Laura C. Huang

Many monoclonal antibodies with an apparent specificity for certain lung cancers are directed against a sugar sequence found in lacto-N-fucopentaose III

Abstract Monoclonal antibodies with an apparent specificity for human small-cell carcinoma, adenocarcinoma, and squamous carcinoma of the lung are produced by some hybridomas obtained from mice and rats immunized with an established line of human small cell lung cancer. Out of 85 of these antibodies produced by independently isolated hybridomas from 15 different fusions, 21 are directed against the sugar sequence which occurs in lacto- N -fucopentaose III ceramide, in several higher glycolipids and in glycoproteins. Specificity was determined by autoradiography of thin-layer chromatograms of glycolipids, by solid-phase radioimmunoassays, and by hapten inhibition studies. All 21 antibodies are of the immunoglobulin M type.

Infusion of pH 2.0 d-chiro-inositol glycan insulin putative mediator normalizes plasma glucose in streptozotocin diabetic rats at a dose equivalent to insulin without inducing hypoglycaemia

SummaryWe compared the effects of infusing a chemically defined chiro-inositol glycan putative insulin mediator with an equivalent dose of insulin in low-dose (45 mg/kg) streptozotocin diabetic rats. Insulin decreased plasma glucose levels from 17.32±0.17 to 3.96±0.064 mmol/l (p<0.0002) in 120 min, a decrease of 77.13%, while the putative mediator promoted a decrease in plasma glucose from 14.85±0.084 to 7.22±0.13 mmol/l (p<0.007) in 60 min. The putative mediator maintained euglycaemia over the ensuing 60 min with a plasma glucose level of 7.01±0.10 mmol/l at 120 min. Thus, insulin further reduced the plasma glucose from euglycaemia at 60 min to produce hypoglycaemia at 120 min. The lack of production of hypoglycaemia by the putative mediator can be explained by its inhibition of glucose-stimulated insulin secretion by the islet beta cells, thus providing a potential negative feedback regulatory mechanism; or by its selective action on muscle to increase glycogen synthesis. The significance of these results in terms of future directions in drug design is herein considered.

Studies on the insulin mediator. II. Separation of two antagonistic biologically active materials from fraction II.

SUMMARY Insulin treatment significantly altered the elution profile of deproteinized muscle extracts chromato-graphed on Sephadex G-25 columns, particularly in fraction II, which contains the insulin mediator. Further purification of fraction II by high-voltage paper electrophoresis at pH 1.9 and 3.5 resulted in two active fractions. Fraction 1 → 4 stimulated the cyclic AMP-dependent protein kinase and inhibited glycogen synthase phosphoprotein phosphatase, and may be a novel substance. Fractions 1 → > 6 and 3 → 6 inhibited the cyclic AMP-dependent protein kinase and stimulated glycogen synthase phosphatase. It is proposed that the insulin mediator is present in fractions 1 → 6 and 3 → 6.

Insulin mediator stimulation of pyruvate dehydrogenase phosphatases

A two stage assay for detecting insulin mediator based upon its stimulation of soluble pyruvate dehydrogenase (PDH) phosphatase to activate soluble pyruvate dehydrogenase complex (PDC) has been developed. This coupled assay determines the activation of PDC by monitoring production of [14C]CO2 from [1-14C]pyruvic acid. In addition to being more sensitive than the rat liver mitoplast assay previously used, it allows for the separation and investigation of the effects of mediator on the PDH phosphatases individually. It has been previously shown that the insulin mediator stimulates the most abundant PDH phosphatase, the divalent cation dependent PDH phosphatase, by decreasing the phosphatases metal requirement (1). A metal independent PDH phosphatase has been found in bovine heart mitochondria. This phosphatase is not immunoprecipitated by antiphosphatase 2A antibody, it is not inhibited by okadaic acid, and it is not stimulated by spermine. However, it is stimulated (more than threefold) by insulin mediator prepared from isolated rat liver membranes. It is inhibited by Mg-ATP, with half-maximal inhibition at 0.3 mM; however, this inhibition is overcome by the insulin mediator.

Cyclic AMP regulation of protein kinase(s) in rabbit skeletal muscle: Nucleoside triphosphate and divalent cation specificity

Abstract Two fractions of protein kinase(s) from rabbit skeletal muscle have been shown to catalyze the phosphorylation of endogenous protein, histone, and glycogen synthase I utilizing UTP, GTP, CTP, and ITP as phosphoryl donors. With one protein kinase fraction, it was found that the enzymatic activity was enhanced by Mn2+ when CTP served as the phosphoryl donor. This is in contrast to the inhibitory effect of Mn2+ on the kinase activity with ATP as the phosphoryl donor. With the second protein kinase fraction, the UTP, GTP, CTP and ITP associated phosphorylation reactions were found to be inhibited by cyclic AMP, whereas the phosphorylation reaction in which ATP served as the phosphoryl donor was found to be stimulated by cyclic AMP. These results suggest that different forms of the protein kinase(s) may exist which have different nucleoside triphosphate donor specificity and metal ion requirement. The serine residues of endogenous protein have been demonstrated to be the sites of phosphorylation when UTP, GTP, CTP and ITP served as the phosphoryl donors.

Insulin unexpectedly increases the glucose 6-phosphate Ka of skeletal muscle glycogen synthase in calorie-restricted monkeys.

In skeletal muscle of normal subjects, the concentration of glucose 6-phosphate (G6P) at which the activity of glycogen synthase (GS) is half maximal (Ka) is decreased by in vivo insulin, and the fractional activity is increased without a change in GS maximal activity (Vmax). We have shown that moderate chronic calorie restriction, previously shown in rodents to be effective in slowing aging, resulted in the prevention of obesity and type 2 diabetes in primates (rhesus monkeys, Macaca mulatta). However, unexpectedly, in a subgroup of calorie-restricted monkeys, insulin during a euglycemic hyperinsulinemic clamp caused an unanticipated decrease in skeletal muscle GS fractional activity. These same monkeys had the lowest whole-body glucose disposal rate (M), the greatest increase in skeletal muscle G6P content and the greatest increase in skeletal muscle glycogen phosphorylase activity during the euglycemic hyperinsulinemic clamp compared to the remaining calorie-restricted monkeys with normal insulin action. To determine whether this highly unusual insulin-mediated decrease in GS fractional activity was due to increased phosphorylation (increased Ka), we measured the activity of skeletal muscle GS at 9 different G6P concentrations before and during the euglycemic hyperinsulinemic clamp in 6 calorie-restricted monkeys. G6P Ka increased (n = 4) and Vmax decreased (n = 5) during the clamp. Basal G6P Ka was inversely related to basal GSfv (r = -0.94, p < 0.002). G6P Ka and skeletal muscle G6P content were positively related under insulin-stimulated conditions (r = 0.93, p < 0.005). The change in G6P Ka (insulin-stimulated minus basal) was inversely related to M (r = -0.94, p < 0.002) and positively related to the change in skeletal muscle G6P content (r = 0.93, p < 0.005). We conclude that moderate calorie restriction results in a reversal of normal insulin action at the skeletal muscle with inactivation of glycogen synthase which is likely to be due to an increase in phosphorylation of GS together with a decrease in Vmax of GS during a euglycemic hyperinsulinemic clamp in most of the calorie-restricted monkeys. These alterations are likely to be involved in the anti-diabetogenic effects of calorie restriction.

Stimulation of muscle glycogen synthase phosphatase by polyamines

The naturally-occurring polyamines were found to stimulate glycogen synthase phosphatase from rat skeletal muscles. The sequence of effectiveness in the stimulation was spermine greater than spermidine greater than putrescine. It was shown that the spermine-sensitive phosphatase was present primarily in the soluble fraction of the muscle extract. In the presence of spermine, the phosphatase activity can be further stimulated by Mn2+; however, a lower Mn2+ concentration is required for the activation of the enzyme in comparison with that in the absence of spermine. Kinetic studies indicated that activation of glycogen synthase phosphatase by spermine was achieved by an increase in its V without significant alteration in the Km, suggesting that spermine directly stimulated the catalytic efficiencly of the phosphatase enzyme.

Fasting Decreases the Content of D-Chiroinositol in Human Skeletal Muscle

Two classes of inositol phosphoglycans have been implicated as second messengers of insulin, one that activates pyruvate dehydrogenase and contains D-chiroinositol, and one that inhibits cyclic AMP–dependent protein kinase and contains myoinositol. We examined the effects of a 3-day fast on muscle contents of inositols in healthy humans. An oral glucose tolerance test was performed and a biopsy was obtained from the quadriceps femoris muscle after an overnight fast and after a 72-hour fast. The 72-hour fast significantly increased plasma glucose (1.5- to 2-fold) and insulin (2- to 4-fold) after glucose ingestion versus the values after the overnight fast, indicating the manifestation of peripheral insulin resistance. The 72-hour fast resulted in an ∼20% decrease in the muscle content of D-chiroinositol (P < 0.02), but no change in the myoinositol content. These data demonstrate that fasting specifically decreases the muscle content of D-chiroinositol in human muscle and this may contribute to the finding that insulin-mediated activation of pyruvate dehydrogenase is attenuated after short-term starvation.

The function of Mg-ATP in interactions between the regulatory and catalytic subunits of type I cAMP-dependent protein kinase from rabbit skeletal muscle

The regulatory subunit of Type I cAMP-dependent protein kinase from rabbit skeletal muscle can bind [3H]cAMP to form the R-[3H]cAMP complex, and the slow phase of the enhanced exchange of free cAMP with [3H]cAMP from the R-[3H]cAMP complexes was studied under various conditions using the equilibrium isotope exchange technique. Results indicate that Mg-ATP and the catalytic subunit are absolutely required for the enhanced exchange reaction to occur, but phosphorylation of the regulatory subunit by Mg-ATP does not play a determining role in the slow rate of the dissociation/association of the Type I protein-kinase in the presence of cAMP and the catalytic subunit. We interpret the role of Mg-ATP as being one in which it may provide the structural attributes required for formation of a stabilized transient state of the cAMP-regulatory subunit-catalytic subunit ternary complex, an obligatory intermediate involved in the dissociation/association of Type I cAMP-dependent protein kinase.

Insulin Mediators and Their Control of Covalent Phosphorylation

We have detailed two separate mechanisms for insulin to activate glycogen synthase. In mechanism I, insulin acts without glucose present via a mediator to convert the cyclic AMP-dependent protein kinase to a desensitized holoenzyme, effectively lowering the response of the cell machinery to existing concentrations of cyclic AMP. Mechanism II, originally discovered in rat adipocytes, is seen in the presence of glucose, or of a hexose whose transport is accelerated by insulin. Enhanced phosphorylation of the 6 position is also required, since the hexose-6-phosphate acts informationally to activate the phospho-protein phosphatase to convert glycogen synthase to its dephospho state. Today I should like to discuss recent findings of our laboratory which demonstrate the presence of mechanism II in muscle, namely, mouse diaphragm, pointing out the generality of this mechanism and discuss our recent studies on the identification of insulin mediators which control protein phosphorylation state.