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In hepatomas, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, is not normally suppressed by cholesterol. To examine the biochemical mechanism of this loss of feedback control of cholesterol synthesis, a comparison was made of the properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase after solubilization and partial purification from microsomes of normal rat liver and of a minimal deviation rat hepatoma. The solubilized enzyme from the two sources behaved identically with respect to substrate kinetics, heat inactivation, cold inactivation, and gel filtration. By the use of a new method that permits assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity after polyacrylamide gel electrophoresis, it was shown that the solubilized hepatoma enzyme and the normal liver enzyme had similar electrophoretic mobility. It is concluded that the failure of hepatomas to suppress 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity is not due to the production of an altered form of the enzyme.

For an electron microscopic analysis of the response of the ACTH-cell to adrenalectomy, pituitaries were taken from normal controls and from rats 24 hr after adrenalectomy and at additional intervals up to 3 weeks. Electron micrographs of ACTH-cells that had been sampled by an objective, blind procedure were measured by planimetry and the measurements submitted to statistical analysis. The ACTH-cell undergoes a significant increase of 30% in cell size by 24 hr after adrenalectomy; a 100% increase in cell size is reached as early as 5 days, and then cell size plateaus through 21 days. The size of the nucleus of the ACTH-cell shows a significant increase by the fifth postoperative day and then remains constant to 21 days. The nucleoli of ACTH-cells are significantly enlarged 24 hr postadrenalectomy, and heterochromatin also shows a transformation to euchromatin at this early interval. Secretory granulation of the ACTH-cells is strikingly reduced at 24 hr, then gradually increases at the later intervals. The...

The effect of the addition of dihydrocholesterol to a high cholesterol diet has been studied in the chicken. It is shown that in this species the elevated plasma cholesterol and resulting atherosclerosis can be reduced to normal levels by such a procedure.

Summary1. Material giving a very strong periodic acid-Schiff reaction was seen in certain structures in sections of mouse pituitaries. This material was shown to be glycogen by its disappearance when exposed to saliva or diastase prior to staining. 2. In mice 8 days and younger, glycogen could be demonstrated in the intercellular spaces and in the cells of the neural lobe, in the cells of the pars tuberalis, and intercellularly in the stalk and median eminence of the hypothalamus. At 5 days and less, glycogen also appeared to be located in irregular patterns in the sinusoids of the anterior lobe. 3. After 8 days of age, glycogen was apparently restricted, in the pituitary system, to the pars tuberalis. After one month the amount of glycogen was considerably reduced, but nevertheless could still be demonstrated at 7 months.

A procedure for the isolation of mevalonate and β-hydroxy-β-methylglutarate from animal tissues is presented. This technique involves differential extraction of the short chain acids with ethyl ether, formation of their dibenzylethylenediamine salts, and final purification of the methyl esters of these acids by gas-liquid chromatography. This technique has been employed for the measurement of mevalonate synthesis in a cell-free liver system, and the validation of such a procedure established by showing the chromatographic purity of the methyl and ethyl derivatives of the isolated mevalonate-14C.

SummaryCholesterol-4-C14 was administered intravenously to normal rats and the C14 end products in feces and bile were fractionated. 1. From 4 to 10% of the total C14 in bile and from 17 to 23% of fecal C14 were recovered in the nonsaponifiable fraction which includes cholesterol, dihydrocholesterol, coprosterol, etc. 2. Little if any of the C14 in either bile or feces was recovered in the fatty acid fraction. 3. Evidence is presented to show that about 80% of the fecal C14 and about 90% of the bile C14 are present in the form of bile acids. 4. The major metabolic pathways for the nucleus carbons of cholesterol are outlined.

University of Minnesota Masters thesis. May 1920. Major: Anatomy. Advisor: Dr. C. M. Jackson. 1 computer file (PDF); 51 pages.

1. By means of a gas-liquid chromatographic technique the effect of exogenous cholesterol on the incorporation of acetate-2-14C into β-hydroxy-β-methylglutarate and mevalonate has been measured both in cell-free systems of liver and in intact liver cells. 2. It has been shown that mevalonate synthesis is markedly suppressed by cholesterol feeding while the synthesis of β-hydroxy-β-methylglutarate is unaffected by this regimen. These results are consistent with the conclusion that the major site of the cholesterol feedback system is located at the reaction responsible for the conversion of β-hydroxy-β-methylglutarate to mevalonate; namely β-hydroxy-β-methylglutaryl reductase. 3. The intracellular localization of mevalonate synthesis and its feedback control have been likewise examined. Mevalonate was found to be synthesized in both microsomal and soluble fractions of the liver cell; however, the major site of mevalonate synthesis, and hence of feedback control of cholesterol synthesis, is localized to the membranous fraction of the microsomes.

A B S T RA C T This report describes a "desmosterol suppression" technique with which it has been possible to demonstrate the operation of the cholesterol negative feedback system in the intact animal. 0.1% triparanol in the diet causes a virtually complete block in the conversion of desmosterol to cholesterol by liver and intestine. Since desmosterol is not consumed in the diet, the level of plasma desmosterol can be employed as an index of endogenous sterol production and release into the bloodstream. With this technique it was shown that the feeding of cholesterol for 8 days to rats decreases blood desmosterol levels to less than 5% of control values. Very similar results were obtained when cholesterol synthesis was assayed in vivo with acetate-"C as a cholesterol precursor. These observations indicate that the cholesterol feedback system operates very effectively in the intact animal in suppressing the endogenous contribution to the circulating cholesterol pool. Since intestinal cholesterol synthesis is only slightly inhibited by exogenous cholesterol, these results also indicate that the intestine does not represent a significant source of plasma sterols in the rat.

A technique is described for the measurement of muscle capillary basement membranes by electron microscopic examination of needle biopsies of the quadriceps muscle. With this procedure it has been possible to obtain an objective evaluation of the significance of capillary basement membrane hypertrophy in diabetic microangiopathy. The results of such studies of muscle capillary basement membrane thickness in 50 normal, 51 diabetic, and 30 prediabetic patients have demonstrated the following. First, that the average capillary basement membrane width of diabetic patients is over twice that of normal subjects; moreover, such basement membrane thickening is a very constant finding among overtly diabetic patients, in that approximately 98% of individual diabetic subjects demonstrated this lesion. The degree of basement membrane thickening in diabetic patients is, however, unrelated to age, weight, severity, or duration of diabetes. Second, capillary basement membrane hypertrophy has been found in approximately 50% of patients who are genetically prediabetic but who have not yet demonstrated evidence of the manifest carbohydrate disturbances of diabetes mellitus. Third, in contrast to the results obtained in genetically diabetic patients, subjects with severe hyperglycemia due to causes other than genetic diabetes only infrequently show basement membrane hypertrophy. These results indicate that thickening of the muscle capillary basement membranes is a characteristic of genetic diabetes mellitus, and further, that the hyperglycemia of diabetes is probably not the factor responsible for the microangiopathy characteristic of diabetes mellitus. Finally, the discovery of thickened capillary basement membranes in prediabetic patients suggests that basement membrane hypertrophy is a relatively early lesion of the diabetic syndrome and provides further support for the conclusion that this vascular defect is independent of carbohydrate derangements of diabetes mellitus.

1. 1. The synthesis of the cardiotonic sterols, marinobufagin and marinobufotoxin, has been demonstrated in the toad, Bufo marinus . 2. 2. Approximately 2% of administered cholesterol-4-C 14 is converted to the sterol lactones in a 75-day period. No incorporation of acetate-C 14 was detected.

3-Hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol biosynthesis in rat liver, has been solubilized and purified 130-fold from rat liver microsomes. Evidence is presented to suggest that the enzyme is localized to the surface of the microsomal membrane. 3-Hydroxy-3-methylglutaryl-CoA reductase can be removed from microsomes by mild treatments such as exposure to high salt or glycerol concentrations which leave the bi-laminar structure of the microsomal membrane intact. The solubilized enzyme is rapidly inactivated by chilling to 4° and is protected from this cold-induced inactivation by salt only at concentrations in the range of 4 m. 3-Hydroxy-3-methylglutaryl-CoA reductase is the first solubilized microsomal enzyme reported to be cold-sensitive.

To provide further evidence for the identification of the cell type responsible for the production of adrenocorticotrophin (ACTH), the effects of acute and chronic administration of cortisol on the cells of the pituitaries of intact and adrenalectomized rats were studied by electron microscopy. Hypertrophied ACTH-cells, previously described as adrenalectomy-cells, were scarce in controls and became more numerous after adrenalectomy. Adrenalectomy-cells are characterized by their secretory granules, which are dense, sparse, and measure 200 μn or more in maximal diameter, and by the irregularity of their cell outlines, determined by their interior position in the cell cords and their tendency to send processes of cytoplasm around neighboring cells toward the capillaries. Normal ACTH-cells are identified by similar criteria, although they are less voluminous and their cell outlines tend to be less complex and varied. Acute administration of cortisol caused a striking accumulation of secretory granules in bot...

ONE of the physical properties of proteins, that of differential solubility, is not usually taken advantage of when the routine methods of fixation are used prior to histological or histochemical staining procedures. However, it should be possible to expose a tissue containing a mixture of proteins to a reagent in which some of the proteins were denatured and rendered insoluble, while other soluble proteins were not denatured and were extracted. Because of the reversibility of denaturation with some reagents and because solvents are employed in the preparation of tissue sections, it is possible to use a second fixative containing a reagent that will combine with the proteins which were not extracted, precipitating them irreversibly so that they can be subsequently stained. In this manner, it should be possible to extract certain proteins from a mixture of proteins in a tissue, following which comparison of stained sections of the extracted tissue with sections of the same tissue in which all the proteins ...

0 VER the course of the past five years a considerable advance has been made in our understanding of how glucose may influence a number of phases of lipid metabolism. The elucidation of this problem has been greatly aided, on the one hand by studies on the details of glucose breakdown, and on the other by a number of fundamental advances in our knowledge of the biochemical mechanisms of lipogenesis and of ketogenesis. It should be emphasized at the outset of this review, however, that there remain today numerous unanswered questions in this field, especially in regard to the relationship between glycolysis and the synthesis of ketone bodies and of cholesterol. There can be little doubt that the most important advance in this field stems from the realization that glucose can be metabolized in most cells of the body by at least two different pathways. This conclusion was first reached independently by Lipmann [I] and by Dickens [z] in 1936; however, it has only been during the past few years that the details of the biochemical reactions involved in the so-called pentose phosphate pathway of glucose breakdown have been fully described [3,4]. Furthermore, only recently have methods been developed for determining the relative quantitative significance of the two major pathways of glucose catabolism.

Studies were performed to examine synthesis, tissue localization, and metabolism of mevalonic acid in normal rats. Circulating mevalonate was found to have a rapid turnover phase of 5 min and a slower phase of 40-50 min. Under in vitro conditions the synthesis of mevalonate is carried out most actively by the liver and only to a minor extent by the other tissues studied. The most unexpected finding of this study was that both in vivo and in vitro the kidneys rather than the liver are the primary site of the metabolism of circulating mevalonate. Whereas mevalonate in the liver is rapidly transformed to cholesterol, the major products of mevalonate metabolism in the renal tissues during the same time period are squalene and lanosterol. Exogenous in contrast to circulating mevalonate is metabolized primarily in the intestine.

The feedback response of mevalonate: NADP oxidoreductase (acylating CoA; EC 1.1.1.34) in two varieties of hepatoma has been examined. In marked contrast to the feedback inhibition of this enzyme that is regularly observed in normal liver, the feedback control is completely lost in minimal-deviation hepatomas 9121 and 3924A. This finding provides the first specific biochemical localization of the defect in cholesterol feedback control, which has been shown previously to be a characteristic of all hepatomas.