Effect of plumbagin on some glucose metabolising enzymes studied in rats in experimental hepatoma

Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) isolated from Plumbago zeylanica Linn, when administered orally, at a dosage of 4 mg/kg body weight induces tumour regression in 3-methyl-4-dimethyl aminoazobenzene (3Me-DAB) induced hepatoma in Wistar male rats. The purpose of this investigation was to identify the changes in the rate of glycolysis and gluconeogenesis in tumour-bearing rats and the effects of treatment with Plumbagin. The levels of certain glycolytic enzymes, namely, hexokinase; phosphoglucoisomerase; and aldolase levels increased (p<0.001) in hepatoma bearing rats, whereas they decreased in Plumbagin administered rats to near normal levels. Certain gluconeogenic enzymes, namely, glucose-6-phosphatase and fructose-1,6-diphosphatase decreased (p<0.001) in tumour hosts, whereas Plumbagin administration increased the gluconeogenic enzyme levels in the treated animals. These investigations indicate the molecular basis of the different biological behaviour of 3MeDAB induced hepatoma and the anticarcinogenic property of Plumbagin against hepatoma studied in rats.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

Differential expression of genes related to glucose metabolism in domesticated pigs and wild boar

Glucose metabolism is a basic biological process that shows substantial variation within and between species. Using pig as a model organism, we investigated differences in glucose metabolic genes in seven tissues from domesticated pigs (Rongchang pig and Tibetan pig, meanwhile, the Tibetan pig just as a special case of the domesticated pig under plateau condition) and wild boar. We found large differences in the expression of genes involved in multiple aspects of glucose metabolism, including genes associated with glucose transport, gluconeogenesis, and glycolysis. In addition, we identified microRNAs (miRNAs) that may be involved in the divergence of glucose metabolism in pig. A combined analysis of mRNA and miRNA expression indicated that some miRNA:mRNA pairs showed ab facto function in it. Our results provide a valuable resource for further determination of miRNA regulatory roles in pig glucose metabolism and reveal the divergence of glucose metabolism in pigs under domestication.     

Role of mitochondria in hepatic fructose metabolism

During metabolism of fructose at concentrations exceeding 5 mM, isolated liver cells accumulate fructose 1-phosphate and lose ATP. At added bicarbonate concentrations below 10 mM in the incubation medium, the addition of atractyloside (or carboxyatractyloside) causes a significant net accumulation of 2-phosphoglycerate, resulting in an increase in the ratio 2-phosphoglycerate: 3-phosphoglycerate from below 1 to greater than 5. Digitonin fractionation revealed that virtually all this 2-phosphoglycerate is associated with the mitochondrial fraction, where it achieves a concentration estimated to be about 40 mM. The amount of 2-phosphoglycerate that accumulates is directly related to the initial concentration of fructose. With DL-glyceraldehyde in place of fructose, an even greater accumulation of 2-phosphoglycerate occurs, and this is also dependent upon both the presence of atractyloside and low bicarbonate. Formation of 2-phosphoglycerate is also observed when isolated mitochondria from rat liver are incubated together with glyceraldehyde and an energy source. The obligatory role of atractyloside for the accumulation of 2-phosphoglycerate within intact cells indicates the involvement of the mitochondrial adenylate translocator in this process, possibly as a carrier directly responsible for 2-phosphoglycerate egress from the mitochondrial matrix. If this is so, competition between 2-phosphoglycerate and ATP for egress from the matrix would be predicted to further exaggerate the fructose-induced depletion of cytosolic ATP.     

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.     

Effects of fructose on hepatic glucose metabolism in humans

Hepatic and extrahepatic insulin sensitivity was assessed in six healthy humans from the insulin infusion required to maintain an 8 mmol/l glucose concentration during hyperglycemic pancreatic clamp with or without infusion of 16.7 micromol. kg(-1). min(-1) fructose. Glucose rate of disappearance (GR(d)), net endogenous glucose production (NEGP), total glucose output (TGO), and glucose cycling (GC) were measured with [6,6-(2)H(2)]- and [2-(2)H(1)]glucose. Hepatic glycogen synthesis was estimated from uridine diphosphoglucose (UDPG) kinetics as assessed with [1-(13)C]galactose and acetaminophen. Fructose infusion increased insulin requirements 2.3-fold to maintain blood glucose. Fructose infusion doubled UDPG turnover, but there was no effect on TGO, GC, NEGP, or GR(d) under hyperglycemic pancreatic clamp protocol conditions. When insulin concentrations were matched during a second hyperglycemic pancreatic clamp protocol, fructose administration was associated with an 11.1 micromol. kg(-1). min(-1) increase in TGO, a 7.8 micromol. kg(-1). min(-1) increase in NEGP, a 2.2 micromol. kg(-1). min(-1) increase in GC, and a 7.2 micromol. kg(-1). min(-1) decrease in GR(d) (P < 0. 05). These results indicate that fructose infusion induces hepatic and extrahepatic insulin resistance in humans.     

菠萝叶片中依赖焦磷酸的磷酸果糖激酶的光/暗调节特性(英文)

植物中D:果糖6—磷酸1—磷酸转移酶(PPi—PFK,EG 2.7.1.90)活性的调节是非常重要的。这主要是因为它能可逆催化糖酵解和生糖两个方向的反应。光照处理菠萝叶片或离体的菠萝叶圆片使PPi—PFK的酶活性增高。与从暗处理的叶片中提取的酶的特性相比,光照处理的叶片中的酶对糖酵解方向催化活性相对增加。暗处理导致酶催化精酵解方向活性的下降。这种反映在酶活性和特性上的变化可为光照重新恢复。结果表明,菠萝叶片的PPi—PFK对体内糖酵解或生糖途径的贡献可能决定于光的状况。     

Gradual transfer to sea water of rainbow trout: effects on liver carbohydrate metabolism

The levels of glycogen and glucose, as well as of the activities of several key enzymes of glycogenolysis, glycolysis, gluconeogenesis and the pentose phosphate shunt were assessed in livers of small (97 g) and large (166 g) rainbow trout Oncorhynchus mykiss during gradual transfer to sea water (salinities of 0, 9, 18 and 28%). Plasma glucose and protein levels were also evaluated. Liver carbohydrate metabolism changed during gradual adaptation of rainbow trout to sea water. Glucose increased with salinity in livers of both sizes of animals, as did glycogenolysis and gluconeogenesis, but this latter only in large animals, and glycolytic rates in small animals. The different responses in metabolic parameters between sizes of animals may reflect a higher stress of the small animals and/or a better adaptation of large animals to increased salinities, in a way similar to that previously suggested by other authors after evaluation of non-metabolic parameters. The changes observed at 28% can also be the result of reduced food consumption.     

Impact of chronic fructose infusion on hepatic metabolism during TPN administration

During chronic total parenteral nutrition (TPN), net hepatic glucose uptake (NHGU) is markedly elevated. However, NHGU is reduced by the presence of an infection. We recently demonstrated that a small, acute (3-h) intraportal fructose infusion can correct the infection-induced impairment in NHGU. The aim of this study was to determine whether the addition of fructose to the TPN persistently enhances NHGU in the presence of an infection. TPN was infused continuously into the inferior vena cava of chronically catheterized dogs for 5 days. On day 3, a bacterial clot was implanted in the peritoneal cavity, and either saline (CON, n = 5) or fructose (+FRUC, 1.0 mg. kg(-1). min(-1), n = 6) infusion was included with the TPN. Forty-two hours after the infection was induced, hepatic glucose metabolism was assessed in conscious dogs with arteriovenous and tracer methods. Arterial plasma glucose concentration was lower with chronic fructose infusion (120 +/- 4 vs. 131 +/- 3 mg/dl, +FRUC vs. CON, P < 0.05); however, NHGU was not enhanced (2.2 +/- 0.5 vs. 2.8 +/- 0.4 mg. kg(-1). min(-1)). Acute removal of the fructose infusion dramatically decreased NHGU (2.2 +/- 0.5 to -0.2 +/- 0.5 mg. kg(-1). min(-1)), and net hepatic lactate release also fell (1.6 +/- 0.3 to 0.5 +/- 0.3 mg. kg(-1). min(-1)). This led to an increase in the arterial plasma glucose (Delta13 +/- 3 mg/dl, P < 0.05) and insulin (Delta5 +/- 2 micro U/ml) concentrations and to a decrease in glucagon (Delta-11 +/- 3 pg/ml) concentration. In conclusion, the addition of chronic fructose infusion to TPN during infection does not lead to a persistent augmentation of NHGU.     

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.     

Zinc uptake by blood cells of rats in zinc deficiency and inflammation

In zinc deficiency, the function of leukocytes is impaired. However, the results of studies on the zinc concentration of blood cells in zinc deficiency are conflicting, probably in part because of technical and analytical problems. The aim of this study was to investigate, under standard conditions, the uptake of⁶⁵Zn-labeled zinc by blood cells, taken from zinc-deficient rats and from rats in which an inflammation is induced. In both conditions, the serum zinc concentration is reduced. In clinical practice, this makes it difficult to determine whether the decrease in serum zinc is the result of a real or an apparent zinc deficiency. In stress, like an inflammatory disease, the decrease of zinc reflects an apparent zinc deficiency because of redistribution of serum zinc into the liver and because of decrease in serum albumin concentration. Over 70% of the serum zinc is bound to albumin. Blood cells from zinc-deficient and control rats were isolated using a discontinuous Percoll gradient and incubated under nearly physiological conditions in a⁶⁵Zn-containing medium. A significant increase in the in vitro uptake of⁶⁵Zn-labeled zinc by the blood cells of zinc-deficient rats was seen: erythrocytes 1.3, mononuclear cells 2.0, and polymorphonuclear cells 2.6 times the control values. During inflammation, no change in⁶⁵Zn-labeled zinc uptake by erythrocytes and mononuclear cells was demonstrated after 2 d, although the serum zinc and albumin concentrations were decreased, but a small but significant increase in zinc uptake by polymorphonuclear cells was observed. This study of⁶⁵Zn uptake in vitro under standard conditions may prove of value for distinguishing in patients real zinc deficiency from apparent zinc deficiency owing to, e.g., stress, although additional experiments should be performed. A part of this study has been presented at the Meeting of The American Gastroenterological Association on May 12–18, 1990, San Antonio, TX, and has been published in abstract inGastroenterology 98 suppl., A423.     

Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats

The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was then infused with glucagon (1 nM), isoproterenol (2 microM), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase.     

Metabolic effects of acetaminophen. Studies in the isolated perfused rat liver

The effects of acetaminophen on the metabolism of the isolated perfused rat liver were investigated. The following results were obtained: (1) Acetaminophen increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). (2) Oxygen uptake, gluconeogenesis from either pyruvate or fructose and glycogen synthesis were inhibited. (3) In isolated rat liver mitochondria acetaminophen decreased state III and state IV respiration; it also decreased the ADP/O ratio and the respiratory control ratio. (4) The action of acetaminophen on glycogenolysis was not affected by N-acetylcysteine; this compound, however, increased glycogen synthesis. (5) The effects of acetaminophen are reversible. It was concluded that glycogen depletion by acetaminophen can be produced by two mechanisms. The first, as previously demonstrated by several workers, depends on irreversible binding of a reactive metabolite. The second, however, is reversible and depends primarily on an inhibition of mitochondrial energy metabolism.     

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.     

Axonal Transport of Zinc Transporter 3 and Zinc Containing Organelles in the Rodent Adrenergic System

Zinc is the second most abundant trace metal (after iron) in mammalian tissues, and it is an essential element for growth, development, DNA synthesis, immunity, and other important cellular processes. A considerable amount of zinc in the brain exists as a pool of free or loosely bound zinc ions in synaptic vesicles with zinc transporter 3 (ZnT3) in their membranes. Here we demonstrate that also in the peripheral sympathetic nervous system zinc handling neurons exist. In autonomic ganglia of rats and mice a subset of neuronal cell bodies contain zinc, visualized by the autometallographic (AMG) and TSQ histochemical methods. The Zn-transporter 3 is, as shown by immunofluorescence, also present in tyrosine hydroxylase (TH)-positive neurons, but rarely in cell bodies with neuropeptide Y (NPY)-immunoreactivity (IR). In axons of crush-operated sciatic nerves a rapid bidirectional accumulation of AMG granules occurred. Also ZnT3-IR was found to accumulate rapidly in anterograde as well as retrograde direction, colocalized with TH-IR. So far nerve terminals with ZnT3-IR have not been observed. The functional significance of zinc ions in the sympathetic system is not known. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.