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M C Alexander

Publications and source records attributed to M C Alexander.

10 recordsLinked to original sources
PubMed1988

Insulin stimulates glyceraldehyde-3-phosphate dehydrogenase gene expression through cis-acting DNA sequences.

Glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] mRNA levels are induced by physiologic concentrations of insulin in cultured 3T3-F442A adipocyte and H35 hepatoma cell lines. To examine the mechanism by which insulin regulates GAPDH mRNA levels in these two insulin-sensitive tissues, we have isolated a functional human GAPDH gene. When stably transfected and expressed in 3T3-F442A preadipocytes and H35 hepatoma cells, the intact human GAPDH gene is induced 10-fold by insulin in 3T3-F442A adipocytes and 3-fold by insulin in H35 hepatoma lines, which is similar to the induction obtained with the endogenous gene. A human GAPDH-chloramphenicol acetyltransferase construct, containing sequences -487 to +20 of the human gene fused to the chloramphenicol acetyltransferase gene, is regulated by insulin in stably transfected 3T3 adipocytes and stably or transiently transfected H35 hepatoma cell lines, whereas the Rous sarcoma virus-chloramphenicol acetyltransferase fusion protein is not. Thus, the inductive effect of insulin on human GAPDH gene expression is mediated through cis-acting sequences located between -487 and +20 of the human GAPDH gene.

Adipose Tissue↗
PubMed1982

Insulin-stimulated phosphorylation of ATP-citrate lyase in isolated hepatocytes. Stoichiometry and relation to the phosphoenzyme intermediate.

We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.

ATP Citrate (pro-S)-Lyase↗
PubMed1982

Role of insulin-stimulated protein phosphorylation in insulin action.

Insulin promotes both the phosphorylation and dephosphorylation of proteins in its target cells. Insulin-induced dephosphorylation has long been thought to serve an important regulatory function; the role of insulin-stimulation phosphorylation is less certain. The proteins known to be substrates for this reaction are ATP citrate (pro-3S)-lyase, acetyl-CoA carboxylase, and the ribosomal subunit S6. The evidence as to the physiological role and mechanism underlying the insulin-stimulated phosphorylation of these proteins is summarized. Present information suggests that insulin-stimulated phosphorylation may serve an important regulatory role in certain actions of insulin.

ATP Citrate (pro-S)-Lyase↗
PubMed1981

The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase.

We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.

ATP Citrate (pro-S)-Lyase↗
PubMed1979

Insulin and the phosphorylation of intracellular proteins.

Protein phosphorylation is a ubiquitous form of posttranslational protein modification in mammalian cells which often serves to regulate protein function. Insulin alters the activity of a number of enzymes known to be regulated via phosphorylation. With the premise that altered protein phosphorylation might be an obligatory intermediate step in insulin action, we have examined the effects of insulin on the phosphorylation of the major phosphopeptides in adipocytes and hepatocytes. Insulin affects overall protein phosphorylation in two ways: 1) Insulin selectively stimulates the phosphorylation of a major peptide in adipose tissue (MW 123,000) and liver (MW 46,000) through a mechanism independent of cAMP and the cAMP-dependent protein kinase. Net dephosphorylation is not observed with insulin as the sole hormone. 2) Insulin antagonizes cAMP-directed protein phosphorylation. The mechanism of insulin-stimulated phosphorylation and the possible role of this phenomenon in overall insulin action is discussed.

Adipose Tissue↗
PubMedDate not supplied

MR imaging of a choledochal cyst.

The preoperative magnetic resonance (MR) appearance of a choledochal cyst using the saturation recovery-spin echo technique is presented along with the CT and ultrasound images. Although CT suggested the correct diagnosis of choledochal cyst, the "solid" characteristics depicted by sonography led to confusion. The MR image demonstrated a mass, similar to the CT image with both a long T1 and T2, thus substantiating the cystic nature of the mass. Hepatoscintigraphy confirmed the obstructive nature of the lesion.

Child↗
PubMedDate not supplied

Interferon therapy.

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Cell Cycle↗