Liver Of Alloxan-diabetic Rats Research Articles

Oxidative stress and changes in liver sialoglycoconjugate metabolic parameters in rats with alloxanic diabetes mellitus

BACKGROUND: Oxidative stress may be one of the mechanisms for the development of complications in DM and many forms of CKD. However, the influence of this factor on the metabolism of sialoglycoconjugates, which actively participates in the regulatory processes of the body, is unknown.AIM: comparative study of the effect of lipoic acid on the parameters of liver sialoglycoconjugate metabolism in rats with alloxan diabetes mellitus.MATERIALS AND METHODS: Studies were conducted on white male rats weighing 180–220 grams. The animals were divided into three groups: in the animals of the first and second groups, alloxan diabetes mellitus (DM) was caused by a single subcutaneous injection of alloxan tetrahydrate (AT). Animals of the second group received lipoic acid intramuscularly; the third group consisted of intact rats. On 5, 10, 20, 30 and 40 days after the injection of AT, a comprehensive examination of the animal’s condition was performed: 1) determination of the level of glycemia; 2) assessment of the degree of development of oxidative stress by the content of TBK-active products in the liver; 3) study of the dynamics of the exchange of sialoglycoconjugates in the liver (free, oligo-and protein-bound sialic acids, sialidase activity).RESULTS: The study was conducted on 106 rats, each experimental group had 48 animals, control — 10. It was found that the administration of α-lipoic acid to rats with alloxan diabetes leads to a decrease in the level of glycemia. The introduction of lipoic acid in experimental animals did not reduce the sialidase activity and the content of all sialic acid fractions in the liver, although it reduced the degree of oxidative stress in the body.CONCLUSION: Supplementation of lipoic acid in experimental animals did not significantly decrease sialidase activity and content of the sialic acid fractions under study in the liver, although it did reduce the degree of oxidative stress development in the organism. The increased rate of sialic acid metabolism in the liver of alloxan-diabetic rats may indicate a restructuring of hepatocyte metabolism to adapt the whole organism to prolonged hyperglycemia under insulin deficiency conditions.

Synthesis, physico-chemical properties and effect of adenosine thiamine triphosphate on vitamin B1 metabolism in the liver of alloxan diabetic rats

BackgroundAdenosine thiamine triphosphate (AThTP) is a nucleotide discovered in bacteria and some other living organisms more than a decade ago. No biochemical function for AThTP has been established yet, however, experimental data available indicate its possible involvement in metabolic regulation or cell signaling. Metabolism of AThTP in mammals, as well as the feasibility of its pharmacological application, is essentially unstudied. MethodsPreparative low-pressure chromatography was employed to purify chemically synthesized AThTP with its further analysis by mass spectrometry, HPLC, UV and fluorescence spectroscopy. Enzyme activity assays along with HPLC were used to examine the effects of AThTP and thiamine on vitamin B1 metabolism in the liver of alloxan-induced diabetic rats. ResultsAn improved procedure for AThTP synthesis and purification is elaborated. Solution stability, optical spectral properties and the molar absorption coefficient for AThTP were determined. The levels of thiamine compounds were found to be increased in the liver of diabetic rats. Neither AThTP nor thiamine treatment affected hepatic vitamin B1 metabolism. Fasting blood glucose concentration was also unchangeable after AThTP or thiamine administration. General significanceContrast to the widespread view about thiamine deficiency in diabetes, our results clearly shows an adaptive increase in the level of B1 vitamers in the liver of alloxan diabetic rats with no further rising after AThTP or thiamine treatment at a moderate dose. Neither AThTP nor thiamine is effective in glycaemic control. These findings are to be considered in future studies dealing with thiamine or its analogues application to correct metabolic disturbances in diabetes.

Biochemical and Immunological Study on the Effects of Barley and its Components as Hypoglycemic Agents in Diabetic Rats

The present study was carried out to investigate the hypoglycemic effect of barley (Hordeum vulgare L) and some of its components such as amino acids (L-leucine and L-glutamine) and chromium picolinate on some biochemical and immunological parameters of alloxan induced diabetic rats. Alloxan-diabetic rats were treated with barley water (10% w/v) at a dose 10 ml Kg -1 b.wt., chromium picolinate at 15 μg Kg -1 b.wt., L-leucine plus L-glutamine at 4.5 mg & 15 mg Kg -1 b.wt., and/or the combination of barley plus chromium plus L-leucine and L-glutamine at the same previous doses in the same water volume, respectively. Rats received the treatments in their drinking water for four weeks. The levels of glucose, immunoglobulin G (IgG), total lipids (TL), cholesterol, triglycerides (TG), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) were significantly (P<0.05) increased, while high-density lipoprotein (HDL) decreased in plasma of alloxan-diabetic rats compared to control group. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase (AcP) and alkaline phosphatase (AlP) were significantly (P<0.05) decreased in both plasma and liver of alloxan-diabetic rats. On the other hand, the activity of lactate dehydrogenase (LDH) decreased in plasma and increased in liver of alloxan- diabetic rats. Treatment of the diabetic rats with repeated doses of any one of the three treatments alone could restore the changes of the above parameters to their normal levels after four weeks of treatment. However, treatment with the combination of all together did not show complete restoration. Furthermore, the electron microscope results were supported biochemical and immunological findings. The present results showed that barley, amino acids and chromium picolinate exerted antihyperglycemic effects and consequently may alleviate liver damage caused by alloxan-induced diabetes.

Biochemical study on the effects of some Egyptian herbs in alloxan-induced diabetic rats

The present study was carried out to investigate the effects of Lupinus albus, L. (Lupinus termis), family L. leguminosae, Cymbopogon proximus, (Halfa barr), family Gramineae, and Zygophyllum coccineum L. (Kammun quaramany), family L. Zygophyllacae on biochemical parameters in alloxan-induced diabetic rats. A dose of 1.5 ml of aqueous suspension of each herb/100 g body weight (equivalent to 75 mg/100 g b.wt.) was orally administered daily to alloxan-diabetic rats for 4 weeks. The levels of glucose, urea, creatinine and bilirubin were significantly ( P<0.05) increased in plasma of alloxan-diabetic rats compared with the control group. In contrast, total protein and albumin were significantly decreased by 25 and 46%, respectively, versus control. Treatment of the diabetic rats with repeated doses of any one of the three herb suspensions could restore the changes of the above parameters to their normal levels after 4 weeks of treatment. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (AlP) activities were significantly ( P<0.05) increased in the plasma of alloxan-diabetic rats. However, acetylcholinesterase activity was significantly ( P<0.05) decreased in the plasma compared with the control group, whereas, such activity did not change in brain. The activities of AST, ALT and LDH were significantly ( P<0.05) decreased in the liver of alloxan-diabetic rats by 58, 21 and 40%, respectively, and such activities increased in testes by 39, 26 and 26%, respectively, compared with the control group. Also, brain LDH was significantly ( P<0.05) increased. Treatment of the diabetic rats with the aqueous suspension of the tested herbs restored the activities of the above enzymes to their normal level in plasma, liver and testes. The present results showed that the herb suspensions exerted antihyperglycemic effects and consequently may alleviate liver and renal damage caused by alloxan-induced diabetes.

Glucose release from isolated, perfused liver of alloxan-diabetic rats.

Glucose release from the isolated, perfused rat liver was observed to a greater extent in alloxan-diabetic rats, compared to normal rats. The addition of acetoacetate (an acetone body) to the perfusate increased the glucose release from the normal rat liver, suggesting that the greater glucose release from the alloxan-diabetic rat liver is due to functional disorder(s) of the liver induced by acetoacetate in the diabetic condition. It was observed that lipoic acid suppressed glucose release from the liver of alloxan-diabetic rats, and that the nonprotein thiol level in alloxan-diabetic rat liver was lower than that in normal rat liver. Addition of acetoacetate in the perfusate caused a decrease in nonprotein thiol level in normal rat liver; suggesting that nonprotein thiol loss may mediate the increase in glucose release caused by an increase in acetoacetate in alloxan diabetic rats.

Acute Alloxan Diabetes Alters the Activity but Not the Total Quantity of Acetyl CoA Carboxylase in Rat Liver

Alloxan diabetes has repeatedly been shown to reduce lipogenesis in rat liver concomitant with decreased activity of acetyl CoA carboxylase. This and other observations led to the deduction that insulin is required for the synthesis of acetyl CoA carboxylase even though the actual amount of enzyme was not measured. We have developed methods to determine the quantity of acetyl CoA carboxylase in crude tissue extracts with which we have reexamined the role of insulin in regulating the amount of the enzyme in liver of acute (3-d) alloxan diabetic rats. The results show that although there was a decrease in the quantity of the active cytoplasmic form of acetyl CoA carboxylase in the liver of alloxan diabetic rats, there was a corresponding increase in the quantity of relatively inactive forms of the enzyme associated with mitochondria. Thus, the total amount of enzyme was minimally affected by the diabetic state. Instead, the results indicate that decreased acetyl CoA carboxylase activity in liver of the diabetic rats was attributable to a shift in the subcellular distribution of the enzyme from the active cytoplasmic to inactive mitochondrial forms. We have shown previously that subcellular distribution of the enzyme is dietary dependent. Results of this study implicate insulin in the mobilization and activation of mitochondrial acetyl CoA carboxylase.

Insulin-induced increases in the activity of the spontaneously active and ATP.Mg-dependent forms of phosphatase-1 in alloxan-diabetic rat liver.

Liver supernatant from normal and alloxan-diabetic rats was fractionated by DEAE-cellulose chromatography and the separated phosphoprotein phosphatase fractions were assayed with [32P]histone f2b, [32P]phosphorylase a and [32P]phosphorylase kinase as substrates. In diabetic rat liver, one of the phosphatase fractions found in the normal liver was significantly reduced. This fraction was identified as a mixture of the spontaneously active form and the ATP . Mg-dependent form of phosphoprotein phosphatase-1 (Fc) based on sensitivity to inhibitor-2, substrate specificity, and the fact that it could be activated 42-70% by glycogen synthase kinase-3 in the presence of ATP . Mg. Further analysis of this fraction showed that liver cytosol from diabetic rats contained 62-79% lower spontaneously active phosphatase-1 activity and 40-51% lower combined spontaneously active and ATP . Mg-dependent protein phosphatase-1 (Fc) activity. Insulin administration increased the spontaneously active and the ATP . Mg-dependent protein phosphatase-1 activities approximately 45% and 36%, respectively, in alloxan-diabetic rats. These data imply that the lower levels of spontaneously active phosphatase-1 activity in diabetic rat liver cannot be explained by presuming phosphatase-1 to have been present as Fc, the inactive form. Moreover, insulin restored the total activity of the spontaneously active and activatable forms of phosphatase-1 to those present in normal liver implying that both forms of phosphatase-1 activity are under hormonal control.

Isolation and characterization of liver glycogen synthase from diabetic rats

Glycogen synthase was isolated from the livers of alloxan diabetic rats by the addition of a high-molecular-weight, polydisperse glycogen, described previously ( D. M. Haverstick and A. H. Gold, 1981, Anal. Biochem. III, 137–145 ), to supernatant extracts of centrifuged liver homogenates. Further purification was achieved by high-speed centrifugation and ion-exchange chromatography. Comparative polyacrylamide gel electrophoresis show that synthase isolated from normal and diabetic rats have the same subunit molecular weight. Enzymes from both sources react with antisynthase antibody in the same manner. Both enzymes have essentially the same affinity toward substrate, UDP-Glc, and activator, Glc-6-P, and both enzymes are capable of undergoing b into a transformation in vitro with exogenous, partly purified synthase phosphatase. These results suggest that diabetes does not result in the in vivo appearance of an “abnormal” type of glycogen synthase which is not capable of interacting with the synthase b to a conversion system.

Changes in peroxisomal fatty acid oxidation in the diabetic rat liver.

Changes in fatty acid oxidation of peroxisomes in the liver of alloxan-diabetic rats were studied. After injection of alloxan (150 mg/kg, subcutaneously), the activity of peroxisomal cyanide-insensitive beta-oxidation increased more rapidly than that of carnitine palmitoyltransferase, which was the rate-limiting step of mitochondrial beta-oxidation, and reached 3 times the control level at 7 days after the treatment. The peroxisomal beta-oxidation activity was more potent toward medium chain acyl-CoAs (C=10 and 12), though it was extremely low for shorter chain lengths. The activity of carnitine acetyltransferase increased to 2.4 times the control level and the change appeared mainly in the peroxisomal fraction. On the other hand, the activity of palmitoyltransferase increased to twice the control level, distributed mostly in the mitochondrial fraction. The activity of carnitine acyltransferase increased mainly in the peroxisomal fraction, and was higher for shorter and medium chain acyl-CoAs. These results suggest that peroxisomal fatty acid oxidation and transport of acetyl-CoA and medium chain acyl-CoA as well as NADH product in peroxisomes may be rapidly enhanced in response to the demand of organs for the urgent supply of energy from fatty acids in the diabetic condition.

Hepatic microsomal glucose-6-phosphatase of normal and alloxan-diabetic rats: Thermotropic effects on kinetics and interaction with deoxycholate and 1-anilino-8-naphthalene sulfonate

Thermotropic effects on the kinetics of glucose-6-phosphatase ( d-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) activity of hepatic microsomes from normal and alloxan-diabetic rat liver were investigated by determining V, K m and K i (substrate inhibition) values. Influence of deoxycholate (0.1%) and 1-anilino-8-naphthalene sulfonate (2.5 mM) on the kinetics was also evaluated. 1. 1.|Substrate inhibition occurred at 0.06 M for the enzyme from normal rats and at 0.02–0.025 M for the enzyme from diabetic rats. 2. 2.|The enzyme from diabetic rats showed a transition that extended between 22.7 and 27°C in the Arrhenius plot (log V vs. T −1) instead of at 19.5°C. 3. 3.|Deoxycholate increased the V value of both enzymes without affecting substrate inhibition at all the temperatures but did not completely abolish the transition in the Arrhenius plot of the enzyme from diabetic rats. 4. 4.|1-Anilino-8-naphthalene sulfonate eliminated substrate inhibition and activated the enzyme of normal rats above 27.5°C by increasing both V and K m values. Below this temperature, the enzyme showed biphasic or allosteric kinetics. At low substrate concentrations the enzyme was inhibited competitively, whereas at high substrate concentrations it was activated as both V and K m values were increased. The enzyme from diabetic rats, on the other hand, was activated at all the temperatures and exhibited linear kinetics. 5. 5.|Binding of 1-anilino-8-naphthalene sulfonate to the microsomal fraction increased with decreasing temperature as revealed by the increase of relative fluorescence. The microsomal fraction of diabetic rats showed a more anomalous fluorescence response between 13–18°C. 6. 6.|Enthalphy changes for glucose 6-phosphate binding to the inhibition site were slightly larger than binding to the active site. Calculated entropies of activation for transition state complex of glucose-6-phosphatase reaction were fairly large and negative. The free energy of activation (28–30 kcal/mol) was independent of temperature and experimental conditions. 7. 7.|In the microsomal fraction (total as well as rought), phospholipid content and fatty acid unsaturation index of phospholipids were decreased after diabetes. The level of free cholesterol remaiined unchanged but the molar ratio of cholesterol to phospholipid increased. The different thermal response and 1-anilino-8-naphthalene sulfonate interaction of the enzyme from diabetic rat liver could be ascribed to the altered lipid environment of the enzyme on the endoplasmic reticulum membrane.

Rapid inactivation of rat liver phospho enolpyruvate carboxykinase by microsomes and reversal by reductants

The hepatic gluconeogenic enzyme phospho enolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lase (transphosphorylating), EC 4.1.1.32) is activated 3–4-fold by micromolar quantities of Fe 2+ and ferroactivator, a cytosol protein that is necessary for this activation. After the carboxykinase in liver cytosol, or the purified carboxykinase, was activated by adding Fe 2+, rat liver microsomes were added to duplicate their ratio to the carboxykinase in liver. Microsomes caused a biphasic inactivation of the carboxykinase in cytosol. All of the activity corresponding to Fe 2+ activation was lost within 10 min ( t 1 2 equaled 3–4 min) and the remaining (basal) activity was lost over hours ( t 1 2 equaled 3–4 h). Adding the reducing agent dithiothreitol (1 mM) after 10 or 60 min of exposure of cytosol to microsomes caused a complete and three-fourths complete restoration of Fe 2+ activation. In preventing the microsome-induced loss of Fe 2+ activation, ascorbate and cysteine were also effective. When the purified carboxykinase was activated by Fe 2+ and the synthetic ferroactivator 3-aminopicolinate, microsomes interfered with the enzyme activity in a manner identical to their effect on the enzyme in cytosol, suggesting that they did not act on ferroactivator protein. When Fe 2+ was added to the crude or the purified carboxykinase 5–60 min after microsomes, the carboxykinase was not activated. If the microsomes were acting by oxidizing or sequestering Fe 2+, the carboxykinase would have been at least transiently activated because it takes only seconds for Fe 2+ to fully activate the enzyme and minutes for microsomes to cause a complete loss of Fe 2+ activation. By the process of elimination, microsomes probably act on the carboxykinase itself. Microsomes did not inactivate five other glycolytic/gluconeogenic enzymes in liver cytosol, including some that have sulfhydryls which are very sensitive to oxidation. The microsomal factor was inactivated by the proteases, chymotrypsin, trypsin and pronase. The activity of the microsomal factor was highest in liver and kidney; almost as high in heart as in liver; and low or negligible in pancreas, spleen, lung and skeletal muscle. The microsomal activity was decreased in livers of alloxan diabetic rats, and the decrease was proportional to their blood glucose concentrations — such that severely diabetic rats had almost none of the activity. Liver microsomes from rats treated with the antioxidant, vitamin E, were completely without effect on Fe 2+ activation. The results indicate that it is possible to alter rapidly the Fe 2+ activation of phospho enolpyruvate carboxykinase by mechahisms that probably involve oxidation/reduction. The decreases in the microsomal factor induced by alloxan diabetes; its high content in the gluconeogenic organs, liver and kidney; and its apparent specificity for the carboxykinase are preliminary evidence of the microsomal factor having a physiological role in the regulation of the carboxykinase.

Decrease of Rat Liver Cysteine Dioxygenase (Cysteine Oxidase) Activity Mediated by Glucagon

The hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5 h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.

Preincubation stimulates fatty acid synthesis by liver slices and isolated hepatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1- 14C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10 −4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1- 14C] acetate into fatty acids or cholesterol. The rate of 14CO 2 synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1- 14C] acetate or [ 3H] H 2O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control

Effect of insulin in vitro on the isolated, perfused alloxan-diabetic rat liver.

Withdrawal of exogenous insulin and a subsequent fast (24 h) of alloxan diabetic rats stimulated rates of gluconeogenesis, urogenesis, ketogenesis, and amino acid release by in situ perfused livers when compared to those from normal, fasted rats. The contribution of liver glycogen to the high rates of gluconeogenesis observed with the diabetic liver could be excluded. Perfusate lactate concentrations remained constant during the period when the elevated rate of gluconeogenesis was observed with diabetic liver. Addition of insulin as a bolus (750 mU) and continuous infusion (12.5 mU/min) to the perfusion medium of diabetic livers resulted in constant perfusate levels of glucose, urea and alpha-amino nitrogen indicating a suppression of the catabolic processes present in the fasted, diabetic liver. The rate of ketogenesis was also slowed by insulin to about half the rate prior to addition of the hormone. These data indicate that insulin has an immediate anti-catabolic effect in the perfused, diabetic liver.

Rapid Conversion by Insulin of Hepatic Intermediary Metabolism from Glucose Production to Glucose Utilization in the Liver of Alloxan-diabetic Rats

The concentration of free fatty adds in the blood, the tissue concentrations of hepatic cyclic AMP, long-chain acyl-CoA (LCA), adenine nucleo tides, inorganic phosphate, the intermediates of the Embden-Meyerhof pathway and the citric acid cycle (including acetyl-CoA and free CoA), and the cytoplasmic and mitochondrial redox couples were determined in alloxan-diabetic rats 15, 30, 60, and 90 minutes after insulin injection. From the meared values, the energy state of the adenine nucleotide system in the liver, the redox state of the NAD system, and the concentrations of malate, oxaloacetate, citrate, and α-ketoglutarate were estimated for the cytoplasmic and mitochondrial compartment by the use of established calculation methods. Insulin injection into alloxan-diabetic rats caused a rapid decline in the concentration of serum free fatty acids and a coordinate decline in hepatic LCA concentration. The latter alteration was accompanied by an increase in the cytoplasmic and a synchronous decrease in the mitochondrial ATP/ADP×P ratio. Simultaneously, the redox state of the cytoplasmic NAD system was shifted towards the oxidized state. When the appropriate data were plotted against each other, a highly significant correlation between the concentration of free fatty acids in the blood and that of LCA in the liver, between the hepatic LCA concentration and the cytoplasmic energy state, and between the cytoplasmic energy state and the cytoplasmic redox state was obtained. These findings are interpreted to support the hypothesis derived from in-vitro experiments that insulin, because of the concentration of hepatic LCA, might affect the translocation of adenine nucleotides between the cytoplasmic and the mitochondrial compartment, thereby regulating the cytoplasmic energy state and, consequently, the redox state of the cytoplasmic NAD system. Insulin injection into alloxan-diabetic rats provoked rapid, coordinate changes in the concentration of the intermediates of both the citric acid cycle and the Embden-Meyerhof chain, indicating the altered substrate flow through these pathways. Thosemetabolit s known to modulate the activity of key regulatory enzymes in vitro were analyzed with respect to their proposed regulatory function. As to the insulin-induced shift from pyruvate carboxylatlon to pyruvate decarboxylatIon, the data reveal that the decrease in pyruvate carboxylase activity can be attributed to the decrease in the intramitochondrial ATP/ADP ratio and the simultaneous fall in acetyl-CoA concentration, while the coordinate increase in pyruvate dehydrogenase activity can be ascribed to the decline in the concentration of LCA and, consequently, in the ratios of ATP/ADP, NADH/NAD, and acetyl-CoA/CoA within the mitochondria.

Changes in Adenylate Energy Charge of the Liver After an Oral Glucose Load

Changes in the adenine nucleotide metabolism after an oral glucose load were studied in the liver of normal and alloxan-diabetic rats. Changes in the energy charge were positively correlated with those in the blood glucose and plasma immunoreactive insulin levels. One hour after an oral glucose load when the plasma immunoreactive insulin levels increased maximally, the energy charge increased maximally from 0.846 to 0.867 (P less than 0.001). The increase in the energy charge was accompanied by a concomitant decrease in the ADP levels (P less than 0.05). The respiratory control ration, state 3 respiration per unit of cytochrome a (+a3), and DNP-induced ATPase activity per unit of cytochrome a (+a3) increased significantly. The adenylate kinase and pyruvate kinase activities in the liver remained unchanged. On the other hand, the energy charge in the liver of alloxan-diabetic rats did not increase significantly after an oral glucose load. It was suggested that an increase in the energy charge of the liver is attributable to the more rapid flux of intermediary metabolism in the enhanced ADP-phosphorylating reactions by mitochondria, owing to an elevated level of insulin available to the hepatic cells.

The effect of diet on hepatic cholesterol synthesis in alloxan diabetic rats.

The effect of dietary composition on cholesterol formation in the liver of alloxan diabetic rats was studied. Cholesterol formation in these animals was maintained at a subnormal level when the diet had a high content of simple sugar (sucrose, glucose or fructose) without fat, but was severely impaired with diets of all other compositions tested. The possible mechanism of maintenance of hepatic cholesterol synthesis in diabetic rats on a diet with a high content of simple sugar without fat is discussed. The rate of cholesterol synthesis in the liver was not always proportional to the plasma cholesterol level.

Synthesis of liver glycogen in starved alloxan diabetic rats.

Alloxan diabetic rats are considered unable to synthesize liver glycogen because of a low activity of glycogen synthetase. The present report shows, however, that livers of alloxan diabetic rats have activities of the I-form of glycogen synthetase intermediate between those of starved normal or adrenalectomized rats, and can synthesize substantial amounts of liver glycogenafter refeeding of a chow diet or administration of hydrocortisone. Neither glycogenic procedure, increased the activity of glycogen synthetase in livers of starved alloxan diabetic rats. In contrast, both normal and adrenalectomized rats had increased activities of the I-form of glycogen synthetase three hours after refeeding. After twenty-four hours of access to food, normal and adrenalectomized rats had liver glycogen contents similar to those found in nonstarved rats, and a markedly decreased activity of synthetase I. Alloxan diabetic rats refed for twenty-four hours also had lowered activity of synthetase I, but the concentration of glycogen was not higher than that found after three hours of refeeding. These results show that livers of alloxan diabetic rats have sufficient glycogen synthetase activity to synthesize glycogen rapidly, but the ability to accumulate larger amounts of glycogen is limited by an alteration in the control of glycogen synthetase activity by the content of liver glycogen. Also, the effect of hydrocortisone on the activity of glycogen synthetase in normal or adrenalectomized rats is the result of insulin release; however, not all the changes in glycogen metabolism produced by adrenalectomy can be explained by insulin deficiency.

Fatty acid oxidation, citric acid cycle activity, and morphology of mitochondria in diabetic rat liver.

SummaryPalmitate and oleate oxidation in isolated mitochondria from alloxan diabetic rat liver were shown to be elevated two or threefold over normal. These increases were accompanied by enlargement of mitochondria as observed by electron microscopy. These metabolic and structural changes were reversed by treating the diabetic rat with insulin. The activities of citric acid cycle enzymes in MT were unchanged in the diabetic state. It is suggested that insulin regulates LCFA oxidation in the liver by an effect on MT structure and function.

Rates of ketone-body formation in the perfused rat liver.

1. The rates of formation of acetoacetate and beta-hydroxybutyrate by the isolated perfused rat liver were measured under various conditions. 2. The rates found after addition of butyrate, octanoate, oleate and linoleate were about 100mumoles/hr./g. wet wt. in the liver of starved rats. These rates are much higher than those found with rat liver slices. 3. The differences between the rates given by slices and by the perfused organ were much higher with the long-chain than with short-chain fatty acids. The increments caused by oleate and linoleate were 12 and 16 times as large in the perfused organ as in the slices, whereas the increments caused by butyrate and octanoate were about four times as large. 4. The rates of ketogenesis in the unsupplemented perfused liver of well-fed rats, and the increments caused by the addition of fatty acids, were about half of those in the liver from starved rats. 5. The value of the [beta-hydroxybutyrate]/[acetoacetate] ratio of the medium was raised by octanoate, oleate and linoleate. 6. Carnitine did not significantly accelerate ketogenesis from fatty acids. 7. Oleate formed up to 82% of the expected yield of ketone bodies. 8. In the liver of alloxan-diabetic rats the endogenous rates of ketogenesis were raised, in some cases as high as in the liver from starved rats, after addition of oleate. 9. On addition of either beta-hydroxybutyrate or acetoacetate to the perfusion medium the liver gradually adjusted the [beta-hydroxybutyrate]/[acetoacetate] ratio towards the normal range. 10. The [beta-hydroxybutyrate]/[acetoacetate] ratio of the medium was about 0.4 when slices were incubated, but near the physiological value of 2 when the liver was perfused. 11. The experiments demonstrate that for the study of ketogenesis slices are in many ways grossly inferior to the perfused liver.