Prolylcarboxypeptidase (PRCP) as a new target for obesity treatment.

Recently, we serendipitously discovered that mice with the deficiency of the enzyme prolylcarboxypeptidase (PRCP) have elevated alpha-melanocyte-stimulating hormone (alpha-MSH) levels which lead to decreased food intake and weight loss. This suggests that PRCP is an endogenous inactivator of alpha-MSH and an appetite stimulant. Since a modest weight loss can have the most profound influence on reducing cardiovascular risk factors, the inhibitors of PRCP would be emerging as a possible alternative for pharmacotherapy in high-risk patients with obesity and obesity-related disorders. The discovery of a new biological activity of PRCP in the PRCP-deficient mice and studies of alpha-MSH function indicate the importance and complexity of the hypothalamic pro-opiomelanocortin (POMC) system in altering food intake. Identifying a role for PRCP in regulating alpha-MSH in the brain may be a critical step in enhancing our understanding of how the brain controls food intake and body weight. In light of recent findings, the potential role of PRCP in regulating fuel homeostasis is critically evaluated. Further studies of the role of PRCP in obesity are much needed.

Effect of aldonic acids on the uptake of ascorbic acid by 3T3 mouse fibroblasts and human T lymphoma cells.

1. Previously, we reported that calcium L-threonate caused a dose-related increase in uptake of ascorbic acid (AA) by human T-lymphoma cells. Preincubation of mouse fibroblasts with calcium L-threonate also resulted in a dose-related augmentation in uptake of AA as compared to non-treated controls. 2. Potassium L-lyxonate increased AA uptake by lymphoma cells, but did not significantly affect uptake by fibroblasts. Tartaric acid decreased uptake of AA by both cell lines. 3. Ouabain and dinitrophenol had no effect on AA uptake nor on the ability of threonate to augment AA uptake by fibroblasts. However, in T-lymphoma cells ouabain and dinitrophenol reduced AA uptake and prevented augmentation of AA uptake by calcium L-threonate.

The effects of various levels of ascorbic acid on the response of the ODS rat to trimethyltin.

The effects of trimethyltin (TMT) on behavioral and histological parameters were investigated in rats maintained on low, mid, and high levels of ascorbic acid (AA). Male osteogenic disorder Shionogi (ODS) rats were used. Like man, ODS rats are unable to synthesize AA. AA was administered in the drinking water. Radial arm maze (RAM) performance and locomotor activity were measured before (i.e., baseline) and after (i.e., retest) TMT administration. During baseline, all rats learned the RAM task. Also during baseline, locomotor activity of rats maintained on high levels of AA was found to be lower than the other groups. After administration of 7.5 mg/kg TMT chloride (p.o.), RAM performance of all the groups declined, but RAM performance of rats maintained on low levels of AA appeared least affected by TMT. Also, rats in the high AA group had a significant increase in locomotor activity compared to baseline. These results suggest that in the ODS rat, TMT toxicity may be influenced by levels of AA intake.

Effects of d-alpha-tocopherol supplementation on experimentally induced primate atherosclerosis.

Prevention and regression of induced atherosclerosis by d-alpha-tocopherol was investigated in 24 male M. fascicularis. One group received a basal diet, while three others consumed an atherogenic diet. Two of the latter groups also received tocopherol, one at the onset of the study (prevention) and the other after atherosclerosis was established by ultrasound evaluation (regression). Atherosclerosis was monitored over a 36-month period by duplex ultrasound imaging of the common carotid arteries. At termination, 24 arterial sites were examined for histopathology. In those animals receiving an atherogenic diet, mean percent ultrasound stenosis at 36 months posttreatment was lower in the tocopherol-supplemented groups (61 and 18%) than in the unsupplemented group (87%). Plasma tocopherol concentration was negatively correlated with percent ultrasound stenosis (p less than 0.002). Percent stenosis in the regression group decreased from 33 to 8% (p less than 0.05) 8 months after tocopherol supplementation. Although not consistently significant, histopathological changes were greater in untreated compared to treated animals. These results, if confirmed, indicate that d-alpha-tocopherol may be prophylactically and therapeutically effective in atherosclerosis.

Inhibition of arylsulfatase B by ascorbic acid.

One hypothesis for atherosclerotic lesion formation is the response to injury hypothesis. If catabolism of the ground substance of the intima of blood vessels is viewed as an injury, then inhibition of this catabolism could lead to a decrease in atherosclerotic lesions. Arylsulfatase B catalyses the desulfation of glycosaminoglycans in the catabolism of the intimal ground substance. We have found that ascorbic acid inhibits arylsulfatase B (Km = 2.06 mM, KI = 4.89 mM), thus providing a possible mechanism by which ascorbic acid may decrease atherosclerosis.

Stimulatory action of calcium L-threonate on ascorbic acid uptake by a human T-lymphoma cell line.

The effects of preincubation of human T-lymphoma cells with increasing concentrations of calcium L-threonate on the uptake of L-[1-14C]ascorbic acid were examined. Calcium L-threonate (0-1,000 mg%) stimulated ascorbic acid (1.25 mg%) uptake in a dose-dependent manner. These results indicate that calcium threonate and possibly other ascorbic acid metabolites have biological activity and potential pharmacological applications.

Comparison of the anti-scorbutic activity of L-ascorbic acid and Ester C in the non-ascorbate synthesizing Osteogenic Disorder Shionogi (ODS) rat.

The Osteogenic Disorder Shionogi (ODS) rat, Clea Inc., Tokyo, Japan lacks the ability to synthesize L-ascorbic acid (AA). As with man, monkey and the guinea pig, this rat lacks L-gulonolactone oxidase necessary for the synthesis of AA from glucose. This study shows this animal to be an alternative to the guinea pig in AA studies. The anti-scorbutic potency of Ester C (EC), a calcium ascorbate and calcium threonate mixture, was compared with an AA dose of equal ascorbate activity equivalents (AAE) for anti-scorbutic activity in the ODS rat. The minimal anti-scorbutic dose of EC was determined to be 0.44 mg/kg/day (AAE), while an AA dose of 0.51 mg/kg/day (AAE) was not anti-scorbutic in a 24 day study. At 24 days EC rats gained 125% of initial body weight (BW) and the AA rats only 45% BW. Scorbutic signs at 24 days were scored on a 0 (min) to 3 (max) scale. The EC/AA ratio scores were: hemorrhage 0/1.4, behavior change 0/2.0, piloerection 0/2.2, mobility 0.4/2.2, dysbasia 0.6/2.8 and ataxia 0.4/1.0. Pearson's correlation coefficient for BW versus AAE was r = .34 for the AA group and r = .90 for the EC group. The morbidity index for EC was 0/5 and for the AA group 2/5. The AAE dose of AA which was 16% higher/day than the EC AAE dose was not anti-scorbutic, while the EC dose was anti-scorbutic. EC rats had 3.5X greater weight gain, a sensitive indicator of scurvy, than the AA rats. EC rats had 3-4 times less, if any, scorbutic signs than AA rats. The results clearly show that, based on ascorbate activity equivalents, EC has more available ascorbate activity/potency than AA. The mechanism of this increased potency is believed to be due to the facilitated transport of AAE into the cell by the threonate (a normal in vivo metabolite of AA) present in the EC product. In addition, previous studies have shown EC (AAE) to be higher in plasma and excreted less rapidly than the AAE derived from AA administered orally.

Effect of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: mechanism of impaired ascorbate transport in diabetes.

Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3H-2-deoxy-D-glucose (2.5 mg%) by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14C-L-ascorbic acid (1.25 mg%). The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus.

Clinical profile of a 4-year primate atherosclerosis model.

Twelve male M. fascicularis monkeys were divided into two groups of 6 animals each. One group (BASAL) was fed a diet containing 24% protein, 38% carbohydrate and 20% fat, while the other group (ATHER) received an identical diet with the addition of 4.08 g/kg diet cholesterol. The animals were studied over a 4-year period. Blood samples were regularly collected, ECGs taken and carotid artery status evaluated by duplex ultrasound scanning. Lipid xanthomas were monitored by visual inspection. The ATHER group experienced a rapid and sustained rise in serum total cholesterol, concomitant with depression of HDL-cholesterol. In general, triglycerides were significantly higher in ATHER animals. Routine clinical analysis revealed lower hematocrit and hemoglobin, and elevated BUN and alkaline phosphatase in the treated group. Lipid xanthomas were detected early in the ATHER animals, progressing until infiltration was evident on the entire body surface. There were no differences in ECGs between the groups. At approximately 17 months posttreatment, stenosis was apparent in the carotid arteries of treated animals, rising to an average of 90% at study termination. These results indicate that diet-induced carotid atherosclerosis can be monitored non-invasively in the primate with minimum risk to the animal.

Intimal permeability evaluated in a short-term organ culture of diabetic guinea pig aorta.

A novel short-term organ culture system was used to evaluate intimal permeability changes by measuring aortic [14C]methylated albumin accumulation. Aortic plugs were removed from the upper thoracic aorta of male guinea pigs and maintained in serum-free media. The accumulation of [14C]albumin in the intimal-medial layer was determined after a 5 h incubation. In preliminary studies, albumin recovered from intimal-injured aortic plugs was significantly greater than those from non-injured plugs. Aortic plugs from streptozotocin-treated guinea pigs, diabetic for 3 weeks, also accumulated significantly more [14C]albumin than plugs from nondiabetic controls. Histological changes were not observed in the aorta of either the diabetic or control group. A strong significant inverse correlation was found between plasma ascorbic acid levels and [14C]-activity recovered from aortic plugs. This study demonstrates a simple and rapid method for assessing aortic permeability changes under a well-defined in vitro system, and suggests that vascular permeability changes in the streptozotocin-diabetic guinea pig may be associated with an ascorbic acid deficit.

Selected physical and biochemical parameters in the streptozotocin-treated guinea pig: insights into the diabetic guinea pig model.

Since evidence suggests that ascorbic acid deficits may provoke certain diabetic complications, it becomes necessary to develop a diabetic animal model which, like man, is unable to synthesize this vitamin. To this end, the present study monitored the diabetogenic effects of streptozotocin (STZ, 150 mg/kg) in the male guinea pig, a species rarely used in diabetes research. Over a 3-week period, body weight and relative food intake were lower in the STZ group compared to controls. The mean daily water intake and urine volume of the STZ group after 1 week were 175 and 270% of their initial pretreatment values, respectively, while control values were unchanged. The STZ group also exhibited a persistent glycosuria throughout the study. At the end of 3 weeks, aldehyde fuchsin staining of pancreatic beta cell granules (an index of stored insulin) was 58% lower in the STZ group compared to controls. Plasma C-peptide (indicator of insulin secretion) was expressed in human equivalents (mean +/- SEM). C-peptide was reduced in the STZ group (103 +/- 65 pg/ml) compared to controls (549 +/- 96 pg/ml); however, no change in plasma glucose was observed. Plasma ascorbic acid levels also were lower for STZ animals (150 +/- 26 micrograms%) versus controls (410 +/- 28 micrograms%). This study 1) demonstrates a diabetic syndrome in the STZ-treated guinea pig based on a reduced growth rate, beta cell dysfunction, polydipsia, polyuria and glycosuria, and 2) suggests the usefulness of this diabetic model in studies of pathologic mechanisms influenced by ascorbic acid.

An acute study on the relative gastro-intestinal absorption of a novel form of calcium ascorbate.

Several functions of L-ascorbic acid (vitamin C) have been suggested in addition to its role in the prevention of scurvy. Consequently, a controversy has arisen over the daily intake of the vitamin which will afford maximum benefits. Rapid cellular uptake and delayed renal excretion of ascorbic acid would be conducive to providing optimum cellular concentration for biochemical activity. ESTER-C (patent pending), a complex consisting of L-ascorbic acid and Ca++, has been recently developed by Inter-Cal Corporation (421 Miller Road, Prescott, AZ 86301). It has been proposed that the structure of ESTER-C may render it more readily absorbed and less rapidly excreted than the acid or salt form of the vitamin. To test this hypothesis, ESTER-C and L-ascorbic acid were administered to two groups of rats. Blood was sampled at 20, 40, 80, 160 and 240 minutes and plasma analyzed for ascorbic acid. As urine appeared in collection cups, it was tested qualitatively for the presence of ascorbic acid. The plasma concentration of ascorbic acid was higher in ESTER-C treated rats at 20, 40 and 80 minutes than in rats given L-ascorbic acid. Ascorbic acid was detected in the urine of animals administered ESTER-C later than in those treated with L-ascorbic acid. These results support the hypothesis that ESTER-C is absorbed more readily and excreted less rapidly than L-ascorbic acid.

The effects of clonidine on food consumption and food competition in male stumptail macaques (Macaca arctoides).

The effects of clonidine on noncompetitive and competitive feeding were examined in three adult male stumptail macaques (Macaca arctoides). Noncompetitive feeding was examined when the subjects were individually-caged or group-caged. Clonidine (1.0 mg/kg) administered IM substantially increased food consumption over control values in all subjects in both housing situations. The effects of clonidine on competitive feeding behavior were evaluated in the group-caged environment. Food competition was assessed by recording the number of chows retrieved from a common food box by each subject. Food retrieval was increased in the lowest-ranking subject when clonidine was administered to all subjects. Retrieval decreased in the middle-ranking subject and was unchanged in the highest-ranking subject. Administration of clonidine to individual subjects did not increase food retrieval. It is concluded that since the hyperphagic property of clonidine was only apparent during noncompetitive food-getting, that other drug-behavioral interactions prevented an increase in food retrieval during competitive situations.

Diet-induced changes in selected clinical chemistry parameters in M. fascicularis.

Thirty-six male M. fascicularis monkeys were fed a basal diet (180 g/day, 24% protein, 38% carbohydrate and 20% fat) for a period of 17 months. At that time, the animals were divided into two groups. One group (6 animals) continued on the basal diet (BASAL group), while the others (30 animals) received an atherogenic diet (ATHER group) containing 4.08 g/kg cholesterol. Blood samples were drawn for clinical chemistry analyses. Serum cholesterol rose dramatically in the ATHER group, an average increase of 350 mg/dl over basal-diet values after two weeks on the atherogenic diet. Serum triglycerides followed the same pattern. HDL-cholesterol in the atherogenic-diet group decreased sharply. These animals also had slightly elevated BUN, uric acid and SGOT levels. Skin lipid xanthomas were also observed in the ATHER group, but were absent in animals fed the basal diet. Body weight among all animals remained relatively constant. Minimal changes in clinical chemistry values other than those noted above are evidence of the low physiological stress placed on the animals by this dietary regimen.

Normal serum biochemical, hematological, and EKG parameters in anesthetized adult male Macaca fascicularis and Macaca arctoides.

Selected serum enzymes, cholesterol, triglycerides, glucose, uric acid, protein, albumin, bilirubin, BUN, hematology, and electrocardiograms (EKG) were obtained from adult male cynomolgus (Macaca fascicularis) and adult male stumptailed (Macaca arctoides) macaques. Serum alkaline phosphatase, uric acid, albumin, bilirubin, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and monocytes were significantly lower in the cynomogus monkeys. This relationship was reversed for serum levels of triglycerides, phosphorus, red blood cell counts and lymphocytes. EKG analysis revealed significantly increased PR interval and QRS wave duration in the cynomolgus species. However, there were no differences in heart rate. Right axis deviation was common in both species.

The interaction of aortic glycosaminoglycans and 3H-inulin endothelial permeability in cholesterol induced rabbit atherogenesis.

Male Dutch-belted rabbits were fed a pelleted 0.5% cholesterol diet for 8 weeks. A control group was maintained on basal pelleted diet for the same period of time. Prior to sacrifice both groups received i.v. 3H-inulin. Plasma and aortic sections were assayed for 3H-inulin activity. Aortic sections were also analyzed for glycosaminoglycan concentrations. A ratio of the aortic 3H-inulin activity to the plasma 3H-inulin activity was calculated and expressed as a permeability index. Mean plasma activity of 3H-inulin was significantly lower in the control group, suggesting increased aortic permeability in the cholesterol-fed animals. Glycosaminoglycan concentrations were significantly higher in the control group, and significant negative correlations were detected between glycosaminoglycan concentrations and permeability index in all animals. These findings are consistent with the hypothesis that sufficient glycosaminoglycan levels are important to maintaining the permeability characteristics of the endothelial layer.

Fruit and vegetable consumption and cardiovascular mortality.

A steady fall in mortality rates from cardiovascular diseases (CVD) has been reported recently in the United States. This study tests the hypothesis that fruits and vegetables had a protective effect against cardiovascular mortality among the American population from 1964-78. Special attention was focused on more specific groups of fruits and vegetables, especially those rich in vitamin C, to examine the effect of consumption of these foods on cardiovascular mortality rates. These data showed that the consumption of fruits and vegetables, particularly those rich in vitamin C (based on content and consumption), may have offered a protective effect against deaths from CVD. Increased fruit and vegetable consumption appeared to contribute significantly to the reduction of CVD mortality in the American population in recent years.

Effects of streptozotocin in the male guinea pig: a potential animal model for studying diabetes.

The effects of acutely administered streptozotocin in the male guinea pig were studied for a period of 18 days following treatment. A single intracardiac injection of streptozotocin (150 mg/kg) was administered on Day 0. On Day 2, plasma glucose concentrations were not significantly different from control levels. On Day 7 and 18, an oral glucose tolerance test was performed with streptozotocin-treated animals receiving an acute injection of either insulin (18 U/kg, i.m.) or saline 90 minutes prior to glucose loading. On Day 7, streptozotocin-treated animals receiving saline had significantly elevated plasma and urine glucose concentrations at 3 hours after glucose loading when compared to controls. Streptozotocin-treated animals receiving insulin however, had significantly lower plasma glucose concentrations at 3 hours while urinary glucose was equal to control values. The second glucose tolerance test performed on Day 18 yielded similar results. Necropsies were performed on animals that died after Day 6. Lesions found in the streptozotocin-treated animals included: small and irregular pancreatic islets, pyknotic nuclei and degranulation of beta cells, renal proximal tubule swelling and vacuolization, adrenal cortical hyperplasia, hepatocyte vacuolization, and visceral fat atrophy. Animals surviving until Day 18 were sacrificed and found to have significantly elevated kidney and adrenal weights compared to controls. These changes illustrate the effectiveness of streptozotocin in the acute chemical induction of diabetes in an animal model (guinea pig) which, like humans, requires a dietary source of ascorbic acid.

The effects of glucose on ascorbic acid uptake in heart endothelial cells: possible pathogenesis of diabetic angiopathies.

Glucose in concentrations of 20 mg% (or greater) significantly inhibited 14C-labelled ascorbic acid (1.25 mg%) uptake in endothelial cells in the presence of insulin (1600 microU/ml). The absence of insulin also significantly reduced ascorbic acid uptake. Furthermore, this reduction could be exacerbated by glucose (40, 160 mg%) but not equimolar concentrations of fructose. Increased ascorbic acid concentrations (two-fold) in the absence of insulin (1) significantly enhanced uptake, and (2) reversed the inhibition of glucose. These findings support earlier reports that ascorbic acid uptake into the cell may be compromised by decreased insulin and/or increased extracellular glucose levels. Since previous animal studies have correlated experimental ascorbic acid deficiencies with atherogenic processes (presumably by altering glycosaminoglycan metabolism), the postulation that the "diabetic condition" (low insulin, hyperglycemia) accelerates the cellular changes leading to atherosclerosis by impairing ascorbic acid uptake into the vascular endothelium, may now be supported.