Effects of eldecalcitol on bone and skeletal muscles in glucocorticoid-treated rats

Glucocorticoids cause secondary osteoporosis and myopathy, characterized by type II muscle fiber atrophy. We examined whether a new vitamin D₃ analogue, eldecalcitol, could inhibit glucocorticoid-induced osteopenia or myopathy in rats, and also determined the effects of prednisolone (PSL) and/or eldecalcitol on muscle-related gene expression. Six-month-old female Wistar rats were randomized into four groups: PSL group (10 mg/kg PSL); E group (0.05 µg/kg eldecalcitol); PSL + E group; and control group. PSL, eldecalcitol, and vehicles were administered daily for 2 or 4 weeks. Right calf muscle strength, muscle fatigue, cross-sectional areas (CSAs) of left tibialis anterior muscle fibers, and bone mineral density (BMD) were measured following administration. Pax7, MyoD, and myogenin mRNA levels in gastrocnemius muscles were also determined. Muscle strength was significantly higher in the PSL + E group than in the PSL group (p < 0.05) after 4 weeks, but not after 2 weeks. No significant difference in muscle fatigue was seen between groups at 2 or 4 weeks. CSAs of type II muscle fibers were significantly larger in the E group and the PSL + E group than in the PSL group at 4 weeks (p = 0.0093, p = 0.0443, respectively). Eldecalcitol treatment for 4 weeks maintained the same BMD as the PSL + E group. After 2 weeks, but not 4 weeks, eldecalcitol treatment significantly increased Pax7 and myogenin mRNA expression in gastrocnemius muscle, and PSL also stimulated myogenin expression. Eldecalcitol appears to increase muscle volume and to protect against femur BMD loss in PSL-administered rats, and it may also stimulate myoblast differentiation into early myotubes.     

Different localizations of drugs simultaneously administered in a strand of hair by micro‐segmental analysis

Segmental hair analysis is used to estimate the time of drug intake at monthly precision in drug‐related crimes. Previously, we advanced this analytical method to specify the day of drug intake by cutting a strand of hair into 0.4‐mm segments, which correspond to daily hair growth. Herein, we investigated the distributions of 7 compounds in a strand of hair using micro‐segmental analysis. Several strands of hair were collected 33.1?229.4 days after subjects were administered 4 pharmaceutical products that contained 10 drugs in single doses within 32 hours. The administered drugs and resulting metabolites were extracted from 0.4‐mm hair segments and quantified using liquid chromatography–tandem mass spectrometry. Acidic and neutral compounds were detected at low amounts in any of the hair segments analyzed. Epinastine, fexofenadine, dihydrocodeine, chlorpheniramine, and the chlorpheniramine metabolite, desmethylchlorpheniramine each was localized to 2 regions within a strand of hair. By contrast, methylephedrine and its metabolite, ephedrine, each was localized to only a region. Among 20 individual strands of hair associated with different subjects and head regions, few differences in the shapes of drug concentration–hair segment curves for each compound were detected. Our data indicated that 2 mechanisms for drug uptake into hair can operate depending on drug properties and that co‐administered drugs can be localized to different regions in a strand of hair. Micro‐segmental analysis may aid in the identification of the day of drug intake and help to elucidate the mechanisms of drug uptake into hair.     

Hepatectomy-Related Hypophosphatemia: A Novel Phosphaturic Factor in the Liver-Kidney Axis

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na⁺-dependent phosphate (Pi) uptake decreased by 50%–60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na⁺-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.Inorganic phosphate (Pi) absorption in the renal proximal tubules and small intestine is important for Pi homeostasis.¹ The Na⁺-dependent Pi (Na/Pi) transport system includes type IIa and type IIc Na/Pi transporters, which are localized in the apical membrane of the proximal tubular cells, and type IIb Na/Pi transporters, which are localized in the apical membrane of the intestinal epithelial cells.^1,2 Pi (re)absorption is regulated by the dietary Pi content, parathyroid hormone (PTH), and the active metabolite of vitamin D, 1α, 25-dihydroxyvitamin D3 [1,25(OH)₂D₃].³ Other phosphaturic hormones, termed phosphatonins, also control renal Pi handling.⁴ The discovery that fibroblast growth factor (FGF) 23, the first identified phosphatonin,⁵ originated from osteocytes established the concept of the bone-kidney axis.^6,7The incidence of liver transplantation has steadily increased and the incidence of partial hepatectomy (PH) has also consequently increased.⁸ Hypophosphatemia frequently occurs after liver resection.^9–11 Acute hypophosphatemia causes septicemia and is associated with a poor prognosis.^11,12 Acute hypophosphatemia is of considerable clinical relevance because many hepatectomized patients develop marked hypophosphatemia and, thus, large doses of Pi replacement are required to maintain metabolic homeostasis.¹³ Urinary Pi excretion is markedly increased in many patients. After hepatectomy, hypophosphatemia is associated with hyperphosphaturia.¹³For many years, the increased metabolic demand of the regenerating liver was considered the underlying pathologic mechanism of hypophosphatemia. The magnitude of Pi uptake by the recovering liver, however, cannot explain the severity of the resulting hypophosphatemia.¹¹ Hepatectomy-induced hypophosphatemia is associated with an increased renal fractional excretion index for Pi unrelated to intact FGF23, FGF7, or secreted frizzled-related protein 4 as a phosphaturic factor,¹⁴ indicating that other factors have a role in the pathogenesis of hypophosphatemia.Nicotinamide (NAM) inhibits intestinal and renal Na/Pi transport activity in normal rats.^15–17 Administration of NAM to rats produces a specific dose-dependent inhibition of Na/Pi transport across the renal brush-border membrane (BBM) and an increase in urinary Pi excretion.^16,17 NAM suppresses hyperphosphatemia in hemodialysis patients.¹⁸ Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first rate-limiting step in converting NAM to NAD,^19,20 which is essential for cellular metabolism, energy production, and DNA repair.^20–22 Nampt exists in two known forms: intracellular Nampt (iNampt) and secreted extracellular Nampt (eNampt).²³ eNampt also generates an intermediate product, nicotinamide mononucleotide (NMN).²³Our findings indicate that the acceleration of NAM metabolism through Nampt function in the kidney is involved in the hepatectomy-induced hypophosphatemia in rodent models. This study also suggests that NAM metabolism through the liver-kidney axis is important in Pi homeostasis.     

Endoscopic ultrasound elastography for small solid pancreatic lesions with or without main pancreatic duct dilatation

Background/Objectives: Endoscopic ultrasound elastography (EUS-EG) is useful for diagnosis of small solid pancreatic lesions (SPLs), particularly in excluding pancreatic cancer (PC), but its dependence on main pancreatic duct dilatation (MPDD) has not been examined. We aimed to investigate EUS-EG for diagnosis of small SPLs with and without MPDD.MethodsPatients with pathologically diagnosed SPLs of ≤20 mm were included and retrospectively analyzed. Using the blue:green ratio, an EUS-EG image was classified as blue-dominant, equivalent, or green-dominant. Using multiple EUS-EG images per patient, a lesion with a greater number of blue-dominant than green-dominant images was classified as stiff, and the others as soft. EUS-EG images in random order were judged by three raters. Considering stiff SPLs as PC, diagnostic performance of EUS-EG was examined for SPLs with and without MPDD.ResultsOf 126 cases analyzed, 65 (52%) were diagnosed as PC, and 63 (50%) had MPDD. A total of 1077 EUS-EG images were examined (kappa coefficient = 0.783). Lesions were classified as stiff in 91 cases and soft in 35 (kappa coefficient = 0.932). The ratio of stiff to soft lesions was significantly higher in PC than in non-PC (62:3 vs. 29:32, P < 0.001). The sensitivity, specificity, and negative predictive value of a stiff lesion with vs. without MPDD for diagnosis of PC were 94%, 23%, and 50% vs. 100%, 60%, and 100%, respectively.ConclusionsUsing the EUS-EG stiffness classification for small SPLs, PC can be excluded with high confidence and concordance for a soft lesion without MPDD.     

An initial trial of quantitative evaluation of autoimmune pancreatitis using shear wave elastography and shear wave dispersion in transabdominal ultrasound

Background/ObjectivesWe aimed to examine therapeutic efficacy and prognosis prediction of autoimmune pancreatitis (AIP) using shear wave elastography (SWE) and shear wave dispersion (SWD) in transabdominal ultrasound (US).MethodsThe subjects were 23 patients with diffuse type 1 AIP who underwent SWE and SWD, and 34 controls with a normal pancreas. Elasticity and dispersion were defined as the pancreatic elastic modulus (PEM) and dispersion slope, respectively. PEM and dispersion slope were compared between AIP and control cases, and the short-term therapeutic effect and long-term prognosis were examined.ResultsPEM (30.9 vs. 6.6 kPa, P < 0.001) and dispersion slope (15.3 vs. 13.0 (m/sec)/kHz, P = 0.011) were significantly higher in AIP cases than in controls. Among the 17 AIP patients followed-up in two weeks after treatment, these parameters were 12.7 kPa and 10.5 (m/sec)/kHz with median decrease rate of 37.2% and 32.8%, respectively, which were significantly higher than the change in the size of pancreatic parenchyma (14.4%, P = 0.026). Fourteen of these subjects were followed up for >12 months, during which 2 had relapse; diabetes improved in 5 and worsened in 2; in 60% of cases, the pancreatic parenchyma was atrophied. The % change in PEM after two weeks was tended to be higher in non-atrophy cases.ConclusionSWE and SWD measurement in US may be useful for quantitative assessment of AIP and evaluation of short-term treatment efficacy.     

Two tumor antigens and their polypeptides in adenovirus type 12-infected and transformed cells

A tumor (T) antigen, designated T antigen g, was visualized as fine fluorescent granules in nuclei of adenovirus type 12 (Ad12)-infected cells by immunofluorescence with sera from rats bearing HY cell tumors (H sera). HY cells are rat cells incompletely transformed by the Acc I-H endonuclease fragment (0-4.7 map units) of Ad12 DNA. The antigen is different from the usually described T antigen, designated T antigen f, which is visualized as fluorescent flecks or filaments in both nucleus and cytoplasm of Ad12-infected cells when tested with narrowly reacting T sera. Extracts of [³⁵S]methioninelabeled infected cells were immunoprecipitated with H sera, and the resultant precipitate was analyzed by the two-dimensional gel electrophoresis technique of O'Farrell. The autoradiogram showed the presence of a cluster of several polypeptides (M_r 35,000-40,000, pI 5.0-5.5) that was absent in extracts of mock-infected cells. A similar autoradiogram of infected cells analyzed with narrowly reacting T sera showed the presence of a small polypeptide (M_r 10,000, pI 6.4), that was absent in extracts of mock-infected cells. The results show that M_r 35,000-40,000 polypeptides are components of T antigen g and a M_r 10,000 polypeptide is a component of T antigen f. Ad12-transformed cells showed a similar result. T antigen g was present and T antigen f was absent in HY cells. Both T antigen g and T antigen f were present in CY cells, which are rat cells completely transformed by the EcoRI-C endonuclease fragment (0-16 map units) of Ad12 DNA. The possible functions of these proteins are discussed.     

Effect of AMPK activation on monocarboxylate transporter (MCT)1 and MCT4 in denervated muscle

It is now evident that exercise training leads to increases in monocarboxylate transporter (MCT)1 and MCT4, but little is known about the mechanisms of coupling muscle contraction with these changes. The aim of this study was to investigate the effect of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) induced activation of AMP-activated protein kinase (AMPK) on MCT1, MCT4, and GLUT4 in denervated muscle. Protein levels of MCT4 and GLUT4 after 10 days of denervation were significantly decreased in mice gastrocnemius muscle, while MCT1 protein levels were not altered. AICAR treatment for 10 days significantly increased MCT4, and GLUT4 protein levels in innervated muscle as shown in previous studies. We found that the MCT1 protein level was also increased in AICAR treated innervated muscle. AICAR treatment prevented the decline in MCT4 and GLUT4 protein levels in denervated muscle. Thus, the current study suggests that MCT1 and MCT4 protein expression in muscles, as well as GLUT4, may be regulated by AMPK-mediated signal pathways, and AMPK activation can prevent denervation-induced decline in MCT4 protein.     

Delayed DNA Synthesis Induced by 3-Aminobenzamide in Partially Hepatectomized Liver of Rats

The possibility of poly(ADP-ribosyl)ation playing a role during liver regeneration induced by partial hepatectomy (PH) in vivo was examined. When rats were given an i.p. injection of 3-antinobenzamide (ABA) at a dose of 600 mg/kg body weight 12 h after PH, the levels of DNA synthesis at 20 h after PH were significantly reduced. The time course of DNA synthesis in regenerating liver was significantly delayed in the ABA-treated group. Enzymatic assay revealed the activity of poly-(ADP-ribose)polymerase (PADPRP) in controls to be increased in parallel with the increase of DNA synthesis induced by PH. This increase in PADPRP activity was delayed and very much weaker after ABA treatment. The results thus suggested that poly (AUP-ribosyl)ation might play an important role in DNA synthesis during liver regeneration in vivo.