Biochemical medicine and metabolic biology最新文献

This study was performed to determine whether vitamin E supplementation in streptozotocin-induced diabetic rats treated by insulin could reduce serum oxidation markers (malondialdehyde:MDA, Schiff bases, anti-protein-MDA adduct antibodies) and modulate lipid changes. After 10 weeks, diabetes induced in rats a significant increase in Schiff bases (P < 0.006) and anti-protein-MDA adduct antibodies (P < 0.01). These alterations were accompanied by a significant rise in serum free fatty acids (225%), triglycerides (35%), and phospholipids (30%) and changes in fatty acid distribution in these fractions and in cholesterol esters. Vitamin E supplementation in diabetic rats reduced Schiff bases and antiprotein-MDA adduct antibodies and tended to restore the fatty acid profile close to control rats without decreasing quantitatively serum lipids enhanced by diabetes. Concerning fatty acids, vitamin E chiefly reduced stearic acid (C18:0) in free fatty acids, cholesterol esters, and phospholipids and cancelled the decrease in low molecular triglycerides observed in diabetic rats. Furthermore, vitamin E maintained the ratio of monounsaturated and polyunsaturated fatty acids, particularly with respect to oleic acid (C18:1), dihomo-γ-linolenic acid (C20:3 n-6), eicosapentaenoic (C20:5 n-3), and docosapentaenoic acid (C22:5 n-3), in serum phospholipids. These changes observed in vitamin E supplemented rats, compared to vitamin E-untreated diabetic rats, could favor prevention of accelerated atherogenesis. Particularly, the decrease of serum peroxides and enhancement in phospholipid fatty acids (C20:3 n-6, C20:5 n-3, and C22:5 n-3) could induce the preferential formation of prostaglandins (PGE₁, PGI₂, PGI₃) which are protective in cardiovascular diseases.

The effects of copper on metal regulatory factors were studied in cultured human fibroblasts using mobility shift assays and protein blotting. A mobility shift was apparent on polyacrylamide gels when fibroblast whole cell extracts were reacted with a ³²P-labeled 14-base-pair MREa sequence of the hMT IIA gene. Binding was specific, since the shifted band was not competed away by MREa analogs containing two base changes. Known transcription factors SP1, AP1, AP2, and OCT1 did not compete with the fibroblast metal regulatory element binding protein (MREBP) for binding to the MREa probe. When fibroblasts were grown in 200 or 800 μM copper, binding of the MREBP was enhanced. Protein blotting experiments were performed by electrophoresing cell extracts, blotting onto nitrocellulose, and probing with the ³²P-labeled MREa 14-mer. A 112-kDa band, apparent in HeLa cell nuclear extract as well as fibroblast whole cell extract, was not influenced by the presence of copper in the growing fibroblasts. A 34-kDa protein, also present in both HeLa cells and fibroblasts, appeared to correspond with the protein causing the mobility shift. Copper added to the fibroblast growth media enhanced binding of the 34-kDa protein to MREa using the blotting procedure. We conclude that the fibroblast 34-kDa MREBP is a positive regulatory factor influenced by copper. Human fibroblasts can be employed to further study metal regulatory factors whose binding to metal regulatory elements is influenced by copper and other heavy metals.

The acute effects of streptozotocin-induced diabetes on several parameters of glucose metabolism were investigated in rat peritoneal macrophages. These cells accumulated in vitro about twofold more fructose 2,6-bisphosphate in the presence of increasing glucose concentration than cells from normal rats, and an increased production of lactate was observed. Phosphofructokinase-1, phosphofructokinase-2, hexokinase, and pyruvate kinase activities were increased in cells from diabetic rats compared with those from normal rats. Transport of 2-deoxy-D-glucose was increased in cells from diabetic rats. [U-¹⁴C]Glucose incorporation into glycogen was also increased in cells from diabetics and the ¹⁴CO₂ liberation was less than in cells from normal animals. Moreover, macrophages from diabetics did not possess a more active pentose phosphate pathway (measure with [1-¹⁴C]glucose oxidation) nor a greater production of superoxide anion (index of activation of macrophages) than in cells from normal animals.

The metabolic, ionic, and secretory response to D-glucose was investigated in islets of adult rats either injected with streptozotocin during the neonatal period (STZ rats) or presenting with inherited diabetes (GK rats). At a high concentration of D-glucose (16.7 mM), the ATP/ADP ratio was lower in islets from STZ and GK than control rats. This coincided with an impaired response of perifused islets to a rise in o-glucose concentration in terms of stimulation of insulin release, suppression of effluent radioactivity from islets prelabeled with [2-³H]adenosine, reduction in ⁸⁶Rb efflux, and induction of a phosphate flush in islets prelabeled with ³²P_i. The ratio in either D-[5-³H]glucose utilization or D-[2-¹⁴C]glucose oxidation at high/low hexose concentration, as well as the paired ratio between D-[2-¹⁴C]glucose oxidation and D-[5-³H]glucose utilization in islets incubated at a high concentration of the hexose, was also lower in STZ and GK rats than in control rats. Such was not the case, however, from the oxidation of [2-¹⁴C]pyruvate. Instead, the latter 2-keto acid, when tested at a 5.0 mM concentration, improved more efficiently the overall oxidative response of the islets to a rise in D-glucose concentration in STZ and GK rats than in control animals. It is proposed, therefore, that in both STZ and GK rats, the B-cell secretory defect is primarily attributable to an anomaly in oxidative glycolysis. In islets exposed to a high concentration of D-glucose, this metabolic deficiency results in impaired ATP generation, altered closing of ATP-responsive K⁺ channels, and, hence, diminished insulin output.

Maple syrup urine disease results from defects in the branched chain α-ketoacid dehydrogenase complex. Cells from seven German, three Turkish, and two Italian families including five consanguineous matings were analyzed for the causative mutations. Enzyme assays were used to confirm the initial clinical diagnosis of all probands. Immunoblots of mitochondrial proteins from these probands revealed reduced expression of the E1 α and β proteins of the complex. Previous studies showed that interaction of α and β was necessary to stabilize both proteins so that defects in either protein can result in decreased presence of both. The E1α Y393N mutation common in the Mennonite population that results in diminished amounts of both α and β proteins was not the cause of the reduction in these European patients.

Renal transport of glycine was studied in control and glutathione-depleted rats. Diethylmaleate (4.0 mmol/kg body wt, ip) was used as a glutathione depletor agent and the studies were carried out 6 and 10 h post-diethylmaleate injection. Renal transport was measured in isolated rat kidney preparations by means of clearance techniques and in brush border membrane vesicles by a rapid filtration method. Tubular reabsorption of glycine, when compared to glomerular filtration rate, measured at different substrate tubular loads, was higher in treated kidneys. Tissue ¹⁴C accumulation was also higher in kidneys from diethylmaleate-treated animals. Studies with brush border membrane vesicles indicated that glutathione depletion induced higher sodium-dependent glycine uptake in contrast with control preparations. This adaptation was not associated with an increment in either τ-glutamyltransferase activity or in protein concentrations. These results could explain in part the replenishment of GSH cellular levels in glutathione-depleted kidneys by means of higher transport capacity for glycine (a glutathione precursor) which was maintained even when GSH levels were restored.

Dopamine plays a major role in the regulation of appetite and growth hormone. Dopaminergic agonists suppress appetite and dopamine D₂ receptor antagonists enhance it. We examined the hypothesis that allelic variants of the DRD2 locus may be associated with weight and height. Sarkar and Sommer described two DRD2 polymorphisms that could be haplotyped by PCR. For weight, the mean Z score (National Center for Health Statistics) for 208 subjects without haplotype 4 was 0.086 versus 0.557 for 280 subjects with haplotype 4, P = 0.0003. Two separate sets of subjects were studied and these results were significant for both, providing an internal replication. For height, the mean Z score for 164 subjects without haplotype 4 was 0.1677 versus 0.6885 for 219 subjects with haplotype 4, P < 0.00001. These and other data suggest that the 4 haplotype is in linkage disequilibrium with allelic variants of the DRD2 gene that play a major role in the regulation of weight (obesity) and height, and may serve as a risk factor in late-onset non-insulin-dependent diabetes mellitus (NIDDM).

Topoisomerase I (Topo I) is involved in many cellular functions that involve unwinding of supercoiled DNA, such as transcription and replication. Topo I is also the target of autoimmune antibodies in progressive systemic sclerosis (scleroderma), and abnormal regulation of Topo I may influence the excessive production of collagen found in scleroderma. Topo I is phosphorylated in vivo at serine residues and, in vitro, the activity of Topo I is increased by phosphorylation by casein kinase type II (CKII) and protein kinase C (PKC). In this study, a protein kinase activity from rat liver nuclei is shown to copurify with Topo I during Bio-Rex 70 cation exchange chromatography. The kinase can phosphorylate Topo I at senile residues, resulting in a threefold increase in topoisomerase activity. A relatively tight association between this kinase and Topo I is demonstrated by the ability to coprecipitate the kinase with scleroderma autoimmune anti-Topo I antibodies. The kinase activity is similar to CKII since it is Ca²⁺ and cyclic nucleotide independent, it can utilize either ATP or GTP as phosphate donor, and it can phosphorylate casein and phosvitin. but not histones. However, unlike typical CKII, the Topo I-associated kinase could utilize Mn²⁺ almost as well as Mg²⁺, it is not stimulated by polyamines, and it does not appear to undergo autophosphorylation. In conclusion, we demonstrate that rat liver Topo I is relatively tightly associated with a CKII-like protein kinase that can phosphorylate and activate Topo I. These findings provide corroborative evidence that CKII, or a CKII-like protein kinase, is a physiologic regulator of Topo I.