Ki-Han Kim

Sp1 mediates glucose activation of the acetyl-CoA carboxylase promoter.

Acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in the biosynthesis of fatty acids, is induced in the presence of high glucose levels. The ACC gene contains two promoters: promoter I (PI) expression is inducible under lipogenic conditions, while promoter II (PII) expression, even though constitutively expressed in all tissues, is also controlled under various physiological conditions. Examination of the expression pattern of a series of deletion constructs of PII showed that the region from −340 to −249 was essential for ACC induction. In addition, by electrophoretic mobility shift assays, supershift assays, and DNase I footprinting studies, we have detected the binding of the transcription factor Sp1 at the two GC-rich sequences located within the −340 to −249 region of promoter II. Mutations at the GC-rich sequences prevented binding of Sp1, and the induction of the PII promoter was no longer observed. Cotransfection studies, in Drosophila Schneider SL2 cells, with the Sp1 expression vector and PII-CAT constructs, have further confirmed the activation of promoter II by Sp1. In addition, we have identified Sp3, another member of the Sp1 family of transcription factors, as a second factor that can bind to the glucose response elements of PII.

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Physiological regulation of acetyl-CoA carboxylase gene expression: Effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA

We measured acetyl-CoA carboxylase mRNA levels in various tissues of the rat under different nutritional and hormonal states using a cDNA probe. We surveyed physiological conditions which are known to alter carboxylase activity, and thus fatty acid synthesis, to determine whether changes in the levels of carboxylase mRNA are involved. The present studies include the effects of fasting and refeeding, diabetes and insulin, and lactation on carboxylase mRNA levels. Northern blot analysis of liver RNA revealed that fasting followed by refeeding animals a fat-free (high carbohydrate) diet dramatically increased the amount of carboxylase mRNA compared to the fasted condition. These changes in the level of mRNA correspond to changes in the activity and amount of acetyl-CoA carboxylase. Acetyl-CoA carboxylase mRNA levels in epididymal fat tissue decreased upon fasting and increased to virtually normal levels after 72 h of refeeding, closely resembling the liver response. The amount of acetyl-CoA carboxylase mRNA decreased markedly in epididymal fat tissue of diabetic rats as compared to nondiabetic animals. However, 6 h after injection of insulin the mRNA level returned to that of the nondiabetic animals. Gestation and lactation also affected the levels of carboxylase mRNA in both liver and mammary gland. Maximum induction in both tissues occurred 5 days postpartum. These studies suggest that these diverse physiological conditions affect fatty acid synthesis in part by altering acetyl-CoA carboxylase gene expression.

Regulation of Acetyl-CoA Carboxylase

Publisher Summary This chapter discusses the regulation of acetyl-CoA carboxylase. Acetyl-CoA carboxylase catalyzes the ATP-dependent carboxylation of acetyl-CoA in the formation of malonyl-CoA. Malonyl-CoA is then condensed to acetyl-CoA in the process of long-chain fatty acid synthesis. Citrate activation of crude or purified carboxylase is accompanied by polymerization of the enzyme. Various methods of dissociating the high-molecular-weight polymeric form into the protomeric form in vitro result in the inactivation of the carboxylase. Acetyl-CoA carboxylase is inhibited by long-chain fatty acyl-CoA, and such inhibition is accompanied by enzyme depolymerisation. Inactivation of the carboxylase through covalent phosphorylation accompanies depolymerization in the absence of CO 2 , and this depolymerization occurs even in the presence of citrate. While covalent modification amplifies the sensitivity of the carboxylase toward the allosteric molecules at physiological concentrations, the allosteric molecules in turn affect the covalent modification.

cAMP Activation of CAAT Enhancer-binding Protein-β Gene Expression and Promoter I of Acetyl-CoA Carboxylase

The acetyl-CoA carboxylase (ACC) gene contains two distinct promoters, denoted PI and PII. PI is responsible for the generation of class I ACC mRNAs which are induced in a tissue-specific manner under lipogenic conditions. PII generates class II ACC mRNAs which are expressed constitutively. During 30A5 preadipocyte differentiation, both promoters are activated; the preadipocytes must be pretreated with cAMP for this activation to occur. In this report, we present evidence that CAAT enhancer-binding protein-β (C/EBP-β) is induced and involved in the PI activation by cAMP. Expression of the reporter gene under the control of the PI promoter is activated within 3 h after treatment of 30A5 cells with a cyclic AMP analogue, 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate, and 3-isobutyl-1-methylxanthine, in association with the accumulation of C/EBP-β mRNA and protein. These accumulations were inhibited in the presence of H8, a protein kinase inhibitor; H8 also inhibited activation of PI by cAMP. However, the induction of reporter gene expression and the increase of C/EBP-β mRNA by cAMP were not affected by treatment with tumor necrosis factor α, which completely inhibited the accumulation of C/EBP-α mRNA. Overexpression of C/EBP-β by transfection with the C/EBP-β gene led to increased binding of C/EBP-β to DNA and partial PI activation. cAMP did not affect the amount of C/EBP-β binding to the DNA but did promote phosphorylation of C/EBP-β and PI activation. As in the case of C/EBP-α, C/EBP-β bound to the CCAAT box of the PI promoter. These results indicate that cAMP not only induces, but also activates, bound C/EBP-β through phosphorylation for PI activation. Our studies also indicate that cAMP induces C/EBP-α. C/EBP-β induction, however, precedes that of C/EBP-α.

Essential Role of Acetyl-CoA Carboxylase in the Glucose-Induced Insulin Secretion in a Pancreatic β-Cell Line

The current model of the nutrient sensing mechanism in pancreatic beta-cells implies that malonyl-CoA plays a key role. According to this hypothesis, glucose activation of acetyl-CoA carboxylase triggers a rapid production of malonyl-CoA which inhibits carnitine palmitoyltransferase 1 and the importation of fatty acyl-CoA into the mitochondria for oxidation. The increase in cytosolic long chain fatty acyl-CoA leads to the exocytosis of insulin by a mechanism which has not yet been clearly defined. To obtain direct evidence that ACC plays a central role in this process, we generated stable transfectants of an insulin secreting cell line (INS-1) that express ACC specific antisense mRNA. The amounts of ACC mRNA and the protein level were specifically decreased in these stable clones compared to those of the control cells. The glucose activation of ACC in these cells was also significantly diminished. Both acute and long-term induction of insulin secretion by glucose were decreased. This decrease was inversely correlated to the levels of ACC activity in clones. In these clones, the insulin secretion induced by other nutrients, amino acids and ketocaproate, is also impaired, while the KCl-induced insulin secretion remains unchanged. Decreased ACC expression was accompanied by impaired malonyl-CoA production and elevated fatty acid oxidation. The expressions of the pancreatic specific glucokinase, glucose transporter 2 or beta-actin in these cells, as well as glucose utilisation were not affected, suggesting that the effect of the expression of the ACC mRNA specific gene on insulin secretion is specifically related to the decrease in the amount of ACC gene products. These results provide direct evidence of a causal relationship between ACC and insulin secretion.

TNF-α inhibits glucose-induced insulin secretion in a pancreatic β-cell line (INS-1)

Recent studies suggest that TNF‐α affects various biochemical and physiological processes which may be linked to the etiology of non‐insulin‐dependent diabetes mellitus (NIDDM). For example, TNF‐α interferes with the signaling of the insulin receptor and the metabolism of glucose transporters. The possibility that TNF‐α might directly reduce glucose‐stimulated insulin secretion in pancreatic β‐cells was examined by using an established pancreatic β‐cell line (INS‐1). TNF‐α did not affect glucose‐induced acute insulin secretion (30 min). However, over a longer time period (24 h), TNF‐α decreased glucose‐induced insulin secretion without affecting the total amount of insulin in the cell. In the presence of TNF‐α levels of 0, 10, 100 and 1000 U/ml, the respective 20 mM glucose‐induced insulin secretion was 1.736 ± 0.166, 1.750 ± 0.302, 1.550 ± 0.200, and 1.400 ± 0.112 mU/ml per 3 × 105 cells in 24 h.

Differential effects of metabolites on the active and inactive forms of hepatic acetyl CoA carboxylase

Abstract Partially purified acetyl CoA carboxylase was converted in vitro to its predominately phosphorylated (less active, b ) or dephosphorylated (active, a ) form. Studies of the properties of the two forms of carboxylase indicated that the a -form had a greater V than the b -form in the presence of different concentrations of citrate, pyruvate, MgATP 2− , MnATP 2− , acetyl CoA, and palmityl CoA. The concentration required for half-maximum stimulation of the a -form was less for citrate and the same as the b -form for MgATP 2− , MnATP 2− , and acetyl CoA. The concentration required for half-maximum inhibition of the a -form was higher for palmityl CoA, avidin, and ATP. The b -form was more strongly inhibited by palmityl CoA and avidin and this inhibition was partially reversed by citrate. These studies indicate that under normal physiological concentrations of metabolites, the b -form is virtually inactive. The physiological significance of the interconversion between the two forms of acetyl CoA carboxylase thus appears to lie in their differential response to the various metabolites which regulate the enzyme activity.

MOLECULAR CLONING AND CHARACTERIZATION OF P113, A MOUSE SNF2/SWI2-RELATED TRANSCRIPTION FACTOR

A full-length cDNA encoding a 113-kDa transcription factor, named P113, was cloned from mouse preadipocyte line 30A5. P113 binds to a 7-bp consensus TNF-response element and a 30-bp fragment from mouse PAI-1 promoter (-88/-59). Sequence analysis indicates that the P113 is highly homologous to HIP116/HLTF (human) and RUSH-1alpha (rabbit). The sequence homology and the fact that P113 contains seven motifs conserved in many DNA-dependent helicases/ATPases indicate that it is a new member of the SNF2/SWI2 protein family. A cysteine-rich motif, called RING finger, was found close to the C-terminus of P113. The expression pattern of P113 mRNA in rat tissues is significantly different from that of HLTF in human tissues. Affinity-purified P113 has an ATPase activity that is activated by DNA in a sequence-specific manner. Using Northern blot analysis and the PAI-1 promoter/luciferase system, we demonstrated that P113 is a transcription factor that activates the transcription of the PAI-1 gene in 30A5 cells.

Control of acetyl-CoA carboxylase by covalent modification.

SummaryIn this review, various experiments which establish the occurrence of covalent modification mechanisms, both in vivo and in vitro, in the control of acetyl-CoA carboxylase have been presented. It is interesting to note that phosphorylation of the carboxylase results in disaggregation of the active species. These studies indicate that aggregation and disaggregation of the enzyme are involved in the control of carboxylase activity. Our covalent modification mechanism and the allosteric control mechanism share a common ground in that both mechanisms affect the equilibrium between protomers and polymers of the enzyme. However, it is clear that the allosteric control mechanism cannot functon alone under normal physiological conditions. Covalent modification of the carboxylase is prerequiste for efficient functioning of the allosteric mechanism.There are many aspects of the regulation of acetyl-CoA carboxylase which require further clarification. However, it is now established that short-term control of acetyl-CoA carboxylase involves the covalent modification mechanism.