Length of lags in responses of milk yield and somatic cell score on test day to heat stress in Holsteins.

We used daily records from provincial Japanese weather stations and monthly test-day records of milk production to investigate the length of the lags in the responses of cows' milk yield and somatic cell score (SCS) to heat stress (HS). We also investigated the HS thresholds in milk yield and SCS. Data were a total of 17,245,709 test-day records for milk and SCS in Holstein cows that had calved for the first time between 2000 and 2015, along with weather records from 60 weather stations. Temperature-humidity index (THI) values were estimated by using average daily temperature and average daily relative humidity. Adjusted THI values were calculated by using temperature, relative humidity, wind speed, and solar radiation. The model contained herd, calving year, month of test day, age group, days in milk, and THI as a fixed effect. THIs for each day from 14 days before the test day until the test day were used to represent the HS effects. The HS occurring 3 days, and between 8 and 10 days, before the test day had the greatest effect on the milk yield and SCS, respectively. The threshold THI values for the HS effect were about 60-65 for both traits.

Identification of Adenine and Benzimidazole Nucleosides as Potent Human Concentrative Nucleoside Transporter 2 Inhibitors: Potential Treatment for Hyperuricemia and Gout.

To test the hypothesis that inhibitors of human concentrative nucleoside transporter 2 (hCNT2) suppress increases in serum urate levels derived from dietary purines, we previously identified adenosine derivative 1 as a potent hCNT2 inhibitor (IC50 = 0.64 µM), but further study was hampered due to its poor solubility. Here we describe the results of subsequent research to identify more soluble and more potent hCNT2 inhibitors, leading to the discovery of the benzimidazole nucleoside 22, which is the most potent hCNT2 inhibitor (IC50 = 0.062 µM) reported to date. Compound 22 significantly suppressed the increase in plasma uric acid levels after oral administration of purine nucleosides in rats. Because compound 22 was poorly absorbed orally in rats (F = 0.51%), its pharmacologic action was mostly limited to the gastrointestinal tract. These findings suggest that inhibition of hCNT2 in the gastrointestinal tract can be a promising approach for the treatment of hyperuricemia.

Design, synthesis, and structure-activity relationships of a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl ß-D-glycopyranosides substituted with novel hydrophilic groups as highly potent inhibitors of sodium glucose co-transporter 1 (SGLT1).

Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of therapeutic options for postprandial hyperglycemia. Previously, we reported potent and selective SGLT1 inhibitors 1 and 2 showing efficacy in oral carbohydrate tolerance tests in diabetic rat models. In a pharmacokinetic (PK) study of 2, excessive systemic exposure to metabolites of 2 was observed, presumably due to the high permeability of its aglycone (2a). To further improve SGLT1 inhibitory activity and reduce aglycone permeability, a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl ß-D-glycopyranoside derivatives bearing novel hydrophilic substitution groups on the phenyl ring were synthesized and their inhibitory activity toward SGLTs was evaluated. Optimized compound 14c showed an improved profile satisfying both higher activity and lower permeability of its aglycone (22f) compared with initial leads 1 and 2. Moreover, the superior efficacy of 14c in various carbohydrate tolerance tests in diabetic rat models was confirmed compared with acarbose, an α-glucosidase inhibitor (α-GI) widely used in the clinic.

Structure-activity relationship studies of 4-benzyl-1H-pyrazol-3-yl ß-d-glucopyranoside derivatives as potent and selective sodium glucose co-transporter 1 (SGLT1) inhibitors with therapeutic activity on postprandial hyperglycemia.

Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of treatments for postprandial hyperglycemia. A series of 4-benzyl-1H-pyrazol-3-yl ß-d-glucopyranoside derivatives have been synthesized, and its inhibitory activity toward SGLTs has been evaluated. By altering the substitution groups at the 5-position of the pyrazole ring, and every position of the phenyl ring, we studied the structure-activity relationship (SAR) profiles and identified a series of potent and selective SGLT1 inhibitors. Representative derivatives showed a dose-dependent suppressing effect on the escalation of blood glucose levels in oral mixed carbohydrate tolerance tests (OCTT) in streptozotocin-nicotinamide-induced diabetic rats (NA-STZ rats).

Hypouricemic effects of novel concentrative nucleoside transporter 2 inhibitors through suppressing intestinal absorption of purine nucleosides.

We have developed concentrative nucleoside transporter 2 (CNT2) inhibitors as a novel pharmacological approach for improving hyperuricemia by inhibiting intestinal absorption of purines. Dietary purine nucleosides are absorbed in the small intestines by CNTs expressed in the apical membrane. In humans, the absorbed purine nucleosides are rapidly degraded to their final end product, uric acid, by xanthine oxidase. Based on the expression profile of human CNTs in digestive tract tissues, we established a working hypothesis that mainly CNT2 contributes to the intestinal absorption of purine nucleosides. In order to confirm this possibility, we developed CNT2 inhibitors and found that (2R,3R,4S,5R)-2-(6-amino-8-{[3'-(3-aminopropoxy)-biphenyl-4-ylmethyl]-amino}-9H-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (KGO-2142) and 1-[3-(5-{[1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-benzimidazol-2-ylamino]-methyl}-2-ethoxyphenoxy)-propyl]-piperidine-4-carboxylic acid amide (KGO-2173) were inhibitory. These CNT2 inhibitors had potent inhibitory activity against inosine uptake via human CNT2, but they did not potently interfere with nucleoside uptake via human CNT1, CNT3 or equilibrative nucleoside transporters (ENTs) in vitro. After oral administration of KGO-2173 along with [(14)C]-inosine, KGO-2173 significantly decreased the urinary excretion of radioactivity at 6 and 24h in rats. Since dietary purine nucleosides are not utilized in the body and are excreted into the urine rapidly, this decrease in radioactivity in the urine represented the inhibitory activity of KGO-2173 toward the absorption of [(14)C]-inosine in the small intestines. KGO-2142 almost completely inhibited dietary RNA-induced hyperuricemia and the increase in urinary excretion of uric acid in cebus monkeys. These novel CNT2 inhibitors, KGO-2142 and KGO-2173, could be useful therapeutic options for the treatment of hyperuricemia.

KGA-2727, a novel selective inhibitor of a high-affinity sodium glucose cotransporter (SGLT1), exhibits antidiabetic efficacy in rodent models.

The high-affinity sodium glucose cotransporter (SGLT1) plays a critical role in glucose absorption from the gastrointestinal tract. We have developed 3-(3-{4-[3-(ß-D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethyl]-3-methylphenoxy}propylamino)propionamide (KGA-2727), which has a pyrazole-O-glucoside structure, as the first selective SGLT1 inhibitor. KGA-2727 inhibited SGLT1 potently and highly selectively in an in vitro assay using cells transiently expressing recombinant SGLTs. In a small intestine closed loop absorption test with normal rats, KGA-2727 inhibited the absorption of glucose but not that of fructose. After oral intake of starch along with KGA-2727 in normal rats, the residual content of glucose in the gastrointestinal tract increased. In the oral glucose tolerance test with streptozotocin-induced diabetic rats, KGA-2727 attenuated the elevation of plasma glucose after glucose loading, indicating that KGA-2727 improved postprandial hyperglycemia. In Zucker diabetic fatty (ZDF) rats, chronic treatments with KGA-2727 reduced the levels of plasma glucose and glycated hemoglobin. Furthermore, KGA-2727 preserved glucose-stimulated insulin secretion and reduced urinary glucose excretion with improved morphological changes of pancreatic islets and renal distal tubules in ZDF rats. In addition, the chronic treatment with KGA-2727 increased the level of glucagon-like peptide-1 in the portal vein. Taken together, our data indicate that the selective SGLT1 inhibitor KGA-2727 had antidiabetic efficacy and allow us to propose KGA-2727 as a candidate for a novel and useful antidiabetic agent.

Effects of amino acids infused into the vein on ghrelin-induced GH, insulin and glucagon secretion in lactating cows.

To investigate the effects of amino acids on ghrelin-induced growth hormone (GH), insulin and glucagon secretion in lactating dairy cattle, six Holstein cows were randomly assigned to two infusion treatments in a cross-over design. Mixture solution of amino acids (AMI) or saline (CON) was continuously infused into the left side jugular vein via catheter for 4 h. At 2 h after the start of infusion, synthetic bovine ghrelin was single injected into the right side jugular vein through the catheter. Ghrelin injection immediately increased plasma GH, glucose and non-esterified fatty acids (P<0.05) with no difference between both treatments. Additionally, plasma insulin and glucagon concentrations were increased by ghrelin injection in both treatments. The peak value of plasma insulin concentration was greater in AMI compared with CON (P<0.05). Plasma glucagon concentration showed no difference in the peak value reached at 5 min between both treatments, and then the plasma levels in AMI compared with CON showed sustained higher values (P<0.05). After plasma glucose concentration reached the peak, the decline was greater in AMI compared with CON (P<0.05). These results showed that the increased plasma amino acids may enhance ghrelin action which in turn enhances insulin and glucagon secretions in lactating cows.

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway.

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

Influence of orally administered bovine lactoferrin on lipid metabolism in lipopolysaccharide-injected preruminant calves.

ABSTRACT The aim of this study was to investigate the influence of oral lactoferrin (LF) administration on lipid metabolism changes in calves given lipopolysaccharide (LPS). Twenty-one 4-day-old Holstein calves were divided into three groups, with each group receiving one of three oral doses of LF (0, 1, 3 g/day) for 10 consecutive days (day -10 to day -1). All calves were intravenously injected with LPS (50 ng/kg BW) on day 0, the day after LF treatment ended. Plasma triglyceride concentrations were lower (P < 0.05) in the LF-treated calves than in the control calves given 0 g/day of LF at 12 and 24 h after LPS injection. Plasma NEFA concentrations were elevated between 6 and 24 h after LPS treatment. At 12 h, the concentration of plasma NEFA was lower (P < 0.05) in the calves given LF 3 g/day than in the control calves. On day 0, plasma total cholesterol and phospholipid concentrations tended to be lower in the LF groups administered 1 and 3 g of LF/day than in the control group, but did not differ significantly among the groups. The plasma very-low-density and low-density lipoprotein concentrations were lower (P < 0.05) at 12, 24, and 72 h in the LF groups than in the control calves. The concentrations of plasma high-density lipoprotein tended to be lower in the LF groups than in the control group between day 0 and 96 h, though there were no significant group differences. The concentration of plasma interleukin-1beta was lower (P < 0.05) in the calves fed LF 3 g/day than in the control calves at 2 and 12-48 h after LPS injection. These data suggest that LF inhibits LPS-induced alterations in lipid metabolism in preruminant calves.

Sergliflozin, a novel selective inhibitor of low-affinity sodium glucose cotransporter (SGLT2), validates the critical role of SGLT2 in renal glucose reabsorption and modulates plasma glucose level.

The low-affinity sodium glucose cotransporter (SGLT2), which is expressed specifically in the kidney, plays a major role in renal glucose reabsorption in the proximal tubule. We have discovered sergliflozin, a prodrug of a novel selective SGLT2 inhibitor, based on benzylphenol glucoside. In structure, it belongs to a new category of SGLT2 inhibitors and its skeleton differs from that of phlorizin, a nonselective SGLT inhibitor. We investigated its pharmacological properties and potencies in vitro and in vivo. By examining a Chinese hamster ovary-K1 cell line stably expressing either human SGLT2 or human high-affinity sodium glucose cotransporter (SGLT1), we found sergliflozin-A (active form) to be a highly selective and potent inhibitor of human SGLT2. At pharmacological doses, sergliflozin, sergliflozin-A, and its aglycon had no effects on facilitative glucose transporter 1 activity, which was inhibited by phloretin (the aglycon of phlorizin). The transport maximum for glucose in the kidney was reduced by sergliflozin-A in normal rats. As a result of this effect, orally administered sergliflozin increased urinary glucose excretion in mice, rats, and dogs in a dose-dependent manner. In an oral glucose tolerance test in diabetic rats, sergliflozin exhibited glucose-lowering effects independently of insulin secretion. Any glucose excretion induced by sergliflozin did not affect normoglycemia or electrolyte balance. These data indicate that selective inhibition of SGLT2 increases urinary glucose excretion by inhibiting renal glucose reabsorption. As a representative of a new category of antidiabetic drugs, sergliflozin may provide a new and unique approach to the treatment of diabetes mellitus.

Effects of ghrelin injection on plasma concentrations of glucose, pancreatic hormones and cortisol in Holstein dairy cattle.

Ghrelin affects not only growth hormone secretion but also nutrient utilization and metabolic hormone secretion in humans and experimental animals. The effects of ghrelin on plasma metabolic hormone and metabolite levels in domestic herbivores remain unclear despite the fact that the physiological characteristics of nutrient digestion and absorption imply specific responses to ghrelin. Therefore, the effects of ghrelin on plasma glucose, pancreatic hormones and cortisol concentrations were investigated in Holstein dairy cattle in various physiological states. Ghrelin (0.3 nmol/kg) or placebo (2% bovine serum albumin in saline) was intravenously injected in pre-ruminant calves (pre-rumen function), adult non-lactating (functional rumen) and lactating cows (functional rumen and lactation), and plasma glucose, insulin, glucagon and cortisol concentrations were then determined. Ghrelin injection increased plasma glucose concentrations in adult cows, especially in lactating cows. No hyperglycemic response was observed in pre-ruminant calves. A transient rise of insulin and glucagon levels was distinctively found in lactating cows in response to the ghrelin administration. Ghrelin injection decreased the insulin level in pre-ruminant calves. Ghrelin increased cortisol secretion independently of the physiological state. The results of the present study suggest that the effects of ghrelin on plasma glucose and pancreatic hormone levels may reflect differences in the physiological states of dairy cattle.

Rat model of the hypercalcaemia induced by parathyroid hormone-related protein: characteristics of three bisphosphonates.

In our preliminary experiment, we found that a constant infusion of a high dose of parathyroid hormone-related protein induced both hyperphosphataemia and hypocalcaemia, secondary to renal dysfunction. Therefore, in this study, we developed two types of parathyroid hormone-related protein-induced hypercalcaemia models. One is the hypercalcaemia model, which did not show renal-dysfunction-induced hypocalcaemia. This model might be suitable for estimating hypocalcaemic activities of drugs, especially of those that act on bone resorption. The other is the model for estimating histological changes, which is associated with renal dysfunction. We then used these models to investigate the effects of three different bisphosphonates. Since the hypercalcaemic effect of parathyroid hormone-related protein infusion plateaued at 20 pmol/h, and higher doses of parathyroid hormone-related protein caused an elevation of blood urea nitrogen, the parathyroid hormone-related protein infusion rate was fixed at 20 pmol/h to avoid renal dysfunction and at 40 pmol/h to elicit renal dysfunction. The hypocalcaemic efficiencies of clodronate and etidronate were almost the same but pamidronate was 17.9 times more potent than clodronate. Additionally, both clodronate and pamidronate decreased the plasma concentrations of blood urea nitrogen and the Ca2+ times inorganic P product, whereas etidronate lacked these effects. Clodronate suppressed renal calcification and tubular dilatation in the renal-dysfunction model. These data indicated that clodronate and pamidronate not only decrease the plasma Ca2+ concentration but also improve the renal dysfunction induced by hypercalcaemia.

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose.

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) >> D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).

Effects of clodronate and alendronate on local and systemic changes in bone metabolism in rats with adjuvant arthritis.

We compared the effects of nonaminobisphosphonate (nonamino-BP; clodronate) and amino-BP (alendronate) on the changes in local and systemic bone metabolism associated with chronic inflammation in adjuvant-arthritis rats. A given rat received one of the BPs orally each day for 28 days from the day of adjuvant inoculation. Hindpaw swelling was observed from day 10 after adjuvant inoculation up to day 28 (peak, day 21). Clodronate slightly decreased the hindpaw swelling at doses of 12.5 and 25 mg/kg, p.o./day; however, alendronate (0.125-0.5 mg/kg) did not. Radiological examination of the distal limb joints revealed that only clodronate decreased bone deformation. Urinary deoxypyridinoline increased as arthritis developed, and it was decreased by clodronate. On day 29, pQCT analysis of the 5th lumbar vertebra revealed trabecular bone loss and cortical bone thinning in the arthritis control group, leading to compressive strength being reduced. Both BPs prevented this bone loss and strength reduction. These data suggest that only clodronate decreases inflammation and local bone deformation, while both BPs inhibit the arthritis-related decreases in systemic bone mass and bone strength. Clodronate would be useful in the treatment of inflammation-induced bone deformation and osteopenia.

Further evidence for the role of glucose as a metabolic regulator of hypothalamic gonadotropin-releasing hormone pulse generator activity in goats.

The present study examined the relative importance of blood glucose vs. free fatty acids as a metabolic signal regulating GnRH release as measured electrophysiologically by multiple-unit activity (MUA) in the arcuate nucleus/median eminence region in ovariectomized, estradiol-treated goats. MUA was recorded before, during, and after: 1) cellular glucoprivation by peripheral infusion of 2-deoxy-d-glucose (2DG; 25, 50, and 75 mg/kg.h, iv); 2) peripheral hypoglycemia in response to various doses (15-195 mU/kg.h, iv) of insulin infusion; and 3) cellular lipoprivation induced by peripheral infusion of sodium mercaptoacetate (MA; 2.4 mg/kg.h alone or combined with 25 mg/kg.h of 2DG, iv), and effects on the interval of characteristic increases in MUA (MUA volleys) were examined. Infusion of the highest dose of 2DG increased the mean interval between MUA volleys, whereas the lower doses of 2DG had no effect on volley interval. The MUA volley intervals lengthened as insulin-induced hypoglycemia became profound. There was a negative correlation between MUA volley intervals and blood glucose concentrations during insulin infusion, and coinfusion of glucose with insulin returned the MUA volley interval to a normal frequency. Infusion of MA alone or MA with 2DG did not increase MUA volley intervals. These findings demonstrate that glucose availability, but not fatty acids, regulates the GnRH pulse generator activity in the ruminant. Glucose is considered a key metabolic regulator that fine-tunes pulsatile GnRH release.

Molecular cloning and expression of HRLRRP, a novel heart-restricted leucine-rich repeat protein.

We isolated a novel leucine-rich repeat protein (LRRP) cDNA from E13 mouse embryos by the in silico approach. The cDNA encoded a protein of 274 amino acids having 7 leucine-rich repeat motifs at the center of the protein. An in vitro transcription/translation study showed that the cDNA coded for a peptide of approximately 31kDa. Northern blot analysis suggested that the mRNA of this novel LRRP was expressed only in the heart, although RT-PCR indicated slight expression in skeletal muscle as well. The transcripts of this gene and Nkx-2.5/Csx were detected in the early stage of cardiac differentiation of P19CL6 embryonal carcinoma cells treated with 1% dimethyl sulfoxide. The fusion protein made between it and GFP was detected at a high level in mitochondria and a low level in the nuclei of COS7 cells. The nuclei of the adult mouse heart were strongly stained with the antibody raised against the synthetic peptide of the protein. Therefore, we designated the gene as heart-restricted leucine-rich repeat protein (HRLRRP) and assume that mouse HRLRRP may play important roles in cardiac development and/or cardiac function.

Gene expression of resistin in adipose tissue and mammary gland of lactating and non-lactating cows.

Resistin has been suggested to induce insulin resistance in obesity and to inhibit adipocyte differentiation. In lactating cows, glucose uptake in the mammary gland is a rate-limiting step in milk synthesis, and to supply glucose to the mammary gland, insulin resistance increases. We examined the expression of the resistin gene by real-time PCR of cDNA in the adipose tIssue and mammary gland of lactating and non-lactating cows. Lactation induced a significant increase of resistin expression in adipose tIssue compared with that in the dry period, and decreased resistin expression in the mammary gland. There were no significant differences in the expression of insulin responsive glucose transporter (GLUT4) mRNA between the adipose tIssue of lactating and non-lactating cows, and GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was lower in lactating cows than in non-lactating cows. These results indicate that the pattern of resistin expression in peripheral tIssues is changed in association with milk production. The increase of resistin expression and maintenance of a lower level of plasma insulin concentration may decrease glucose availability by increasing insulin resistance in adipose tIssue. Additionally, our results suggest that the decrease of resistin expression in the mammary gland may influence on the insulin-dependent glucose uptake in mammary epithelial cells during lactation.

Clodronate stimulates osteoblast differentiation in ST2 and MC3T3-E1 cells and rat organ cultures.

We investigated the direct effects of various bisphosphonates on osteoblasts. At 10(-5) M, clodronate increased alkaline phosphatase activity in cultured MC3T3-E1 (osteoblast-like line) and ST2 (pluripotent mesenchymal line) cells. Etidronate significantly increased alkaline phosphatase activity at 10(-5) M only in MC3T3-E1 cells. These effects were due to an increase in alkaline phosphatase-positive cell numbers, and the differentiation-enhanced cells were capable of mineralization (von Kossa stain). Other bisphosphonates (pamidronate, alendronate, and incadronate) did not increase alkaline phosphatase activity in either cell line. In cultured rat calvariae, clodronate stimulated the expression of genes for alkaline phosphatase and osteocalcin (osteoblast-differentiation markers), but decreased the expression of the gene for tartrate-resistant acid phosphatase (osteoclast marker). Clodronate, etidronate, and incadronate inhibited protein Tyr phosphatase and Ser/Thr phosphatase activities in MC3T3-E1 cells. These data suggest that clodronate acts directly on mesenchymal cells to enhance osteoblast differentiation, and this effect may be partly expressed through inhibition of protein Tyr phosphatase and/or Ser/Thr phosphatase activity.

Bovine apolipoprotein E in plasma: increase of ApoE concentration induced by fasting and distribution in lipoprotein fractions.

Apolipoprotein E (apoE) is a protein constituent of lipoproteins, and acts as a receptor-binding ligand. Although the existence of bovine apoE in lipoprotein fractions has already been reported, quantitative studies on the changes of apoE in plasma and lipoprotein fractions are lacking. In the present study, an increase of a 38 kDa protein in the very low-density lipoprotein (VLDL) fraction obtained from fasted calves was detected. This 38 kDa protein was identified as bovine apoE by determination of the N-terminal amino acid sequence. Bovine apoE was purified and an enzyme-linked immunosorbent assay (ELISA) was developed. Using this system, the effect of fasting on the concentration of apoE in plasma and the distribution of apoE in lipoprotein fractions were investigated. After 3 days of fasting, the concentration of plasma apoE increased significantly (p<0.05) by 280 %, and was returned to the basal level by 3 days of refeeding. The lipoprotein fractions obtained from before and after fasting was separated by ultracentrifugation. ApoE was significantly increased in VLDL, low-density lipoprotein (LDL) and non-lipoprotein fractions by fasting (p<0.05). On the other hand, in high-density lipoprotein (HDL) fractions obtained from both before and after fasting, the level of apoE was very low compared to the other fractions. These results suggested that bovine apoE contents in triglyceride-rich lipoproteins are modulated by nutritional treatment and closely associated with triglyceride-rich lipoprotein metabolism.

Reductions in bone mass, structure, and strength in axial and appendicular skeletons associated with increased turnover after ovariectomy in mature cynomolgus monkeys and preventive effects of clodronate.

Over 16 months, we evaluated the effects of ovariectomy (OVX) and bisphosphonate clodronate (CLO) on bone in 48 cynomolgus monkeys (9-15 years old) fed a normal calcium diet. We established three OVX groups (oral CLO at 0 [OVX control], 12, or 60 mg/kg per day) and one sham-operated (SHAM) group. At 16 months, the bone mineral density (BMD) values (percentage of group baseline; OVX control vs. SHAM) for lumbar bone (L3-L5), proximal femur, midfemur, radius, and tibia were -2.6% versus 11.2%, -3.5% versus 8.9%, -3.0% versus 9.0%, -5.5% versus 15.7%, and -6.7% versus 13.9%, respectively. In OVX control (i) tibia showed significant loss of bone mineral content (BMC; vs. baseline), (ii) urinary deoxypyridinoline (DPD) and serum osteocalcin (OC) levels increased (peak = 182% and 168%, respectively, of SHAM), (iii) in lumbar bone and midfemur, ultimate load (UL) was reduced (vs. SHAM), (iv) in lumbar bone, trabecular bone-formation rates (BFRs) were not changed significantly, but tibial endocortical and intracortical bone formation rates were significantly raised (vs. SHAM), (v) the volumetric BMD (vBMD) and geometry of the tibial cortex (measured by peripheral quantitative computed tomography [pQCT]) were significantly reduced (vs. SHAM). CLO, 60 mg/kg per day but not 12 mg/kg per day, significantly inhibited OVX-induced changes, age-dependent increases in bone mass, and ability to maintain structure. Thus, in OVX mature cynomolgus monkeys (possibly, a unique model of the cortical bone loss secondary to estrogen deficiency), the post-OVX increases in systemic bone markers were slight, but stimulation of local turnover in the cortical envelope was enough to cause bone loss (more so in tibia than in lumbar trabecular bone). High-dose CLO prevented these changes.