tissue and concomitant lower plasma levels in a dietary model of obesity as well as in insulin resistant 3T3-L1 adipocytes. We show reduced adiponectin expression and phosphorylation of AMPK in white adipose tissue of L-PGDS KO mice after 14 weeks on a high fat diet as compared to control C57BL/6 mice. We also observe an increased fat content in L-PGDS KO mice as demonstrated by adipocyte hypertrophy and increased expression of lipogenenic genes. We confirmed our in vivo findings in in vitro 3T3-L1 adipocytes, using an enzymatic inhibitor of L-PGDS (AT56). Rosiglitazone treatment drastically increased L-PGDS expression in insulin resistant 3T3-L1 adipocytes and increased adiponectin expression and AMPK phosphorylation in AT56 treated 3T3-L1 adipocytes. We conclude that the absence of L-PGDS has a deleterious effect on adipose tissue functioning, which further reduces insulin sensitivity in adipose tissue. Consequently, we propose L-PGDS appears to function as a potential member of the adipokine secretome involved in the regulation of the obesity-associated metabolic syndrome.

Effects of Fermented Houttuynia cordata Thunb. on Diabetic Rats Induced by a High-Fat Diet with Streptozotocin and on Insulin Resistance in 3T3-L1 Adipocytes.

Houttuynia cordata Thunb. (plukaow in Thai language) exhibits several biological properties, and many products of H. cordata are therefore commercially available for human consumption, such as fermented juice or tablets as food supplements. This study aimed to investigate the antidiabetic effects of fermented H. cordata (HC) in high-fat diets and streptozotocin-induced diabetic rats. Oral administration of HC at a dose of 100 mg/kg.bw not only maintained bodyweight, food intake, and water consumption but also reduced blood glucose levels and improved glucose tolerance ability in the diabetic rats. Moreover, HC also decreased oxidative stress markers in serum and inflammatory-related mediators in pancreas tissues, indicating the improvement of pancreatic beta-cell function in the diabetic rats. In order to clarify the mechanism of HC, the effects of ethanolic extract of HC (HCE) on insulin resistance were determined in 3T3-L1 adipocytes. FHE could recover glucose uptake and decrease lipolysis in palmitate-treated 3T3-L1 adipocytes. Taken together, these results demonstrate that HC can improve diabetic symptoms by enhancing insulin sensitivity, reducing oxidative stress, and suppressing inflammation.

Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes.

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein–protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort17b. Further, results demonstrate that over-expression of Sort17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.

MiR-223-3p as a potential biomarker and player for adipose tissue dysfunction preceding type 2 diabetes onset

Circulating microRNAs (miRNAs) have been proposed as biomarkers for type 2 diabetes (T2D). Adipose tissue (AT), for which dysfunction is widely associated with T2D development, has been reported as a major source of circulating miRNAs. However, the role of dysfunctional AT in the altered pattern of circulating miRNAs associated with T2D onset remains unexplored. Herein, we investigated the relationship between T2D-associated circulating miRNAs and AT function, as well as the role of preadipocytes and adipocytes as secreting cells of candidate circulating miRNAs. Among the plasma miRNAs related to T2D onset in the CORonary Diet Intervention with Olive oil and cardiovascular PREVention (CORDIOPREV) cohort, baseline miR-223-3p levels (diminished in patients who next developed T2D [incident-T2D]) were significantly related to AT insulin resistance (IR). Baseline serum from incident-T2D participants induced inflammation and IR in 3T3-L1 adipocytes. We demonstrated that tumor necrosis factor (TNF)-α inhibited miR-223-3p secretion while enhancing miR-223-3p intracellular accumulation in 3T3-L1 (pre)adipocytes. Overexpression studies showed that an intracellular increase of miR-223-3p impaired glucose and lipid metabolism in these cells. Our findings provide mechanistic insights into the alteration of circulating miRNAs preceding T2D, unveiling both preadipocytes and adipocytes as miR-223-3p-secreting cells and suggesting that inflammation promotes miR-223-3p intracellular accumulation, which might contribute to (pre)adipocyte dysfunction and body metabolic dysregulation.