Alkaline lipolytic activity of rabbit adipose tissue: Genotypes and their inheritance

A genetic variant of alkaline lipolytic activity (ALA), a soluble esterase found in mammalian adipose tissue, has previously been described. This slow variant was characterized by slower migration during starch gel electrophoresis, a decreased tendency to dimerize, and reduced enzymatic activity in constrast to the fast electrophoretic phenotype. The present paper identifies a homozygous (FF) and a heterozygous (Ff) fast phenotype which are differentiated after starch gel electrophoresis by the presence or absence of a slow band identical to that of the homozygous slow (ff)/it phenotype. In addition, ALA of homozygous and heterozygous fast rabbits differed in total enzymatic activity and its tendency to dimerize. The inheritance of this variant was proven to be Mendelian.This work was supported in part by United States Public Health Service Grants AM-06872 from the National Institute of Arthritis and Metabolic Diseases and GM-15874 from the National Institute of General Medical Sciences; and Project #417 of the Children's Bureau, Department of Health, Education and Welfare.     

Insulin and lipolytic hormones stimulate the same phosphodiesterase isoform in rat adipose tissue

Insulin sensitive phosphodiesterase from rat adipocytes is found in particulate fractions. Solubilisation of the enzyme with triton X-100 yields a preparation containing more than one phosphodiesterase activity as judged by its rate of thermal denaturation at 45 degrees C and by its non-linear kinetic plots. Immunoprecipitation of solubilised activity with a polyclonal antiserum raised against purified insulin-sensitive rat liver phosphodiesterase selected a form of the enzyme which showed a single exponential decay of enzyme activity when heated at 45 degrees C and linear low Km kinetics. Treatment of adipocytes with insulin ACTH, glucagon or isoproterenol stimulated the low Km particulate phosphodiesterase. The hormonal activation was retained following solubilisation and was also seen when activity was immunoprecipitated. It is suggested that all four hormones activate the same form of phosphodiesterase.     

Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.     

The lipolytic proteome of mouse adipose tissue

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.     

In vivo human lipolytic activity in preperitoneal and subdivisions of subcutaneous abdominal adipose tissue

We studied eight normal-weight male subjects to examine whether the lipolytic rate of deep subcutaneous and preperitoneal adipose tissues differs from that of superficial abdominal subcutaneous adipose tissue. The lipolytic rates in the superficial anterior and deep posterior subcutaneous abdominal adipose tissues and in the preperitoneal adipose tissue in the round ligament were measured by microdialysis and (133)Xe washout under basal, postabsorptive conditions and during intravenous epinephrine infusion (0.15 nmol. kg(-1). min(-1)). Both in the basal state and during epinephrine stimulation, the superficial subcutaneous adipose tissue had higher interstitial glycerol concentrations than the two other depots. Similarly, the calculated glycerol outputs from the superficial depot were significantly higher than those from the deep subcutaneous and the preperitoneal depots. Thus, it is concluded that the lipolytic rate of the superficial subcutaneous adipose tissue on the anterior abdominal wall is higher than that of the deep subcutaneous adipose tissue on the posterior abdominal wall and that of the preperitoneal adipose tissue in the round ligament.     

Insulin signalling in human adipose tissue

Adipose tissue is a critical regulator of energy balance and substrate metabolism, and synthesizes several different substances with endocrine or paracrine functions, which regulate the overall energetic homeostasis. An excessive amount of adipose tissue has been associated with the development of type 2 diabetes, premature atherosclerosis, and cardiovascular disease. It is believed that the adverse metabolic impact of visceral fat relies on a relative resistance to the action of insulin in this depot compared to other adipose tissue depots. However, information on insulin signalling reactions in human fat is limited. In this paper, we review the major insulin signalling pathways in adipocytes and their relevance for metabolic regulation, and discuss recent data indicating different signalling properties of visceral fat as compared to other fat depots, which may explain the metabolic and hormonal specificity of this fat tissue depot in humans.     

YKL-40 secreted from adipose tissue inhibits degradation of type I collagen

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.     

Insulin action rapidly decreases multifunctional protein kinase activity in rat adipose tissue

ATP-citrate lyase in vivo contains three phosphorylation sites on two tryptic peptides (peptides A and B). These phosphorylation sites are under hormonal control. Multifunctional protein kinase (MFPK) from rat liver phosphorylates peptide B on serine and threonine residues whereas cAMP-dependent protein kinase phosphorylates peptide A on a serine residue (Ramakrishna, S., and Benjamin, W. B. (1985) J. Biol. Chem. 260, 12280-12286). We now report that rat adipose tissue MFPK also phosphorylates serine and threonine residues of peptide B of ATP-citrate lyase. When the activity of MFPK was assayed using partially purified (by chromatography on phosphocellulose) cytosol fractions from insulin-treated adipose tissue, it was found that MFPK activity was decreased by over 55%. This decrease in MFPK activity occurs at physiological concentrations of insulin (EC50 = 1 x 10(-10) M). Its onset is rapid and almost maximal at 5 min after the addition of insulin. Even when new protein synthesis is inhibited by cycloheximide, extracts from insulin-treated fat pads have less MFPK activity compared to the control. The insulin effect is maintained after further chromatography on a gel filtration column suggesting that the decrease in MFPK activity is not due to a low molecular weight inhibitor. The insulin-induced decrease in MFPK activity is due to a decrease in Vmax whereas the affinity of this enzyme toward ATP-citrate lyase or ATP is unchanged.     

Hyperthermia effect on lipolytic processes in rat blood and adipose tissue

Rats anaesthetized with Brevinarcon were placed in a high-temperature chamber (air temperature 50 degrees C, relative humidity 50%) for induction of hyperthermia (rectal temperature 41.0 +/- +/- 0.5 degrees C). The control group comprised rats anaesthetized in the same way but kept at room temperature. In the serum in both groups glucose, free fatty acids, immunoreactive insulin, lipolytic activity and ability to mobilize lipids in vitro were determined. It was shown that the glucose and free fatty acid levels and the activity mobilizing serum lipids in vitro in the rats subjected to hyperthermia were lower than in the control group by 12%, 23% and 150% respectively. The lipolytic activity of the serum of rats subjected to hyperthermia was 42% higher, and the level of immunoreactive insulin rose by about 224% in relation to the control group. These results point to inhibition of lipolysis in the adipose tissue with simultaneous activation of intravascular lipolysis during hyperthermia in rats.     

Lipogenesis in rabbit adipose tissue.

Previous reports that rabbit adipose tissue does not synthesize fatty acids at significant rates led us to study in detail the pathways of lipogenesis and glyceroneogenesis in this tissue. We found that rabbit adipose tissue has a low capacity for denovo fatty acid synthesis from glucose but a high capacity for synthesis from pyruvate and acetate. The tissue can also convert pyruvate to glyceride-glycerol via the dicarboxylic acid shuttle and gluconeogenic pathways. Experiments with hydroxycitrate, a potent inhibitor of citrate cleavage enzyme, demonstrated that this is an obligatory enzyme in lipogenesis from pyruvate. The lipogenic system of rabbit adipose tissue resembles that of a ruminant in that it is adapted to utilize acetate rather than glucose. However, in contrast to ruminant tissues, the limited ability to convert glucose to fatty acid results not from a deficiency in the enzymes concerned with the transport of acetyl units out of the mitochondria but from a block prior to the level of pyruvate, most likely at the hexokinase and pyruvate kinase reactions.     

Involvement of the sympathetic nervous system in lipolytic activity in brown adipose tissue of cold acclimated rats

In cold acclimated rats, in vitro, NE led to a significant increase in release of FFA and glycerol in denervated IBAT. In vivo, study of arteriovenous differences showed that the denervated BAT loses its full capacity to utilize FFA and glycerol released by NE. After denervation an increase of blood flow in Sulzer's vein was observed. This effect appeared immediately after intervention whereas the effect on fat metabolism appeared later. In cold acclimated rats, the sympathetic nervous system appears to be an important regulator of fatty acid metabolism in BAT.     

Insulin degradation by adipose tissue. Studies at several levels of cellular organization.

A systematic study of the degradation of physiological concentrations of 125I-labelled insulin was performed in intact fat-pads, isolated adipocytes and subcellular fractions of isolated adipocytes. The findings indicate that insulin is rapidly degraded to low-molecular-weight peptides and/or amino acids by the intact tissue and isolated cells. Of the total insulin-degradation products present after incubation with an intact fat-pad, 94% is recovered in the medium, indicating that these products are not retained by the cells or tissue. The plasma membranes do not degrade insulin significantly in the absence of reduced glutathione, and over 99% of the cellular degradative capacity is found in the postmicrosomal supernatant (cytosol). The cytosol degrades insulin to several labelled fragments that are intermediate in size between insulin and insulin A chain, as well as to the low-molecular-weight tissue degradation products. Inclusion of plasma membranes with cytosol accelerates the cleavage of the intermediate fragments to the size of the small products seen with the intact tissue. However, plasma membranes do not increase the initial step in the degradation of insulin when incubated with cytosol, suggesting that the insulin receptor is not involved with the direct cleavage of insulin. This study supports the hypothesis that the bulk of insulin degradation occurs in the adipocyte cytosol, where intermediate-sized fragments are generated and rapidly cleaved to smaller products by the plasma membrane and quickly released into the surrounding medium.