Interactions between insulin and thyroid hormone in the control of lipogenesis.

1. The effects of intragastric glucose feeding and L-tri-iodothyronine (T3) administration on rates of hepatic and brown-fat lipogenesis in vivo were examined in fed and 48 h-starved rats. 2. T3 treatment increased hepatic lipogenesis in the fed but not the starved animals. Brown-fat lipogenesis was unaffected or slightly decreased by T3 treatment of fed or starved rats. 3. Intragastric glucose feeding increased hepatic lipogenesis in control or T3-treated fed rats, but did not increase hepatic lipogenesis in starved control rats. Glucose feeding increased hepatic lipogenesis if the starved rats were treated with T3. Glucose feeding increased rates of brown-fat lipogenesis in all experimental groups. The effects of glucose feeding on liver and brown-fat lipogenesis were mimicked by insulin injection. 4. The increase in hepatic lipogenesis in T3-treated 48 h-starved rats after intragastric glucose feeding was prevented by short-term insulin deficiency, but not by (-)-hydroxycitrate, an inhibitor of ATP citrate lyase. The increase in lipogenesis in brown adipose tissue in response to glucose feeding was inhibited by both short-term insulin deficiency and (-)-hydroxycitrate. 5. The results tend to preclude pyruvate kinase and acetyl-CoA carboxylase as the sites of interaction of insulin and T3 in the regulation of hepatic lipogenesis in 48 h-starved rats. Other potential sites of interaction are discussed.     

Regulation of hepatic lipogenesis in starved and diabetic animals by thyroid hormone

The effects of intragastric feeding with glucose and of the administration of L-triiodothyronine (T₃) on in vivo rates of hepatic lipogenesis were investigated in control (fed ad libitum on norrnal diet), diabetic (fed ad libitum on normal diet), fat-fed (fed ad libitum on high-fat diet), and starved (food removed for 48 h) rats. Two days of T₃ treatment increased hepatic lipogenesis in control and fat-fed animals but not in the diabetic or starved animals, although increases in lipogenesis in diabetic animals were observed after 4 days of T₃ treatment. Intragastric glucose feeding increased hepatic lipogenesis in the livers of control animals and T₃-treated control animals. Such increases are mediated by an increase in the circulating insulin concentration, as increases are not observed in diabetic rats or T₃-treated diabetic rats. Glucose feeding failed to increase hepatic lipogenesis in fat-fed rats or starved rats. Insulin injection together with glucose feeding increased lipogenesis in the fat-fed group but not the starved group; i.e., impaired insulin secretion following an oral glucose load may in part explain the lack of response in the fat-fed but not the starved animals. Marked increases in hepatic ]ipogenesis after glucose feeding were, however, observed if either the starved or the fat-fed animals were treated with T₃, The physiological implications of these observations are discussed.     

Possible mechanisms of the inhibiting effect of nicotinamide on lipogenesis in rat liver

The activity of some NAD- and NADP-dependent dehydrogenases involved in generation of the reducing equivalents for lipogenesis and the activity and some kinetic parameters of ATP-citrate (pro-3S)-lyase from rat liver, i. e. the enzyme involved in the formation of CoASAc, the primary substrate of fatty acid biosynthesis, were studied. The changes in the activity of NADP-dependent dehydrogenase and ATP-citrate(pro-3S)-lyase, as well as the affinity of the latter for sitrate and CoA and the rate of lipogenesis in starved rats and in rats kept on a carbohydrate-rich diet after starvation appeared to be parallel. Nicotinamide decreased the activity of all NADP-dependent dehydrogenases under study, which was especially well-pronounced after nicotinamide addition against increased lipogenesis. The affinity of ATP-citrate(pro-3S)-lyase for citrate and CoA decreased simultaneously with the decrease in the concentration of the latter. These changes can possibly induce the decrease of lipogenesis rate in rat liver after addition of nicotinamide.     

Regulation of enzymes of glucose metabolism and lipogenesis in diabetic rat liver by thyroid hormones

Experimental diabetes in rats is associated with a degree of hypothyroidism. Hepatic enzymes involved in carbohydrate and lipid metabolism were estimated in control (untreated), control +T3 treated, alloxan diabetic and alloxan diabetic + T3-treated rats. The key glycolytic enzymes, phosphofructokinase and pyruvate kinase, were decreased in activity in diabetes and unchanged by further treatment with T3. In contrast, certain enzymes involve in lipogenesis, ATP-citrate lyase 'malic' enzyme and 6-phosphogluconate dehydrogenase, which were decreased in activity in diabetes, were increased to, or above, control values when diabetic rats were treated with T3. It is suggested that T3 deficiency may play a role in the decrease in enzyme activities observed in experimental diabetes, in particular, some enzymes associated with lipogenesis and the provision of NADPH.     

Inhibition of rat liver acetyl-CoA carboxylase by beta, beta'-tetramethyl-substituted hexadecanedioic acid (MEDICA 16)

Rat liver acetyl-CoA carboxylase activity was inhibited by the free as well as the CoA monothioester of beta, beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) (Bar-Tana, J., Rose-Kahn, G. and Srebnik, M. (1985) J. Biol. Chem. 260, 8404-8410 (1985). (1) The CoA monothioester of MEDICA 16 served as a dead-end inhibitor with an apparent Ki of 2 microM and 58 microM for the biotin-carboxylated and noncarboxylated enzyme forms, respectively. MEDICA 16-CoA binding was not mutually exclusive with that of citrate and did not affect the avidin-resistance of rat liver acetyl-CoA carboxylase. (2) The free dioic acid of MEDICA 16 was competitive to citrate, having an apparent Ki of about 70 microM, as compared to a Ka of 2-8 mM for the citrate activator. Inhibition of the carboxylase by the free dioic acid of MEDICA 16 was accompanied by an increase in its avidin resistance. The resultant inhibition of acetyl-CoA carboxylase by MEDICA 16 and its CoA thioester, together with the previously reported citrate-competitive inhibition of ATP-citrate lyase by MEDICA 16, may account for the observed hypolipidemic effect of MEDICA 16 under dietary conditions where liver lipogenesis constitutes a major flux of liver lipid synthesis.     

Direction of carbon flux in starvation and after refeeding: in vitro and in vivo effects of 3-mercaptopicolinate

3-Mercaptopicolinate (3-MPA) is a specific inhibitor of phosphoenolpyruvate carboxykinase (PEP CK). In vivo the hypoglycaemic action of 3-MPA in 24 h-starved rats was abolished on intragastric glucose refeeding. Nonetheless, 3-MPA decreased hepatic glycogen content and rate of synthesis in starved animals re-fed glucose. The inference is that on re-feeding after starvation hepatic glycogen is synthesised mainly de novo via glyconeogenesis involving PEP CK. 3-MPA increased hepatic lipogenesis in water- and glucose-fed normal and diabetic rats. This increase is presumed to result from inhibition of PEP CK and consequent diversion of pyruvate from gluconeogenesis to lipogenesis. In contrast, 3-MPA inhibited brown-fat lipogenesis in water- and glucose-fed rats.     

Nonalcoholic Hepatic Steatosis in Zucker Diabetic Rats: Spontaneous Evolution and Effects of Metformin and Fenofibrate

No specific treatment for nonalcoholic hepatic fatty liver disease has been defined. We followed the spontaneous evolution of liver steatosis and tested the therapeutic usefulness of metformin and fenofibrate in a model of steatosis, the Zucker diabetic fatty (ZDF) rat. ZDF and control rats were studied at 7, 14, and 21 weeks. After initial study at 7 weeks, ZDF rats received no treatment, metformin or fenofibrate until studies at 14 or 21 weeks. ZDF rats were obese, hypertriglyceridemic, insulin resistant at 7 weeks, type 2 diabetic at 14, diabetic with insulin deficiency at 21. They had steatosis at 7 weeks with increased hepatic expression and activity of lipogenesis. Steatosis was unchanged at 14 and 21 weeks despite lower expression and activity of lipogenesis. Metformin and fenofibrate did not modify energy intake or expenditure or the evolution of diabetes. Both compounds decreased plasma triacylglycerol (TAG) concentrations. Hepatic TAG content was reduced by fenofibrate at 14 and 21 weeks but only at 21 weeks by metformin. Metformin had no significant effects on the expression in liver of genes of fatty acids metabolism. The beneficial effect of fenofibrate occurred despite increased expression of genes involved in the uptake and activation of fatty acids. Acyl‐CoA oxidase (ACO) and carnitine palmitoyltransferase I (CPTI) mRNA levels were increased by fenofibrate showing evidence of increased lipid oxidation. To conclude, metformin had only moderate effects on liver steatosis. The effects of fenofibrate was more marked but remained mild.     

Induction of hepatocyte stimulating activity by T3 and appearance of the activity despite inhibition of DNA synthesis by adriamycin

A hepatocyte stimulating activity (HSA) has been extracted from rats that had received an injection of a pharmacological dose of T3 20 hours earlier. The injection of HSA from T3-treated rats into different recipient rats that had previously had 40% of their liver removed resulted in a significant increase in hepatic DNA synthesis. The injection of saline or HSA from normal rat liver had little or no effect on hepatic DNA synthesis in recipient rats. HSA from the T3-treated rats also stimulated DNA synthesis in Novikoff hepatoma cells and primary hepatocytes in culture, and in isolated normal rat liver nuclei in a nuclear incorporating system. In further experiments in which the increased DNA synthesis that follows partial hepatectomy was blocked by adriamycin, HSA appeared in these non-regenerating livers. This latter observation had indicated that the development of HSA is not merely an accompaniment of DNA synthesis.     

Carnitine and creatine content of tissues of normal and alloxan-diabetic sheep and rats

The concentration of carnitine in liver increased 28-fold and urinary carnitine excretion 5-fold in alloxan-diabetic sheep. In contrast there were no similar increases in alloxan-diabetic rats. The creatine content of liver decreased 3-fold and creatine excretion decreased 2-fold in diabetic sheep. In contrast the creatine content of liver increased nearly 4-fold in diabetic rats with no change in creatine excretion. The marked increased in production of carnitine by the liver of the diabetic sheep appears possible because of decreased production and excretion of creatine.     

Deregulation of hepatic lipid metabolism associated with insulin resistance in rats subjected to chronic monocrotophos exposure

In our previous study, we demonstrated the potential of monocrotophos (MCP), an organophosphorus insecticide (OPI), to induce glucose intolerance, insulin resistance (IR), and dyslipidemia with hyperinsulinemia in rats after chronic exposure. As hyperinsulinemia is likely to exert an impact on hepatic lipid metabolism, we carried out this study to establish the effect of chronic MCP exposure (0.9 and 1.8 mg/kg/day for 180 days) on hepatic lipid metabolism in rats. The state of IR induced by MCP in rats was associated with an increase in the liver lipid content (triglyceride and cholesterol) and expression levels of sterol regulatory element‐binding proteins, PPARγ, acetyl‐CoA carboxylase, and fatty acid synthase in the liver. Similarly, activities of key enzymes (acetyl‐COA carboxylase, fatty acid synthase, lipin 1, malic enzyme, glucose‐6‐phosphate dehydrogenase, and glycerol‐3‐phosphate dehydrogenase), which regulate lipogenesis, were enhanced in livers of pesticide‐treated rats. A strong correlation was observed between insulin levels, hepatic lipid content, and plasma lipid profile in treated rats. Our study suggests that long‐term exposure to OPIs not only has a propensity to induce a state of hyperinsulinemic IR, but it is also associated with augmented hepatic lipogenesis, which may explain dyslipidemia induced by chronic exposure to MCP.     

Changes in the content and structure of the coenzyme A moiety in the liver of diabetic mice (db/db) administered a regimen of nicotinamide

Alterations in the content and structure of CoA moiety typical of hyperlipogenesis (a rise in total and free CoA levels, a drop in short-chained fatty acyl-CoA/CoA and long-chained fatty acyl-CoA/CoA ratios) were found in the liver of obese mice with non-insulin-dependent diabetes (db/db). The treatment of diabetic mice with nicotinamide, an antilipemic drug, was accompanied by a decrease in total and free CoA levels and a rise in short-chained fatty acyl-CoA content and short-chained fatty acyl-CoA/CoA and long-chained fatty acyl-CoA/CoA ratios, probably leading to the inhibition of the enzymes of primary lipogenesis steps. It is suggested that CoA moiety structure is essential as an integral index regulating the rate of fatty acid biosynthesis in diabetes mellitus.     

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.     

Effects of fasting on the control of fatty-acid synthesis in hepatoma 7777 and host liver. Role of long-chain fatty acyl-CoA,, the mitochondrial citrate transporter and pyruvate dehydrogenase activity.

The effects of fasting on the rate of fatty acid synthesis, the properties of the mitochondrial citrate transporter and on pyruvate dehydrogenase activity were investigated in "poorly-differentiated" tmorris hepatoma 7777 and in host liver preparations. The properties of the citrate transporter from hepatoma mitochondria were similar to those of host liver mitochondria, with the exception that the Km for the liver mitochondrial citrate transporter was 248 plus or minus 20 mu M while that in hepatoma mitochondria was less than 75 mu M. The acid-insoluble CoA content was 180 plus or minus 20 pmol/mg protein in the hepatoma and remained essentially unchanged in the fasted state, while the acid-insoluble CoA levels in livers from fed rats was 720 plus or minus 80 pmol/mg protein and were increased to 1050 plus or minus 50 pmol/mg protein during fasting. After a 36-h fast, the rate of lipogenesis and the percentage of pyruvate dehydrogenase present in the active form were each decreased by approximately 80% in host liver preparations. In contrast, the rate of lipogenesis by hepatoma slices did not decrease during fasting, and essentially all pyruvate dehydrogenase present was in the active form of hepatomas obtained from either fed or fasted animals. Implications concerning the identification of possible regulatory sites in the control of lipogenesis were discussed in relation to the above observations.     

Altered interactions between lipogenesis and fatty acid oxidation in regenerating rat liver.

The concentrations of malonyl-CoA, citrate, ketone bodies and long-chain acylcarnitine were measured in freeze-clamped liver samples from fed or starved normal, partially hepatectomized or sham-operated rats. These parameters were used in conjunction with measurements of the concentration of plasma non-esterified fatty acids and the rates of hepatic lipogenesis to obtain correlations between rates of fatty acid delivery to the liver, lipogenesis and fatty acid oxidation to ketone bodies and CO2. These correlations indicated that the development of fatty liver after partial hepatectomy is due to an increased partitioning of long-chain acyl-CoA towards acylglycerol synthesis and away from acylcarnitine formation. However, this did not appear to be due to an altered relationship between hepatic malonyl-CoA concentration and acylcarnitine formation. For any concentration of long-chain acylcarnitine, the concentrations of both hepatic and blood ketone bodies were significantly lower in partially hepatectomized rats than in normal or sham-operated animals. This indicated that a lower proportion of the product of beta-oxidation was used for ketone-body formation and more for citrate synthesis in the regenerating liver, especially during the first 24 h after resection. This inference was supported by the changes in hepatic citrate concentrations observed. The high rates of lipogenesis that occurred in the liver remnant were accompanied by an altered relationship between lipogenic rate and hepatic malonyl-CoA concentration, such that much lower concentrations of malonyl-CoA were associated with any given rate of lipogenesis. These adaptations are discussed in relation to the requirements by the remnant for high rates of energy formation through the tricarboxylic acid cycle during the first 24 h after resection, and the possibility that cycling between fatty acid oxidation and synthesis may occur to a greater degree in regenerating liver.     

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, Km and Ki (substrate inhibition) values. Influence of deoxycholate (0.1%) and 1-anilino-8-naphthalene sulfonate (2.5 mM) on the kinetics was also evaluated. 1. Substrate inhibition occurred at 0.06 M for the enzyme from normal rats and at 0.0-0.025 M for the enzyme from diabetic rats. 2. The enzyme from diabetic rats showed a transition that extended between 22.7 and 27 degrees C in the Arrhenius plot (log V vs. T-1) instead of at 19.5 degrees C. 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. 1-Anilino-8-naphthalene sulfonate eliminated substrate inhibition and activated the enzyme of normal rats above 27.5 degrees C by increasing both V and Km values. Below this temperature, the enzyme showed biphasic or allosteric kinetics. At low substrate concentrations it was activated as both V and Km values were increased. The enzyme from diabetic rats, on the other hand, was activated at all the temperatures and exhibited linear kinetics. 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 degrees C. 6. Enthalpy 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. In the microsomal fraction (total as well as rough), phospholipid content and fatty acid unsaturation index of phospholipids were decreased after diabetes. The level of free cholesterol remained unchanged but the molar ratio of cholesterol to phospholipid increased. The different thermal response and 1-anilino-8-naphthalene sulfonate interaction to the enzyme from diabetic rat and liver could be ascribed to the altered lipid environment of the enzyme on the endoplasmic reticulum membrane.     

Citrate content of liver and kidney of rat in various metabolic states and in fluoracetate poisoning

The citrate content of rat liver changes little when normal rats are starved, when starved rats are re-fed with various diets and when normal animals are made diabetic with alloxan. The citrate content of rat kidney changes little on starvation, but it doubles on induction of diabetes. Fluoroacetate poisoning has relatively little effect on the citrate content of liver under a variety of conditions except that normal female rats show a 2·4-fold increase. Fluoroacetate poisoning leads to increases in the citrate content of kidney under all conditions. The relevance of these observations to the regulation of fatty acid synthesis is discussed. The acetic anhydride–pyridine method and the pentabromoacetone method for the estimation of citrate are compared.     

Inhibition of phosphoenolpyruvate transport via the tricarboxylate and adenine nucleotide carrier systems of rat liver mitochondria

The translocation of phosphoenolpyruvate by the tricarboxylate carrier system in rat liver mitochondria was shown to be inhibited by atractyloside and long chain fatty acyl CoA esters as well as benzene, 1, 2, 3 tricarboxylate. By contrast benzene 1, 2, 3 tricarboxylate did not inhibit atractyloside sensitive adenine nucleotide translocation catalyzed by phosphoenolpyruvate. These results indicate that although phosphoenoppyruvate is preferentially transported by the tricarboxylate carrier system, it may also be transported by the adenine nucleotide translocase. The inhibition of the adenine nucleotide and tricarboxylate carrier systems by atractyloside and long chain acyl CoA esters indicates a close functional interrelation-ship of these transport carriers in the inner mitochondrial membrane. Moreover, the potent inhibition of phosphoenolpyruvate, citrate, and adenine nucleotide transport by long chain acyl CoA's provides further evidence that these esters are natural effectors which participate in the regulation of gluconeogenesis, lipogenesis, and energy-linked respiration.     

The Effects of Insulin on Hepatic Glucocorticoid Receptor Content in the Diabetic Rat

Abstract

Streptozotocin-induced diabetic rat liver was analyzed for glucocorticoid receptor (GR) content by saturation and Scatchard analysis. The hepatic GR content of streptozotocin-induced diabetic rats was significantly decreased from a control level of 0.17 ± .01 pmol/mg protein to 0.11 ± .01 pmol/mg protein. Insulin replacement therapy to the diabetic rat dramatically increased the hepatic GR content to 0.26 ± 0.02 pmol/mg protein as compared to the diabetic value of 0.11 ± 0.01 pmol/mg protein.

A time course study of GR content in the diabetic rat liver demonstrated that after an initial decrease in hepatic GR content at 14 days, the 25-day diabetic receptor level elevated back to control levels. A significant increase in GR content over controls was observed in the 110-day diabetic rats. These results suggest that insulin has a role in the regulation of hepatic GR content.     

Influence of pancreatic hormones on enzymes concerned with urea synthesis in rat liver

1. The activities of enzymes of the urea cycle [carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (these last two comprising the arginine-synthetase system) and arginase] have been measured in control, alloxan-diabetic and glucagon-treated rats. In addition, measurements were made on alloxan-diabetic rats treated with protamine–zinc–insulin. 2. Treatment of rats with glucagon for 3 days results in a marked increase in the activities of three enzymes of the urea cycle (carbamoyl phosphate synthetase, argininosuccinate synthetase and argininosuccinase). The pattern of change in the alloxan-diabetic group is very similar to that of the glucagon-treated group, although the magnitude of the change was much greater. 3. Comparison was made of the actual and potential rate of urea synthesis in normal and diabetic rats. In both groups the potential rate of urea production, as measured by the activity of the rate-limiting enzyme, argininosuccinate synthetase, slightly exceeds the actual rate of synthesis by liver slices in the presence of substrates. The relative activities of the actual and potential rates were similar in the two groups of animals, this ratio being 1:0·70. 4. In the alloxan-diabetic rats treated with protamine–zinc–insulin for 2·5 or 4 days there was a marked increase in liver weight. This was associated with a rise in the total hepatic activity of the urea-cycle enzymes located in the soluble fraction of the cell (the arginine-synthetase system and arginase) after 2·5 days of treatment. After 4 days of treatment the concentration of these enzymes/g. of liver decreased, and the total hepatic content then reverted to the untreated alloxan-diabetic value. 5. No effects of glucagon or of insulin in vitro could be found on the rate of urea production by liver slices. 6. The present results are discussed in relation to how far this pattern of change is typical of conditions resulting in a high urea output, and comparison has been made with other values in the literature.     

The effect of starvation and alloxan-diabetes on the contents of citrate and other metabolic intermediates in rat liver

1. The content of citrate in ;freeze-clamped' livers from starved and alloxan-diabetic rats was measured by using the specific citrate assay method of Gruber & Moellering (1966). 2. The content of citrate fell progressively during a period of 48hr. starvation to reach a plateau value that is 50% of the value for livers from fed rats. Some possible explanations for the conflicting reports of changes in hepatic citrate content during starvation are discussed. 3. The hepatic contents of ATP, pyruvate, lactate, glycogen and the hexose phosphates were decreased during starvation, whereas those of acetyl-CoA and AMP were increased. 4. Acute alloxan-diabetes produced similar changes in the contents of these metabolic intermediates. 5. The effects of starvation and diabetes on the citrate and acetyl-CoA contents are discussed in relation to control of gluconeogenesis, fatty acid synthesis and the activity of citrate synthase.