Effect of plasma triglyceride metabolism on lipid storage in adipose tissue: Studies using genetically engineered mouse models

The obesity epidemic is associated with an increased incidence of type 2 diabetes, cardiovascular morbidity and various types of cancer. A better insight into the molecular mechanisms that underlie adipogenesis and obesity may result in novel therapeutic handles to fight obesity and these associated diseases. Adipogenesis is determined by the balance between uptake of fatty acids (FA) from plasma into adipocytes, intracellular FA oxidation versus esterification of FA into triglycerides (TG), lipolysis of TG by intracellular lipases, and secretion of FA from adipocytes. Here, we review the mechanisms that are specifically involved in the entry of FA into adipose tissue. In plasma, these originating FA are either present as TG within apoB-containing lipoproteins (i.e. chylomicrons and VLDL) or as free FA bound to albumin. Kinetic studies, however, have revealed that TG are the major source of FA entering adipose tissue, both in the fed and fasted condition. In fact, studies with genetically engineered mice have revealed that the activity of lipoprotein lipase (LPL) is a major determinant for the development of obesity. As a general rule, high fat diet-induced adipogenesis is aggravated by stimulated LPL activity (e.g. by adipose tissue-specific overexpression of LPL or deficiency for apoCIII), and attenuated by inhibited LPL activity (e.g. by adipose-specific deficiency for LPL, overexpression of apoCI or angptl4, or by deficiency for apoE or the VLDL receptor). In addition, we describe that the trans-membrane transport of FA and cytoplasmic binding of FA in adipocytes can also dramatically affect adipogenesis. The relevance of these findings for human pathophysiology is discussed.     

Lysophosphatidylserine regulates blood glucose by enhancing glucose transport in myotubes and adipocytes

Lysophosphatidylserine (LPS) is known to have diverse cellular effects, but although LPS is present in many biological fluids, its in vivo effects have not been elucidated. In the present study, we investigated the effects of LPS on glucose metabolism in vivo, and how skeletal muscle cells respond to LPS stimulation. LPS enhanced glucose uptake in a dose- and time-dependent manner in L6 GLUT4myc myotubes, and this effect of LPS on glucose uptake was mediated by a Gα_i and PI 3-kinase dependent signal pathway. LPS increased the level of GLUT4 on the cell surface of L6 GLUT4myc myotubes, and enhanced glucose uptake in 3T3-L1 adipocytes. In line with its cellular functions, LPS lowered blood glucose levels in normal mice, while leaving insulin secretion unaffected. LPS also had a glucose-lowering effect in STZ-treated type 1 diabetic mice and in obese db/db type 2 diabetic mice. This study shows that LPS-stimulated glucose transport both in skeletal muscle cells and adipocytes, and significantly lowered blood glucose levels both in type 1 and 2 diabetic mice. Our results suggest that LPS is involved in the regulation of glucose homeostasis in skeletal muscle and adipose tissue.     

Differences in transport of fatty acids and expression of fatty acid transporting proteins in adipose tissue of obese black and white women

We have reported that the rate of de novo triglyceride (TG) synthesis by omental, but not subcutaneous, adipose tissue was higher in African-American women (AAW) than in Caucasian women (CAW). The purpose of this study was to explore the potential mechanisms underlying this increase. Toward that end, we determined the activities of key enzymes in the pathway of TG synthesis, the rates of uptake of fatty acids by adipocytes, mRNA and protein levels of the fatty acid-transporting proteins FAT/CD36 and FATP, and mRNA and protein levels of PPARgamma in omental fat of AAW and CAW. The results showed 1) no difference in the activity of phosphofructokinase, glycerol-3-phosphate dehydrogenase, or diacylglycerol acyltransferase; 2) a higher rate of fatty acid uptake by adipocytes of the AAW; 3) an increase in the mRNA and protein levels of CD36 and FATP4 in the fat of the AAW; and 4) an increase in the mRNA and protein levels of PPARgamma, which can stimulate the expression of CD36 and FATP. These results suggest that the increase in the transport of fatty acid, which is mediated by the overexpression of the transport proteins in the omental adipose tissue of the AAW, might contribute to the higher prevalence of obesity in AAW.     

Endothelium,the dynamic interface in cardiac lipid transport

Vascular endothelium is the dynamic interface in transport of lipid from blood to myocytes in heart and arteries. The luminal surface of endothelium is the site of action of lipoprotein lipase on chylomicrons and VLDL and the site of uptake of fatty acids from albumin. Fatty acids and monoacylglycerols are transported from the lumen in an interfacial continuum of endothelial and myocyte membranes. Lipoprotein lipase is transferred from myocytes to the vascular lumen, and is anchored there, by proteoheparan sulfate in cell membranes. Insulin, needed for synthesis of lipoprotein lipase and esterfication of fatty acids, is captured from the blood stream and delivered to myocytes by endothelial insulin receptors. Fatty acids, monoacylglycerols, lipoprotein lipase and insulin are transported along the same route, but by different mechanisms. The route involves the plasma membrane of endothelium and myocytes, the membrane lining transendothelial channels, and intercellular contacts. (Mol Cell Biochem116: 181–191, 1992)     

Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone Fiber Matrices

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Expression of Adipocyte Biomarkers in a Primary Cell Culture Models Reflects Preweaning Adipobiology

A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning.     

Ectopic recombination in the central and peripheral nervous system by aP2/FABP4-Cre mice: Implications for metabolism research

aP2-Cre mice have amply been used to generate conditional adipose selective inactivation of important signaling molecules. We show that the efficiency of Cre mediated recombination in adipocytes and adipose selectivity is not always guaranteed. In particular, Cre activity was found in ganglia of the peripheral nervous system (PNS), in adrenal medulla and in neurons throughout the central nervous system (CNS). Because these tissues have an important impact on adipose tissue, care should be taken when using aP2-Cre mice to define the role of the targeted genes in adipose tissue function.     

Liposomes to Target Peripheral Neurons and Schwann Cells

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.     

Ghrelin is a signal to facilitate the utilization of fatty acids and save glucose by the liver,skeletal muscle,and adipose tissues in chicks

Ghrelin, classically known as a central appetite-stimulating hormone, has recently been recognized to play an important role in peripheral tissue energy metabolism. In chicken, contrary to mammal, ghrelin acts as an anorexia signal, increased by fasting and further elevated after refed. In the present study, the effect of ghrelin on glucose/lipid utilization by peripheral tissues was investigated. Injection of exogenous acyl ghrelin reduced plasma triglyceride and glucose levels of chickens at both fasting and fed status. In the in vitro cultured chicken primary hepatocytes, adipocytes, and myoblasts, ghrelin suppressed glucose uptake, stimulated fatty acids uptake and oxidation, and decreased TG content. In hepatocyte, ghrelin increased the activities of LPL and HL, and upregulated the expression levels of gene ACC, CPT1, and PPARα. Ghrelin treatment markedly increased the protein level of p-ACC, PPARγ, PGC1α, and CPT1 in hepatocytes, adipocytes and myoblasts. Inhibition of AMPK activity by Compound C had no influence on glucose uptake by hepatocyte, adipocyte, and myoblast, but further amplified the stimulated fatty acid uptake of adipocyte by ghrelin. The present result demonstrates that ghrelin facilitates the uptake and oxidation of fatty acid and cut down the utilization of glucose by the liver, muscle, and adipose tissues. The result suggests that ghrelin functions as a signal of fatty acid oxidation. The study provides a vital framework for understanding the intrinsic role of ghrelin as a crucial factor in the concerted regulation of metabolic substrate of hepatocytes, adipocytes, and myoblasts.     

Seizure-susceptibility and decreased taurine transport in the genetically epileptic rat

P₂ fractions from brains of genetically seizure-susceptible (SS) rats as compared to seizure-resistant (SR) rats show decreased high affinity uptake of taurine. Uptakes of GABA and glutamate into P₂ fractions did not differ between the substrains. In neonatal SS rats that had never had a seizure, the uptake of taurine is decreased both into the whole brain in vivo and into P₂ fraction in vitro, as compared to age-matched SR rats. This indicates that decreased uptake is not a consequence of seizure activity per se. In non-seizure susceptible progeny of SS rats, the uptake of taurine into P₂ fraction did not differ significantly from that of SR rats. In kidney cortex slices from SS rats, taurine uptake is slightly greater than in slices from SR rats. We propose that the decreased taurine transport in the P₂ fraction of the brains of SS rats may reflect a defect in transport in vivo that contributes to seizure-susceptibility.     

Insulin induces Thr484 phosphorylation and stabilization of SIK2 in adipocytes

Aims/hypothesisSalt-inducible kinase 2 (SIK2) is downregulated in adipose tissue from obese or insulin-resistant individuals and inhibition of SIK isoforms results in reduced glucose uptake and insulin signalling in adipocytes. However, the regulation of SIK2 itself in response to insulin in adipocytes has not been studied in detail. The aim of our work was to investigate effects of insulin on various aspects of SIK2 function in adipocytes.MethodsPrimary adipocytes were isolated from human subcutaneous and rat epididymal adipose tissue. Insulin-induced phosphorylation of SIK2 and HDAC4 was analyzed using phosphospecific antibodies and changes in the catalytic activity of SIK2 with in vitro kinase assay. SIK2 protein levels were analyzed in primary adipocytes treated with the proteasome inhibitor MG132.ResultsWe have identified a novel regulatory pathway of SIK2 in adipocytes, which involves insulin-induced phosphorylation at Thr484. This phosphorylation is impaired in individuals with a reduced insulin action. Insulin stimulation does not affect SIK2 catalytic activity or cellular activity towards HDAC4, but is associated with increased SIK2 protein levels in adipocytes.Conclusion/interpretationOur data suggest that downregulation of SIK2 in the adipose tissue of insulin-resistant individuals can partially be caused by impaired insulin signalling, which might result in defects in SIK2 expression and function.     

Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.     

Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.     

Reversal of Inflammation‐Induced Impairment of Glucose Uptake in Adipocytes by Direct Effect of CB1 Antagonism on Adipose Tissue Macrophages

Macrophage infiltration into adipose tissue (AT‐MP) is thought to induce insulin resistance and diabetes in obesity. Here, we investigated the effect of the antiobesity drug SR141716 (a CB1 antagonist) on macrophage‐mediated inhibition of insulin signaling in adipocytes. THP1 macrophages (THP1) were stimulated in vitro with lipopolysaccharide (LPS) and SR141716 or vehicle. The resulting conditioned medium (CM) was analyzed and incubated on human adipocytes. CM from LPS‐stimulated THP1 inhibited insulin‐induced AKT phosphorylation in adipocytes, in contrast to CM from nonactivated THP1. Moreover, it contained higher concentrations of tumor necrosis factor‐α (TNFα) and lower levels of the anti‐inflammatory cytokine IL‐10. SR141716 reduced TNFα production and increased IL‐10 secretion, resulting in a rescue of insulin signaling in adipocytes. To confirm these findings in vivo, AT‐MP CM from cafeteria diet‐fed or Zucker diabetic fatty (ZDF) rats that had received SR141716 for 3 weeks were isolated, analyzed, and incubated with adipocytes. Cafeteria diet induced macrophage‐mediated inhibition of insulin signaling in adipocytes. Interestingly, SR141716 rescued insulin‐induced glucose uptake in adipocytes. Finally, AT‐MP CM from obese ZDF rats inhibited insulin‐stimulated glucose uptake in adipocytes in contrast to AT‐MP CM from lean ZDF rats. After treatment with SR141716, AT‐MP CM rescued insulin‐induced glucose uptake in adipocytes. In summary, our data indicate that CB1 receptor antagonism in macrophages modified their cytokine production and improved the insulin responsiveness of adipocytes that had been incubated with macrophage CM. Thus, SR141716 ameliorated adipose tissue insulin resistance by direct action on AT‐MP demonstrating a novel peripheral mode of action of CB1 antagonism.