Metabolism

Postprandial hyperglycemia and hyperinsulinemia are often present in obese subjects with glucose intolerance in whom insufficient early phase insulin secretion and subsequent delayed hyperinsulin response are observed. To address this problem, a novel palatinose-based enteral formula designated as MHN-01 was developed for the prevention of postprandial hyperglycemia and hyperinsulinemia. The effects of MHN-01 on carbohydrate and lipid metabolism in Sprague-Dawley (SD) rats were compared with those of the standard balanced formula (SBF). After a bolus intragastric injection of each formula equivalent to 0.9 g/kg carbohydrate, the peak levels of plasma glucose (PG) and insulin (IRI) in peripheral and portal veins of the MHN-01 group were significantly lower than those of the SBF group. The areas under the curve of PG and IRI in the MHN-01 group were 58.0% and 43.1% of those in the SBF group in the femoral vein and 65.0% and 69.3% in the portal vein, respectively. In the 2-month study, serum levels of IRI and triglyceride in peripheral blood in the MHN-01 group decreased and those in the SBF group increased compared with initial levels. Consequently, both levels in the MHN-01 group were significantly lower than those in the SBF group. In addition, the amount of accumulated fat in abdominal adipose tissue and liver tissue of the MHN-01 group was markedly reduced in comparison to that of the SBF group. Insulin sensitivity, evaluated as glucose infusion rate using the hyperinsulinemic euglycemic clamp technique, in the MHN-01 group was higher than that in the SBF group. Thus, in comparison to SBF, MHN-01 suppressed postprandial hyperglycemia and hyperinsulinemia, reduced visceral fat accumulation, and improved insulin sensitivity. Therefore, human study on the effects of MHN-01 on carbohydrate and lipid metabolism will be recommended to confirm whether MHN-01 may be a useful functional food for the treatment and prevention of insulin resistance.

The association of hypertension with obesity has been well recognized, but the etiology remains poorly understood. Obesity is characterized by hyperinsulinemia, which reflects peripheral insulin resistance. Insulin resistance may participate in the development of hypertension with obesity. CS-045 [(I)-5-[4-(5-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy)be nzy l]-2,4- thiazolidiendion] is a new orally effective antidiabetic agent that potentiates insulin action and reduces insulin resistance in obese Zucker rats and other obese diabetic animals. In this study, we examined the antihypertensive effect of CS-045 in obese male and female Zucker rats as a model of hypertension associated with obesity. CS-045 was administered as a food admixture (approximately 16 and approximately 70 mg/kg/d) for 4 weeks (female) and (approximately 15 and approximately 67 mg/kg/d) 8 weeks (male) in obese Zucker rats at 5 to 7 months of age. CS-045 slightly but significantly decreased plasma glucose levels. Plasma insulin levels were significantly decreased in obese male rats, but were not significantly decreased in obese female rats. Drug administration led to significant decreases in plasma triglyceride and cholesterol levels and systolic blood pressure (SBP) in a dose-dependent manner from 1 week after administration in obese Zucker rats. CS-045 increased urinary sodium excretion, sodium/potassium ratio, and creatine clearance in a dose-dependent manner, and also led to a remarkable decrease in urinary protein excretion. However, CS-045 did not reduce urinary catecholamine excretion. These data indicate that CS-045 may promote renal sodium excretion and improve decreased glomerular filtration rates, which may reflect the amelioration of insulin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats fed a sucrose-rich diet ([SRD] 63% wt/wt) up to 270 days develop stable hypertriglyceridemia, impaired glucose tolerance, and insulin insensitivity. The aim of the present study is to investigate whether the hypoglycemic agent troglitazone introduced as a pharmacologic intervention could improve and/or reverse the whole-body insulin insensitivity and related abnormalities present after feeding normal rats with a SRD long-term. For this purpose, male Wistar rats were fed a SRD for 210 days. While half of the animals continued with this diet for up to 270 days, troglitazone (0.2 g/dL wt/wt) was added to the SRD of the other half for up to 270 days. Troglitazone markedly reduced in vivo the hepatic triglyceride secretion rate (TGSR) and enhanced its removal from the circulation, leading to a normalization of plasma triglyceride levels. It also normalized the whole-body peripheral insulin resistance, the glucose homeostasis, and the elevated free fatty acids (FFAs) without detectable changes in plasma insulin levels. The clear alteration of the biphasic pattern of glucose-stimulated insulin secretion in the in vitro perfused beta-cell islets of rats fed the SRD long-term (270 days) was also completely normalized when the SRD was supplemented with troglitazone for 2 months. The normalization of the altered patterns of glucose-stimulated insulin secretion, as well as the enhancement of peripheral insulin sensitivity without detectable changes in plasma insulin, might be largely a result of the significant action of troglitazone in the decrease of circulating lipids and enhancement of whole-body glucose metabolism.

We studied the effect of troglitazone, a new oral antidiabetic agent that potentiates insulin action and reduces insulin resistance, on albuminuria in streptozotocin (STZ)-treated diabetic rats. Without affecting blood glucose level, blood pressure, and creatinine clearance, troglitazone treatment of diabetic rats significantly decreased the diabetes-associated albuminuria at all time points studied 14 to 12 weeks of treatment: diabetic 510 +/- 161 micrograms/24 h v diabetic treated 112 +/- 34 micrograms/24 h at 12 weeks, P < .05). These data suggest that troglitazone has potential in the treatment of diabetic nephropathy.

Antidiabetic effects of CS-045 were evaluated in 5-month-old C57BL/KsJ-db/db mice (db/db). CS-045 administered for 3 weeks to diabetic db/db mice as a 0.2% food admixture improved hyperglycemia (855 +/- 25 v 298 +/- 62 mg/dL, P less than .01) and glucose intolerance, and lowered plasma triglyceride (299.6 +/- 28.7 v 76.3 +/- 20.7 mg/dL, P less than .01) and free fatty acid (FFA) levels (1.16 +/- 0.14 v 0.57 +/- 0.07 mEq/L, P less than .01). Food intake was not changed, while a small but significant increase in body weight was observed in CS-045-treated mice. Plasma insulin levels gradually increased after 5 days of CS-045 treatment, and a nonsignificant increase was observed in plasma insulin levels after 3 weeks (1.85 +/- 0.50 v 4.54 +/- 1.47 mg/mL). In contrast, the plasma glucagon levels decreased after 3 weeks of CS-045 treatment. Histological examination by aldehyde-fucshin staining demonstrated that pancreatic beta cells in CS-045-treated db/db mice were heavily regranulated, whereas most of the beta cells were extensively degranulated in nontreated db/db mice. The heavily regranulated state of beta cells was also compatible with an increase in pancreatic insulin content in CS-045-treated db/db mice. Electron microscopic analysis showed a well-developed endoplasmic reticulum and the accumulation of much amorphous structural material in the intracisternal space of beta cells from CS-045-treated db/db mice, which were suggestive of an increase in insulin synthesis. Moreover, CS-045 treatment decreased exocrine-containing islets, which was associated with the islets' degeneration process. Immunohistochemical staining of islets showed that CS-045 treatment normalized the distribution pattern of endocrine cells in the islets of db/db mice, reflected by a predominantly peripheral location of alpha and delta cells.(ABSTRACT TRUNCATED AT 250 WORDS)

To clarify the relationship between lipid and glucose metabolism abnormalities in fructose-fed rats, we examined whether an improvement of insulin sensitivity by troglitazone (CS-045) or a decrease in plasma lipids by bezafibrate affects the relationship between serum levels of lipid and glucose. In addition, we also examined changes in liver glycogen metabolism and beta-oxidation in fructose-fed rats. Troglitazone ameliorated fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia. In addition, it augmented glycogen synthase activity by 53%, and decreased the mitochondrial palmitic acid beta-oxidation rate and ketone body production rate by 27% and 55%, respectively. However, hyperglycemia and liver glycogen synthase activity were not improved by bezafibrate treatment despite a marked reduction of serum triglyceride (TG) levels resulting from a 1.76-fold increase in mitochondrial oxidation and a 2.04-fold increase in hepatic ketone body production. These results suggest that abnormalities in glucose and lipid metabolism in fructose-fed rats, which are ameliorated by troglitazone, may be closely linked to reduced glycogen synthase activity in the liver.

We recently discovered that glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide can both prevent the development of atherosclerosis in apolipoprotein E-null (Apoe(-/-)) mice. In the present study, we attempted to extend these findings to orally administered dipeptidyl peptidase (DPP)-4 inhibitor. Seventeen-week-old Apoe(-/-) mice fed an atherogenic diet were administered a DPP-4 inhibitor, vildagliptin analogue (PKF275-055 [PKF], 100 µm/[kg d]), in drinking water over a period of 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation were determined. Orally administered PKF increased plasma levels of active glucagon-like peptide-1 by 3.5-fold, increased total glucose-dependent insulinotropic polypeptide levels by 2-fold, reduced body weight by 13%, and reduced plasma cholesterol levels by 30%. Compared with drinking water controls, PKF significantly suppressed total aortic atherosclerotic lesions, atheromatous plaque in the aortic root, and macrophage accumulation in the aortic wall by 30% to 40% (P < .001). None of these changes were associated with the PKF-induced reductions in body weight and plasma cholesterol levels. Foam cell formation was suppressed by 40% in the exudate peritoneal macrophages obtained from the PKF-treated mice. The DPP-4 inhibitor prevents the development of atherosclerotic lesions by suppressing macrophage foam cell formation.

The increment for the prevalence of diabetes mellitus and impaired glucose tolerance warrants lowering the cutoffs of normoglycemia to help predict the future development of diabetes. The aim of this study was to find out whether insulin resistance and high-sensitivity C-reactive protein (hsCRP), a nontraditional cardiovascular risk factor, were related to the fasting glucose level, even in normoglycemic range that was categorized by the newly recommended criteria by the American Diabetes Association. Among the participants undergoing medical checkup program at Kangbuk Samsung Hospital, 10059 subjects (5535 men and 4524 women; mean age, 45 years) with normal fasting glucose levels, as defined by the newly recommended criteria (<5.6 mmol/L), were enrolled in this study. The blood pressures, body mass index (BMI), fasting blood glucose, fasting insulin, lipid batteries, and hsCRP levels were checked. The homeostatic model assessment-insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check indexes (QUICKI) were calculated. All subjects were subdivided into 4 groups according to the fasting glucose level. The HOMA-IR, QUICKI, and log-transformed (log) hsCRP, or log(hsCRP), level significantly increased according to the increment in fasting glucose, and these associations were consistent after adjustment for age and BMI, except for the log(hsCRP) (P = .124 after adjustment). Log(hsCRP) increased as the HOMA-IR increased and as the QUICKI decreased, and when multiple regression analysis was done with log(hsCRP) as the dependent variable, age, high BMI, male sex, high HOMA-IR, hypertriglyceridemia, and low high-density lipoprotein cholesterol were the significant predictor for log(hsCRP). In conclusion, the insulin resistance indexes and hsCRP increased gradually even in the normal fasting glucose range, as categorized by the newly recommended criteria for abnormal fasting glucose levels, supporting the rationale for expanding the range of fasting hyperglycemia.

The objective was to investigate circulating concentrations of bone formation markers (undercarboxylated osteocalcin [Glu-OC], an established marker of bone formation during fetal and early postnatal life], and Dickkopf-1 [DKK-1], a natural inhibitor of osteoblastogenesis during fetal development]) in intrauterine-growth-restricted (IUGR; associated with impaired fetal skeletal development) and appropriate-for-gestational-age (AGA) pregnancies. Circulating concentrations of Glu-OC and DKK-1 were determined by enzyme immunoassay in 40 mothers and their 20 asymmetric IUGR and 20 AGA singleton full-term fetuses and neonates on postnatal day 1 (N1) and 4 (N4). Parametric tests were applied in the statistical analysis. No significant differences in Glu-OC concentrations were observed between IUGR and AGA groups, whereas fetal DKK-1 concentrations were lower in the IUGR group (P = .028). In both groups, maternal Glu-OC and DKK-1 concentrations were lower than fetal, N1, and N4 concentrations (P ≤ .012 in all cases), whereas fetal Glu-OC concentrations were higher than N1 and N4 ones (P ≤ .037 in all cases). In addition, N1 Glu-OC concentrations were higher than N4 concentrations (P = .047). Finally, maternal Glu-OC and DKK-1 concentrations positively correlated with fetal, N1, and N4 ones (r ≥ 0.404, P ≤ .01 in all cases). Fetal/neonatal bone formation may not be impaired in full-term asymmetric IUGR infants, as indicated by the similar Glu-OC concentrations in both groups. Fetal DDK-1 concentrations are lower in the IUGR group, representing probably a compensatory mechanism, favoring the formation of mineralized bone. Fetal/neonatal bone turnover is markedly enhanced compared with maternal one and seems to be associated with the latter in both late pregnancy and early postpartum.

Agonists of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) are pharmacologically active antihyperglycemic agents that act by increasing peripheral tissue sensitivity to insulin. Many of these agonists have antihyperglycemic activity that is directly proportional to their ability to bind and activate PPAR gamma; however, recent data bring this relationship into question. In this report we describe a new PPAR gamma agonist, CLX-0921, that is derived from a natural product. This thiazolidinedione (TZD) has a spectrum of activity that differs from commercially available TZDs. It is a weak activator of PPAR gamma (EC(50) of 0.284 micromol/L) compared to rosiglitazone (EC(50) 0.009 micromol/L). Despite this difference, the drug maintains potent glucose uptake activity in vitro and glucose-lowering activity in vivo that is equipotent to that of rosiglitazone. Moreover, CLX-0921 showed a 10-fold reduction in in vitro adipogenic potential compared to rosiglitazone. CLX-0921 also increases glycogen synthesis, an activity not typically associated with rosiglitazone or pioglitazone. Thus CLX-0921 appears to have a distinct spectrum of activity relative to other TZDs.

Acute osteoporosis is known to occur after immobilization in spinal cord injured patients and is related to an early increase in osteoclastic bone resorption. Whether osteoporosis develops in healthy immobilized human patients is still a matter of controversy. Furthermore, acute osteoporosis was thought to be a good model to study the effects of weightlessness on the human skeleton and to adapt preventive procedures. A bed rest experiment was developed in the USSR on 15 healthy human volunteers to determine the precise effects on bone structure and cell activities. A preventive protocol, including an anti-osteoclastic drug (1-hydroxyethylidene-1,1 bisphosphonic acid; K salt) was investigated. Two transiliac bone biopsies were performed on the 15 individuals before and at the end of the 120-day bed rest period. Undecalcified bone biopsies were studied with automatic and semi-automatic image analyzers specially devoted to bone histomorphometry. Trabecular bone volume, osteoid amount, and eroded surfaces were measured. Osteoclast number was measured after histochemical identification of tartrate-resistant acid phosphatase. After the bed rest period, an insignificant bone loss was observed in healthy humans while osteoclast number was highly increased. In bisphosphonate-treated subjects, osteoclast number was markedly reduced and so was osteoid amount. Bisphosphonates were shown to present a highly cytotoxic activity on osteoclasts, a finding that has never been demonstrated in normal subjects.

Experiments in humans and rodents using oral doses of glycine and phenylalanine have suggested that the metabolism of these amino acids contributes to urinary oxalate excretion. To better define this contribution, we have examined the primed, constant infusion of [1-(13)C(1)] phenylalanine and [1,2-(13)C(2)] glycine in the postabsorptive state in healthy adults. Subjects were infused for 5 hours, hourly urines were collected, and blood was drawn every 30 minutes. Ion chromatography/mass spectrometry was used to measure [(13)C] enrichment in urinary oxalate, glycolate, and hippurate; and the enrichment of (13)C-amino acids in plasma samples was measured by gas chromatography/mass spectrometry. Following infusion with either 6 μmol/(kg h) [1-(13)C(1)] phenylalanine or 6 μmol/(kg h) [1,2-(13)C(2)] glycine, no isotopic glycolate or oxalate was detected in urine. Based on the limits of detection of our ion chromatography/mass spectroscopy method, these data indicate that less than 0.7% of the urinary oxalate could be derived from phenylalanine catabolism and less than 5% from glycine catabolism. Infusions with high levels of [1,2-(13)C(2)] glycine, 60 μmol/(kg h), increased mean plasma glycine by 29% and the whole-body flux of glycine by 72%. Under these conditions, glycine contributed 16.0% ± 1.6% and 16.6% ± 3.2% to urinary oxalate and glycolate excretion, respectively. Experiments using cultured hepatoma cells demonstrated that only at supraphysiological levels (>1 mmol/L) did glycine and phenylalanine metabolism increase oxalate synthesis. These data suggest that glycine and phenylalanine metabolism make only minor contributions to oxalate synthesis and urinary oxalate excretion.

In order to determine the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on bone matrix appositional rate (Ma AR) and bone mineral appositional rate (Mi AR), three doses (0.06, 0.13 and 0.20 microgram/kg/d) of 1,25(OH)2D3 were continuously infused for seven days in young mice. Histologic parameters of bone formation and resorption were evaluated by morphometric and autoradiographic methods. All doses of 1,25(OH)2D3 increased serum calcium and produced a dose-related increase in the metaphyseal osteoclastic surface and in the number of acid phosphatase-stained osteoclasts. The Mi AR evaluated by double tetracycline labeling was enhanced at all dosage levels. By contrast the Ma AR evaluated by double 3H-proline labeling was decreased at the two highest doses of 1,25(OH)2D3 which also produced growth impairment. We concluded that the continuous administration of 1,25(OH)2D3 in the mouse produces contrasting effects on bone matrix synthesis and calcification, resulting in a dose-related reduction in the amount of osteoid.

In diabetic animals, there is a decrease in serum 1,25-dihydroxyvitamin D [1,25(OH)2D] and in renal production of 1,25(OH)2D. In nondiabetic animals, renal 1,25(OH)2D production is markedly stimulated by parathyroid hormone (PTH) and calcitonin (CT). There is evidence that diabetes impairs the responsiveness of the kidney to PTH. The effect of diabetes on responsiveness to CT is unknown. The studies reported here determined the effect of streptozotocin-induced diabetes on renal responsiveness to PTH and CT. Experiments were performed in 7- to 8-week-old rats that were fed a diet sufficient in calcium and vitamin D and were thyroparathyroidectomized (TPTX) 5 days before hormone treatment. PTH (0.33 U/g body weight at 24, 12, and 2 hours before death) significantly increased renal 1,25(OH)2D production by threefold in nondiabetic rats. This effect was markedly attenuated by diabetes. On the other hand, CT (20 U/100 g body weight at 12 and 2 hours before death) produced a maximal response in both groups of animals. In diabetic rats, CT stimulated renal 1,25(OH)2D production fivefold, whereas PTH stimulated production only 1.5-fold. Diabetes did not affect the capacity of PTH to increase serum calcium or decrease renal tubular reabsorption of phosphorus (TRP). These findings suggest that the decrease in renal 1,25(OH)2D production seen in experimental diabetes may be due to decreased renal responsiveness to PTH, but not to decreased responsiveness to CT.

We examined ten cellular or tissue sources of lymphocytes for specific binding of 1,25(OH)2D3, the hormonally active form of vitamin D3. A specific-binding protein was found in three of these sources. Scatchard analysis of cytosol from a follicular lymphoma cell line revealed binding sites with a Kd of 7.0 X 10(-11) and a receptor concentration of 6.6 fmol/mg protein. Sucrose density centrifugation of 3H-1,25(OH)2D3 labeled cytosol showed a 3.75 peak which was absent in cytosols incubated with excess nonradioactive 1,25(OH)2D3. The relative amounts of vitamin D3 metabolites required to displace 50% of the specifically bound 3H-1,25(OH)2D3 were 1,25(OH)2D3: 1,24,25(OH)3D3: 25(OH)D3: 24,25(OH)2D3 = 1: 180: 1000: 2700. Excess vitamin D3, cortisol, and estradiol failed to displace 3H-1,25(OH)2D3. Scatchard analysis of spleen cytosol from a patient with prolymphocytic transformation of chronic lymphocytic leukemia demonstrated a binding protein with a Kd of 1.2 X 10(-10) and a receptor concentration of 0.2 fmol/mg protein. DNA cellulose binding confirmed the presence of the specific-binding protein in this cytosol. Specific binding of 3H-1,25(OH)2D3 was also quantitated in a cell line from a patient with Burkitt's lymphoma with a Kd of 0.3 X 10(-10) and a receptor concentration of 29.6 fmol/mg protein. No specific binding of 3H-1,25(OH)2D3 was observed in lymphocytes from seven other malignant and nonmalignant sources. These results are the first to demonstrate a specific-binding protein for 1,25(OH)2D3 in lymphocytes from tissue and from these specific cell lines. The presence of this protein in some lymphocytes but not others may reflect the state of activation of the lymphocytes.

Six patients with Paget disease of bone were treated with a 6-mo course of disodium-ethane-1-hydroxy-1, 1-diphosphate (EHDP) at a dosage of 5 mg/kg/day (one patient) or 20 mg/kg/day (five patients). In addition to symptomatic and biochemical improvement which persisted 3 mo after discontinuance of therapy, EHDP resulted in a significant increase in intestinal calcium absorption that was not correlated with a change in serum 1 alpha, 25-(OH)2-vitamin D concentration. EHDP appears to stimulate intestinal calcium transport by either a vitamin D independent process or by an increased intestinal mucosal cell sensitivity to vitamin D.

To clarify the mechanisms of hypocalcemia with renal insufficiency and to gain more insight into the mechanisms of secondary hyperparathyroidism in these patients, an 85-day study was conducted to examine the effect of dietary phosphate restriction on divalent ion metabolism in patients with early renal insufficiency. The study was conducted on four male patients with stable mild renal insufficiency who had creatinine clearances of 55 to 60 mL/min. Our results correspond with those of other studies that indicate that phosphate restriction is adequate to reverse and correct secondary hyperparathyroidism as well as other abnormalities in divalent ion metabolism. Because dietary phosphate restriction appears to exert its effect through the increased production of 1,25(OH)2D, an alternative therapeutic approach would be supplementation of 1,25(OH)2D3 (calcitriol). To test this, another study was conducted evaluating the effect of 1-year therapy with 1,25(OH)2D3 on blood levels of parathyroid hormone (PTH) and on various parameters of bone pathology in patients with creatinine clearances of 15 to 55 mL/min. Our results showed that the use of calcitriol is safe and effective in the management of secondary hyperparathyroidism and bone disease in patients with moderate renal failure.

In postmenopausal osteoporotics, malabsorption of calcium is associated with reduced levels of serum 1,25-dihydroxyvitamin D. Metabolic studies have shown that calcium absorption can be normalized and calcium balance improved after administration of oral doses of synthetic 1,25-dihydroxyvitamin D3 (Rocaltrol) 0.25 micrograms twice daily. Further studies performed at two centers compared the effect of Rocaltrol 0.25 micrograms twice daily versus placebo on vertebral fracture rates in osteoporotics. A significant reduction in vertebral fracture rates was seen at the end of 1 year. Those patients who continued on Rocaltrol for a second and third year showed a progressive decrease in vertebral fractures. Rocaltrol, administered at a dose of 0.25 micrograms twice daily, seldom causes hypercalcuria or hypercalcemia in osteoporotic patients on a typical calcium intake of 700 to 800 mg/d. Careful measurements of renal function over a period of 3 years in patients treated with Rocaltrol, 0.25 micrograms twice daily, showed no deterioration in renal function. These data suggest that 1,25-dihydroxyvitamin D3 is a useful therapy in the management of patients with postmenopausal osteoporosis, particularly those who have malabsorption of calcium. We found that it improves calcium balance, reduces the vertebral fracture rate, and is safe to use provided that the dietary calcium is monitored and does not exceed 800 mg/d.

The effects of 1alpha-OH D3 or 1,25-(OH)2D3 on calcium and phosphorus metabolism have been evaluated in five hypoparathyroid patients to establish the direct effects of these compounds in adult humans, uncomplicated by compensatory changes in parathyroid hormone secretion. Doses of 1-2.5 mug/day in four patients (5 mug/day in a fifth patient on diphenylhydantoin and phenobarbital) caused a marked increase in serum calcium concentration and urinary calcium excretion, without significant changes in renal calcium clearance or urinary hydroxyproline excretion. These results suggest that the correction of hypocalcemia involved primarily a stimulation of intestinal calcium absorption rather than a stimulation of skeletal calcium resorption. Simultaneously, there were increases in urinary phosphorus excretion and variable changes in serum inorganic phosphate concentration. These effects were produced by doses of 1alpha-OH D3 and 1,25-(OH)2D3 which approach the dose needed to prevent rickets, in contrast to the very large doses of vitamin D or 25-OH D3 required for comparable effects in hypoparathyroid patients. The increased relative effectiveness of these one-hydroxylated forms of vitamin D reveals a deficiency of vitamin D one-hydroxylation in hypoparathyroidism. The rapidity of action of 1alpha-OH D3 and 1,25-(OH)2D3 was also striking. Apart from its physiologic implications, the potency of the one-hydroxylated forms of vitamin D offers significant therapeutic advantages in some patients whose hypoparathyroidism is difficult to control with vitamin D itself.

Human colon carcinoma (HT-29) cells were examined for their capacity to bind and respond to 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. These cells are known to differentiate and increase their population doubling time when galactose is substituted for glucose in their media. High-affinity and specific binding of 1,25-(OH)2[3H]D3 was observed in extracts of these cells grown in glucose. The binder sedimented in sucrose gradients and eluted from DEAE-cellulose columns in a manner indistinguishable from rabbit intestinal 1,25-(OH)2D3-receptor. Smaller amounts of this binder were seen in HT-29 cells grown in galactose. Both glucose-fed and galactose-fed cells exhibited a dose-dependent decrease in growth rate on exposure to 10(-12) to 10(-6) mol/L 1,25-(OH)2D3. Ultrastructural examination of galactose-fed and glucose + 1,25-(OH)2D3-treated cells showed enterocytic differentiation and features that were not distinguishable between these groups. Sucrase activity was higher in galactose-fed cells and did not change with 1,25-(OH)2D3 treatment. However, the lower sucrase activity in glucose-fed cells increased after exposure to 10(-8) mol/L 1,25-(OH)2D3. These results indicate receptor content and bioresponsivity to 1,25-(OH)2D3 in a human enterocytic cell line, suggesting that it will be a useful model for the study of the mechanisms of action of this sterol.

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I alpha 2-procollagen, and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGF beta decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels, but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors.

To investigate the effect of dietary 1,3-diacylglycerol (DAG) on the development of insulin resistance (IR) and obesity, brown adipose tissue-deficient mice, a model of high-fat diet-induced IR and obesity, were fed Western-type diets (WTD) containing either DAG oil (n = 8) or standard triacylglycerol (TAG) oil (n = 9) for 15 weeks, beginning at 8 weeks of age. Although brown adipose tissue-deficient mice became obese on both TAG- and DAG-enriched WTD (TAG-WTD and DAG-WTD), the mice eating DAG-WTD gained less weight and had less body fat accumulation. The results of glucose tolerance tests conducted after 5 weeks of each WTD were not different. However, after 10 weeks of each WTD, impaired glucose tolerance developed in the TAG-WTD group but was prevented by DAG-WTD. Exploratory analyses of gene expression suggested that consumption of DAG-WTD was associated with reduced phosphoenolpyruvate carboxykinase gene expression in liver and increased expression of the genes for peroxisome proliferator-activated receptor alpha, lipoprotein lipase, and uncoupling proteins 2 and 3 in skeletal muscle. There were no effects of the DAG-WTD on fasting and postprandial plasma triglyceride (TG) levels, hepatic TG content, or the rate of secretion of TG from the liver. These findings suggest that diets enriched in 1,3-DAG oil may reduce WTD-induced IR and body fat accumulation by suppressing gluconeogenesis in liver and stimulating fat oxidation in skeletal muscle.

Diets containing 1,3 butanediol (BD) as a replacement of carbohydrate were fed to normal, diabetic, and diabetic insulintreated rats for two weeks. The diabetic animals fed BD survived longer than the diabetic animals on essentially the same diet without BD. The activities of malic enzyme in 105,000 × g. (1 hour) supernatant fractions of liver were determined. The PEPcK activity in liver was increased by 320 per cent in diabetic and by 240 per cent in diabetic rats fed the BD diet. Insulin decreased the PEPcK activity toward normal. In liver and adipose tissues malic enzyme activity was greatly decreased in diabetic and increased in diabetic insulin treated rats. The incorporation of radioactive bicarbonate into organic acids by liver mitochondrial pyruvate carboxylase and the formation of intermediates for gluconeogenesis were examined. The concentration of metabolites in liver was also examined and all changes were found to be minimal. Oral administration of BD greatly increased blood ketone levels and the ratio of β-hydroxybutyrate to acetoacetate. Normal animals fed BD had significantly lower blood glucose levels. Liver perfused with BD also showed decreased glucose production from lactate. Analysis of metabolites from livers perfused with BD showed a large increase in the lactate to pyruvate ratios (from 13.1 to 81.0). Malate and aspartate were increased, whereas pyruvate, PEP, 2 PGA, and 3 PGA were decreased. It is concluded that BD exerts its hypoglycemic effect at the conversion of malate to oxalacetate and hence to PEP.

The effects of 2-ethylamino-1,3,4-thiadiazole have been assessed in two patients with a disorder of uric acid metabolism and cerebral function. Patients with this inborn error of metabolism have marked overproduction of purine, and absent activity of hypoxanthine, guanine phosphoribosyl transferase. It was found that this uricogenic thiadiazole increased further, the elevated concentrations of uric acid in blood and urine. Clinical reactions were dramatic, required vigorous therapy, and suggested that the investigation of this compound should not be undertaken lightly in patients with this disease. There was an increase in the cumulative conversion of 14C-labeled glycine into urinary uric acid. These observations indicate that the increased de novo purine synthesis of this condition is capable of further increase. They suggest the possibility that the overproduction of purine found in the disease and that which follows thiadiazoles, are both related to the role of guanine nucleotide in the feedback regulation of purine synthesis.