Activity, hexokinase isoenzyme spectrum and various factors of their regulation in liver and skeletal muscles of young piglets]

Hexokinase in the liver of 1- and 5-day-old piglets is presented by four isoforms and in the skeletal muscles--by two ones. The enzyme activity in the liver and skeletal muscles of 5-day-old piglets is much higher than in 1-day-old ones. The increased hexokinase activity in the tissues of piglets during the first days of life appears to be due to the changes in their isoenzyme spectrum. The hexokinase activity and isoenzyme spectrum in the investigated tissue were affected by insulin, cortisol and 24 hours long starvation. These changes depended upon the age of the animals and differed in various organs and tissues: in 1-day-old piglets they were more pronounced in the skeletal muscles, while in 5-days-old animals--in the liver.     

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.     

Hexokinase isoenzymes in diabetes

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.     

A comparative study on the effect of homobrassinolide and gibberellic acid on lipid peroxidation and antioxidant status in normal and diabetic rats

Dietary content of phytohormones may potentially influence metabolic processes in animal cells. This study therefore aimed to investigate the effect of two plant growth regulators homobrassinolide (HB) and gibberellic acid (GBA) on the antioxidant defense status and lipid peroxidation level in the tissues of normal and streptozotocin- induced diabetic rats. Normal and diabetic rats (Albino –wistar strain) were administered 50μg HB and GBA intradermally each day for seven days and their tissue and blood levels of malondialdehyde (MDA), 4-hydroxy-2-nonenol (4-HNE), reduced glutathione (GSH) content and catalase (CAT) activity were determined. Subchronic treatment of rats with HB reduced lipid perioxidation and elevated antioxidant defense whereas GBA caused enhancement of lipid peroxidation and reduction of antioxidant defense in treated animals compared to the control rats.     

Insulin effect on [14C]-valine incorporation and its relation to hexokinase activity in developing brain

Using minced brain cortex from fetal and postnatal rats, we studied the incorporation of [14C]-valine into protein in the presence of insulin. We also assayed the "particle bound" and soluble hexokinase in these tissues. Insulin significantly stimulated the incorporation of [14C]-valine into brain proteins from fetal stage upto 2 days of life. After this period the insulin effect was minimal, with no effect by day 5. The "particle bound" (40,000g pellet) brain hexokinase, on the other hand, remained low till about 2 days of life and then increased to almost adult level by 5 days. Our results show that there is an inverse relation between this anabolic effect of insulin and the "particle bound" hexokinase activity in the cortex of developing rat brain.     

Antidiabetic effect of a leucocyanidin derivative isolated from the bark of Ficus bengalensis Linn

A dimethoxy derivative of leucocyandin 3-O-beta-D-galactosyl cellobioside isolated from the bark of F. bengalensis Linn demonstrated antidiabetic action. On oral administration, it decreased blood sugar very significantly both in normal and moderately diabetic rats and increased serum insulin significantly in the latter at a dosage of 250 mg/kg for a 2 hr period. During one month treatment of the diabetic rats orally with the active principle, at a dosage of 100 mg/kg, there was a significant decrease in blood and urine sugar, certain lipid components in serum and tissues and glucose-6-phosphatase activity in liver, but significant increase in body weight and the activities of hexokinase and HMGCOA reductase in tissues as compared to diabetic control. The mechanism of action of the principle may be related to its protective/inhibitory action against the insulin degradative processes.     

Homobrassinolide induced conformational changes in hexokinase: a possible mechanism for its antidiabetic potential

Hormonal regulation of cell growth and development, tissue morphology, metabolism and physiological function in animals and man is a well‐established knowledge domain in modern biological science. The present study was carried out to investigate the structural stability of hexokinase when exposed to diabetic levels of glucose and its binding efficiency. The fluorescence study indicated that 28‐homobrassinolide was able to protect or restore the native structure of hexokinase. Proteins are synthesized and fold into the native form to become active. The inability of a protein molecule to remain in its native form is called as protein misfolding and this is because of several factors. Protein aggregation and misfolding are known to play a critical role in several human diseases including diabetes. Homobrassinolide interaction with hexokinase was studied by UV–Vis spectrophotometer and fluorescence spectrophotometer. Results were suggested that the denatured hexokinase was renatured upon binding with homobrassinolide. In silico, docking study was performed to recognize the binding activity of homobrassinolide against a subunit of the glucokinase, and homobrassinolide was able to bind to the drug binding pocket of glucokinase. The glide energy is ?7.1 kcal/mol, suggesting the high binding affinity of homobrassinolide to glucokinase. Overall, these studies predict that the phytohormone 28‐homobrassinolide would function as an anti‐diabetic when present in human and animal diet by augmenting the hexokinase enzyme activity in the animal cell. Copyright © 2015 John Wiley & Sons, Ltd.     

Testosterone deficiency impairs glucose oxidation through defective insulin and its receptor gene expression in target tissues of adult male rats

Testosterone and insulin interact in their actions on target tissues. Most of the studies that address this issue have focused on the physiological concentration of testosterone, which maintains normal insulin sensitivity but has deleterious effects on the same when the concentration of testosterone is out of this range. However, molecular basis of the action of testosterone in the early step of insulin action is not known. The present study has been designed to assess the impact of testosterone on insulin receptor gene expression and glucose oxidation in target tissues of adult male rat. Adult male albino rats were orchidectomized and supplemented with testosterone (100 microg/100 g b. wt., twice daily) for 15 days from the 11th day of post orchidectomy. On the day after the last treatment, animals were euthanized and blood was collected for the assay of plasma glucose, serum testosterone and insulin. Skeletal muscles, such as gracilis and quadriceps, liver and adipose tissue were dissected out and used for the assay of various parameters such as insulin receptor concentration, insulin receptor mRNA level and glucose oxidation. Testosterone deprivation due to orchidectomy decreased serum insulin concentration. In addition to this, insulin receptor number and its mRNA level and glucose oxidation in target tissues were significantly decreased (p<0.05) when compared to control. However, testosterone replacement in orchidectomized rats restored all these parameters to control level. It is concluded from this study that testosterone deficiency-induced defective glucose oxidation in skeletal muscles, liver and adipose tissue is mediated through impaired expression of insulin and its receptor gene.     

The pentose phosphate pathway of glucose metabolism. Influence of a growth-hormone-secreting pituitary tumour on the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the activities of enzymes of the pentose phosphate cycle, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase, as well as of the related or competing enzymes glucokinase, hexokinase, phosphoglucose isomerase and phosphoglucomutase, in control rats and in rats bearing the growth-hormone- and prolactin-secreting pituitary tumour MtTW5, to study the effect of high endogenous concentrations of growth hormone on this pathway in liver. 2. There was a twofold increase in liver weight. Glucokinase activity/g. of liver decreased to half the control value in the experimental group, although on a total liver basis it remained unchanged. Hexokinase activity increased in parallel with the liver weight, so that the total activity was doubled in rats with a high endogenous concentration of growth hormone. No differences in response were found between heat-stable and heat-labile forms of hexokinase. 3. The activity/g. of liver of the two oxidative enzymes of the pathway decreased slightly in the experimental group, but this was offset by the increase in liver weight, and the resultant effect was a 50% increase in the total activity. 4. Of the non-oxidative enzymes of the cycle the most marked increase on a total liver basis was in ribose 5-phosphate isomerase activity, to 2.5 times the control value. Ribulose 5-phosphate epimerase activity showed the smallest increase. Transketolase and transaldolase activities were also increased. The latter is the rate-limiting enzyme of the non-oxidative reactions of the cycle in these animals. 5. The results are discussed in relation to the glycolytic pathway and synthesis of glycogen, and more particularly to the increased requirement for ribose 5-phosphate for RNA synthesis.     

Changes in hexokinase isoenzymes in regions of rat brain during thyroid deficiency

In this work, activities of hexokinase isoenzymes Type I and Type II were measured in the soluble and particulate fractions from the brain regions (cerebral hemispheres (cerebrum), cerebellum and brain stem) of the thyroidectomized adult rats as well as of the thyroidectomized rats administered with triiodothyronine. Thyroidectomy generally decreased the hexokinase activity associated with particulate and soluble fractions. Hexokinase Type II isoenzyme was more affected than the Type I isoenzyme. Administration of triiodothyronine to the hypothyroid rats abolished the effect of thyroidectomy. Adult brain enzymes have been generally considered not be affected by thyroid hormones. The data obtained in this work are suggestive of an effect of thyroid hormones on hexokinase in the adult brain. Since the effects of thyroidectomy on the energy metabolism of the heart tissue are well known, the heart tissue was also studied for comparison.     

The development of hepatic glucokinase in the neonatal rat

1. Glucokinase and hexokinase activities have been determined in the livers of newborn rats and attempts made to influence in vivo the development of the glucokinase. 2. Glucokinase first appears in rat liver about 16 days after birth and adult activities are reached 10–12 days later. Evidence is presented which indicates that this represents synthesis of new protein. Hexokinase activities remain constant throughout the period of glucokinase development. 3. Both exogenous glucose and insulin are necessary for the natural development of glucokinase, for this is retarded in starved and alloxan-diabetic neonatal rats. 4. The absence of glucokinase during the first 2 weeks of extrauterine life in the rat is not due to lack of insulin. 5. Attempts to advance the time at which glucokinase first appears by infusions of glucose, insulin and chlorpropamide alone and in various combinations have resulted in marginal effects only. 6. When rats are starved for 3 days during the period of glucokinase development and then re-fed, glucokinase is more rapidly synthesized, indicating that the potential ability to synthesize glucokinase continues to develop throughout the period of starvation. 7. Some possible reasons for the comparatively late development of glucokinase are discussed.     

Mitochondrial hexokinase from small-intestinal mucosa and brain

1. The submitochondrial localization of hexokinase activity in preparations of mitochondria from the small intestine of the guinea pig was studied by conventional methods. 2. Hexokinase activity in this tissue was predominantly associated with the outer mitochondrial membrane. 3. The inactivation of mitochondrial enzymes by trypsin in iso-osmotic and hypo-osmotic conditions was also used to determine the submitochondrial localization of hexokinase activity. 4. Hexokinase activity was found to be on the outside of the outer mitochondrial membrane. 5. It was shown that both type I and type II hexokinase activities are bound to the outside of the outer mitochondrial membrane. The types are present in the same ratio as that in which they occur in the cytosol of the cell. 6. Mitochondrial hexokinase from the small intestine did not show the latency phenomenon demonstrated by mitochondrial hexokinase from brain when subjected to a variety of treatments. However, hexokinase activity was solubilized from preparations of mitochondria from the small intestine by the same treatments as for mitochondrial hexokinase from brain. 7. The submitochondrial distribution of hexokinase activity in mitochondrial preparations from rat brain was determined by the trypsin inactivation method. 8. Hexokinase activity in preparations of mitochondria from rat brain was found on the outside of the outer membrane, between the mitochondrial membranes, and within the inner mitochondrial membrane. 9. Hexokinase from rat brain showed latency properties irrespective of its submitochondrial location.     

植物己糖激酶的信号转导作用

己糖激酶在植物细胞的信号转导中起着重要的作用。近年来,有关植物己糖激酶的研究工作已经较多,受到足够的重视。现对植物己糖激酶的特性、亚细胞定位、编码基因分子特征、感受己糖与信号转导功能、依赖己糖激酶的糖信号转导途径及其调控作用进行介绍。     

Hexokinase receptors: Preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.     

Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, alpha-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic alpha-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyceride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyceride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.     

Maximal activities of key enzymes of glutaminolysis, glycolysis, Krebs cycle and pentose-phosphate pathway of several tissues in mature and aged rats

1. The maximum activities of some key enzymes, which provide a quantitative indices of flux through several important pathways have been measured in brain, liver, muscle, white and brown adipose tissue and lymphocytes of mature and aged rats. 2. The results were expressed as mumol/min per g fresh weight and nmol/min per mg protein. 3. On the both basis, as compared to mature rats, hexokinase activity is decreased in brown adipose tissue and increased in soleus muscle. 4. Glucose-6-phosphate dehydrogenase activity is decreased in most tissues and increased in brain. 5. Citrate synthase activity, which provides a qualitative index of the Krebs cycle, is decreased in white adipose tissues and lymphocytes. 6. Glutaminase activity is decreased in brain, white and brown adipose tissues but is increased in lymphocytes.     

Comparison of ochratoxin A levels in edible pig tissues and in biological fluids after exposure to a contaminated diet

The aim of this study was to compare ochratoxin A (OTA) levels in pig tissues and biological fluids after animal exposure to contaminated diet (250 μg OTA/kg of feed) during 4 weeks of fattening. OTA concentrations were quantified using a validated immunoassay method (ELISA) and high-performance liquid chromatography with fluorescence detector (HPLC-FD). The highest mean OTA concentration in pig tissues was determined in kidneys of exposed animals (13.87?±?1.41 μg/kg), followed by lungs (10.47?±?1.97 μg/kg), liver (7.28?±?1.75 μg/kg), spleen (4.81?±?0.99 μg/kg), muscle tissue (4.72?±?0.86 μg/kg), fat tissue (4.11?±?0.88 μg/kg), heart (3.71?±?1.09 μg/kg), and brain (3.01?±?0.25 μg/kg). Furthermore, on the last day of exposure (day 28), significantly higher mean OTA levels were determined in urine (16.06?±?3.09 μg/L) in comparison to serum (4.77?±?1.57 μg/L) showing that OTA urine analysis could be a good marker to identify elevated levels of this contaminant in porcine tissues used for human consumption. This study gave guidelines for the most efficient OTA control in pig-derived biological materials that can be exercised at slaughterhouses.     

Distribution of 125I-labeled rat growth hormone in regional brain areas and peripheral tissue of the rat.

The uptake of intraperitoneally injected 125I-labeled rat growth hormone into brain and peripheral tissues was measured in normal and hypophysectomized adult rats. A significant level of radioactivity was observed in the seven brain regions examined -- the telencephalon, diencephalon, midbrain, pons-medulla, cerebellum, pineal and pituitary glands. The pineal and pituitary glands, which are outside the blood-brain barrier, contained three to four times the concentration of radioactivity of the other brain regions. Compared to brain, the level of radioactivity was much higher in peripheral tissues (the diaphragm, kidney, serum and liver). For example, the serum contained ten times the level of radioactivity of most brain regions. For a given tissue, however, the normal and hypophysectomized rats showed a comparable amount of 125I-growth hormone. Trichloroacetic acid precipitates from each tissue sample showed that peripheral tissues had a higher proportion of radioactivity (35-48% of total tissue radioactivity) than the brain samples (13-26%). The data support the view that growth hormone, or a metabolite can enter the central nervous system and may directly affect on-going metabolic processes.     

Effect of prodigiozan on the glucocorticoid and insulin ratio in the blood serum of white rats

Significant hypertrophy of the adrenal glands was observed in Wistar rats the day after a single prodigiozan administration. This did not change the level of glucocorticoids in blood serum, but increased the concentration in insulin. Analysis of steroidogenesis in the adrenal glands indicated significant increase in steroid's production in the experimental group of rats as compared to the control. An increase of glucoso-6-phosphate dehydrogenase and hexokinase activity in adrenal tissue was also observed. The results suggest that stimulation of corticosteroid's secretion was accompanied by an increase in their reception to tissues which caused an increase of nonspecific resistance of the organism.