The effect of mevalonic acid deprivation on enzymes of DNA replication in cells emerging from quiescence.

We have investigated the biochemical basis of the mevalonate dependence of DNA replication. Stimulating quiescent rat hepatoma cells to proliferate in the presence of compactin, an inhibitor of mevalonate synthesis, prevented DNA replication in as many as 80% of these cells. The percentage of cells that failed to replicate DNA increased with the increased duration of quiescence. Aphidicolin-sensitive DNA polymerase and ornithine decarboxylase activities were selectively decreased in compactin-treated cells, whereas RNA and protein synthesis, the level of dihydrofolate reductase and aphidicolin-resistant DNA polymerase activity were unaffected. Adding putrescine, the product of ornithine decarboxylase and the precursor of other polyamines, did not restore DNA replication. Our results demonstrate that the decreased activities of at least two DNA-replication enzymes are among the proximal causes of the failure of mevalonate-deprived cells to synthesize DNA. More importantly, our data indicate that a mevalonate-dependent factor(s) is progressively depleted during quiescence, and that inability to resynthesize this factor(s) may be the ultimate cause of the failure of resting cells to replicate DNA when stimulated to proliferate in the absence of mevalonate.

Diabetic ketoacidosis and hyperosmolar coma.

DKA-hyperosmolar coma is a readily diagnosed and easily treated, potentially catastrophic emergency that regularly occurs in both Type I and Type II diabetics. This review emphasized that diabetic ketoacidosis and hyperosmolar coma can, and very frequently do, occur concurrently, but it is the hyperosmolar state rather than the DKA that is the primary cause of coma and death in this condition. One must therefore vigorously treat the hyperosmolarity and resulting dehydration, especially when total calculated osmolarity exceeds 230 to 240 mOsm/L. The major aim of treatment is to rapidly replace the major water loss that is responsible for this clinical condition and to stimulate glucose metabolism with insulin. The diagnosis of this dangerous condition is relatively simple. The therapy, in most regards, is equally apparent. There are good data demonstrating that the prompt recognition of DKA-hyperosmolar coma and the simple institution of rapid rehydration have continued to reduce the mortality and complications of this potentially disastrous complication of diabetes mellitus.

Prospective studies of muscle capillary basement membrane width in prediabetics.

In 1968, this laboratory reported that muscle capillary basement membrane thickening was present in approximately 50% of nondiabetic individuals who had a strong genetic predisposition for developing diabetes mellitus. In the present manuscript we report the 20-yr follow-up observations in this group of prediabetics. We were able to obtain information regarding the presence or absence of diabetes in 17 of 33 subjects (51.5%). In these 17 individuals, 8 (47%) developed diabetes over the ensuing 20+ yr of observation. Initial (1964) glucose tolerance tests in the subjects that developed diabetes were not significantly different from those that did not develop diabetes. One- and 2-h glucose values on follow-up (4-8 yr later) glucose tolerance tests were significantly higher in the group that developed diabetes. On initial biopsy, muscle capillary basement membrane width was similar in the 2 groups; in the follow-up specimens the mean muscle capillary basement membrane width tended to be greater in the group that ultimately developed diabetes, but this difference was not statistically significant. However, in the group that developed diabetes the follow-up muscle capillary basement membrane width increased significantly compared to the initial measurement, whereas in the nondiabetic group the muscle capillary basement membrane width decreased with time (diabetics, +75.50 +/- 23.66; nondiabetic, -14.44 +/- 28.71 A/yr; P less than 0.05). Seven of 8 (87%) of the individuals who developed diabetes showed progressive thickening of their muscle capillary basement membrane thickness, whereas in the nondiabetic group 5 of 8 had a decrease in width. These results suggest that an increase in muscle capillary basement membrane width over time might serve as a marker to detect individuals who are at increased risk to develop diabetes. The detection of an increased susceptibility for diabetes could potentially allow for interventions that could delay or prevent the development of diabetes.

Diabetic microangiopathy, genetics, environment, and treatment.

As the major cause of disability and death in insulin-dependent diabetes, microangiopathy is obviously of major concern to diabetologists. Unlike macroangiopathy, which can readily be prevented by means that are currently on hand, the origin and treatment of microangiopathy remain far more problematical. The complexity of this lesion is indicated by the findings in this laboratory that hyperglycemia induced by the rodenticide, vacor, can cause microangiopathy independent of genetic diabetes, yet significant microangiopathic lesions can be detected in genetic diabetic patients before the appearance of hyperglycemia. Further, there is now intriguing evidence based both on basement membrane measurements from our laboratory and on clinical studies showing that significant microangiopathy only rarely occurs prior to the onset of puberty. The evidence that control or even normalization of blood glucose levels does not influence the course of established microangiopathy is becoming increasingly convincing. Five prospective, randomized studies over the past five years have shown that strict regulation of glucose has no consistent benefit on, and in some studies may, at least transiently, accelerate, the retinopathy of diabetes. Moreover, the first controlled study of successful pancreatic transplantation to achieve normalization of blood glucose levels has again demonstrated that established retinopathy is neither prevented nor even delayed by normal glucose levels. This review, therefore, emphasizes that, though hyperglycemia is required for clinically significant microangiopathy to occur, clearly other factors, genetic, environmental, or both, must play major roles in determining the course of microangiopathy. It is toward these nonglycemic factors in the development of diabetic microangiopathy that future research should increasingly be directed.

Regulation of cholesterol metabolism in a slow-growing hepatoma in vivo.

Cholesterol metabolism and its regulation are altered in hepatomas as compared to normal liver. We investigated parameters of cholesterol metabolism and their regulation in rats bearing the well-differentiated Morris hepatoma 9108. The numbers of membrane associated receptors recognizing chylomicron remnants, the lipoproteins that deliver dietary lipid to the liver, were substantially decreased in the 9108 tumor relative to the host liver. Cholesterol synthetic rates were 2-3-fold higher in the tumor, while the activity of 3-hydroxy-3-methylglutarylcoenzyme A reductase (EC 1.1.1.88), a rate-limiting enzyme for sterol synthesis, was elevated 6-14-fold. Although tumor free and esterified cholesterol contents were elevated, the activity of acylcoenzyme A:cholesterol acyltransferase (EC 2.3.1.26), the enzyme responsible for intracellular sterol esterification, was unchanged. Similar to the host liver, cholesterol synthesis and 3-hydroxy-3-methylglutarylcoenzyme A reductase were inhibited in the tumor when rats were fed a diet containing cholesterol, cholate and lard, and there was no effect on the numbers of chylomicron remnant receptors. Administering an intravenous bolus of very low density lipoproteins obtained from hypercholesterolemic rats caused an inhibition of tumor reductase activity, but had little effect on cholesterol content or cholesterol esterification. Thus, hepatoma 9108 expressed quantitative differences in cellular parameters involved in the uptake, metabolism, and synthesis of cholesterol and their susceptibility to regulation when compared with the host liver. These differences are best explained by changes in the hepatoma of multiple factors involved in the regulation of normal hepatic cholesterol metabolism.

Relationship of muscle capillary basement membrane thickness and diabetic retinopathy.

We examined the relationship between the thickness of the quadriceps muscle capillary basement membrane and diabetic retinopathy. Basement membrane thickness was measured in two groups of patients with long-standing type II diabetes mellitus. One group of patients (N = 13) had no evidence of diabetic retinopathy on fluorescein angiography, whereas the other (N = 12) had proliferative microvascular disease. All the patients were male, and both groups were of similar ages, duration of diabetes, serum creatinines, and glycemic control as reflected by HbA1 levels. Mean muscle capillary basement membrane width (+/- SE) of the patients with proliferative retinopathy (3346 +/- 262) was significantly greater (P less than .05) than that observed in the patients without retinopathy (2660 +/- 177). The results of this study suggest that there is a relationship between capillary basement membrane thickness in skeletal muscle and the severity of microangiopathy in the eye. However, there was a substantial overlap between the two groups, indicating that for any individual patient the measurement of muscle capillary basement width will probably not be useful in identifying the presence or absence of retinopathy.

The role of circulating mevalonate in nephrotic hypercholesterolemia in the rat.

The cause of the hypercholesterolemia that characterizes the nephrotic syndrome has never been adequately explained. The present study examines the possibility that enhanced availability of the cholesterol precursor, mevalonic acid, to the liver in the nephrotic state may result in increased hepatic cholesterogenesis. In normal animals, the kidneys are known to be the major site of the metabolism of circulating mevalonate to both cholesterol and CO2. Previous studies, using perfusion of isolated, intact kidneys, have shown that the excretion and metabolism of mevalonate are both impaired in nephrosis. The present investigation has demonstrated in vivo that puromycin aminonucleoside nephrosis results in a 25% reduction in the oxidation of mevalonate to CO2. In the same nephrotic animals, cholesterogenesis from circulating mevalonate was significantly increased in both liver and carcass. In addition, liver slices from nephrotic animals incorporated increased amounts of [5-14C]mevalonate into cholesterol when calculated per whole liver, but not per gram of liver. Oxidation of mevalonic acid by kidney slices was significantly reduced, whether expressed as per gram of tissue or per whole organ. HMG-CoA (3-hydroxy-3-methylglutaryl) reductase activity in liver of nephrotic animals was significantly increased. We conclude that, in the nephrotic state, impaired mevalonate metabolism by the kidney may contribute to enhanced cholesterogenesis by increasing delivery of mevalonate to liver and carcass; in addition, nephrosis appears to provide an undefined stimulus for HMG-CoA reductase activity in the liver, thereby providing an additional enhancement of hepatic cholesterogenesis.

Muscle capillary basement membrane width in patients with vacor-induced diabetes mellitus.

Muscle capillary basement membrane width is a sensitive marker for the presence of diabetic microangiopathy. Studies have indicated that genetic factors and alterations in glucose metabolism influence muscle capillary basement membrane width. To define the role of these factors we have measured muscle capillary basement membrane thickness in controls, insulin dependent diabetics, and individuals with diabetes secondary to the ingestion of Vacor, a rat poison, which results in hyperglycemia. Hemoglobin A1 concentrations were increased in both diabetic groups, but hemoglobin A1 levels and the duration of diabetes were similar in the two diabetic groups. The muscle capillary basement membrane width was increased to a similar extent in the insulin-dependent diabetics (control, 1,781 +/- 46 vs. IDD, 2,287 +/- 144 A, P less than 0.001) and in the Vacor diabetic group (2,320 +/- 149 A, P less than 0.001). In the insulin-dependent diabetic group, 63% of the patients had a muscle capillary basement membrane width greater than two standard deviations above the mean of the controls, while in the Vacor diabetic group this figure was 56%. Despite the relatively short duration of diabetes (6.2 +/- 0.3 yr), 44% of the Vacor diabetic patients had retinopathy and 28% had proteinuria. The present study provides strong evidence that even in the absence of genetic diabetes mellitus, hyperglycemia or some other abnormality related to insulin lack can cause microvascular changes.

Role of the kidneys in the metabolism of circulating mevalonate in humans.

Previous studies in several animal species have demonstrated that the kidneys are the primary site of mevalonate metabolism by the oxidative or shunt pathway. To determine the role of the human kidney in mevalonate oxidation, we studied mevalonate shunt activity in patients undergoing hemodialysis for varying degrees of renal failure. Surprisingly, at least half of the uremic patients and even anephric patients had normal ability to oxidize mevalonate by the shunt pathway. In addition, we found a strong negative correlation (R = -0.94) between mevalonate shunt activity and serum phosphorus levels in uremic patients. The resulting inhibition of mevalonate oxidation by high serum phosphorus levels was reversed by lowering the serum phosphorus in one patient. Finally, a positive correlation was found between mevalonate oxidation and serum PTH levels. The results of this study suggest that, in humans, extrarenal tissues can be major contributors to mevalonate oxidation. It is therefore probable that in humans, in contrast to other animals, the kidney is not the primary site of mevalonate metabolism by this oxidative pathway. Finally, the strong negative correlation between serum phosphorus levels and the ability of uremic patients to oxidize mevalonate suggests a regulatory role for the phosphate ion in the mevalonate shunt pathway.

Type II diabetes: some problems in diagnosis and treatment.

This case is a rather typical presentation of a patient with mild, type II diabetes. His case illustrates some of the pitfalls in the dietary management of diabetes, in treating the glucose abnormality and hypertension, and in diagnosing the mental obtundation that can occur in type II disease. This patient¿s glucose abnormality can probably be treated adequately with a normal- or low-calorie diet that is appropriately high in carbohydrates and low in fats. His hypertension must be treated adequately, preferably with lower doses of diuretics or, if other drugs are necessary, one other than a beta-adrenergic blocking drug. Clearly, his cholesterol should be treated too, but the high-carbohydrate, low-fat diet now prescribed for diabetics will probably be adequate to reduce this risk factor for cardiovascular disease. Finally, the last major risk factor for cardiovascular disease, namely smoking, should unquestionably be removed.

Role of cholesterogenesis and isoprenoid synthesis in DNA replication and cell growth.

In summary, there is now ample evidence that cell growth, as well as DNA replication, is closely linked to cholesterogenesis in a manner that has only recently been appreciated. Mevalonic acid, the product of HMG-CoA reductase, the rate-limiting enzyme of cholesterogenesis, is now known to serve at least two functions in the cell cycle. First, mevalonate acts as a precursor of the cholesterol that is needed for cell membrane synthesis, and second, independent of cholesterogenesis, mevalonate has been shown to serve a rapid and essential initiator function in DNA replication. These findings, coupled with the fact that loss of the cholesterol feedback control of mevalonic acid represents the most consistent biochemical defect so far identified in the malignant and premalignant states, suggests the possibility that uncontrolled synthesis of mevalonate may play a role in deranged DNA synthesis and malignant transformation.

Altered activation state of hydroxymethylglutaryl-coenzyme A reductase in liver tumors.

Extensive studies have demonstrated that the normal inhibition of cholesterol synthesis by cholesterol feeding is decreased in all hepatomas studied in vivo. This loss of the normal feedback regulation of cholesterol synthesis has been shown to be due to the failure of cholesterol ingestion to inhibit the activity of hydroxymethylglutaryl (HMG)-CoA reductase. The basis for this absence of feedback control of cholesterogenesis is unknown. Studies to date have not demonstrated structural or kinetic differences between the HMG-CoA reductase of normal liver and hepatoma. The present study, however, demonstrates significant differences in the activation state of HMG-CoA reductase from normal liver and hepatoma. In normal liver only approximately 10-20% of the microsomal HMG-CoA reductase is in the dephosphorylated, active form while 80-90% is in the phosphorylated, inactive state. In contrast, in three different Morris hepatomas in vivo, from 53 to 73% of the HMG-CoA reductase is in the active state. That the increased activation state in hepatomas is a property of tumor tissue and is not solely due to rapid growth is demonstrated by the fact that in both fetal and regenerating liver an enhanced activation state of HMG-CoA reductase is not observed. Additionally, preincubation with magnesium and ATP results in the inhibition of HMG-CoA reductase both in tumor and in liver. Presumably, this decrease in HMG-CoA reductase activity is due to the phosphorylation of the enzyme. Similarly, the preincubation of tumor and liver microsomes with phosphatase results in an increase in HMG-CoA reductase activity presumably by the dephosphorylation of the enzyme to its active form. The relationship between the altered activation state of HMG-CoA reductase in hepatomas and the reduction in the feedback regulation of this enzyme in liver tumors remains to be explored.

Normalization of fasting blood glucose levels in insulin-requiring diabetes: the role of ethanol abstention.

The long-term remission of insulin-dependent diabetes mellitus is an unusual occurrence. The present report describes three cases of typical insulin-dependent diabetes in which the metabolic abnormalities greatly improved after the patients abstained from excessive ethanol intake. Our patients presented with polyuria, polydipsea, and weight loss of several weeks duration and laboratory studies revealed marked elevations in blood glucose concentrations and, in two of the three patients, mild ketosis. When these patients subsequently abstained from excessive ethanol intake, the elevations in blood glucose levels and the clinical symptoms of diabetes resolved. Two of our three patients have a strong family history of diabetes mellitus and we have therefore hypothesized that excessive ethanol intake only results in hyperglycemia in individuals with either an underlying predisposition to develop diabetes or mild unrecognized preexisting diabetes. It is important that clinicians recognize that excessive ethanol intake can lead to hyperglycemia and that abstention from ethanol may result in the marked improvement of the metabolic abnormalities.

Activation of HMG-CoA reductase by microsomal phosphatase.

HMG-CoA reductase activity can be modulated by a reversible phosphorylation-dephosphorylation with the phosphorylated form of the enzyme being inactive and the dephosphorylated form, active. Phosphatases from diverse sources, including cytosol, have been shown to dephosphorylate and activate HMG-CoA reductase. The present study demonstrates phosphatase activity capable of activating HMG-CoA reductase that is associated with purified microsomes. The incubation of microsomes at 37 degrees C for 40 min results in a twofold stimulation of HMG-CoA reductase activity, and this stimulation is blocked by sodium fluoride or phosphate. The ability of microsomes to increase HMG-CoA reductase activity occurs regardless of whether microsomes are prepared by ultracentrifugation or calcium precipitation. Additionally, phosphatases capable of activating HMG-CoA reductase are present in both the smooth and rough endoplasmic reticulum. Freeze-thawing does not prevent microsomes from activating HMG-CoA reductase but preincubation results in a significant decrease in the ability of microsomes to increase HMG-CoA reductase activity. Thus, the present study demonstrates that purified liver microsomes contain phosphatase activity capable of activating HMG-CoA reductase.

Differences in de novo cholesterol synthesis between the intact male and female rat.

Three different isotopes were used to quantify de novo sterologenesis in intact male and female rats. All three substrates (i.e. [14C]acetate, [14C]octanoate, and [3H]water) were incorporated into nonsaponifiable lipids and cholesterol at significantly greater rates in males than in females. Even with cholesterol feeding, male animals synthesized significantly more cholesterol and nonsaponifiable lipids than females. The primary site of this sex difference in sterologenesis is extrahepatic, extraintestinal tissues (e.g. carcass). In the carcass this sex difference is chiefly due to an enhancement of sterol synthesis in the skin of male rats. Cholesterol synthesis is 73% greater and nonsaponifiable lipid synthesis is 85% greater in the skin of males than in females. Moreover, de novo sterologenesis in skin is hormonally dependent. In castrated females, testosterone treatment results in a 2-fold stimulation of skin sterol synthesis compared to that in animals administered estradiol or oil vehicle alone. In castrated males, estradiol treatment caused a 30% reduction in skin cholesterol and nonsaponifiable lipid synthesis compared to that in animals administered testosterone. The effects of sex steroid hormones on skin are, therefore, probably responsible for mediating the observed sex difference in de novo sterol synthesis. Additionally, this study demonstrates that the administration of estradiol and testosterone in physiological doses to castrated animals has no effect on cholesterol or nonsaponifiable lipid synthesis in liver, intestine, or other nondermal tissues.

The dual role of mevalonate in the cell cycle.

It is well established that either exogenous or endogenous cholesterol is required for both cell growth and proliferation. This laboratory has recently discovered that, in baby hamster kidney-21 cells, independent of its role as a cholesterol precursor, mevalonic acid plays an essential role in S phase DNA replication. It was later shown that isopentenyl adenine, a known product of mevalonate in prokaryotes and lower eukaryotes, is 100 to 200 times more effective than mevalonate in restoring DNA replication in cells in which mevalonic acid synthesis is blocked with the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor, compactin. The present study was designed to determine the relationship in the cell cycle between the known requirement for cholesterol and the newly discovered effect of mevalonic acid and isopentenyl adenine on S phase DNA synthesis. Employing cells arrested by serum depletion, it was shown that the cholesterol requirement is limited to the early and mid-G1 phases, whereas the isopentenyl effect is required at the late G1-S interphase of the cell cycle. The evidence supporting these conclusions involves: first, in serum-arrested cells blocked early in G1 by compactin, only the combination of cholesterol added in early G1 and either mevalonate or isopentenyl adenine in late G1 permitted progression through the G1 and S phase DNA synthesis. Neither isopentenyl adenine added early in G1 nor cholesterol in late G1 was capable of restoring DNA synthesis in this system. Second, in accord with the above formulation, inhibition of cholesterol synthesis with the oxidosqualene cyclase inhibitor, dl-4,4,10 beta-trimethyl-trans-decal-3 beta-ol, affected only the early G1 phase of the cell cycle, but had no late G1 effect on DNA replication.