[Evaluation of the Japanese version of HLS-Q12 in older adults].

Objectives　The purpose of this study was to evaluate the internal consistency and factorial validity of the Japanese version of HLS-Q12 health literacy rating scale when used with older adults.Methods　A questionnaire-based mail survey was conducted among older adults living in a community. The data collection period was from January to February 2022. The Japanese version of HLS-Q12 was used to obtain data on health literacy of older adults, and the internal consistency and factorial validity of the scale were evaluated using Cronbach's alpha coefficient and confirmatory factor analysis, respectively. In addition, a Rasch model was used to conduct a detailed analysis on each questionnaire item.Results　Questionnaires were distributed among 3,572 people, out of which, 1,082 responses were received from older adults (over 65 years of age). Cronbach's alpha coefficient was 0.8 or higher, indicating no internal consistency issues. According to the confirmatory factor analysis, CFI was equal to 0.933, AGFI, 0.876, and RMSEA, 0.092; thus, all indicators met certain evaluation levels. However, deviation from one of the multiple fit criteria was confirmed for the value of RMSEA, indicating that the error between the estimated value calculated by the scale and the true value was relatively large. Analysis of each question item using the Rasch model showed that all the questions met the criteria of Infit MSQ and were suitable in their question structure.Conclusion　Following the evaluation of the HLS-Q12 scale in older adults, the scale was found to be reliable and valid at a certain level. However, the value of RMSEA indicated that it was relatively large in terms of the error between the estimated value calculated by the scale and the true value.

Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired ß-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver.

Growth hormone (GH) activates multiple signal transduction pathways. To investigate these pathways, we identified novel genes whose transcription was induced by GH in the liver of hypophysectomized (HPX) rats using the suppression subtractive hybridization technique. We found that regulator of calcineurin 1 (Rcan1) mRNA was upregulated by GH administration. RCAN1 regulates the activity of calcineurin, a Ca/calmodulin-dependent phosphatase. Rcan1 encodes two major transcripts, Rcan1-1 and Rcan1-4, resulting from differential promoter use and first exon choice. We found that a single injection of GH increased the levels of Rcan1-4 mRNA and RCAN1-4 protein transiently, but did not increase Rcan1-1 mRNA in HPX rat liver. Then the molecular mechanism of GH to induce Rcan1-4 transcription was examined in rat hepatoma H4IIE cells. Experiments using inhibitors suggested that c-JUN N-terminal kinase was required for the induction of Rcan1-4 mRNA by GH. GH increased the levels of phosphorylated c-JUN protein and c-Jun mRNA in HPX rat liver. The luciferase and electrophoretic mobility shift assays showed that c-JUN upregulated Rcan1-4 mRNA by binding to the cAMP-responsive element in the upstream of Rcan1 exon 4. These results indicate that GH activates c-JUN to affect the activity of calcineurin by the induction of Rcan1-4 in rat liver.

A translation repressor, 4E-BP1, regulates the triglyceride level in rat liver during protein deprivation.

Protein deprivation has been shown to induce fatty liver in humans and animals, but the molecular mechanisms underlying such induction are largely unknown. Our previous studies have shown that a low-protein diet increases eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein and triglyceride (TG) levels in rat liver. 4E-BP1 is known to repress translation by binding to eIF4E. There is also evidence indicating that 4E-BP1 regulates lipid metabolism. Here, we examined the role of 4E-BP1 on TG accumulation in the livers of rats under protein deprivation. The low-protein diet rapidly increased the hepatic 4E-BP1 mRNA level within 1 day, followed by the induction of hepatic TG accumulation. The knockdown of hepatic 4E-BP1 attenuated the TG accumulation in rat liver induced by the low-protein diet. 4E-BP1 knockdown also increased the protein level of carnitine palmitoyltransferase 1A (CPT1A), a regulator of fatty acid oxidation, in the liver of rats fed a low-protein diet. These results indicate that a low-protein diet increases the amount of 4E-BP1, leading to TG accumulation in rat liver. We thus conclude that 4E-BP1 plays an important role in inducing hepatic steatosis under protein deprivation.

Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein ß pathway in rat liver.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPß pathway.

Low-protein diet enhances adiponectin secretion in rats.

Previous studies including ours have shown that a low-protein diet up-regulates insulin signaling in the liver and muscle and induces fatty liver in rats. Adiponectin is known as an insulin-sensitizing adipocytokine. We, therefore, examined the effect of a low-protein diet on the adiponectin levels in rats. The low-protein diet significantly increased serum adiponectin level. However, mRNA and protein levels of adiponectin in white adipose tissue (WAT) were not changed by the low-protein diet. Since it is known that oligomerization is important to control serum adiponectin level, we examined the population of adiponectin oligomeric forms in WAT and found that low-protein diet did not change it. Despite these events, the amount of its secretion was significantly increased in the adipocytes isolated from WAT of low-protein diet-fed rats. These results indicate that a low-protein diet enhances adiponectin secretion, which is not due to the increased intracellular amount and oligomerization of adiponectin.

Triglyceride synthesis in hepatocytes isolated from rats fed a low-protein diet is enhanced independently of upregulation of insulin signaling.

It is known that protein malnutrition develops fatty liver in rats. However, the mechanisms by which protein malnutrition enhances lipid accumulation in the liver are not fully understood. Our previous studies have demonstrated that protein malnutrition upregulates insulin signaling with an increase in TG levels in rat livers. Here, we examined whether the upregulated insulin signaling contributes to an enhancement of TG accumulation under protein malnutrition. As it is difficult to analyze insulin-induced hepatic TG synthesis in vivo, the isolated hepatocytes derived from rats fed a low-protein diet were used. The hepatocytes were isolated from rats fed a 15% casein diet (15C) as a control diet or a 5% casein diet (5C) as a low-protein diet and then treated with insulin. As shown in vivo, insulin signaling was upregulated in isolated hepatocytes from 5C-fed rats (5C hepatocytes). However, the insulin-induced increase in the mRNA levels of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS), was similar in both groups. The amounts of TG synthesized from both glucose and palmitate, as well as ACC1 and FAS protein levels, were increased at the basal state in 5C hepatocytes, but were not further increased by insulin. These results indicate that TG synthesis via both de novo fatty acid synthesis and esterification is enhanced in 5C hepatocytes, which is independent of the upregulation of insulin signaling.

Differentiation behavior of iPS cells cultured on PLGA with osteoinduction medium.

In the present report, we have generated osteoblast-like cells derived from mouse induced-pluripotent stem (iPS) cells on PLGA with osteoinduction medium in vitro and in vivo. The cell culture period was 2 weeks. At 2 weeks, mRNA level of type I collagen was significantly higher than at 1 week. Osteocalcin mRNA level at 2 weeks was tendency to increase compared with at 1 week. And the cells cultured on PLGA were positive for immunofluorescent staining of osteocalcin and alizarin red S staining. The scaffold and osteogenic-like cells induced in vitro were implanted subcutaneously into SCID mice. In resected teratoma, hard tissues resembling bone were observed mixed with other tissues on the scaffold. The sum of these findings suggests that PLGA does not disturb the osteogenesis of iPS cells.

Tissue-specific effects of protein malnutrition on insulin signaling pathway and lipid accumulation in growing rats.

Our previous studies have revealed that protein malnutrition enhances insulin signaling in rat liver and muscle in response to a bolus insulin injection. However, it has not been established whether protein malnutrition up-regulates insulin signaling under physiological conditions, such as feeding. Here, we studied the effects of protein malnutrition on insulin signaling after feeding in rat liver, muscle and white adipose tissue (WAT). Six-week-old rats were fed a 15% casein diet (15C) or a calorie-matched 5% casein diet (5C) for 8 h/day during 14 days. On the 15th day, blood and tissues were collected at various time points after feeding. Feeding-induced insulin secretion was reduced in 5C-fed rats compared to 15C-fed rats. The 5C-feeding suppressed immediate activation of insulin receptor after feeding in the liver, muscle, and WAT. However, 5C-feeding constantly increased tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and threonine phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in the liver during the examined periods, corresponding to the changes of their amounts. In skeletal muscle, 5C-feeding did not appreciably alter insulin signaling. In WAT, 5C-feeding decreased tyrosine phosphorylation of IRS-1 compared to 15C-feeding. Furthermore, hepatic triglyceride content was increased and feeding-induced acetyl-CoA carboxylase 1 gene expression was enhanced in 5C-fed rats. The 5C-feeding decreased insulin-dependent glucose uptake in adipocytes. These results suggest that enhanced insulin signaling through increased IRS-2 and 4E-BP1 levels in the liver and repressed insulin signaling through decreased IRS-1 levels in WAT contribute to the preferential hepatic lipid accumulation under protein malnutrition.

Second primary squamous cell carcinoma arising in a skin flap: a case report and literature review on etiologic factors and treatment strategy.

The number of reports describing malignant tumors arising in flaps has been increasing recently. This report describes the case of a patient who had a second squamous cell carcinoma (SCC) in the center of the skin island of a forearm flap raised approximately 6 years previously. Histopathologic examination suggested human papillomavirus infection and "mucosalization" of the flap. In addition, the authors review previous cases, with special attention to etiologic factors and surgical strategies. There might be a causal relation between second primary SCC in the flap and the carcinogenic effect (chronic inflammation) induced by the environment surrounding the squamous epithelium of the flap. If environmental factors have greatly contributed to the occurrence of a tumor, there is a potential for tumor occurrence in the remaining flap. There also might be a potential for generating second primary SCC in these foci. The results suggested that total resection of the flap might be recommended in the surgical resection of second primary SCC arising in a flap.

Dietary protein deprivation upregulates insulin signaling and inhibits gluconeogenesis in rat liver.

This study was undertaken to elucidate the effects of dietary protein deprivation on glucose metabolism and hepatic insulin signaling in rats. The results of glucose and pyruvate tolerance tests in rats fed with a 12% casein diet (12C) and a protein-free diet (PF) indicated that protein deprivation enhanced clearance of blood glucose and suppressed gluconeogenesis. Correspondingly, the mRNA level of hepatic phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme, was suppressed by dietary protein deprivation. In PF-fed rats, total tyrosine phosphorylation of insulin receptor (IR) in the liver induced by insulin injection was enhanced compared with 12C pair-fed rats due to an increase in IR protein level. In addition, protein deprivation caused an increase in protein levels of IR substrate 1 (IRS1) and IRS2, leading to the marked enhancement of insulin-induced tyrosine phosphorylation of IRS2 and its binding to the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). Based on these results, we conclude that protein deprivation suppresses gluconeogenesis by a mechanism primarily mediated by the enhancement of the insulin signals through the IR/IRS/PI3K/mammalian target of rapamycin complex 1 pathway in the liver. Taken together with our previous report, these findings suggest that tissue-specific potentiation of insulin action in the liver and the skeletal muscle plays important roles in maintaining glucose homeostasis even when energy usage is reduced by dietary protein deprivation.

Activation of leukemia-associated RhoGEF by Galpha13 with significant conformational rearrangements in the interface.

The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).

Human growth hormone induces SOCS3 and CIS mRNA increase in the hypothalamic neurons of hypophysectomized rats.

The activation of the growth hormone (GH) receptor is followed by activation of the JAK2-STAT system in peripheral tissues, which in turn induces the expression of suppressors of cytokine signaling (SOCS) and/or cytokine-inducible SH2 protein (CIS) to achieve the attenuation of the signaling. To examine whether GH involves the SOCS/CIS system as intracellular negative regulators in the hypothalamus, we observed the effects of human GH on the gene expression of SOCS/CIS in the rat hypothalamus. The mRNAs of CIS, SOCS2, and SOCS3 in the hypothalamus of hypophysectomized male rats were examined by Northern analysis following the intravenous administration of recombinant human GH (hGH), 50 microg/100 g BW. The SOCS3 and CIS mRNAs were increased transiently with maximum expression at 1 h after hGH administration. The intravenous hGH did not induce SOCS2 mRNA expression in the hypothalamus. In situ hybridization demonstrated the increase of SOCS3 and CIS mRNAs in the arcuate nucleus after hGH administration, and the increase of SOCS3 mRNA in the periventricular nucleus. The hGH applied to primary cultured hypothalamic neurons at 500 ng/ml induced transient increase of SOCS3 and CIS mRNAs, but not SOCS2 mRNA. The results show that hGH acts directly on the neurons in the hypothalamus, and increases SOCS3 and CIS mRNAs, suggesting that these negative regulators may be involved in the mechanism that turns off the hGH action in the hypothalamic neurons.

Microinjection of dihydrotestosterone into the medial preoptic area produces male-like pattern of growth hormone secretion in ovariectomized female rats.

The pattern of growth hormone (GH) secretion is sexually dimorphic in rats. We have previously shown that the secretory pattern in adult ovariectomized (OVX) female rats is masculinized by the administration of a single dose of dihydrotestosterone (DHT), a nonaromatizable androgen. To investigate the primary site of action of DHT in the brain, a small amount of DHT was injected directly into a defined area of the brain, and the blood GH profile was observed for 18 h in conscious adult OVX female rats. The bilateral direct injection of 1 microg DHT into the medial preoptic area (MPA) produced a male-like secretory pattern of GH in OVX rats. The masculinizing effects became apparent at 9 h after injection, from which time the episodic GH secretion was produced regularly at intervals of about 150 min, the amplitude of the peak increased and baseline levels were lowered. These parameters, analyzed during 9-18 h after DHT injection, were not different from those in adult male rats. On the contrary, microinjection of DHT into the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, or the hypothalamic arcuate-ventromedial nucleus did not affect the secretory pattern of GH. The data indicate that DHT primarily acts on cells in the MPA through androgen receptors and modulates the secretion of somatostatin and/or GH-releasing hormone secondarily to masculinize the GH secretory pattern in OVX rats.