Drug distribution studies with microdialysis: I. Tissue dependent difference in recovery between caffeine and theophylline

Microdialysis was applied to estimate concentrations of caffeine and theophylline in vitro or in vivo in blood, adipose tissue, muscle, liver and brain of rats. The in vivo and in vitro recovery of a compound was estimated by perfusing the dialysis probe with varying concentrations of caffeine and theophylline. The difference between the concentration in the dialysate and the concentration in the perfusion medium was plotted against the concentration in the perfusion medium and the slope of the resulting line was taken as an estimate of the recovery (difference method). In all experiments caffeine (20 mg/kg sc) and theophylline (20 mg/kg sc) were administered simultaneously. The recovery in vitro was virtually identical for caffeine and theophylline. The in vivo recovery of theophylline was significantly smaller than the recovery of caffeine in brain, liver, muscle and adipose tissue. The difference in recovery was significantly larger in the brain than in other tissues. The results show that the transport of a substance from the tissue to the dialysis probe may differ between tissues and between chemically very similar compounds. It is shown that the recovery of theophylline rapidly declines after death ensues which shows that energy-dependent processes are involved in the transport to the dialysis probe and not solely passive diffusion. It is suggested the differences in transport over brain capillaries explain the difference between caffeine and theophylline. It is concluded that the use of internal standards in microdialysis experiments requires validation in every specific application.     

Longitudinal studies on adipose tissue and its distribution in human subjects.

On 27 men and 6 women, total body density and 10 skinfolds were measured 12 yr apart, with the mean age increasing from 31 to 43 yr. The increase in skinfold thickness was found to be related to the increase in total body adiposity, calculated from hydrostatic weighing. The external adipose tissue was calculated from the mean skinfold thickness and body surface area. Variations in total adiposity among the population studied as well as changes in total adiposity with age showed a characteristic distribution with approximately two-thirds on the surface and one-third in the interior. The essential body mass or total adipose mass determined by hydrostatic weighing was compared with the values obtained by water-immersion volumetry, total body potassium counting, and skinfold measurements. Teh volumetric and skinfold determinations gave better estimates of these parameters than total body potassium counting.     

Drug distribution studies with microdialysis. II. Caffeine and theophylline in blood, brain and other tissues in rats

Microdialysis was applied to estimate the pharmacologically active concentration of caffeine and theophylline in blood, adipose tissue, muscle, liver and brain of rats. The concentration of the drugs in the extracellular space was estimated by perfusion with varying concentrations of the drug through the microdialysis probe (difference method). Caffeine (20 mg/kg) was found to be evenly distributed with a free concentration of approximately 120 microM. Theophylline concentration in the brain was 91 microM and in other tissues approximately 120 microM. The rate of penetration into brain extracellular space was higher for caffeine than for theophylline. It is suggested that the lower levels of theophylline attained in the brain may to some extent explain the differences in clinical action profile between caffeine and theophylline.     

Lipolysis in human adipose tissue during exercise: comparison of microdialysis and a-v measurements.

Subcutaneous adipose tissue lipolysis was studied in vivo by Fick's arteriovenous (a-v) principle using either calculated (microdialysis) or directly measured (catheterization) adipose tissue venous glycerol concentration. We compared results during steady-state (rest and prolonged continuous exercise), as well as during non-steady-state (onset of exercise and early exercise) experimental settings. Fourteen healthy women [age: 74 +/- 1 (SE) yr] were studied at rest and during 60-min continuous bicycling at 60% of peak O(2) uptake. Calculated and measured subcutaneous abdominal adipose tissue venous glycerol concentrations increased substantially from rest to exercise but were similar both at rest and during later stages of exercise. In contrast, during the initial approximately 40 min of exercise, calculated glycerol concentration was significantly lower (approximately 40%) than measured adipose tissue venous glycerol concentration. Despite several methodological limitations inherent to both techniques, the results strongly suggest that microdialysis and catheterization provide similar estimates of subcutaneous adipose tissue lipolysis in steady-state experimental settings like rest and continuous prolonged exercise. However, during shorter periods of exercise (<40 min), the results from the two techniques may differ quantitatively in the studied subjects. Caution should, therefore, be taken when lipolysis is evaluated, based on results obtained by the two techniques under non-steady-state conditions.     

Extracellular degradation of lipoprotein lipase in rat adipose tissue

Background  

Recent studies in vivo indicate that short-term regulation of lipoprotein lipase (LPL) in rat adipose tissue is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product. It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process.     

Application of in vivo microdialysis to measure leptin concentrations in adipose tissue

Microdialysis is a relatively new in vivo sampling technique, which allows repeated collecting of interstitial fluid and infusion of effector molecules into the tissue without influencing whole body function. The possibility of using microdialysis catheter with a large-pore size dialysis membrane (100 kDa) to measure concentrations of the adipocyte-derived peptide hormone leptin in interstitial fluid of adipose tissue was explored. Krebs–Henseleit buffer with 40 g/l dextran-70 was used to prevent perfusion fluid loss across the dialysis membrane. The relative recovery of leptin in vitro was determined using CMA/65 microdialysis catheter (100 kDa cut-off, membrane length 30 mm; CMA, Stockholm, Sweden) and four perfusion rates were tested (0.5, 1.0, 2.0, 5.0 μl/min). Furthermore, the microdialysis catheter CMA/65 was inserted into subcutaneous abdominal adipose tissue of 11 healthy human subjects and leptin concentrations in the interstitial fluid of adipose tissue in vivo were measured. The present findings are the first documentation on the use of microdialysis to study local leptin concentrations in the interstitial fluid of adipose tissue.     

Brown adipose tissue thermogenesis: interdisciplinary studies

Energy expenditure for thermogenesis in brown adipose tissue (BAT) serves either to maintain body temperature in the cold or to waste food energy. It has roles in thermal balance and energy balance, and when defective, is usually associated with obesity. BAT can grow or atrophy; it is usually atrophied in obese animals. Control of BAT thermogenesis and growth is by the sympathetic nervous system, with integration of signals in the hypothalamus. Sensory nerves may also be involved. Understanding the control of growth and differentiation of BAT is important for discovering how to reactivate it is obesity. Studies on control of gene expression in BAT are concentrating on thermogenically important components such as the uncoupling protein (which allows BAT mitochondria to operate in a thermogenic uncoupled mode), lipoprotein lipase (which allows BAT to compete with white adipose tissue for dietary lipid), and thyroxine 5'-deiodinase (which allows endogenous triiodothyronine generation, part of the control of differentiation and growth of BAT). Differentiation of BAT cell precursors in culture has recently been achieved. BAT is present in adult humans and some anti-obesity drugs are targeted to stimulation of BAT thermogenesis. However, extrapolation to humans of results of studies of BAT requires the development of novel approaches to the noninvasive assessment of amount and function of human BAT.     

Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue.

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.     

Dehydroepiandrosterone concentration in adipose tissue of normal and overweight subjects.

Unconjugated dehydroepiandrosterone has been isolated from human abdominal adipose tissue and identified by double isotope derivatization and recrystallization to constant isotope ratio. Gas chromatography with electron capture detection revealed an actual dehydroepiandrosterone concentration between 0.32 to 2.82 mug/g in adipose tissue of normal and overweight subjects. The approximate dehydroepiandrosterone content of the total adipose tissue mass varied between 30 and 173 mg in subjects with severe obesity.     

Ultrasonic measurements of subcutaneous adipose tissue thickness in man

Subcutaneous adipose tissue thickness (SCATT) has for many years been measured using skinfold calipers, but calipers have several disadvantages, not least that they compress the skinfold. Measuring SCATT using ultrasound was validated by comparing thicknesses at 12 sites with soft-tissue radiographs in 24 adults and with depth gauge measurements on a cadaver. Correlation coefficients ranged from 0.87 to 0.99, and the regression statistics showed no significant difference between ultrasound and the other procedures. The standard errors of estimate of body density from SCATT measured by calipers and ultrasound in 63 young men was +/- 7.8 and 7.3 kg X m-3, respectively. Although problems in the identification of the adipose tissue-muscle interface can arise, ultrasound is a viable alternative to skinfold calipers and is to be preferred when measuring uncompressed SCATT.     

Studies of phosphodiesterase effects on adipose tissue metabolism in obese subjects by the microdialysis technique.

The effect of non-selective (theophylline) inhibition of cyclic AMP breakdown on norepinephrine stimulated lipolysis rate was investigated in subcutaneous adipose tissue of obese subjects. In addition, changes in interstitial glucose and lactate concentration were assessed by means of the microdialysis technique. The interaction of endogenous released insulin and theophylline on adipocyte metabolism was determined. Theophylline and norepinephrine alone increased glycerol outflow significantly. When both agents were perfused in combination, interstitial glycerol concentration increased further. The enhanced glycerol level due to theophylline application was slightly decreased by insulin. In the presence of theophylline, extracellular glucose concentration increased, in contrast to the catecholamine. Norepinephrine decreased interstitial glucose level. When both drugs were added in combination, the level of interstitial glucose increased to about 1 mM, greater than with theophylline alone. With each intervention, lactate was synthesized. Local adipose tissue blood flow was increased by theophylline and theophylline plus norepinephrine. In conclusion, post-receptor mechanisms increased norepinephrine maximal stimulated lipolysis rate in subcutaneous adipose tissue. Glucose uptake was inhibited by the non-specific inhibitor of phosphodiesterase. The effect of insulin on inhibition of lipolysis was modest but sustained in the presence of high theophylline (10(-4) M) concentration. Phosphodiesterase activity may be relatively low in obese subjects in comparison with lean subjects. In lean subjects theophylline caused a transient reversal of the antilipolytic effect of insulin.     

Long-term alcohol consumption and brown adipose tissue in man

The purpose of the present work was to study whether long-term alcohol consumption in man affects the development of brown adipose tissue. The adipose tissue around the thoracic aorta and common carotid arteries was collected at medicolegal autopsies on adults with a positive record of heavy alcohol consumption. Adults without any evident history of alcohol consumption served as controls. Histochemical reactions of the oxidative mitochondrial enzymes, cytochrome oxidase and succinate dehydrogenase were studied in samples of this adipose tissue and the activities of the enzymes were measured biochemically. There was histological evidence of some multilocular adipose tissue around the thoracic aorta and common carotid tissue from the non-drinkers was mostly unilocular resembling white adipose tissue. Histochemical evidence of brown adipose tissue was found in all alcohol consumers, but also in some of the controls. Biochemical cytochrome oxidase (CYO) and succinate dehydrogenase measurements in isolated mitochondria showed activity in 70% of the cases of drinkers and in one of the eight controls. Activity of CYO was measurable in the mitochondria from two other controls. The protein content of the samples from the alcoholics was twice that of the controls. The results suggest that chronic alcohol intake may induce a change in the white adipose tissue around the thoracic aorta and common carotid arteries of human adults into brown fat.     

In vivo lipolysis in adipose tissue from two anatomical locations measured by microdialysis

In this study, the difference in lipolytic response in inguinal subcutaneous and epididymal adipose tissues of male Sprague-Dawley rats was assessed in vivo by microdialysis. Probes were perfused with Ringer solution in which increasing concentrations of isoproterenol (10(-7) - 10(-4) mol/L) were added. Glycerol release, expressed as extracellular glycerol concentration, was used as lipolytic index. The effect of isoproterenol on local blood flow was investigated using the ethanol technique. No differences were found in the interstitial glycerol concentration between both adipose tissues under basal conditions. When isoproterenol was perfused, a dose-response increase in glycerol production was induced in both tissues. Interstitial glycerol concentration from epididymal adipose tissue was higher than that of inguinal subcutaneous depot at all isoproterenol concentrations. No vasodilatory effect of isoproterenol was found. These results suggest that epididymal adipose tissue is more responsive in vivo to beta-adrenergic lipolysis stimulation than is subcutaneous fat pad from the inguinal region.     

Fatty acids synthesis in rat adipose tissue. Tracer concentration effects in vitro

1. The use of labelled acetate for studying the synthesis of long-chain fatty acids in rat adipose tissue in vitro has been examined, with special reference to the effect of acetate concentration. 2. The incorporation of acetate into fatty acids is proportional to the concentration of acetate in the medium when the latter does not exceed about 10mum. Above this concentration, the relative incorporation becomes progressively less, and reasons for this are discussed. 3. In particular it is shown that this is not necessarily due to disturbance of the endogenous rate of fatty acid synthesis by a relatively large amount of acetyl-CoA derived from added acetate. 4. However, to ensure that the added acetate does not cause such a disturbance its concentration must be kept sufficiently low. For labelled acetate used under present conditions, this concentration should not be more than about 10mum.     

Leptin concentrations in the abdominal subcutaneous adipose tissue of patients with anorexia nervosa assessed by in vivo microdialysis

OBJECTIVE: The adipocyte-derived hormone leptin is involved in energy metabolism and body weight regulation. Plasma leptin concentrations are significantly reduced in patients with anorexia nervosa (AN) and with severe malnutrition. Whether reduced plasma leptin is reflected by its decreased production by the adipose tissue is unknown. METHODS: In the present study we measured leptin concentrations locally in the abdominal subcutaneous adipose tissue of 9 female AN patients and 11 healthy controls by in vivo microdialysis. RESULTS: Adipose tissue free leptin levels were not different in patients with AN compared to controls (2.59+/-1.99 vs 2.36+/-0.25 ng/ml, P>0.05). Plasma leptin soluble receptor (sOb-R) levels were significantly higher in patients with AN than in healthy subjects (58.05+/-38.69 vs 12.79+/-5.08 U/ml, P<0.01). The area of adipocyte in AN was considerably smaller than in the controls (183+/-104.01 microm2 compared to 2145.8+/-1003.41). CONCLUSIONS: We conclude that decreased plasma leptin levels in patients with AN are not directly related to dialysate leptin levels in the abdominal subcutaneous adipose tissue.