Structure-function relationships in the adipose cell. 3. Effects of bovine serum albumin on the metabolism of glucose and the release of nonesterified fatty acids and glycerol by the isolated adipose cell.

Serum albumin stimulates the uptake of U-glucose-(14)C and the incorporation of (14)C-counts into triglyceride glycerol and inhibits the incorporation of (14)C-counts into triglyceride fatty acids by isolated adipose cells; insulin and epinephrine enhance these effects. In the absence of hormones, these responses to albumin increase with increasing albumin concentration. In the presence of insulin, a qualitatively similar pattern of increasing responses to albumin is observed; the enhancement of each response by insulin is, however, only slightly potentiated by higher albumin concentrations. In contrast, in the presence of epinephrine, these responses to albumin are maximal at the lowest albumin concentration tested, 0.1%; the enhancement of each response by epinephrine is similarly maximal at 0.1% albumin, but decreases rapidly as the albumin concentration is raised. Increasing serum albumin concentrations do, however, stimulate the release of fatty acids and glycerol by epinephrine-treated cells increasingly until a plateau, determined by the epinephrine dose, is reached. These data support the suggestion that intracellular fatty acid levels function in the regulation of adipose cell activity, and further suggest that serum albumin plays a role in determining the metabolic fate of these fatty acids.

Evidence for separate peptide sequences related to the lipolytic and magnesium-accumulating activities of ACTH. Analogy with adrenergic receptors.

Native adrenocorticotropin [ACTH-(1-39)] and ACTH-(1-24) stimulate both lipolysis and magnesium accumulation in rat adipocyte plasma membrane vesicles. ACTH-(1-20) retains full lipolytic activity but has a minimal effect on magnesium accumulation. In contrast ACTH-(11-24) stimulates magnesium accumulation but not lipolysis. These findings indicate that within the ACTH molecule the peptide sequence responsible for stimulation of magnesium accumulation is distinctly separate from the core sequence (residues 4-10) essential for stimulation of adenylyl cyclase activity and cAMP mediated lipolysis. Phentolamine, an alpha-adrenergic antagonist, blocks the bulk of magnesium accumulation stimulated by native ACTH and norepinephrine; propranolol, a beta-adrenergic antagonist, blocks the earliest phase of Mg2+ uptake by these hormones but has little effect on net uptake. Isoproterenol, a beta-adrenergic agonist, stimulates magnesium uptake only minimally. The pattern of uptake stimulated by methoxamine, an alpha-adrenergic agonist, or ACTH-(11-24) is quite similar to that produced by native ACTH in the presence of propranolol. The receptor through which ACTH mediates stimulation of the bulk of magnesium appears to be analogous to the alpha-adrenergic receptor through which norepinephrine stimulates this same process.

Lipolytic and adenyl-cyclase-stimulating activity of N alpha-trinitrophenyl glucagon: comparison with other glucagon derivatives modified at the amino terminus.

The trinitrophenyl (TNP) derivative of glucagon has less lipolytic activity and potency than the carbamyl derivative. The N alpha, e-acetyl derivative has slightly less activity than the TNP derivative. In contrast to liver, where the TNP derivative fails to stimulate adenyl cyclase, all the derivatives stimulate this enzyme in the adipocyte.

Lipolytic and adenyl-cyclase-stimulating activity of glucagon 1-6: comparison with glucagon derivatives chemically modified in the 7-29 sequence.

Glucagon 1-6 has a maximum lipolytic activity (Lmax) in the rat adipocyte which is 66% of that of glucagon. The N epsilon-guanidyl derivative, modified at Lys12, has about the same Lmax as glucagon 1-6. Modifying the carboxyl groups of glucagon with glycinamide or removing the COOH-terminal residues with cyanogen bromide reduces Lmax to less than 25% of the level of glucagon. The potency of each of these analogs (A50) in microM is as follows: glucagon 6 X 10(-3); glucagon 1-6 2 X 10(-2); N epsilon-guanidyl glucagon 9 X 10(-3); glycinamide glucagon 10(-2); cyanogen bromide peptide of glucagon 2 X 10(-1). The ability of all of the glucagon analogs to stimulate adenyl cyclase was somewhat less than their lipolytic activities with the exception of the glycinamide derivative and the cyanogen bromide peptide, which were slightly more active in stimulating adenyl cyclase than in lipolysis. Glucagon 1-6 is much more potent in stimulating adipocyte than liver adenyl cyclase.

Synthetic position 5 analogs of adrenocorticotropin fragments and their in vitro lipolytic activity.

Twenty-three analogs of the ACTH-(4-10)-heptapeptide sequence, which forms the "active core" of adrenocorticotropin (ACTH) and related hormones, have been synthesized by the solid-phase method. These analogs all contain structural modifications at or near the 5-glutamic acid residue of ACTH. The peptides were purified to electrophoretic and chromatographic homogeneity. The peptides were assayed for lipolytic activity in an isolated cell system derived from rabbit adipose tissue. In this system, it was determined that residue 5 plays a very important "spacer" role in the peptide, but that this spacer function is not very dependent on the nature of the side chain of the position 5 amino acid. It was found, however, that a number of analogs containing basic residues (arginine or lysine) in position 3 and/or position 5 of ACTH-(3-10) and ACTH-(4-10) fragments have 5 to 10 times the activity of the respective parent peptides. The presence of a latent anionic locus in the rabbit fat-cell receptor for ACTH is suggested by this study.

Steroidogenic activity of fragments of adrenocorticotropin with arginine in residue positions 3 and 5.

The maximal steroidogenic response (Smax) to adrenocorticotropin (ACTH) is enhanced by the substitution of arginine in position 3. Though the Smax of adrenocorticotropin-(3-10)-octapeptide [ACTH-(3-10)] is only 20% of ACTH-(1-24) in isolated rabbit adrenal cells and 10% in rat cells, [Arg3]ACTH-(3-10) has an Smax comparable to that of the longer peptide. Activity determined from the midpoints of dose-response curves (A50), however, is several orders of magnitude less. The Smax of ACTH-(4-10) is not significantly different from that of the 3-10 analog. Substitution of arginine in the 5 position of ACTH-(4-10) decreases activity for rabbit cells, but does not affect activity for rat cells. The substitution of arginine in the 5 position of ACTH-(1-20) markedly decreases Smax in rabbit but not rat cells. The Smax of [Arg 3,5)]ACTH-(3-10), containing both substitutions, is greater than that of ACTH-(3-10), but much less than that of [Arg3]acth-(3-10) in both species studied. These findings contrast with those in the rabbit adipocyte, where both arginine-containing ACTH analogs have enhanced activity and the effects of both substitutions in a single peptide are additive.