Objective:
Circulating ANGPTL8 has recently been used as a marker of insulin action. We studied expression and insulin regulation of ANGPTL8 and ANGPTL3 in vivo and in vitro.
Design and Methods:
...Expression of ANGPTL8 and ANGPTL3 was studied in 34 paired samples of human liver and adipose tissue. Effects of insulin on 1) plasma concentrations and adipose tissue expression of ANGPTL8 and ANGPTL3 (in vivo 6-h euglycemic hyperinsulinemia; n = 18), and 2) ANGPTL8 and ANGPTL3 gene and protein expression in immortalized human hepatocytes (IHH) and adipocytes were measured. Effect of ANGPTL3 on secretion of ANGPTL8 in cells stably overexpressing ANGPTL3, -8, or both was determined.
Results:
ANGPTL3 was only expressed in the liver, whereas ANGPTL8 was expressed in both tissues. In vivo hyperinsulinemia significantly decreased both plasma ANGPTL8 and ANGPTL3 at 3 and 6 hours. Insulin increased ANGPTL8 expression in human adipose tissue 14- and 18-fold at 3 and 6 hours and ANGPTL8 was the most insulin-responsive transcript on microarray. Insulin also increased ANPGTL8 in cultured adipocytes and IHH but the protein mainly remained intracellular. In vitro in IHH, insulin decreased ANGPTL3 gene expression and secretion of ANGPTL3 into growth medium. Overexpression of ANGPTL8 in CHO cells did not result in its release into culture medium while abundant secretion occurred in cells co-expressing ANGPTL3 and -8.
Conclusions:
Insulin decreases plasma ANGPTL3 by decreasing ANGPTL3 expression in the liver. Insulin markedly increases ANGPTL8 in adipose tissue and the liver but not in plasma. These data show that measurement of plasma ANGPTL3 but not -8 reflects insulin action in target tissues.
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Adipose tissue (AT) expansion through hyperplasia or hypertrophy requires vascular remodeling that involves angiogenesis. There is quite some evidence that obese white AT (WAT) ...displays altered vasculature. Some studies suggest that this is associated with hypoxia, which is thought to play a role in inducing inflammatory activation of the excessively expanding WAT. Increasing evidence, based on genetic manipulations or treatments with inhibitory or activator pharmaceuticals, demonstrates that AT angiogenesis is crucial for AT metabolic function, and thereby for whole body metabolism and metabolic health. Despite some contradiction between studies, disturbance of WAT angiogenesis in obesity could be an important factor driving WAT dysfunction and the comorbidities of obesity. Endothelial cells (ECs) contribute to healthy WAT metabolism via transport of fatty acids and other plasma components, secretory signaling molecules, and extracellular vesicles (EVs). This communication is crucial for adipocyte metabolism and underscores the key role that the AT endothelium plays in systemic energy homeostasis and healthy metabolism. Adipocytes communicate towards the neighboring endothelium through several mechanisms. The pro-inflammatory status of hypertrophic adipocytes in obesity is reflected in ECs activation, which promotes chronic inflammation. On the other hand, adiponectin secreted by the adipocytes is important for healthy endothelial function, and adipocytes also secrete other pro- or anti-angiogenic effector molecules and a wealth of EVs – however, their detailed roles in signaling towards the endothelium are yet poorly understood. To conclude, targeting AT angiogenesis and promoting the healthy communication between adipocytes and ECs represent potentially promising strategies to treat obesity and its comorbidities.
Angiopoietin like protein 3 (ANGPTL3) is best known for its function as an inhibitor of lipoprotein and endothelial lipases. Due to the capacity of genetic or pharmacologic ANGPTL3 suppression to ...markedly reduce circulating lipoproteins, and the documented cardioprotection upon such suppression, ANGPTL3 has become an emerging therapy target for which both antibody and antisense oligonucleotide (ASO) therapeutics are being clinically tested. While the antibody is relatively selective for circulating ANGPTL3, the ASO also depletes the intra-hepatocellular protein, and there is emerging evidence for cell-autonomous functions of ANGPTL3 in the liver. These include regulation of hepatocyte glucose and fatty acid uptake, insulin sensitivity, LDL/VLDL remnant uptake, VLDL assembly/secretion, polyunsaturated fatty acid (PUFA) and PUFA-derived lipid mediator content, and gene expression. In this review we elaborate on (i) why ANGPTL3 is considered one of the most promising new cardiometabolic therapy targets, and (ii) the present evidences for its intra-hepatocellular or cell-autonomous functions.
•Suppression of ANGPTL3 expression or activity markedly reduces plasma lipoproteins.•ANGPTL3 is an emerging CVD therapy target inhibited by antibody or ASO drugs.•In addition to function as a lipase inhibitor, ANGPTL3 has cell autonomous functions.•Here we summarize why ANGPTL3 is considered as a promising therapy target.•We discuss the evidence and implications of its cell autonomous functions in hepatocytes.
Abstract
Context
Angiopoietin-like 8 (ANGPTL8) has been identified as a key regulator of lipid metabolism.
Design
We addressed the correlation between ANGPTL8 messenger RNA (mRNA) with hallmark ...insulin-regulated and lipogenic genes in human adipose tissue (AT). The regulation of ANGPTL8 expression in adipocytes was studied after inflammatory challenge, and the role of microRNA (miRNA)-221-3p therein was investigated.
Results
ANGPTL8 gene expression in subcutaneous AT (SAT) and visceral AT (VAT) was highly correlated with SLC2A4/GLUT4, ADIPOQ, fatty acyl synthase, and diacylglycerol O-acyltransferase 1. ANGPTL8 mRNA in human adipocytes was suppressed by the inflammatory impact of conditioned medium of lipopolysaccharide-stimulated macrophages, which markedly induced miR-221-3p. MiR-221-3p was shown to target the ANGPTL8 mRNA, and to reduce adipocyte ANGPTL8 protein expression. Analysis of SAT biopsies from 69 subjects ranging from lean to morbidly obese and of VAT of 19 female subjects biopsied during gynecologic surgery demonstrated a trend of negative correlation between ANGPTL8 and miR-221-3p. Significant negative correlation of ANGPTL8 and miR-221-3p was identified in presurgery SAT samples from 22 morbidly obese subjects undergoing bariatric surgery, but vanished after ∼2-year surgery-induced weight loss, which also resulted in a marked reduction of miR-221-3p. ANGPTL8 correlated negatively with the AT inflammatory gene phospholipase A2 G7, whereas miR-221-3p showed a significant positive correlation with this marker. Of note, no correlation was found between AT ANGPTL8 mRNA expression and plasma ANGPTL8.
Conclusions
The inflammation-induced miR-221-3p regulates ANGPTL8 expression in adipocytes. This miRNA impact may become especially prominent under pathologic conditions such as morbid obesity, putatively contributing to the impaired AT lipid metabolism in metabolic disease.
ANGPTL8 mRNA in human AT correlates with insulin-sensitive genes and is reduced by inflammation. ANGPTL8 is targeted by miR-221 and correlates negatively with it in the SAT of morbidly obese subjects.
Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral ...infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.
MicroRNA-107 (miR-107) plays a regulatory role in obesity and insulin resistance, but the mechanisms of its function in adipocytes have not been elucidated in detail. Here we show that overexpression ...of miR-107 in pre- and mature human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes attenuates differentiation and lipid accumulation. Our results suggest that miR-107 controls adipocyte differentiation via CDK6 and Notch signaling. CDK6 is a validated target of miR-107 and was downregulated upon miR-107 overexpression. Notch3, a signaling receptor involved in adipocyte differentiation, has been shown to decrease upon CDK6 depletion; Here Notch3 and its target Hes1 were downregulated by miR-107 overexpression. In mature adipocytes, miR-107 induces a triglyceride storage defect by impairing glucose uptake and triglyceride synthesis. To conclude, our data suggests that miR-107 has distinct functional roles in preadipocytes and mature adipocytes; Its elevated expression at these different stages of adipocytes may on one hand dampen adipogenesis, and on the other, promote ectopic fatty acid accumulation and reduced glucose tolerance.
•miR-107 inhibits differentiation of human adipocytes.•miR-107 downregulates CDK6 expression and Notch3 signaling.•miR-107 impairs lipid storage in mature human adipocytes.•miR-107 regulates genes involved in lipid storage and inflammation.
Communication between adipocytes and endothelial cells (EC) is suggested to play an important role in the metabolic function of white adipose tissue. In order to generate tools to investigate in ...detail the physiology and communication of EC and adipocytes, a method for isolation of adipose microvascular EC from visceral adipose tissue (VAT) biopsies of subjects with obesity was developed. Moreover, mature white adipocytes were isolated from the VAT biopsies by a method adapted from a previously published Membrane aggregate adipocytes culture (MAAC) protocol. The identity and functionality of the cultivated and isolated adipose microvascular EC (AMvEC) was validated by imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting (FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, we established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for the analysis of communication between the two cell types. EC-adipocyte communication in this system was validated by omics analyses, revealing several altered proteins belonging to pathways such as metabolism, intracellular transport and signal transduction in adipocytes co-cultured with AMvEC. In reverse experiments, induction of several pathways including endothelial development and functions was found in AMvEC co-cultured with adipocytes. In conclusion, we developed a robust method to isolate EC from small quantities of human VAT. Furthermore, the MAAECC system established during the study enables one to study the communication between primary white adipocytes and EC or vice-versa and could also be employed for drug screening.
MicroRNA-221-3p (miR-221-3p) is associated with both metabolic diseases and cancers. However, its role in terminal adipocyte differentiation and lipid metabolism are uncharacterized. miR-221-3p or ...its inhibitor was transfected into differentiating or mature human adipocytes. Triglyceride (TG) content and adipogenic gene expression were monitored, global lipidome analysis was carried out, and mechanisms underlying the effects of miR-221-3p were investigated. Finally, cross-talk between miR-221-3p expressing adipocytes and MCF-7 breast carcinoma (BC) cells was studied, and miR-221-3p expression in tumor-proximal adipose biopsies from BC patients analyzed. miR-221-3p overexpression inhibited terminal differentiation of adipocytes, as judged from reduced TG storage and gene expression of the adipogenic markers SCD1, GLUT4, FAS, DGAT1/2, AP2, ATGL and AdipoQ, whereas the miR-221-3p inhibitor increased TG storage. Knockdown of the predicted miR-221-3p target, 14-3-3γ, had similar antiadipogenic effects as miR-221-3p overexpression, indicating it as a potential mediator of mir-221-3p function. Importantly, miR-221-3p overexpression inhibited de novo lipogenesis but increased the concentrations of ceramides and sphingomyelins, while reducing diacylglycerols, concomitant with suppression of sphingomyelin phosphodiesterase, ATP citrate lyase, and acid ceramidase. miR-221-3p expression was elevated in tumor proximal adipose tissue from patients with invasive BC. Conditioned medium of miR-221-3p overexpressing adipocytes stimulated the invasion and proliferation of BC cells, while medium of the BC cells enhanced miR-221-3p expression in adipocytes. Elevated miR-221-3p impairs adipocyte lipid storage and differentiation, and modifies their ceramide, sphingomyelin, and diacylglycerol content. These alterations are relevant for metabolic diseases but may also affect cancer progression.
•miR-221-3p suppresses adipogenesis, putatively mediated by 14-3-3γ downregulation.•miR-221-3p suppresses de novo lipogenesis in adipocytes.•miR-221-3p alters adipocyte ceramide, sphingomyelin and diacylglycerol content.•miR-221-3p expression is elevated in adipocytes adjacent to breast carcinoma.•Adipocyte miR-221-3p elevation may promote metabolic disease and cancer progression.
A polymorphism of TM6SF2 associates with hepatic lipid accumulation and reduction of triacylglycerol (TAG) secretion, but the function of the encoded protein has remained enigmatic. We studied the ...effect of stable TM6SF2 knock-down on the lipid content and composition, mitochondrial fatty acid oxidation and organelle structure of HuH7 hepatoma cells. Knock-down of TM6SF2 resulted in intracellular accumulation of TAGs, cholesterol esters, phosphatidylcholine (PC) and phosphatidylethanolamine. In all of these lipid classes, polyunsaturated lipid species were significantly reduced while saturated and monounsaturated species increased their proportions. The PCs encountered relative and absolute arachidonic acid (AA, 20:4n-6) depletion, and AA was also reduced in the total cellular fatty acid pool. Synthesis and turnover of the hepatocellular glycerolipids was enhanced. The TM6SF2 knock-down cells secreted lipoprotein-like particles with a smaller diameter than in the controls, and more lysosome/endosome structures appeared in the knock-down cells. The mitochondrial capacity for palmitate oxidation was significantly reduced. These observations provide novel clues to TM6SF2 function and raise altered mebrane lipid composition and dynamics among the mechanism(s) by which the protein deficiency disturbs hepatic TAG secretion.
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•TM6SF2 depletion in HuH7 cells induced TAG and cholesterol ester accumulation.•TM6SF2 depletion resulted in relative enrichment of lipids with SFAs and MUFAs.•TM6SF2 depletion caused loss of arachidonic acid (20:4n-6) from PCs.•TM6SF2 depletion was associated with a reduced rate of mitochondrial FA oxidation.•TM6SF2 depletion altered membrane and organelle dynamics of HuH7 cells.
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