Mary P. Thompson

Increased expression of the mRNA for hormone-sensitive lipase in adipose tissue of cancer patients

The expression of genes coding for regulatory enzymes involved in the uptake, synthesis and mobilisation of lipid was measured in adipose tissue of cancer patients. Total RNA was isolated from subcutaneous adipose tissue of control and cancer patients and the various mRNAs measured by Northern blot analysis. The total lipoprotein lipase enzymic activity and the relative levels of the mRNAs for lipoprotein lipase and for fatty acid synthase were not significantly different between cancer patients and control patients. However, there was a significant two-fold increase in the relative level of mRNA for hormone-sensitive lipase (HSL) in adipose tissue of cancer patients compared with control patients. The cancer patients also exhibited a two-fold elevation in serum triacylglycerol levels and serum free fatty acid levels. There was a significant correlation between the serum free fatty acid level and expression of HSL mRNA in the adipose tissue. The serum levels of insulin and tumour necrosis factor-alpha were not different between cancer and control patients. The results suggest that at least one of the mechanisms for depletion of lipid from adipose tissue in cancer patients operates at the level of increased expression of mRNA of the lipolytic regulatory enzyme, hormone-sensitive lipase.

Links between fatty acids and expression of UCP2 and UCP3 mRNAs

Physiological and pathological states that are associated with elevated plasma fatty acids (FAs) increase uncoupling protein 2 (UCP2) mRNA in white adipose tissue and UCP3 mRNA in skeletal muscle and heart. A direct effect of unsaturated fatty acids from all classes has been shown in various cultured cells. There is evidence that FAs could induce expression of UCPs by acting as ligands for peroxisome proliferator‐activated receptors, influencing the function of sterol responsive element binding protein or activating 5′‐AMP‐activated protein kinase. Oleic acid has been shown to stimulate the activity of the promoter regions of UCP2 and UCP3 genes and the FA responsive regions are beginning to be characterised.

Expression of obese mRNA in genetically lean and fat selection lines of sheep

Genetically separate lines of Coopworth sheep have been bred by selecting for (fat genotype) or against (lean genotype) backfat depth. Typically, the total fat content, adjusted for carcass weight, is 21.2 and 29.3% for the lean and fat lines, respectively. As a homologue of the obese gene, which shows altered expression in several forms of obesity, is also expressed in sheep, it was decided to determine whether the obese gene was differentially expressed in each line of sheep. The relative level of expression of obese mRNA was approximately twofold higher in the fat line compared with the lean line in back, omental and perirenal fat depots of ram lambs fed ad libitum or fasted for 48 h. This elevation in the fat line is most likely a secondary consequence of obesity rather than a cause. Fasting for 48 h decreased obese mRNA levels by 8.9-, 8.5-, and 4.2-fold in back, omental and perirenal fat, respectively, in the lean line, and by 8.3-, 5.7-, and 3.5-fold in back, omental and perirenal fat, respectively, in the fat line. The lean and fat lines of sheep, therefore, responded in a similar way to fasting.

Adiponutrin mRNA expression in white adipose tissue is rapidly induced by meal-feeding a high-sucrose diet

Adiponutrin is a recently identified gene of unknown function that is expressed exclusively in adipose tissues. To provide information about its physiological regulation and possible function, the effect of meal-feeding rats on the expression of adiponutrin mRNA in white adipose tissue was studied. A high-sucrose meal increased adiponutrin mRNA levels by at least 5-fold within 3 h. Post-meal levels returned to pre-meal levels with a half-life of about 5 h. The induction was prevented by injection of actinomycin-D prior to the meal. This pattern of expression was very similar to that seen for leptin mRNA. There were only minimal, or no, effects on acrp30/adiponectin, resistin, adipsin, or stearoyl-CoA desaturase 1. Adiponutrin appears more like leptin with respect to its acute regulation by meal-feeding than to any of the other adipokines or to enzymes directly involved in lipogenesis. This suggests that adiponutrin could be involved in overall energy homeostasis, as is leptin.

Fasting-induced adipose factor identified as a key adipokine that is up-regulated in white adipose tissue during pregnancy and lactation in the rat

Adipokines, which are expressed and secreted from white adipose tissue (WAT), are potential factors that could contribute to the changes in energy homeostasis that occurs in pregnancy and lactation to meet the nutrient demands of fetal growth and milk production. The aim was to identify adipokines that could be involved by measuring the pattern of their mRNA expression in adipose tissue. Adipokine mRNAs were measured by quantitative RT-PCR in RNA isolated from white and brown adipose tissue (BAT) of rats at days 7, 14 and 21 of pregnancy, day 7 of lactation and virgin at dioestrus phase. The results for leptin, adiponectin and resistin expression in WAT essentially confirmed previous studies and it is unlikely that they are directly involved in the metabolic adaptations. The relative amounts of the mRNAs of the adipokines in BAT were comparable with those in WAT, but the patterns of expression did not follow those in WAT, except for apelin. Visfatin mRNA in WAT was elevated 2.5-fold only at day 21 of pregnancy. Apelin mRNA in WAT was increased 2.2-fold at day 7 of pregnancy. Retinol-binding protein 4 mRNA in WAT decreased to 46% of control at day 14 of pregnancy. Fasting-induced adipose factor (FIAF) mRNA in WAT was 2.2- to 2.5-fold higher throughout pregnancy and lactation. The marked induction of FIAF identifies this adipokine as a potential regulator of the metabolic adaptations that occur during pregnancy and lactation.

Glucose tolerance and hormonal changes in rats bearing a transplantable sarcoma

Abstract 1. 1. In tumour-bearing rats as the tumour grew the level of insulin declined to 15 μU/ml, compared to normal basal plasma insulin level of 46 μU/ml. 2. 2. The level of glucagon increased from 262 to 1110 pg/ml as the tumour grew. 3. 3. There was an inverse relationship between the molar insulin/glucagon ratio and the tumour/body wt ratio. 4. 4. The glucose tolerance of tumour-bearing rats was apparently normal. The rate of disappearance of glucose in tumour-bearing rats was within the normal range, but the insulin response to the glucose load was significantly impaired. 5. 5. Tolbutamide did not elicit any greater insulin response than glucose.

Brown adipose tissue in lean and fat selection lines of sheep identified by immunodetection of uncoupling protein in western blots of tissue homogenates

1. Uncoupling protein (UCP) was purified from perirenal adipose tissue of 2-day-old lambs by a procedure involving Triton solubilization and hydroxyapatite treatment. It has an apparent Mr of 34,000. 2. Rabbit anti-sheep UCP and rabbit anti-rat UCP each cross-reacted with both rat and sheep UCP in Western blots, indicating that the major antigenic determinants of the sheep UCP and rat UCP are similar. 3. In Western blots, the anti-sheep UCP showed tissue specificity by detecting a band corresponding to UCP only in brown adipose tissue, but not in heart or liver homogenates. 4. The Western blotting procedure was used to analyse sheep tissues. UCP was detected in samples of perirenal, omental, back and lymph node fat from 2-day-old lambs, but not in heart, liver, muscle or kidney samples. 5. UCP was not detected in any tissue samples from 34-day-old or 7-month-old lambs. 6. Comparison of the amount of UCP in perirenal fat of 2-day-old lambs from lean, fat and control selection lines, using the Western blotting procedure, showed no apparent difference.

A nitrogen balance experiment using simulated urine samples

We describe an undergraduate laboratory experiment that combines the advantages of problem‐based learning with the need for biochemistry students to become proficient in practical laboratory skills. It also avoids the need to obtain ethical approval for recruiting volunteers and eliminates any possible biosafety issues with the handling and disposal of large amounts of urine. Simulated human urine samples are prepared that contain urea, uric acid, and creatinine at concentrations that represent the levels expected in 2 liters of urine collected over 24 h from subjects on various protein diets or during different physiological states. The students measure the nitrogen‐containing compounds in the “urine samples” using specific colorimetric assays and use the data they generate to derive knowledge about nitrogen balance and the excretory metabolism of amino acids.

The ability of the calcium ionophore A-23187 to mimic some of the effects of adrenaline on the metabolism of rat submaxillary gland

Exogenous Ca*+ is required for carbamylcholine to stimulate glycogenolysis [l] and for noradrenaline to stimulate glucose oxidation [2] in rat submaxillary gland slices. It is also required for adrenaline to completely overcome the inhibition of glucose utilization caused by acetoacetate [3]. This latter effect of adrenaline is associated with a decrease of tissue [ATP] which occurs only when exogenous Ca*+ is present [3] . These findings suggest that Ca2+ might be involved in the mechanism by which adrenaline exerts some of its metabolic effects. Ca*+ may simply be required for the hormone to bind to its receptor in the membrane or the hormonereceptor interaction may stimulate movement of Ca*+ into the cell and thus initiate the metabolic response. The ionophore A-23 187 is capable of facilitating the movement of Ca*‘and other divalent cations across biological membranes [4] and can be used as a tool to investigate whether changes in Ca*+ flux can alter cellular metabolism. Indeed A-23187 stimulates glycogen breakdown in submaxillary gland slices [S] and the results reported here show that A-23 187 can also stimulate glucose uptake and cause the tissue [ATP] to decrease. On the other hand, adrenaline was no longer able to stimulate glucose metabolism in the presence of tetracaine or Ni*‘, which appear’ to inhibit Ca*+ flux into cells.

The effects of meal-feeding and the diurnal cycle on lipogenesis in brown adipose tissue of rats

A significant diurnal variation in the rates of lipogenesisin vivo in brown adipose tissue occurred in both virgin and lactating rats. On a meal-feeding regime of either a chow, high-sucrose, or high-lipid diet, there was a very large increase in BAT lipogenesis following the meal. The rates observed after the sucrose meal are the highest so far reported. There was no significant difference in BAT lipogenesis between lactating and virgin rats, contrary to previous reports by others. The pattern of stimulation of BAT lipogenesis by these feeding regimes was different from that for white adipose tissue and liver and was not correlated with plasma insulin levels.