Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white adipocytes to analyze cell-cell communication.

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.

Insulin-inducible THRSP maintains mitochondrial function and regulates sphingolipid metabolism in human adipocytes.

BACKGROUND: Thyroid hormone responsive protein (THRSP) is a lipogenic nuclear protein that is highly expressed in murine adipose tissue, but its role in humans remains unknown. METHODS: We characterized the insulin regulation of THRSP in vivo in human adipose tissue biopsies and in vitro in Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. To this end, we measured whole-body insulin sensitivity using the euglycemic insulin clamp technique in 36 subjects [age 40 ± 9 years, body mass index (BMI) 27.3 ± 5.0 kg/m2]. Adipose tissue biopsies were obtained at baseline and after 180 and 360 min of euglycemic hyperinsulinemia for measurement of THRSP mRNA concentrations. To identify functions affected by THRSP, we performed a transcriptomic analysis of THRSP-silenced SGBS adipocytes. Mitochondrial function was assessed by measuring mitochondrial respiration as well as oxidation and uptake of radiolabeled oleate and glucose. Lipid composition in THRSP silencing was studied by lipidomic analysis. RESULTS: We found insulin to increase THRSP mRNA expression 5- and 8-fold after 180 and 360 min of in vivo euglycemic hyperinsulinemia. This induction was impaired in insulin-resistant subjects, and THRSP expression was closely correlated with whole-body insulin sensitivity. In vitro, insulin increased both THRSP mRNA and protein concentrations in SGBS adipocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. A transcriptomic analysis of THRSP-silenced adipocytes showed alterations in mitochondrial functions and pathways of lipid metabolism, which were corroborated by significantly impaired mitochondrial respiration and fatty acid oxidation. A lipidomic analysis revealed decreased hexosylceramide concentrations, supported by the transcript concentrations of enzymes regulating sphingolipid metabolism. CONCLUSIONS: THRSP is regulated by insulin both in vivo in human adipose tissue and in vitro in adipocytes, and its expression is downregulated by insulin resistance. As THRSP silencing decreases mitochondrial respiration and fatty acid oxidation, its downregulation in human adipose tissue could contribute to mitochondrial dysfunction. Furthermore, disturbed sphingolipid metabolism could add to metabolic dysfunction in obese adipose tissue.

Human adipocyte differentiation and composition of disease-relevant lipids are regulated by miR-221-3p.

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.

Angiopoietin-like protein 3, an emerging cardiometabolic therapy target with systemic and cell-autonomous functions.

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.

Hydroxysteroid 17-ß dehydrogenase 13 variant increases phospholipids and protects against fibrosis in nonalcoholic fatty liver disease.

Carriers of the hydroxysteroid 17-ß dehydrogenase 13 (HSD17B13) gene variant (rs72613567:TA) have a reduced risk of NASH and cirrhosis but not steatosis. We determined its effect on liver histology, lipidome, and transcriptome using ultra performance liquid chromatography-mass spectrometry and RNA-seq. In carriers and noncarriers of the gene variant, we also measured pathways of hepatic fatty acids (de novo lipogenesis [DNL] and adipose tissue lipolysis [ATL] using 2H2O and 2H-glycerol) and insulin sensitivity using 3H-glucose and euglycemic-hyperinsulinemic clamp) and plasma cytokines. Carriers and noncarriers had similar age, sex and BMI. Fibrosis was significantly less frequent while phospholipids, but not other lipids, were enriched in the liver in carriers compared with noncarriers. Expression of 274 genes was altered in carriers compared with noncarriers, consisting predominantly of downregulated inflammation-related gene sets. Plasma IL-6 concentrations were lower, but DNL, ATL and hepatic insulin sensitivity were similar between the groups. In conclusion, carriers of the HSD17B13 variant have decreased fibrosis and expression of inflammation-related genes but increased phospholipids in the liver. These changes are not secondary to steatosis, DNL, ATL, or hepatic insulin sensitivity. The increase in phospholipids and decrease in fibrosis are opposite to features of choline-deficient models of liver disease and suggest HSD17B13 as an attractive therapeutic target.

ANGPTL3 deficiency alters the lipid profile and metabolism of cultured hepatocytes and human lipoproteins.

Loss-of-function (LOF) mutations in ANGPTL3, an inhibitor of lipoprotein lipase (LPL), cause a drastic reduction of serum lipoproteins and protect against the development of atherosclerotic cardiovascular disease. Therefore, ANGPTL3 is a promising therapy target. We characterized the impacts of ANGPTL3 depletion on the immortalized human hepatocyte (IHH) transcriptome, lipidome and human plasma lipoprotein lipidome. The transcriptome of ANGPTL3 knock-down (KD) cells showed altered expression of several pathways related to lipid metabolism. Accordingly, ANGPTL3 depleted IHH displayed changes in cellular overall fatty acid (FA) composition and in the lipid species composition of several lipid classes, characterized by abundant n-6 and n-3 polyunsaturated FAs (PUFAs). This PUFA increase coincided with an elevation of lipid mediators, among which there were species relevant for resolution of inflammation, protection from lipotoxic and hypoxia-induced ER stress, hepatic steatosis and insulin resistance or for the recovery from cardiovascular events. Cholesterol esters were markedly reduced in ANGPTL3 KD IHH, coinciding with suppression of the SOAT1 mRNA and protein. ANGPTL3 LOF caused alterations in plasma lipoprotein FA and lipid species composition. All lipoprotein fractions of the ANGPTL3 LOF subjects displayed a marked drop of 18:2n-6, while several highly unsaturated triacylglycerol (TAG) species were enriched. The present work reveals distinct impacts of ANGPTL3 depletion on the hepatocellular lipidome, transcriptome and lipid mediators, as well as on the lipidome of lipoproteins isolated from plasma of ANGPTL3-deficient human subjects. It is important to consider these lipidomics and transcriptomics findings when targeting ANGPTL3 for therapy and translating it to the human context.

Short-Term Cooling Increases Plasma ANGPTL3 and ANGPTL8 in Young Healthy Lean Men but Not in Middle-Aged Men with Overweight and Prediabetes.

Angiopoietin-like proteins (ANGPTLs) regulate triglyceride (TG)-rich lipoprotein distribution via inhibiting TG hydrolysis by lipoprotein lipase in metabolic tissues. Brown adipose tissue combusts TG-derived fatty acids to enhance thermogenesis during cold exposure. It has been shown that cold exposure regulates ANGPTL4, but its effects on ANGPTL3 and ANGPTL8 in humans have not been elucidated. We therefore investigated the effect of short-term cooling on plasma ANGPTL3 and ANGPTL8, besides ANGPTL4. Twenty-four young, healthy, lean men and 20 middle-aged men with overweight and prediabetes were subjected to 2 h of mild cooling just above their individual shivering threshold. Before and after short-term cooling, plasma ANGPTL3, ANGPTL4, and ANGPTL8 were determined by ELISA. In young, healthy, lean men, short-term cooling increased plasma ANGPTL3 (+16%, p < 0.05), ANGPTL4 (+15%, p < 0.05), and ANGPTL8 levels (+28%, p < 0.001). In middle-aged men with overweight and prediabetes, short-term cooling only significantly increased plasma ANGPTL4 levels (+15%, p < 0.05), but not ANGPTL3 (230 ± 9 vs. 251 ± 13 ng/mL, p = 0.051) or ANGPTL8 (2.2 ± 0.5 vs. 2.3 ± 0.5 µg/mL, p = 0.46). We show that short-term cooling increases plasma ANGPTL4 levels in men, regardless of age and metabolic status, but only overtly increases ANGPTL3 and ANGPTL8 levels in young, healthy, lean men.

miR-107 inhibits CDK6 expression, differentiation, and lipid storage in human adipocytes.

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.

MicroRNA-221-3p Regulates Angiopoietin-Like 8 (ANGPTL8) Expression in Adipocytes.

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.