J. Vinten

A procedure for measurement of distribution spaces in isolated fat cells.

1. 1. Aliquots of homogenous suspensions of isolated rat epididymal fat cells were rapidly transferred to polyethylene tubes containing an oil lighter than buffer and heavier than fat cells. After centrifugation for 30 s the tube was cut through the oil layer which clearly separated the buffer from the cells. 2. 2. 5 different oils were tested of which dinonyl phthalate satisfied all of the following criteria: (a) fat cells were recovered quantitatively; (b) little quenching of 3H radioactivity occured; (c) labelled glucose, inulin, insulin, water and triglyceride did not dissolve in the oil to any significant extent. (d) it was convenient to handle. 3. 3. Fat cells which had passed dinonyl phthalate exhibited normal lipogenesis from glucose and normal sensitivity to insulin. 4. 4. After incubation at 37°C for 2–10 min followed by passage of the cells through dinonyl phthalate, the distribution spaces of the packed cells for [G-3H]-inulin, [I-14C]mannitol and [U-14C]sucrose were about 1.0 μl/100 μl cells and the distribution spaces for 3H2O and 3-O-[14C]methylglucose were about 2.3 μl/100 μl cells. The coefficient of variation for the measurement of these spaces was about 5%. 5. 5. 3H and 14C activity could be removed completely after washing of cells incubated with 3H2O and 3-O-[14C]methylglucose (40 mM). The intracellular 3H activity was almost zero after 30 s whereas the 14C activity had decreased to about half at this point.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

GLUT 4 and insulin receptor binding and kinase activity in trained human muscle.

1. Physical training enhances sensitivity and responsiveness of insulin‐mediated glucose uptake in human muscle. This study examines if this effect of physical training is due to increased insulin receptor function or increased total concentration of insulin‐recruitable glucose transporter protein (GLUT 4). 2. Seven healthy young subjects carried out single leg bicycle training for 10 weeks at 70% of one leg maximal oxygen uptake (VO2,max). Subsequently biopsies were taken from the vastus lateralis muscle of both legs. 3. Single leg VO2,max increased for the trained leg (46 +/‐ 3 to 52 +/‐ 2 ml min‐1 kg‐1 (means +/‐ S.E.M., P < 0.05), and cytochrome c oxidase activity was higher in this compared to the untrained leg (2.0 +/‐ 0.1 vs. 1.4 +/‐ 0.1 nmol s‐1 (mg muscle)‐1, P < 0.05). Insulin binding as well as basal‐ and insulin‐stimulated receptor kinase activity did not differ between trained and untrained muscle. The concentration of GLUT 4 protein was higher in the former (14.9 +/‐ 1.9 vs. 11.6 +/‐ 1.0 arbitrary units (micrograms protein)‐1 in crude membranes, P < 0.05). The training‐induced increase in GLUT 4 (26 +/‐ 11%) matched a previously reported increase in maximum insulin‐stimulated leg glucose uptake (25 +/‐ 7%) in the same subjects, and individual values of the two variables correlated (correlation coefficient (r) = 0.84, P < 0.05). 4. In conclusion, in human muscle training induces a local contraction‐dependent increase in GLUT 4 protein, which enhances the effect of insulin on glucose uptake. On the other hand, insulin receptor function in muscle is unlikely to be affected by training.

Cholesterol Depletion Disrupts Caveolae and Differentially Impairs Agonist-Induced Arterial Contraction

Objective—This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results—Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-&bgr;-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to &agr;1-adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 &mgr;mol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not &agr;1-adrenergic receptors, in the caveolin-1–containing fractions, suggesting localization of the former to caveolae. Conclusions—These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae.

Expression of insulin regulatable glucose transporters in skeletal muscle from Type 2 (non-insulin-dependent) diabetic patients

SummaryA prominent feature of Type 2 (non-insulin-dependent) diabetes mellitus is the inability of insulin to appropiately increase the transport of glucose into target tissue. In adipocytes from individuals with Type 2 diabetes, insulin resistance has been shown to be associated with a depletion of glucose transporters. Similarly, streptozotocin induced diabetes causes a diminished expression of the insulin regulatable glucose transporter in rat adipocytes. The expression of this glucose transporter isoform has not yet been investigated in muscle tissue from patients with Type 2 diabetes. We have measured the content of the insulin regulatable glucose transporter in a vesicular fraction isolated from muscle biopsies from fasting individuals with Type 2 diabetes and control subjects, and we found that the number of the insulin regulatable glucose transporters expressed in skeletal muscle was unaffected by Type 2 diabetes (0.208 vs 0.205, arbitrary units, p>0.5, control subjects and diabetic patients). Thus, the decreased glucose disposal in Type 2 diabetes is not associated with a diminished number of insulin regulatable glucose transporters.

Diurnal variation of the serum leptin concentration in patients with anorexia nervosa

In rodents, leptin is involved in regulating eating behaviour, fat storage, and reproductive function. In humans, the serum leptin concen_tration in obese and normal weight subjects correlates with body mass index, reflecting the body fat store. The serum leptin exhibit diurnal variation, however, this has been reported to be absent in normal weighted amenorrheic athletes. Anorexia nervosa is associated with multiple endocrine abnormalities. Hypothalamic amenorrhoea often precedes the weight loss and may persist after weight recovery. We hypothesized that leptin could be involved in the regulation of eating behaviour and gonadal function in anorexia nervosa.

Decreased tyrosine kinase activity in partially purified insulin receptors from muscle of young, non-obese first degree relatives of patients with Type 2 (non-insulin-dependent) diabetes mellitus

SummaryRecently, we demonstrated insulin resistance due to reduced glucose storage in young relatives of Type 2 diabetic patients. To investigate whether this was associated with a defective insulin receptor kinase, we studied ten of these young (27±1 years old) non-obese glucose tolerant first degree relatives of patients with Type 2 diabetes and eight matched control subjects with no family history of diabetes. Insulin sensitivity was assessed by a hyperinsulinaemic, euglycaemic clamp. Insulin receptors were partially purified from muscle biopsies obtained in the basal and the insulin-stimulated state during the clamp. Insulin binding capacity was decreased by 28% in the relatives (p<0.05) in the basal biopsy. Tyrosine kinase activity in the receptor preparation was decreased by 50% in both basal and insulin-stimulated biopsies from the relatives. After stimulation with insulin “in vitro”, kinase activity was reduced in the relatives in basal (p<0.005) and insulin-stimulated (p<0.01) biopsies and also when expressed per insulin binding capacity (p≈0.05). Insulin stimulation of non-oxidative glucose metabolism correlated with “in vitro” insulin-stimulated tyrosine kinase activity (r=0.61, p<0.01) and also when expressed per binding capacity (r=0.53, p<0.025). We suggest that the marked defect in tyrosine kinase activity in partially purified insulin receptors from skeletal muscle is an early event in the development of insulin resistance and contributes to the pathophysiology of Type 2 diabetes.

A rapid technique for separation of thymocytes from suspensions by centrifugation through silicone oil

Abstract A method is described for rapid separation of thymocytes from suspensions by centrifugation of the cells through silicone oil. The usefulness of several radioactive compounds as markers for the trapped extracellular water space and the total water space of the cell pellets was investigated. According to the results obtained with [ 3 H]inulin and [ 3 H]methoxyinulin, the extracellular water space comprised approximately 12% of the total water space as determined by 3 H 2 O or C 35 S(NH 2 ) 2 . The thymocytes appeared to be undamaged after the separation as judged by their oxygen consumption, sodium content, and dexamethasone binding.

cav-p60 expression in rat muscle tissues

Abstract. Caveolae are plasmalemmal invaginations of uncertain function. In view of the large number of hypotheses on caveolar functions, it is important to identify which components of caveolae are tissue specific and which are general. The only well-characterized major protein of caveolae is caveolin, which exists in three tissue-specific isoforms: caveolin-1, -2, and -3. Recently cav-p60 was characterized as a 60-kDa caveola-specific protein in adipocytes. The distributions of cav-p60 and caveolin isoforms in different rat muscle tissues were examined by immunofluorescence and immunoelectron microscopy. Cav-p60 was present in caveolae of skeletal and heart muscle, in vascular and intestinal smooth muscle, and in adipocyte caveolae. Furthermore cav-p60 was present in endothelial cells and cells of perineural sheaths. Caveolin-1 and -2 were present in adipocytes, endothelial cells, and cells of perineural sheaths. In all kinds of vascular and intestinal smooth muscle, caveolin-1 and -2 were present at high levels, whereas caveolin-3 expression was low or undetectable, depending on the specific smooth muscle subtype. High levels of caveolin-3 were found only in caveolae and T tubules of skeletal and heart muscle. We conclude that cav-p60 is a highly specific marker of caveolae in many if not all cell types having caveolae.

Stimulating effect of hyperosmolarity on glucose transport in adipocytes and muscle cells

Abstract 1. 1. The effect of hyperosmolarity on sugar permeability was assessed in isolated fat cells, epididynaml fat pads and soleus muscles of the rat. 2. 2. Hyperosmolarity, whether induced by the addition of sucrose, mannitol or sorbitol, gave an up to 15-fold increase in glucose metabolism of isolated fat cells. The stimulating effect of mannitol (400 mM) and a submaximal concentration of insulin (5 μunits/ml) were similar with respect to the rates of onsent and reversal, kinetics and sensitivity towards 3-O- methylglucose or phlorizin. 3. 3. In whole epididymal fat pads and soleus muscles hyperosmolarity produced a marked increase in the release of 3-O- methylglucose without causing any decrease in K+ content. This effect could be abolished by phlorizin (5 mM) and showed nearly the same time-course as that induced by a submaximal concentration of insulin. 4. 4. It is concluded that hyperosmolarity stimulates the carrier system mediating glucose transport without impairing the overall integrity of the plasma membrane.