Effect of tumor necrosis factor administration in vivo on lipoprotein lipase activity in various tissues of the rat

When added to murine adipocytes in culture, tumor necrosis factor (TNF) decreases the levels of lipoprotein lipase (LPL). Semb et al (1987. J. Biol Chem. 262: 8390-8394) have shown that administration of murine TNF to rats decreases lipoprotein lipase (LPL) in the epididymal fat pad with maximal inhibition requiring several hours. We have now tested the effects of treatment of rats with TNF on LPL activity in a variety of tissues and find that few show decreases in LPL under conditions that acutely increase serum triglycerides. Ninety minutes after treatment of male rats with human TNF (25 micrograms/200 g, i.v.), serum triglycerides rose 2.2-fold but there was no decrease in LPL activity in epididymal fat. Sixteen hours after TNF treatment LPL activity had decreased by 44% in epididymal fat, consistent with the previously reported data. In contrast, in female rats, no significant decrease was seen in LPL activity in parametrial adipose tissue at either 90 min or 16 hr after TNF administration despite increases in serum triglycerides (1.8-fold and 1.5-fold, respectively). There was little change in LPL activity in most other adipose tissue sites of male or female rats at either time after TNF treatment. No effect of TNF was seen on heart or diaphragm muscle LPL at any time. TNF treatment of both male and female rats produces consistent increases in de novo hepatic lipogenesis in vivo under conditions that increase serum triglycerides. It is unlikely that the limited effects of TNF on LPL in vivo can account for the rapid and sustained increase in serum triglycerides.     

Recombinant human tumor necrosis factor does not inhibit lipoprotein lipase in primary cultures of isolated human adipocytes

Previous studies have demonstrated that cachectin/tumor necrosis factor (TNF) inhibits lipoprotein lipase (LPL) activity in cultures of 3T3-L1 cells. To determine whether TNF also inhibits LPL in human adipocytes, primary cultures of isolated human adipocytes were exposed to a spectrum of concentrations of recombinant human TNF. TNF concentrations up to 1000 pM had no effect on either LPL activity or LPL immunoreactive mass in the human adipocytes. Specific binding of 125I-labeled TNF was demonstrated in human adipocytes, and a TNF concentration of 100 pM competed for approximately 50% of the 125I-labeled TNF binding sites. In contrast, the same TNF in the same concentrations progressively inhibited LPL activity and immunoreactive mass in 3T3-L1 cells. Thus, human adipocytes respond to TNF in a different manner than 3T3-L1 cells. TNF may not cause the cachexia of cancer or chronic infection by directly inhibiting LPL in adipose tissue.     

Rat adipocyte lipoprotein lipase activity is inhibited by theophylline and stimulated by inosine through adenosine-independent mechanisms

Incubation of rat adipose tissue or isolated rat adipocytes with high (50 mM) but not with low concentrations (0.5 mM) of theophylline results in a decrease of lipoprotein lipase (LPL) activity. This effect is not altered by the addition of adenosine deaminase, indicating that the decrease of adipose LPL activity by theophylline is not due to the competition of theophylline with adenosine. On the contrary, incubation of isolated fat cells with adenosine (0.1 – 100 μM) results in an increase of the intracellular form of LPL activity. As this effect is also observed in cells incubated with adenosine deaminase (40 mU/ml) or with inosine (0.1 – 100 μM) but not in cells incubated with the adenosine analog N⁶-phenylisopropyladenosine, it is concluded that the increase in the intracellular form of LPL found after incubation with adenosine is not due to adenosine $\text{per se}$ but to inosine generated from the breakdown of endogenous adenosine by adenosine deaminase.     

Isoproterenol increases active lipoprotein lipase in adipocyte medium and in rat plasma

White adipose tissue (WAT) lipoprotein lipase (LPL) activity channels diet fat towards storage in adipocytes. Adrenaline (ADR) is accepted to reduce WAT or adipocyte LPL activity (LPLa), but available data are not clear-cut regarding long exposure to ADR in vitro or in vivo. We studied the effects of long exposures to ADR or beta-adrenergic agonist on LPL: in isolated rat adipocytes (3 h) and in rats (>1 day). Isoproterenol (ISO) (1 microM) did not alter LPLmRNA nor LPLa in adipocytes, but increased LPLa in medium more than twofold (3.58 +/- 0.35 vs. 1.32 +/- 0.35 mU/10(6) adipocytes, P < 0.001). Effect was time (not present at 1 h, clear at 2 h) and concentration dependent (high sensitivity from 10 to 100 nM, max at 1 microM). Adenylate cyclase activator or cyclic AMP (cAMP) analogue produced a similar increase. Thus in adipocytes ISO produced an increase in LPLa release and/or a decrease in extracellular LPLa degradation. ADR or ISO treated rats had a two to fourfold decrease in WAT LPLa vs. unchanged LPLmRNA. This decrease was 10-fold in WAT heparin-releasable LPLa (5.7 +/- 0.6 vs. 57.3 +/- 10.2 mU/g, P < 0.001), which represents peri/extracellular LPLa. Plasma LPLa was increased 11-fold by ADR (3.30 +/- 0.58 vs. 0.32 +/- 0.08 mU/ml, P < 0.001) whereas only threefold by ISO (P > 0.01). We suggest that in vivo ADR increased release of active LPL to plasma from endothelial cells of LPL-rich tissue(s)-WAT was probably one of these tissues releasing LPL since it lost 90% of its peri/extracellular LPLa-and/or decreased degradation of plasma active LPL. Since liver LPLa was not increased, plasma active LPL might be kept away from hepatic degradation by binding to stabilising entities in plasma (fatty acids (FA), lipoproteins or soluble heparan sulphates (HS)). In conclusion, we believe this is the first report stating that: (a) ISO increases LPLa in isolated adipocyte medium, and (b) ADR administration to rats decreases WAT extracellular active LPL and increases preheparin plasma active LPL.     

Cachectin/tumor necrosis factor decreases human adipose tissue lipoprotein lipase mRNA levels, synthesis, and activity

The effects of the cytokine cachectin/tumor necrosis factor (TNF) on human adipose tissue lipoprotein lipase (LPL) were studied. TNF is produced by activated macrophages and is thought to play a role in mediating hypertriglyceridemia and wasting of adipose tissue triglyceride stores (cachexia) that often accompany infection and malignancy. TNF effects were studied in human adipose tissue fragments maintained in organ culture in the presence of insulin and dexamethasone to induce high LPL activity. Addition of TNF to the culture medium for 20 h caused a dose-dependent inhibition of LPL activity to an average of 37% of controls at 50 U/ml TNF. This inhibition of LPL activity was explained by specific decreases in levels of LPL mRNA (to 40% of controls) and rates of LPL synthesis determined by biosynthetic labeling and immunoprecipitation (to 32% of controls). The decline in LPL synthesis was specific, as it occurred despite a small increase in overall protein synthesis in the presence of TNF. Comparable decreases in LPL activity were observed when TNF was added to adipose tissue cultured solely in the presence of insulin. Thus, similar to results in rodent models, TNF is a potent inhibitor of LPL gene expression in human adipose tissue. TNF may therefore play a role in the disorders of triglyceride catabolism and the pathogenesis of cachexia that occur with stimulation of the immune system in humans.     

The acute phase response stimulates the expression of angiopoietin like protein 4

The acute phase response is characterized by elevations in serum triglyceride levels due to both an increase in hepatic VLDL production and a delay in the clearance of triglyceride rich lipoproteins secondary to a decrease in lipoprotein lipase (LPL) activity. Recently there has been a marked increase in our understanding of factors that regulate LPL activity. GPIHBP1 facilitates the interaction of LPL and lipoproteins thereby allowing lipolysis to occur. Angiopoietin like proteins (ANGPTL) 3 and 4 inhibit LPL activity. In the present study, treatment of mice with LPS, an activator of TLR4 and a model of Gram-negative infections, did not alter the expression of GPIHBP1 in heart or adipose tissue. However, LPS decreased the expression of ANGPTL3 in liver and increased the expression of ANGPTL4 in heart, muscle, and adipose tissue. Serum ANGPTL4 protein levels were markedly increased at 8 and 16 h following LPS treatment. Administration of zymosan, an activator of TLR2 and a model of fungal infections, also increased serum ANGPTL4 protein and mRNA levels in liver, heart, muscle, and adipose tissue. Finally, treatment of 3T3-L1 adipocytes with LPS or cytokines (TNF alpha, IL-1 beta, and interferon gamma) stimulated ANGPTL4 expression. These studies demonstrate that ANGPTL4 is a positive acute phase protein and the increase in ANGPTL4 could contribute to the hypertriglyceridemia that characteristically occurs during the acute phase response by inhibiting LPL activity.     

Role of A kinase anchor proteins in the tissue-specific regulation of lipoprotein lipase

Activation of protein kinase A by catecholamines inhibits lipoprotein lipase (LPL) activity through the elaboration of an RNA binding complex, which inhibits LPL translation by binding to the 3'-untranslated region of the LPL mRNA. To better define this process, we reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the K homology (KH) domain of A kinase anchor protein (AKAP) 121/149 plays a vital role in the inhibition of LPL translation. Inhibition of LPL translation occurred in vitro only when the Calpha subunit, R subunit, and AKAP 149 were present. Using different glutathione-S-transferase fusion proteins of AKAP 149, sequences containing the KH domain were required for inhibition of LPL translation, and the inhibition of AKAP 121 expression in 3T3-F442A adipocytes with short interfering RNA resulted in loss of epinephrine-mediated translation inhibition. After epinephrine injection into mice, LPL activity was inhibited in white adipose tissue but not in brown adipose tissue (BAT) or muscle. LPL activity and synthetic rate were inhibited in vitro by the addition of epinephrine to 3T3-F442A adipocytes, but there was no effect in L6 muscle cells and cultures of brown adipocytes. Corresponding with these differences in LPL translation, AKAP 121 protein and mRNA were abundantly expressed in mouse white adipose tissue, but was either very low or undetectable in BAT and muscle. Thus, AKAP 121/149 contains a KH region that is essential to the translation inhibition of LPL in response to epinephrine. BAT and muscle do not express significant AKAP 121/149, and this likely explains some of the tissue-specific differences in LPL regulation.     

Regulation of lipoprotein lipase in muscle and adipose tissue during exercise.

Lipoprotein lipase (LPL) is regulated in a tissue-specific manner; exercise increases LPL activity in muscle at the same time it is reduced in adipose tissue. The purpose of this study was to determine the relationship between LPL activity and LPL mRNA in muscle and adipose tissue in rats exposed to one bout of exercise. Immediately after a 2-h swim, LPL activity [pmol free fatty acids (FFA).min-1.mg tissue-1] in the exercised animals was reduced 43% in adipose tissue (110 +/- 26 to 63 +/- 17) and increased almost twofold in the soleus muscle (203 +/- 26 to 383 +/- 59) compared with sedentary control animals. At the same time, LPL mRNA was reduced 42% in adipose tissue and increased 50 and 100% in the red vastus and white vastus muscles, respectively. Twenty-four hours after the swim, LPL activity had returned to control levels in adipose tissue and the soleus muscle. At hour 24 of recovery, LPL mRNA was still reduced 23% in the adipose tissue of exercised animals but was not significantly different between exercised and control animals in any of the muscle tissues analyzed. Changes in total RNA concentration could not account for the changes in relative LPL mRNA expression. The relationship between LPL enzyme activity and LPL mRNA in muscle and adipose tissue was +0.86 and +0.93 at 0 and 24 h postexercise, respectively. Thus the tissue-specific changes in enzyme activity induced by exercise could be mediated, in part, through pretranslational control.     

Adipose, muscle and lung tissue lipoprotein lipase activities in young streptozotocin treated rats

Lipoprotein lipase (LPL) activity was studied in adipose, muscle and lung tissues of post-weanling rats 48 and 96 hours after experimentally induced diabetes by streptozotocin administration. Weight gain was reduced, and blood glucose level increased about 3-4 fold above the control level as an indication of the diabetic state. LPL activity in brown and white adipose tissues decreased in diabetic rats to 10-30% of the control level. In soleus muscle the LPL activity was slightly enhanced 96 hours after the streptozotocin injection. In cardiac muscle the LPL activity was markedly increased already 48 hours after the administration of streptozotocin and the increase remained significant until 96 hours. There was in the pulmonary tissue also an increase of LPL activity of diabetic rats, although this was significant only 96 hours after streptozotocin treatment. The results suggest marked tissue specific variation in the LPL activity. Moreover, tissue responses to experimentally induced diabetes vary. In adipose tissue the decrease in the LPL activity suggests that lipid transport to adipocytes is decreased while an increase in skeletal and cardiac muscles and in lung tissue proposes that their lipid utilization is enhanced.     

Insulin regulation of lipoprotein lipase (LPL) activity and expression in gilthead sea bream (Sparus aurata)

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism by virtue of its capacity to hydrolyze triglycerides circulating in the form of lipoprotein particles. Here we analyzed the fasting effects of LPL in gilthead sea bream (Sparus aurata) and also present the first study in fish of the role of insulin as a potential modulator of both LPL activity and expression. Fasting for 2 weeks provoked a clear decrease in adipose tissue LPL activity, concomitant with lower levels of plasma insulin, while no effects were observed in red muscle. To elucidate the specific role of insulin, increases of plasma insulin were experimentally induced by arginine and insulin injections. However, arginine predominantly stimulated glucagon over insulin secretion in this fish species while LPL activity did not change significantly in adipose tissue. Instead, insulin administration induced an increase in adipose tissue LPL activity 3 h after the injection, whereas LPL activity in red muscle was not affected. Changes in LPL activity were accompanied by an increase in LPL mRNA levels in the adipose tissue of insulin-injected gilthead sea bream, although changes in LPL expression were delayed in time with respect to variations in LPL activity. Finally, LPL mRNA levels in red muscle were similar between control and insulin-injected gilthead sea bream, suggesting that insulin does not play a direct role in the regulation of LPL in this tissue. The current study shows that LPL activity is regulated by nutritional condition and underscores the importance of insulin as a modulator of LPL activity and expression in the adipose tissue of gilthead sea bream.     

Endothelial cell specific molecule-1--a newly identified protein in adipocytes.

Expression of the endothelial cell-specific molecule (ESM)-1 was originally identified in lung and kidney endothelial cells, where its expression is regulated by cytokines. In vitro, ESM-1 interferes with the molecular mechanisms of immune cell migration by binding to adhesion molecules. In this study, we have explored the expression of ESM-1 in isolated human adipocytes and in rat adipose tissue depots. Human primary adipocytes were cultivated after collagenase digestion and used for in vitro incubation studies. Adipocytes were also isolated from different fat depots of Sprague-Dawley rats. Gene expression was quantified by TaqMan RT-PCR using specific human and rat ESM-1 primers. The cellular localisation of ESM-1 was determined by confocal microscopy using a specific antibody. ESM-1 expression in human adipocytes was stimulated by phorbol ester, an activator of protein kinase C, and by retinoic acid, an activator of nuclear receptors. The maximum increase in gene expression was 3.2-fold after 72 h treatment with phorbol ester and 4.6-fold after 72 h treatment with retinoic acid. The highest expression was found in subcutaneous rat adipose tissue - two-fold compared to epididymal and six-fold compared to intrascapular brown adipose tissue. As obesity is related to systemic inflammation (examplified by increased circulating levels of C-reactive protein and interleukin-6), the formation of ESM-1 in adipocytes and its activation by protein kinase C may play a role in the regulation of inflammatory processes.     

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.