Highlights d Zfp516 binds UCP1 promoter to drive transcription d Zfp516 expression is regulated b... more Highlights d Zfp516 binds UCP1 promoter to drive transcription d Zfp516 expression is regulated by cold/sympathetic stimuli d Zfp516 promotes the browning of iWAT and prevents dietinduced obesity d Zfp516 ablation leads to defective brown fat formation in mice
The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits o... more The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits of the cardiac isozyme of phosphorylase kinase. beta-Subunit phosphorylation achieves a maximum level of between 1 to 2 mol of phosphate/mol of phosphorylase kinase, a value less than the stoichiometric content of beta-subunits in the enzyme. This, less than stoichiometric incorporation, is not a result of the presence of endogenous phosphate in equivalent sites in the remaining beta-subunit moieties. Pretreatment of phosphorylase kinase with phosphoprotein phosphatase, under conditions proven to dephosphorylate such sites, does not modify the observed extent of beta-subunit phosphorylation. alpha'-Subunit phosphorylation is initiated at a slower rate than beta but achieves a higher maximum level of incorporation. alpha'-Subunit phosphorylation, but not the extent of beta-subunit phosphorylation, is stimulated by MnCl2 and partially inhibited by NaF; neither is effected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The activation of cardiac phosphorylase kinase that occurs concomitantly with phosphorylation appears to be dependent upon phosphate incorporation into both the alpha- and beta-subunits. At low levels of activation a close correlation is observed between activation and either alpha-subunit phosphorylation, beta-subunit phosphorylation, or total phosphorylation. However, the cAMP-dependent catalyzed phosphorylation of alpha, at a time after which beta-subunit phosphorylation is already maximal, also results in activation of cardiac phosphorylase kinase.
The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation... more The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.
This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (a... more This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (approximately 1240-fold), as based upon SDS gel electrophoresis and sucrose density gradient ultracentrifugation (co-migration of enzyme activity and protein with constant specific activity throughout the peak). The molecular weight of the enzyme following purification is identical with that in the soluble extract from beef hearts, and similar to that of the rabbit skeletal muscle isoenzyme (i.e. approximately 1.3 X 10(6)) based on co-migration in sucrose density gradient ultracentrifugation. The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 134,000; beta, 125,000; gamma, 48,000. The subunit stoichiometry is determined to be alpha 1 beta 1.01 gamma 1.35 with a faint band co-migrating with purified bovine brain calmodulin. The enzyme displays little, if any, activity below pH 6.0, but activity increases markedly in the pH range of 6.8 to 8.2. After phosphorylation with pure cAMP-dependent protein kinase catalytic subunit and [gamma-32P]ATP (0.14 mM ATP, 4 mM magnesium acetate) in which the beta subunit was maximally phosphorylated (stoichiometry, 0.25 mol/mol; ratio of 32P in the alpha' and beta subunits, 1.90:1), enzyme activity was increased approximately 2-fold at pH 6.8, while at higher pH values, the effect of phosphorylation was less marked (20 to 50% increase at pH 8.2). Ca2+ is required for enzyme activity; ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.5 mM) completely inhibited the activity of th nonphosphorylated and phosphorylated enzyme, and activity was restored by the addition of Ca2+ (Ka for Ca2+, 1.94 and 1.35 microM for the nonphosphorylated and phosphorylated enzyme, respectively.
Blot analysis of poly(A+) RNA showed a rapid and dramatic insulin induction of fatty acid synthas... more Blot analysis of poly(A+) RNA showed a rapid and dramatic insulin induction of fatty acid synthase (FAS) mRNA in diabetic mouse liver. Insulin administration to diabetic mice increased the mRNA level 4-fold within 1 h, and a maximum of 19-fold was reached in 6 h. The nutritional induction of FAS mRNA by fastinglrefeeding was abolished by streptozotocin-induced diabetes and also by dibutyryl cAMP administered during refeeding in normal animals, demonstrating the roles of insulin and cAMP during nutritional manipulation. Run-on transcription analysis with isolated nuclei showed that the transcription rate of the FAS gene increased 3.5-fold after 30 min, reached a maximum of 7-fold after 2 h of insulin administration in diabetic animals, and was maintained at the maximum level to 6 h. The CAMP, administered to previously fasted normal mice during refeeding of a high carbohydrate diet, abolished the increased transcription of the FAS gene caused by nutritional manipulation. These results indicate positive control by insulin and negative control by dibutyryl cAMP of FAS gene transcription. Furthermore, the effect of insulin on the FAS mRNA level was abolished by cycloheximide administration. This indicates that ongoing protein synthesis is necessary for the transcriptional activation of the FAS gene by insulin. Fatty acid synthase (FAS)' plays a central role in de novo lipogenesis in mammals and birds by catalyzing all the reactions in conversion of acetyl-coA and malonyl-CoA to palmitate. The dimeric enzyme with a subunit M, of 250,000
Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) th... more Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte development.
Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was st... more Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.
Expression of critical enzymes in fatty acid and fat biosynthesis is tightly controlled by nutrit... more Expression of critical enzymes in fatty acid and fat biosynthesis is tightly controlled by nutritional and hormonal stimuli. The expression of fatty acid synthase, which catalyzes all reactions for synthesis of palmitate from acetyl-CoA and malonyl-CoA, and of mitochondrial glycerol-3-phosphate acyltransferase, which catalyzes the first acylation step in glycerophospholipid synthesis, is decreased to an undetectable level during fasting. Food intake, especially a high carbohydrate, fat-free diet after fasting, causes a dramatic increase in the transcription of these genes. Insulin secretion is increased during feeding and has a positive effect on expression. By using adipocytes in culture and transgenic mice that express the reporter gene driven by the fatty acid synthase promoter, the cis-acting sequence that mediates insulin regulation of the fatty acid synthase promoter was defined. Upstream stimulatory factors (USF) that bind to the Ϫ65 E-box are required for insulin-mediated transcriptional activation of the fatty acid symthase gene. Sterol regulatory element binding protein (SREBP)-1 may be also involved in induction of these genes during feeding. Using specific inhibitors and expressing various signaling molecules, we found that insulin regulation of the fatty acid synthase promoter is mediated by the phosphatidylinositol (PI)3-kinase signaling pathway and that protein kinase B/akt is a downstream effector. J. Nutr. 130: 315S-320S, 2000. KEY WORDS: • fatty acid synthase • glycerol-3 phosphate acyltransferase • insulin • USF • transcription 1 Presented at the symposium entitled "The Role of Long Chain Fatty Acyl-CoAs as Signaling Molecules in Cellular Metabolism" as part of the Experimental Biology 99 meeting held April 17-21 in Washington, DC. This symposium was part of the metabolic and disease processes theme sponsored by the American Society for Nutritional Sciences. Symposium proceedings are published as a supplement to
Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation... more Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation. Physiol. Rev. 78: 783–809, 1998. — The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional ...
pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes ... more pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state. pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation. Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation. For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa. Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain. We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overrid...
Long chain fatty acyl-CoA esters are potent in vitro and in vivo inhibitors of adenine nucleotide... more Long chain fatty acyl-CoA esters are potent in vitro and in vivo inhibitors of adenine nucleotide translocation in heart mitochondria. Within a short time following production of myocardial ischemia, the dog heart exhibits an increased concentration of long chain acyl-CoA esters associated with a decrease in adenine nucleotide translocase activity. In contrast to liver, the tricarboxylate carrier system for citrate and phosphoenolpyruvate is rather low in heart mitochondria. Phosphoenolpyruvate-stimulated calcium egress from heart mitochondria may, therefore, result from transport of this anion on the adenine nucleotide translocase. During myocardial ischemia effective modulation of these metabolite trasnport systems are disrupted by accumulation of long chain acyl-CoA esters which leads to a decrease in the overall energy charge of the cell.
Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protei... more Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protein found in the preadipocytes, inhibits adipocyte differentiation in vitro and in vivo. Here, we examined the processing of membrane form of Pref-1A to release the 50-kDa soluble form that inhibits adipocyte differentiation. The ectodomain cleavage of Pref-1 is markedly enhanced by phorbol 12-myristate 13-acetate in a dose- and time-dependent manner. The basal and stimulated cleavage is inhibited by the broad metalloproteinase inhibitor GM6001, a fact that suggests that cleavage of membrane Pref-1A is dependent on a metalloproteinase. Next, we showed that release of soluble Pref-1A is inhibited by TAPI-0 and by a tissue inhibitor of metalloproteinase-3, TIMP-3, that can inhibit tumor necrosis factor alpha converting enzyme (TACE), but not by TIMP-1 or TIMP-2. On the other hand, overexpression of TACE increases Pref-1 cleavage to produce the 50-kDa soluble form. Furthermore, this cleavage ...
Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth ... more Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34 þ cells in 10 of 12 MDS samples compared with CD34 þ cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34 þ hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.
Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown t... more Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown that FAS transcription rate is induced dramatically when fasted animals are refed with a high carbohydrate diet or when streptozotocin-diabetic mice are given insulin. We also reported that FAS gene transcription was up-regulated by insulin through the proximal promoter region from ØŠ71 to ØŠ50 and that upstream stimulatory factors (USFs), including USF1 and USF2, interact with this region in vitro. In the present study, by using site-directed mutagenesis of the ØŠ71/ØŠ50 region and correlating functional assays of the mutated promoter with USF binding activities, we demonstrate that the ØŠ65/ØŠ60 Ebox motif (5-CATGTG-3) is functionally required for insulin regulation and that USFs are in vivo components of the insulin response complex. Mutation of the ØŠ65/ ØŠ60 E-box sequence abolished insulin response in both transiently and stably transfected 3T3-L1 adipocytes in the ØŠ2.1 kb promoter context, which contains all the necessary regulatory elements of the promoter based on our previous transgenic mice studies, and in the minimal ØŠ67 promoter context. Gel mobility shift assays demonstrated that USFs can no longer bind to the ØŠ71/ ØŠ50 promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with the FAS promoter-luciferase reporter constructs increased insulin-stimulated FAS promoter activity. Moreover, cotransfection of dominant negative USF1 and USF2 mutants lacking the DNA binding domain inhibited the insulin stimulation of the FAS promoter activity. On the other hand, site-directed mutagenesis of the ØŠ65/ØŠ60 E-box surrounding sequences within the overlapped tandem copies of sterol regulatory elementbinding protein (SREBP) binding sites prevented SREBP from binding to ØŠ71/ØŠ50 promoter region in vitro but had no effect on insulin regulation of the FAS promoter in vivo. When rat liver nuclear extracts were used in gel mobility shift assays, only USF-containing protein-DNA complexes that can be supershifted by specific USF antibodies were observed. These results demonstrate that upstream stimulatory factor binding to the E-box at ØŠ65 is required for insulin regulation of the fatty acid synthase promoter.
To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty ... more To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5-flanking region (؊2100 to ؉67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene.
Highlights d Zfp516 binds UCP1 promoter to drive transcription d Zfp516 expression is regulated b... more Highlights d Zfp516 binds UCP1 promoter to drive transcription d Zfp516 expression is regulated by cold/sympathetic stimuli d Zfp516 promotes the browning of iWAT and prevents dietinduced obesity d Zfp516 ablation leads to defective brown fat formation in mice
The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits o... more The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits of the cardiac isozyme of phosphorylase kinase. beta-Subunit phosphorylation achieves a maximum level of between 1 to 2 mol of phosphate/mol of phosphorylase kinase, a value less than the stoichiometric content of beta-subunits in the enzyme. This, less than stoichiometric incorporation, is not a result of the presence of endogenous phosphate in equivalent sites in the remaining beta-subunit moieties. Pretreatment of phosphorylase kinase with phosphoprotein phosphatase, under conditions proven to dephosphorylate such sites, does not modify the observed extent of beta-subunit phosphorylation. alpha'-Subunit phosphorylation is initiated at a slower rate than beta but achieves a higher maximum level of incorporation. alpha'-Subunit phosphorylation, but not the extent of beta-subunit phosphorylation, is stimulated by MnCl2 and partially inhibited by NaF; neither is effected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The activation of cardiac phosphorylase kinase that occurs concomitantly with phosphorylation appears to be dependent upon phosphate incorporation into both the alpha- and beta-subunits. At low levels of activation a close correlation is observed between activation and either alpha-subunit phosphorylation, beta-subunit phosphorylation, or total phosphorylation. However, the cAMP-dependent catalyzed phosphorylation of alpha, at a time after which beta-subunit phosphorylation is already maximal, also results in activation of cardiac phosphorylase kinase.
The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation... more The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.
This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (a... more This study reports the purification of bovine cardiac phosphorylase kinase to near homogeneity (approximately 1240-fold), as based upon SDS gel electrophoresis and sucrose density gradient ultracentrifugation (co-migration of enzyme activity and protein with constant specific activity throughout the peak). The molecular weight of the enzyme following purification is identical with that in the soluble extract from beef hearts, and similar to that of the rabbit skeletal muscle isoenzyme (i.e. approximately 1.3 X 10(6)) based on co-migration in sucrose density gradient ultracentrifugation. The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 134,000; beta, 125,000; gamma, 48,000. The subunit stoichiometry is determined to be alpha 1 beta 1.01 gamma 1.35 with a faint band co-migrating with purified bovine brain calmodulin. The enzyme displays little, if any, activity below pH 6.0, but activity increases markedly in the pH range of 6.8 to 8.2. After phosphorylation with pure cAMP-dependent protein kinase catalytic subunit and [gamma-32P]ATP (0.14 mM ATP, 4 mM magnesium acetate) in which the beta subunit was maximally phosphorylated (stoichiometry, 0.25 mol/mol; ratio of 32P in the alpha' and beta subunits, 1.90:1), enzyme activity was increased approximately 2-fold at pH 6.8, while at higher pH values, the effect of phosphorylation was less marked (20 to 50% increase at pH 8.2). Ca2+ is required for enzyme activity; ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.5 mM) completely inhibited the activity of th nonphosphorylated and phosphorylated enzyme, and activity was restored by the addition of Ca2+ (Ka for Ca2+, 1.94 and 1.35 microM for the nonphosphorylated and phosphorylated enzyme, respectively.
Blot analysis of poly(A+) RNA showed a rapid and dramatic insulin induction of fatty acid synthas... more Blot analysis of poly(A+) RNA showed a rapid and dramatic insulin induction of fatty acid synthase (FAS) mRNA in diabetic mouse liver. Insulin administration to diabetic mice increased the mRNA level 4-fold within 1 h, and a maximum of 19-fold was reached in 6 h. The nutritional induction of FAS mRNA by fastinglrefeeding was abolished by streptozotocin-induced diabetes and also by dibutyryl cAMP administered during refeeding in normal animals, demonstrating the roles of insulin and cAMP during nutritional manipulation. Run-on transcription analysis with isolated nuclei showed that the transcription rate of the FAS gene increased 3.5-fold after 30 min, reached a maximum of 7-fold after 2 h of insulin administration in diabetic animals, and was maintained at the maximum level to 6 h. The CAMP, administered to previously fasted normal mice during refeeding of a high carbohydrate diet, abolished the increased transcription of the FAS gene caused by nutritional manipulation. These results indicate positive control by insulin and negative control by dibutyryl cAMP of FAS gene transcription. Furthermore, the effect of insulin on the FAS mRNA level was abolished by cycloheximide administration. This indicates that ongoing protein synthesis is necessary for the transcriptional activation of the FAS gene by insulin. Fatty acid synthase (FAS)' plays a central role in de novo lipogenesis in mammals and birds by catalyzing all the reactions in conversion of acetyl-coA and malonyl-CoA to palmitate. The dimeric enzyme with a subunit M, of 250,000
Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) th... more Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte development.
Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was st... more Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.
Expression of critical enzymes in fatty acid and fat biosynthesis is tightly controlled by nutrit... more Expression of critical enzymes in fatty acid and fat biosynthesis is tightly controlled by nutritional and hormonal stimuli. The expression of fatty acid synthase, which catalyzes all reactions for synthesis of palmitate from acetyl-CoA and malonyl-CoA, and of mitochondrial glycerol-3-phosphate acyltransferase, which catalyzes the first acylation step in glycerophospholipid synthesis, is decreased to an undetectable level during fasting. Food intake, especially a high carbohydrate, fat-free diet after fasting, causes a dramatic increase in the transcription of these genes. Insulin secretion is increased during feeding and has a positive effect on expression. By using adipocytes in culture and transgenic mice that express the reporter gene driven by the fatty acid synthase promoter, the cis-acting sequence that mediates insulin regulation of the fatty acid synthase promoter was defined. Upstream stimulatory factors (USF) that bind to the Ϫ65 E-box are required for insulin-mediated transcriptional activation of the fatty acid symthase gene. Sterol regulatory element binding protein (SREBP)-1 may be also involved in induction of these genes during feeding. Using specific inhibitors and expressing various signaling molecules, we found that insulin regulation of the fatty acid synthase promoter is mediated by the phosphatidylinositol (PI)3-kinase signaling pathway and that protein kinase B/akt is a downstream effector. J. Nutr. 130: 315S-320S, 2000. KEY WORDS: • fatty acid synthase • glycerol-3 phosphate acyltransferase • insulin • USF • transcription 1 Presented at the symposium entitled "The Role of Long Chain Fatty Acyl-CoAs as Signaling Molecules in Cellular Metabolism" as part of the Experimental Biology 99 meeting held April 17-21 in Washington, DC. This symposium was part of the metabolic and disease processes theme sponsored by the American Society for Nutritional Sciences. Symposium proceedings are published as a supplement to
Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation... more Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation. Physiol. Rev. 78: 783–809, 1998. — The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional ...
pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes ... more pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state. pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation. Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation. For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa. Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain. We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overrid...
Long chain fatty acyl-CoA esters are potent in vitro and in vivo inhibitors of adenine nucleotide... more Long chain fatty acyl-CoA esters are potent in vitro and in vivo inhibitors of adenine nucleotide translocation in heart mitochondria. Within a short time following production of myocardial ischemia, the dog heart exhibits an increased concentration of long chain acyl-CoA esters associated with a decrease in adenine nucleotide translocase activity. In contrast to liver, the tricarboxylate carrier system for citrate and phosphoenolpyruvate is rather low in heart mitochondria. Phosphoenolpyruvate-stimulated calcium egress from heart mitochondria may, therefore, result from transport of this anion on the adenine nucleotide translocase. During myocardial ischemia effective modulation of these metabolite trasnport systems are disrupted by accumulation of long chain acyl-CoA esters which leads to a decrease in the overall energy charge of the cell.
Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protei... more Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protein found in the preadipocytes, inhibits adipocyte differentiation in vitro and in vivo. Here, we examined the processing of membrane form of Pref-1A to release the 50-kDa soluble form that inhibits adipocyte differentiation. The ectodomain cleavage of Pref-1 is markedly enhanced by phorbol 12-myristate 13-acetate in a dose- and time-dependent manner. The basal and stimulated cleavage is inhibited by the broad metalloproteinase inhibitor GM6001, a fact that suggests that cleavage of membrane Pref-1A is dependent on a metalloproteinase. Next, we showed that release of soluble Pref-1A is inhibited by TAPI-0 and by a tissue inhibitor of metalloproteinase-3, TIMP-3, that can inhibit tumor necrosis factor alpha converting enzyme (TACE), but not by TIMP-1 or TIMP-2. On the other hand, overexpression of TACE increases Pref-1 cleavage to produce the 50-kDa soluble form. Furthermore, this cleavage ...
Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth ... more Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34 þ cells in 10 of 12 MDS samples compared with CD34 þ cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34 þ hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.
Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown t... more Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown that FAS transcription rate is induced dramatically when fasted animals are refed with a high carbohydrate diet or when streptozotocin-diabetic mice are given insulin. We also reported that FAS gene transcription was up-regulated by insulin through the proximal promoter region from ØŠ71 to ØŠ50 and that upstream stimulatory factors (USFs), including USF1 and USF2, interact with this region in vitro. In the present study, by using site-directed mutagenesis of the ØŠ71/ØŠ50 region and correlating functional assays of the mutated promoter with USF binding activities, we demonstrate that the ØŠ65/ØŠ60 Ebox motif (5-CATGTG-3) is functionally required for insulin regulation and that USFs are in vivo components of the insulin response complex. Mutation of the ØŠ65/ ØŠ60 E-box sequence abolished insulin response in both transiently and stably transfected 3T3-L1 adipocytes in the ØŠ2.1 kb promoter context, which contains all the necessary regulatory elements of the promoter based on our previous transgenic mice studies, and in the minimal ØŠ67 promoter context. Gel mobility shift assays demonstrated that USFs can no longer bind to the ØŠ71/ ØŠ50 promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with the FAS promoter-luciferase reporter constructs increased insulin-stimulated FAS promoter activity. Moreover, cotransfection of dominant negative USF1 and USF2 mutants lacking the DNA binding domain inhibited the insulin stimulation of the FAS promoter activity. On the other hand, site-directed mutagenesis of the ØŠ65/ØŠ60 E-box surrounding sequences within the overlapped tandem copies of sterol regulatory elementbinding protein (SREBP) binding sites prevented SREBP from binding to ØŠ71/ØŠ50 promoter region in vitro but had no effect on insulin regulation of the FAS promoter in vivo. When rat liver nuclear extracts were used in gel mobility shift assays, only USF-containing protein-DNA complexes that can be supershifted by specific USF antibodies were observed. These results demonstrate that upstream stimulatory factor binding to the E-box at ØŠ65 is required for insulin regulation of the fatty acid synthase promoter.
To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty ... more To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5-flanking region (؊2100 to ؉67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene.
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Papers by H. Sul