Humerus of Robust Australopithecus

128

Section: Discussionmentioning

confidence: 99%

Protein-free cell culture on an artificial substrate with covalently immobilized insulin.

Ito

Zheng²,

Imanishi³

et al. 1996

128

“…This binding fulfilled the re- quirements generally accepted to define a biologically important hormone receptor (4)(5)(6). The (42); however, these data have been questioned (43,44), and it is now known that the insulin-agarose complex is unstable in the presence of biological fluids (45)(46)(47). The solubilized insulin released from such preparations of insulin-agarose is sufficient to account for all of the activity detected (46,47).…”

Section: Discussionmentioning

confidence: 99%

Binding of insulin to isolated nuclei.

Goldfine¹,

Smith²

1976

135

(4)(5)(6).In tissues such as liver and fat, insulin has long-term effects on the synthesis of certain enzymes regulating the intracellular metabolism of glucose (7-9). These effects are-believed to result from an increased production of messenger RNA (7,(10)(11)(12) (27). Radioiodinated T3 (200,gCi/,ug) was purchased from Abbott Laboratories, and neutral-pH, carrier-free Na125I (300-500 mCi/ml) was purchased from New England Nuclear.Todination of Insulin and Glucagon. Insulin was radioiodinated by the stoichiometric chloramine-T technique under the conditions described for bovine thyrotropin (27). The initial concentrations of reactants were 15 MAM insulin, 30,gM Na251I, and 30,gM chloramine-T. After the preliminary purification step with Sephadex G-25 (27), the hormone was purified further on Sephadex G-50 (regular). This radioiodinated insulin (80-190 gCi/,gg) was 95-97% precipitable by 10% trichloroacetic acid and greater than 90% precipitable by excess insulin antibodies.Glucagon was radioiodinated as previously described (28). Preparation of Nuclei and Plasma Membranes. Nuclei were prepared by a'combination of the sucrose-density centrifugation (29, 30) and the Triton X-100 techniques (31). Female Sprague-Dawley rats, 80-120 g, fed ad lib, were anesthetized with ether and decapitated. After exsanguination the liver was rapidly removed, freed of connective tissue, placed in a chilled beaker, and minced. The following steps were performed at 4°. Ten grams of liver was added to 100 ml of buffer containing 0.25 M sucrose, 10 mM MgC12, and'20 mM Tris-HCI, pH 7.85 (STM buffer). The tissue was then homogenized with 10 strokes with a Potter-Elvehjem homogenizer (2000 rpm), filtered through four layers of cheesecloth, and centrifuged at 800 X g for 10 min. The pellet was then suspended in 60 ml of 2.2 M sucrose plus 1 mM MgCl2, divided into six portions, and centrifuged at 53,000 X g for 45 min. The combined pellets were then suspended in STM buffer plus 0.5% Triton X-100 (10 ml/g wet weight of liver) and centrifuged at

“…The metabolic effects of insulin are thought to be produced by the interaction of insulin with its receptors on the plasma membranes (1,2). This concept necessitates the involvement of a second messenger or an effector system to explain the mechanism of the complex cellular pleiotypic response to insulin.…”

mentioning

confidence: 99%

Ability of insulin to increase calcium binding by adipocyte plasma membranes.

McDonald¹,

Bruns²,

Jarett³

1976

Calcium specifically binds to adipocyte plasma membranes, demonstrating two classes of binding sites having affinity constants of 4.5 X 104 M-and 2.0 X 1O0 M-'. Insulin (100 microunits/ml) added directly to the isolated plasma membranes caused no alteration in calcium binding, whereas incubation of the adipocytes with 100 microunits/m of insulin resulted in a 25.0 k 1.6% increase in calcium binding to the subsequently isolated plasma membranes. The increase in calcium binding produced by insulin resulted from an increase in the maximum binding capacities of both classes of binding sites without alteration in their affinity constants. Additionally, a second pool of calcium in adipocyte plasma membranes has been identified by atomic absorption analysis; it was more than two times larger than the maximum binding capacity of the calcium binding system. This pool of calcium was stable, did not participate in the 45Ca2+ exchange, and was unaltered by insulin treatment. A similar stable pool of magnesium exists in plasma membranes and was also unaffected by insulin treatment. The increased capacity of the isolated plasma membranes to bind calcium after insulin treatment of the cells may represent an important bioregulating mechanism and supports the concept that calcium may play an important role in the effector system for insulin.The metabolic effects of insulin are thought to be produced by the interaction of insulin with its receptors on the plasma membranes (1, 2). This concept necessitates the involvement of a second messenger or an effector system to explain the mechanism of the complex cellular pleiotypic response to insulin. Considerable evidence suggests that adenosine 3':5'-cyclic monophosphate (cyclic AMP) does not fulfill the criteria of a second messenger for insulin in adipocytes (3, 4) and other cells (5, 6). Alternatively, the divalent cations, calcium and magnesium, have been proposed as possible second messengers for insulin (7-10), with a large body of indirect evidence to support this theory. Krahl has reported that insulin caused an increased intracellular magnesium concentration in rat hemiuteri (11) and adipose tissue (12) and that calcium and magnesium were necessary for maximum insulin stimulation of protein synthesis (12). Agents which promote an increased concentration of intracellular calcium, such as ouabain, procaine, lanthanum, and calcium ionophores, have been reported to mimic the antilipolytic action of insulin (9, 13, 14). Insulin was found to alter efflux of 45Ca2+ from preloaded fat pads and adipocytes (8, 10). Numerous intracellular enzymes have been reported to be both insulin and calcium sensitive (15)(16)(17)(18) (UDP glucose:glycogen 4-a-glucosyltransferase, EC 2.4.1.11) (18) isolated from adipocytes. Thus, small changes of calcium concentration at critical intracellular loci could have significant metabolic impact.The evaluation of cellular calcium distribution and regulation is difficult because intracellular calcium is highly compartmentalized with estimated concent...