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

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

Effects of pre-exercise carbohydrate consumption on metabolism during exercise

It is well documented that consuming carbohydrates (CHO) prior to exercise has been shown to alter metabolism. There are many ways that CHO ingestion affects substrate utilization and blood glucose dynamics at the start of exercise. Changes in the concentrations of blood glucose, insulin, glucagon, free fatty acids (FFAs) as well as varying utilizations of different substrates have been observed. Each of these responses is reflective of the body’s capacity to maintain homeostasis through different physiological conditions and demands. PURPOSE: The aim is to deduce how varying amounts (approximately 0, 12.5, 25 and 50g for a 70 kg person) of pre-exercise sucrose ingestion effects metabolism including blood glucose concentration and fat oxidation during 30 minutes of moderate intense exercise. METHODS: This will be a randomized crossover study. After the initial assessment of baseline data (VO2peak), participants will be asked to perform four cycling trials at 50% of VO2peak for 30 minutes. Forty-five minutes before each exercise trial, participants will consume 0, 12.5, 25 and 50g (for a 70kg person) of sucrose. VO2 and VCO2 will be collected for 15 minutes prior to exercise and for the entire 30 minutes of cycling. Blood glucose will be obtained through the finger prick method and collected directly prior to exercise, 5, 15 and 30 minutes into cycling. Heart rate and rate of perceived exertion will be measured every 5 minutes of exercise. DISCUSSION: It is speculated that a small dose (12.5g for a 70kg person) of pre-exercise sucrose consumption will be able to demonstrate a decline in blood glucose concentration during exercise with a step-wise reduction in fat oxidation. This dose response curve will display the sensitivity of metabolism to ingested sucrose.Kinesiology and Health Educatio

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

Glucocorticoid Receptor and Adipocyte Biology.

Glucocorticoids are steroid hormones that play a key role in metabolic adaptations during stress, such as fasting and starvation, in order to maintain plasma glucose levels. Excess and chronic glucocorticoid exposure, however, causes metabolic syndrome including insulin resistance, dyslipidemia, and hyperglycemia. Studies in animal models of metabolic disorders frequently demonstrate that suppressing glucocorticoid signaling improves insulin sensitivity and metabolic profiles. Glucocorticoids convey their signals through an intracellular glucocorticoid receptor (GR), which is a transcriptional regulator. The adipocyte is one cell type that contributes to whole body metabolic homeostasis under the influence of GR. Glucocorticoids' functions on adipose tissues are complex. Depending on various physiological or pathophysiological states as well as distinct fat depots, glucocorticoids can either increase or decrease lipid storage in adipose tissues. In rodents, glucocorticoids have been shown to reduce the thermogenic activity of brown adipocytes. However, in human acute glucocorticoid exposure, glucocorticoids act to promote thermogenesis. In this article, we will review the recent studies on the mechanisms underlying the complex metabolic functions of GR in adipocytes. These include studies of the metabolic outcomes of adipocyte specific GR knockout mice and identification of novel GR primary target genes that mediate glucocorticoid action in 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

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