Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Primary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Gal beta 1-3GalNAc alpha R beta 1-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of O-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats.

Adenine nucleotide pool size, adenine nucleotide translocase activity, and respiratory activity in newborn rabbit liver mitochondria.

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0 +/- 0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5 +/- 1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8 +/- 0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r = 0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation fo adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.

Acute postnatal regulation of pyruvate carboxylase activity by compartmentation of mitochondrial adenine nucleotides.

The total adenine nucleotide content of the mitochondrial matrix increases 3--4-fold within a few hours of birth in rat liver. This provides a mechanism for the acute postnatal regulation of pyruvate carboxylase, which is located in the matrix compartment and which requires ATP as a substrate. The sudden increase in pyruvate carboxylase activity is proposed to account for a rapid 4--5-fold increase in the gluconeogenic rate of the newborn rat.

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1.

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.

Comparison of chondrogensis in static and perfused bioreactor culture.

As a result of the low yield of cartilage from primary patient harvests and a high demand for autologous cartilage for reconstructive surgery and structural repair, primary explant cartilage must be augmented by tissue engineering techniques. In this study, chondrocytes seeded on PLLA/PGA scaffolds in static culture and a direct perfusion bioreactor were biochemically and histologically analyzed to determine the effects of fluid flow and media pH on matrix assembly. A gradual media pH change was maintained in the bioreactor within 7.4-6.96 over 2 weeks compared to a more rapid decrease from 7.4 to 6.58 in static culture over 3 days. Seeded scaffolds subjected to 1 microm/s flow demonstrated a 118% increase (p < 0.05) in DNA content, a 184% increase (p < 0.05) in GAG content, and a 155% (p < 0.05) increase in hydroxyproline content compared to static culture. Distinct differences were noted in tissue morphology, including more intense staining for proteoglycans by safranin-O and alignment of cells in the direction of media flow. Culture of chondrocyte seeded matrices thus offers the possibility of rapid in vitro expansion of donor cartilage for the repair of structural defects, tracheal injury, and vascularized tissue damage.

Dietary lipid and postnatal development. I. A model for neonatal studies requiring high and low fat diets from birth.

As a prerequisite to a biochemical investigation into the role of dietary lipids in regulating perinatal fatty acid metabolism, we developed and evaluated a model in which the amount of lipid in the diet can be easily controlled from birth to at least 10 days of age. Neonatal rabbits were fed purified equicaloric diets in which lipid supplied either 14.2% or 77.6% of the total calories. Mortality due to all causes was about 40% in both groups. Some differences in physical development between the two groups were noted. Animals receiving the smaller amount of lipid (LF group) gained weight to 150% birth weight day 10, whereas the group receiving the larger amoung of lipid (HF group) did not gain. In addition, the HF group had lower body temperatures, smaller liver and kidney weights, and greater brain weights relative to body weight. These differences are discussed in relation to the composition of the HF and LF diets. The model promises to provide a direct approach to a more precise evaluation of varied dietary regimens in the neonatal period. In particular, the model lends itself to studies relating diet to developing biochemical functions. In an accompanying paper, we report the results of an investigation comparing fatty acid oxidation in heart and liver of neonates fed the HF and LF diets described here.

Liver and skeletal muscle mitochondrial function following burn injury.

The possibility of altered mitochondrial function consequent to burn injury was investigated. Mitochondria isolated from liver or skeletal muscle of burn-injured rats (20% tbs) were compared at 3 days postburn to shams and normal controls. Mitochondrial yields were the same for all groups. ADP;O ratios were in the theoretical ranges expected and did not differ among burn, sham, and normal animals. Respiratory control ratios (RCR's) were decreased in liver mitochondria, averaging 71.7% of normal for burned animals compared to 95.8% for the sham group. The loss of respiratory control in liver mitochondria implies inefficient use of substrate chemical energy and could contribute to postburn hypermetabolism. The different response of muscle mitochondria as compared to liver suggests that alterations may be organ specific.

Phosphorylase synthesis in diabetic hepatocytes and cardiomyocytes.

Whereas total cardiac glycogen phosphorylase activity appears to be unaffected by severe insulin deficiency, a diabetes-induced decreased in hepatic glycogen phosphorylase activity has been demonstrated by our laboratory and others using liver extracts, isolated perfused liver, and cultured hepatocytes. The loss of activity in diabetic liver can be correlated with a drop in protein levels. Using primary cultures of cells from normal and diabetic rats and phosphorylase specific antibodies, we found a corresponding decrease in phosphorylase synthesis in diabetic hepatocytes cultured for 2 days in a serum-free, chemically defined medium. When hepatocytes are cultured in the presence of insulin, triiodothyronine, and cortisol, there is a significant recovery in the rate of phosphorylase synthesis after 3 days. Over the 3-day time period, there is no significant difference in the rate of phosphorylase degradation in normal compared with diabetic hepatocytes. Total protein synthesis in both hepatocytes and cardiomyocytes is unaffected by diabetes, as is phosphorylase synthesis in cultured cardiomyocytes.

Transformation of adult ventricular myocytes with the temperature sensitive A58 (tsA58) mutant of the SV40 large T antigen.

Freshly isolated ventricular myocytes have been used extensively as an adult cardiac model system. Due to their inability to undergo cytokinesis in vitro and their dedifferentiated properties in long-term culture, they can not be used for extended studies. Recent reports tell of the establishment of fetal and neonatal cardiac cell lines and the development of adult cardiomyocytes from transgenic animals. A recent report by Kirshenbaum [1], is the first to demonstrate insertion of genes in to adult ventricular myocytes using viral infection. This paper discusses the infection of primary adult differentiated cardiomyocytes with the SV40 large T antigen and subsequent proliferation under temperature sensitive control. Upon further characterization, the cells could be used as a model to study muscle differentiation and repair as well as adult cardiac cell physiology.

Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control.

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.

Improved hepatocyte culture system for studying the regulation of glycogen synthase and the effects of diabetes.

When 3-4-week-old rats (young rats) are used as a source of hepatocytes, primary culture cells express the adult, differentiated, liver-specific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adult (> 150 g rat) hepatocyte cultures. Corresponding synthase enzyme activity and mRNA levels decrease over time in culture in adult but not in young hepatocyte cultures. Young rat hepatocytes also have the ability to proliferate in chemically defined medium in the absence of added mitogens. A diabetes-induced increase in total synthase activity has been demonstrated by our lab and others, using cultured hepatocytes, liver homogenates, and perfused livers. In the present study, utilizing synthase-specific antibody and primary cultures of cells from young normal and alloxan diabetic rats, we found that greater total synthase activity in the diabetic cells was associated with higher levels of enzyme protein. Immuneprecipitation of 35S methionine-labeled freshly plated cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are correspondingly increased in the diabetic relative to normal cells. Chronic exposure of young, normal hepatocytes to increasing levels of glucose induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells from diabetic animals do not respond by any of these measures to increased glucose concentrations. We conclude that this defined primary culture system represents a useful model for investigating the regulation of hepatic glycogen synthase and the defects which occur as a result of diabetes.

Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver.

A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.

Wortmannin inhibits insulin-stimulated activation of protein phosphatase 1 in rat cardiomyocytes.

A major function of insulin in target tissues is the activation of glycogen synthase. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the insulin-induced activation of glycogen synthase, although the true function of this enzyme remains unclear. Data presented here demonstrate that the PI3K inhibitors wortmannin and LY-294002 block the insulin-stimulated activation of protein phosphatase 1 (PP1) in rat ventricular cardiomyocytes. This loss of phosphatase activation mimics that seen in diabetic cardiomyocytes, in which insulin stimulation fails to activate both PP1 and glycogen synthase. Interestingly, in diabetic cells, insulin stimulated PI3K activity to 300% of that in untreated controls, whereas this activity was increased by only 77% in normal cells. PI3K protein levels, however, were similar in normal and diabetic cells. Our results indicate that PI3K is involved in the stimulation of glycogen synthase activity by insulin through the regulation of PP1. The inability of insulin to stimulate phosphatase activity in diabetic cells, despite a significant increase in PI3K activity, suggests a defect in the insulin signaling pathway that contributes to the pathology of insulin-dependent diabetes.