O. Neal Miller

Inhibition of lipogenesis in rat liver by (−)-hydroxycitrate

Abstract The purpose of these investigations was to determine the effect of the stereoisomers of hydroxycitrate on the rate of lipogenesis in rat liver. These observations were made under conditions in which there was an induced rate of lipid synthesis. Only (−)-hydroxycitrate significantly decreased the conversion of [14C]citrate into lipid in liver high speed supernatant and the conversion of [14C]alanine into lipid in vivo. The in vivo rate of lipogenesis was markedly decreased for 150 min following the administration of (−)-hydroxycitrate either iv (0.017 mmoles/kg) or ip (0.0263 mmoles/kg). Fatty acid and cholesterol synthesis were significantly inhibited by the oral administration of (−)-hydroxycitrate (5.26, 3.95 and 2.63 mmoles/kg) only when the compound was given before the feeding period.

Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: I. Lipogenesis

The purpose of these investigations was to ascertain the effect of (−)-hydroxycitrate on the accumulation of lipid in the meal fed rat by examining the rates of lipogenesis after acute and chronic treatment. Oral administration of (−)-hydroxycitrate depressed significantly the in vivo lipogenic rates in a dose-dependent manner in the liver, adipose tissue, and small intestine. The hepatic inhibition was significant for the 8 hr period, when control animals demonstrated elevated rates of lipid synthesis. The kinetics of this reduction of in vivo hepatic lipogenesis were identical after acute or chronic administration of (−)-hydroxycitrate. However, in vitro rates of lipogenesis were elevated after chronic administration of (−)-hydroxycitrate for 30 days. Rats receiving (−)-hydroxycitrate consumed less food than the untreated controls; however, this decreased caloric intake was not responsible for the drug induced depression of hepatic lipogenesis, as shown by studies using pair fed rats.

Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: II. Appetite

These studies were designed to determine the effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat by examining appetite, wt gain, and total body lipid profiles. The chronic oral administration of a nontoxic dose of (−)-hydroxycitrate to growing rats for 11–30 days caused a significant reduction in body wt gain, food consumption, and total body lipid. The administration of equimolar amounts of citrate did not alter wt gain, appetite, or body lipid. No increase in liver size or liver lipid content occurred with either treatment. Pair feeding studies demonstrated that the reduction in food intake accounted for the decrease in wt gain and body lipid observed with (−)-hydroxycitrate treatment.

Dietary and hormonal influences on rat liver polysome profiles; fat, glucose and insulin☆

Abstract 1. 1. To determine which dietary components cause a restoration of disaggregated liver polysomes from fasted rats, polysome profiles from the livers of rats which had been fasted or fasted and refed diets of glucose or fat were prepared and evaluated. To investigate a possible mechanism through which dietary components act, polysome profiles on alloxan-diabetic and insulin-treated rats were prepared and analyzed. 2. 2. The gradual disaggregation of liver polysomes caused by fasting is virtually complete in 48 h. Ten h after refeeding glucose the polysome profiles of 60-h fasted rats are restored. Refeeding isocaloric amounts of fat to fasted rats is without effect. 3. 3. Production of experimental diabetes with alloxan is accompanied by a gradual disaggregation of the liver polysomes. Intraperitoneal administration of insulin restores the polysomes of an alloxan-diabetic rat within 1 h. 4. 4. Administration of insulin to fasted or fat-refed animals is without effect. 5. 5. The findings reported here appear to be unrelated to the reported effects of amino acid supply on liver polysome profiles. 6. 6. Insulin, in concert with glucose, has a rapid effect on the liver resulting in changes in the polysome profile.

An improved competitive protein binding assay for 1,25-dihydroxyvitamin D

Abstract A practical procedure for the routine analysis of blood levels of 1,25-dihydroxyvitamin D has been developed. 1,25-Dihydroxyvitamin D was extracted from serum with methylene chloride:methanol. The concentration of acidic lipids in the extract was reduced with an alkaline wash. The serum extract was chromatographed on Sephadex LH-20, which was extracted with methanol prior to use in the columns. The chromatography was aided by a device which simultaneously collected and evaporated the eluates. High pressure liquid chromatography to further purify 1,25-dihydroxyvitamin D was found to be unnecessary. These modifications resulted in negligible 1,25-dihydroxyvitamin D values ( 3 standard curve capable of measuring 1.5 pg of 1,25-dihydroxyvitamin D 3 was achieved with the use of high specific activity 3 H-labeled 1,25-dihydroxyvitamin D 3 (92 Ci/mmol). The sensitivity of the standard curve, combined with changes in the resuspension of the assay sample, reduced the required amount of serum for an assay from 5 to 2 ml. A simplified centrifugation procedure for the separation of bound from free 1,25-dihydroxyvitamin D was attained by the inclusion of bovine γ-globulin in the polyethylene glycol precipitation.

Determination of bound vitamin B12

Abstract A simple method has been developed for the determination of bound vitamin B12 in serum which uses activated charcoal to remove the free vitamin. Total and bound vitamin B12 activity was followed as a function of time for 24 hr. after oral or parenteral administration of the vitamin. It was found that 80–85% of the B12 activity is bound in serum from normal subjects. The data suggest that vitamin B12 is more rapidly bound in serum after parenteral administration than after oral administration of the vitamin.

PROPANEDIOL METABOLISM AND ITS RELATION TO LACTIC ACID METABOLISM

Interest in propanediol as related to lactate metabolism began 15 or more years ago when, in experiments with heart muscle slices, it was observed tha t lactic acid utilization by rat ventricle slices could not be completely inhibited by concentrations of sodium fluoride, which is known to block enolease activity. Although greater amounts of lactate were utilized than could be accounted for on the basis of respiration of the heart muscle slices, this increased lactate utilization was not explicable by formation of glycogen or known glycolytic intermediates. Therefore, the interest in propanediol phosphate (PDP) arose from the strictly theoretical consideration that lactate was disappearing to a nonreducing-type compound, which possibly was phosphorylated. Search of the literature for such a compound revealed the work of Lindberg,* who first isolated propanediol phosphate from cow brain and sea urchin eggs. The ester was found to comprise 16 per cent of the total acid soluble phosphorus of sea urchin eggs and as high as 70 per cent of the phosphorus of eggs of Emumena crassa and 5 per cent of the organic phosphorus of cow brain. Subsequently, LePage also found P D P to occur in rat carcinoma, and Sacks4 found it as a normal constituent of rat liver to the extent of one and one-half per cent of the organic phosphates of these tissues. The first metabolic studies were made by Lindberg who administered P32-labeled P D P to rats. I t could be shown tha t the radioactivity appeared in the liver of experimental animals more rapidly than the administration of a corresponding amount of radioactive orthophosphate. Also, a rapid exchange of the radioactivity with the terminal phosphate of A T P could be demonstrated, indicating a rapid turnover rate of the administered PDP. In a series of experiments designed to investigate the in uitro metabolism of P D P by animal tissue preparations, my associates and I succeeded in demonstrating that P D P is able to undergo oxidative changes, presumably resulting in the formation of acetol p h o ~ p h a t e . ~ In these studies it was shown that a Myogen A preparation, which is known to consist of at least three enzyme activities-isomerase, a-glycerophosphate dehydrogenase, and aldolasewas found to oxidize P D P in the presence of NAD. At tha t time it was thought that a-glycerophosphate dehydrogenase might be the responsible enzyme. Later, however, it was shown that a specific enzyme for the dehydrogenation of P D P to form acetol phosphate was also present in the Myogen A prepar-

Effect of Malnutrition on Susceptibility of Rats to Trypanosoma cruzi- II. Riboflavin Deficiency.

Abstract Rats maintained on a diet from which pyridoxine was omitted were more susceptible to Trypanosoma cruzi infection than were normal or pair-fed control rats. A higher, more persistent parasitemia and greater degree of myocarditis were observed most frequently in pyridoxine-deficient rats. Although increased age resulted in a reduced susceptibility of control animals to infection, rats inoculated after 6 weeks on a pyridoxine-deficient diet were equally as susceptible to infection as those which were infected after 3 weeks on diet. Spontaneous respiratory infection occurred more frequently in the pyridoxine-deficient animals. The incidence of this infection was the same whether or not the rats were inoculated with T. cruzi .

Distribution and metabolism of two orally administered esters of tocopherol

A comparison of the distribution of total radioactivity in rat tissue lipids after the oral administration of d,1-3,4-3H2-α-tocopheryl nicotinate and d,1-α-tocopheryl-1’,2’-3H2-acetate in equimolar concentrations has demonstrated that there is considerable variation in the concentration of vitamin E in organs at different times after dosing. A higher total radioactivity was found in the tissues of animals receiving α-tocopheryl nicotinate than after α-tocopheryl acetate 12 hr after feeding with an emulsion, but not at most other time intervals studied. These findings indicate that the tissue uptake of vitamin E after oral dosage with nicotinate ester is, perhaps, poorer than that occurring after feeding with tocopheryl acetate, or that α-tocopheryl nicotinate has a faster turnover than the acetate ester. Although total radioactivity in the blood and liver of those animals dosed with α-tocopheryl acetate varied slightly with time, there was a high peak of radioactivity at 12 hr after dosage with nicotinate ester. In both groups of rats, the adrenals, ovaries, adipose tissue and heart appeared to extract vitamin E from the blood for a period of up to 48 hr postabsorptively. Metabolic products of tocopherol detected by glass-fiber paper chromatography were found in both instances. This analysis revealed that, when orally administered, both α-tocopheryl nicotinate and α-tocopheryl acetate are extensively metabolized by the tissues of the rat. The metabolite most abundantly occurring under these conditions was α-tocopheryl quinone. In the adrenal glands, however, the most highly labeled compound was unesterified tocopherol, which increased with time and comprised up to 90% of the chromatographed radioactivity. From the data obtained, it can be assumed that the adrenal tissue plays a definite role in the metabolism of vitamin E.A comparison of the distribution of total radioactivity in rat tissue lipids after the oral administration of d,1-3,4-3H2-α-tocopheryl nicotinate and d,1-α-tocopheryl-1’,2’-3H2-acetate in equimolar concentrations has demonstrated that there is considerable variation in the concentration of vitamin E in organs at different times after dosing. A higher total radioactivity was found in the tissues of animals receiving α-tocopheryl nicotinate than after α-tocopheryl acetate 12 hr after feeding with an emulsion, but not at most other time intervals studied. These findings indicate that the tissue uptake of vitamin E after oral dosage with nicotinate ester is, perhaps, poorer than that occurring after feeding with tocopheryl acetate, or that α-tocopheryl nicotinate has a faster turnover than the acetate ester. Although total radioactivity in the blood and liver of those animals dosed with α-tocopheryl acetate varied slightly with time, there was a high peak of radioactivity at 12 hr after dosage with nicotinate ester. In both groups of rats, the adrenals, ovaries, adipose tissue and heart appeared to extract vitamin E from the blood for a period of up to 48 hr postabsorptively. Metabolic products of tocopherol detected by glass-fiber paper chromatography were found in both instances. This analysis revealed that, when orally administered, both α-tocopheryl nicotinate and α-tocopheryl acetate are extensively metabolized by the tissues of the rat. The metabolite most abundantly occurring under these conditions was α-tocopheryl quinone. In the adrenal glands, however, the most highly labeled compound was unesterified tocopherol, which increased with time and comprised up to 90% of the chromatographed radioactivity. From the data obtained, it can be assumed that the adrenal tissue plays a definite role in the metabolism of vitamin E.

Liquid scintillation counting with glass fiber paper

Abstract The following conclusions appear warranted: Radioactivity is linearly proportional to the number of disks (up to 25) added to the counting vial over a range of 100 to 40,000 cpm. This relationship seems to hold for both lipid-soluble and water-soluble compounds. Glass fiber paper has a 60–70% higher counting efficiency than cellulose paper. The specific activity can be calculated by using the guide strip for determination of the amount of cholesterol and cholesterol esters; and, by using this data as well as the counts, one can calculate the specific activity of these compounds.