Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting (∼70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase (∼3.0°C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response

Pik3r1 Is Required for Glucocorticoid-Induced Perilipin 1 Phosphorylation in Lipid Droplet for Adipocyte Lipolysis.

Glucocorticoids promote lipolysis in white adipose tissue (WAT) to adapt to energy demands under stress, whereas superfluous lipolysis causes metabolic disorders, including dyslipidemia and hepatic steatosis. Glucocorticoid-induced lipolysis requires the phosphorylation of cytosolic hormone-sensitive lipase (HSL) and perilipin 1 (Plin1) in the lipid droplet by protein kinase A (PKA). We previously identified Pik3r1 (also called p85α) as a glucocorticoid receptor target gene. Here, we found that glucocorticoids increased HSL phosphorylation, but not Plin1 phosphorylation, in adipose tissue-specific Pik3r1-null (AKO) mice. Furthermore, in lipid droplets, the phosphorylation of HSL and Plin1 and the levels of catalytic and regulatory subunits of PKA were increased by glucocorticoids in wild-type mice. However, these effects were attenuated in AKO mice. In agreement with reduced WAT lipolysis, glucocorticoid- initiated hepatic steatosis and hypertriglyceridemia were improved in AKO mice. Our data demonstrated a novel role of Pik3r1 that was independent of the regulatory function of phosphoinositide 3-kinase in mediating the metabolic action of glucocorticoids. Thus, the inhibition of Pik3r1 in adipocytes could alleviate lipid disorders caused by excess glucocorticoid exposure

FAM13A and POM121C are candidate genes for fasting insulin: functional follow-up analysis of a genome-wide association study

Aims/hypothesis: By genome-wide association meta-analysis, 17 genetic loci associated with fasting serum insulin (FSI), a marker of systemic insulin resistance, have been identified. To define potential culprit genes in these loci, in a cross-sectional study we analysed white adipose tissue (WAT) expression of 120 genes in these loci in relation to systemic and adipose tissue variables, and functionally evaluated genes demonstrating genotype-specific expression in WAT (eQTLs). Methods: Abdominal subcutaneous adipose tissue biopsies were obtained from 114 women. Basal lipolytic activity was measured as glycerol release from adipose tissue explants. Adipocytes were isolated and insulin-stimulated incorporation of radiolabelled glucose into lipids was used to quantify adipocyte insulin sensitivity. Small interfering RNA-mediated knockout in human mesenchymal stem cells was used for functional evaluation of genes. Results: Adipose expression of 48 of the studied candidate genes associated significantly with FSI, whereas expression of 24, 17 and 2 genes, respectively, associated with adipocyte insulin sensitivity, lipolysis and/or WAT morphology (i.e. fat cell size relative to total body fat mass). Four genetic loci contained eQTLs. In one chromosome 4 locus (rs3822072), the FSI-increasing allele associated with lower FAM13A expression and FAM13A expression associated with a beneficial metabolic profile including decreased WAT lipolysis (regression coefficient, R = −0.50, p = 5.6 × 10−7). Knockdown of FAM13A increased lipolysis by ~1.5- fold and the expression of LIPE (encoding hormone-sensitive lipase, a rate-limiting enzyme in lipolysis). At the chromosome 7 locus (rs1167800), the FSI-increasing allele associated with lower POM121C expression. Consistent with an insulin-sensitising function, POM121C expression associated with systemic insulin sensitivity (R = −0.22, p = 2.0 × 10−2), adipocyte insulin sensitivity (R = 0.28, p = 3.4 × 10−3) and adipose hyperplasia (R = −0.29, p = 2.6 × 10−2). POM121C knockdown decreased expression of all adipocyte-specific markers by 25–50%, suggesting that POM121C is necessary for adipogenesis. Conclusions/interpretation: Gene expression and adipocyte functional studies support the notion that FAM13A and POM121C control adipocyte lipolysis and adipogenesis, respectively, and might thereby be involved in genetic control of systemic insulin sensitivity

Effects of genetic loci associated with central obesity on adipocyte lipolysis

Objectives: Numerous genetic loci have been associated with measures of central fat accumulation, such as waist-to-hip ratio adjusted for body mass index (WHRadjBMI). However the mechanisms by which genetic variations influence obesity remain largely elusive. Lipolysis is a key process for regulation of lipid storage in adipocytes, thus is implicated in obesity and its metabolic complications. Here, genetic variants at 36 WHRadjBMI-associated loci were examined for their influence on abdominal subcutaneous adipocyte lipolysis. Subjects and Methods: Fasting subcutaneous adipose tissue biopsies were collected from 789 volunteers (587 women and 202 men, body mass index (BMI) range 17.7–62.3 kg/m2). We quantified subcutaneous adipocyte lipolysis, both spontaneous and stimulated by the catecholamine isoprenaline or a cyclic AMP analogue. DNA was extracted from peripheral blood mononuclear cells and genotyping of SNPs associated with WHRadjBMI conducted. The effects on adipocyte lipolysis measures were assessed for SNPs individually and combined in a SNP score. Results: The WHRadjBMI-associated loci CMIP, PLXND1, VEGFA and ZNRF3-KREMEN1 demonstrated nominal associations with spontaneous and/or stimulated lipolysis. Candidate genes in these loci have been reported to influence NFκB-signaling, fat cell size and Wnt signalling, all of which may influence lipolysis. Significance: This report provides evidence for specific WHRadjBMI-associated loci as candidates to modulate adipocyte lipolysis. Additionally, our data suggests that genetically increased central fat accumulation is unlikely to be a major cause of altered lipolysis in abdominal adipocytes

The Effect of a Reduced-Calorie Diet on alpha-2 Adrenergic Receptor Responsiveness in Abdominal Adipose Tissue in Obese Men During Exercise

There is at present an imperfect understanding of the effect of diet on availability of inhibitory receptors in fat cells during exercise among obese men. 

*Objective:* The purpose of this study was to determine whether diet results in downregulation of alpha-2 adrenergic receptor ([alpha]~2-AR~) messenger RNA (mRNA), improving metabolism in exercise in obese men. 

*Design:* One group, pre-test, post-test design.

*Measurements:* Subcutaneous abdominal adipose tissue was tested for physiologic response, such as changes in catecholamines and other markers of lipolysis measured during periods of exercise, before and after a 12-week diet. Plasma markers of lipolysis/antilipolytic activity (catecholamines [adrenaline and noradrenaline], NEFA, lactate, glucose, hematocrit, or insulin levels) were analyzed at four points in time in order to determine the effect of exercise on [alpha]~2-AR~ and [beta]-AR responsiveness to sympathetic stimulation.

*Subjects:* Otherwise healthy 18 to 45 year old obese men (defined as a body mass index (BMI) over 33 kg/m^2^).

*Results:* The 12-week reduced calorie diet did not result in improved metabolism. Instead, upregulation of alpha-2 adrenergic receptor ([alpha]~2-AR~) messenger RNA (mRNA) was observed. On average, [alpha]~2-AR~ mRNA levels (ratio of [alpha]~2-AR~ to cyclophilin) in subjects increased by 0.022-0.023 after the diet. The average differences in of [alpha]~2-AR~ mRNA and [beta]-AR mRNA measured before and after diet were both insignificant (M = 0.015) t(4) = -0.911; _P_ > 0.05; (M = 0.0139; t(4) = 0.077; _P_ > 0.05). 

*Conclusion:* The observed direction of change in [alpha]~2-AR~ mRNA levels, when viewed together with the stability of [beta]-AR mRNA levels, suggests that upregulation of [alpha]~2-AR~ rather than downregulation occurred. Downregulation would account for decreased lipolytic activity during exercise, future study is needed

Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages

学位記番号：医博甲174

Adiponectin Deficiency Impairs Maternal Metabolic Adaptation to Pregnancy in Mice.

Hypoadiponectinemia has been widely observed in patients with gestational diabetes mellitus (GDM). To investigate the causal role of hypoadiponectinemia in GDM, adiponectin gene knockout (Adipoq-/- ) and wild-type (WT) mice were crossed to produce pregnant mouse models with or without adiponectin deficiency. Adenoviral vector-mediated in vivo transduction was used to reconstitute adiponectin during late pregnancy. Results showed that Adipoq-/- dams developed glucose intolerance and hyperlipidemia in late pregnancy. Increased fetal body weight was detected in Adipoq-/- dams. Adiponectin reconstitution abolished these metabolic defects in Adipoq-/- dams. Hepatic glucose and triglyceride production rates of Adipoq-/- dams were significantly higher than those of WT dams. Robustly enhanced lipolysis was found in gonadal fat of Adipoq-/- dams. Interestingly, similar levels of insulin-induced glucose disposal and insulin signaling in metabolically active tissues in Adipoq-/- and WT dams indicated that maternal adiponectin deficiency does not reduce insulin sensitivity. However, remarkably decreased serum insulin concentrations were observed in Adipoq-/- dams. Furthermore, β-cell mass, but not glucose-stimulated insulin release, in Adipoq-/- dams was significantly reduced compared with WT dams. Together, these results demonstrate that adiponectin plays an important role in controlling maternal metabolic adaptation to pregnancy

Preparation, characterisation and application of naturally derived polar lipids through lipolysis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

Monoglycerides are lipid based emulsifiers extensively used for their broad technical function in the food industry. Commercial monoglycerides are generally manufactured through chemical synthesis; however, lipolysis of triglycerides by lipase enzyme provides a biochemical pathway by which monoglycerides may be produced. This is particularly appealing for consumers for whom all natural and clean labelled food products are a particular driver. Accordingly, rather than replacing monoglycerides from formulations with other types of emulsifiers (and that may lack the requisite functionality), an alternative approach may be to develop a non-chemical and more natural pathway to produce the emulsifier, thereby allowing the particular monoglyceride functionality to be retained within products. Therefore, this study was conducted to investigate the feasibility of using lipase enzyme as a processing tool to synthesise polar lipids, namely monoglycerides, in situ of the manufacture of whippable food emulsions, such as cream and ice cream. This concept idea was initially proven viable through interfacial tension (IFT) measurements obtained using a straight-forward surface characterisation technique. R. miehei lipase was found to competitively bind at the interface of vegetable oils-water and that the adsorbed protein (sodium caseinate and whey protein isolate) or surfactant layer (lecithin and Tween 80) did not act as a barrier to lipase adsorption at the oil-water interface. IFT measurements were also able to demonstrate the progressive accumulation of polar lipids at the oil-water interface arising from lipolysis, and were additionally used to indicate how thermal treatment of the enzyme could be used to terminate activity. In considering how the requisite functionality could be achieved for whippable emulsion formulations, emulsion droplet size, type of emulsifiers used as well as lipase concentration were shown to be key variables by which the extent and rate of lipolysis could be manipulated and controlled. The results showed that formulation (emulsifier types and oil content) and processing conditions (Microfluidizer® pressure and number of passes) had significant effects on the emulsion droplet size. As part of controlling the extent of lipolysis, the conditions by which the reaction could be terminated were investigated by measuring the viability of R. miehei lipase against thermal treatment. Results showed that the R. miehei lipase was thermostable up to temperatures of 70 °C. Above this temperature, substantial reduction of the residual activity occurred. However, even elevated temperature of between 90 and 100 oC did not immediately inactivate the lipase, with heating for ~ 2 min required before activity was no longer detected. In terms of emulsion stability, the palm oil emulsion tested in this study was found to be thermostable up to 100 oC, thus allowing development of a thermalisation step that was able to inactivate the enzyme without compromising the stability of the emulsion. The shear stability analyses on lipolysed O/W emulsion showed the lipolysed emulsions were susceptible to shear-induced aggregation, and that the degree of aggregation could be manipulated as a consequence of controlling the extent of lipolysis through either enzyme concentration or holding time. The drastic increase in the viscosity curve between the nonlipolysed and lipolysed emulsion suggested that the shear–induced partial coalescence was primarily due to the lipolysis reaction and was not as a result of the high fat content (30 %). The findings elucidate the ability of the generated polar lipids in the emulsion to displace the existing sodium caseinate adsorbed layer, thus compromising emulsion stability upon shearing. The quantification of synthesised polar lipids from the triglyceride component of fat droplets by the lipolysis reaction showed a mixture of fatty acids, di- and mono-glycerides being produced. Palmitic acid was observed to be the main liberated fatty acids. While, monoolein and monopalmitin were the most prominent monoglycerides, with measured concentrations of 3.755 ± 0.895 and 1.660 ± 0.657 mg / g fat respectively after 15 min with lipase concentration of 50 mg /g fat. The relative concentration of polar lipids produced was found to be dependent on the lipase concentration as well as time of lipolysis. Furthermore, up to 30 min of lipolysis (concentration 50 mg /g fat) were seen to have no observable effect on the droplet size distribution of the emulsion suggesting that quiescently stable emulsions could be produced. The results show the importance of controlling reaction conditions (both enzyme concentration and reaction time) in order to provide requisite functionality without excessively destabilising emulsions such that droplet structuring can occur under quiescent conditions. The generation of monoglycerides at quantum satis levels able to impart critical functionality was demonstrated in whipped cream and ice cream. The addition of R. miehei lipase at very low concentration of 5 mg /g fat was able to produce a rigid and stable whipped cream with overrun exceeding 100 %. However, good stability of the whipped cream over time was achievable with concentration above 10 mg /g fat. Similarly, ice cream made with the addition of 5 mg /g fat exhibited good melt stability and firmness. The findings proved the feasibility of in situ production of polar lipids, namely monoglycerides and fatty acids, in replicating the functionality imparted by commercial monoglycerides in whippable emulsions. Thus, the findings in this thesis offer an alternative biochemical pathway for the generation of polar lipids to that of commercially available monoglycerides, which are currently produced synthetically. The potential for using this approach as part of the processing step for food emulsion manufacture has also been demonstrated. The concept can be tailored for various emulsion based food products

A systems biology analysis of brain microvascular endothelial cell lipotoxicity.

BackgroundNeurovascular inflammation is associated with a number of neurological diseases including vascular dementia and Alzheimer's disease, which are increasingly important causes of morbidity and mortality around the world. Lipotoxicity is a metabolic disorder that results from accumulation of lipids, particularly fatty acids, in non-adipose tissue leading to cellular dysfunction, lipid droplet formation, and cell death.ResultsOur studies indicate for the first time that the neurovascular circulation also can manifest lipotoxicity, which could have major effects on cognitive function. The penetration of integrative systems biology approaches is limited in this area of research, which reduces our capacity to gain an objective insight into the signal transduction and regulation dynamics at a systems level. To address this question, we treated human microvascular endothelial cells with triglyceride-rich lipoprotein (TGRL) lipolysis products and then we used genome-wide transcriptional profiling to obtain transcript abundances over four conditions. We then identified regulatory genes and their targets that have been differentially expressed through analysis of the datasets with various statistical methods. We created a functional gene network by exploiting co-expression observations through a guilt-by-association assumption. Concomitantly, we used various network inference algorithms to identify putative regulatory interactions and we integrated all predictions to construct a consensus gene regulatory network that is TGRL lipolysis product specific.ConclusionSystem biology analysis has led to the validation of putative lipid-related targets and the discovery of several genes that may be implicated in lipotoxic-related brain microvascular endothelial cell responses. Here, we report that activating transcription factors 3 (ATF3) is a principal regulator of TGRL lipolysis products-induced gene expression in human brain microvascular endothelial cell