Protein acylations induced by a ketogenic diet demonstrate diverse patterns depending on organs and differ between histones and global proteins.

An essential ketone body, ß-hydroxybutyrate (BOHB), plays various roles in physiological regulations via protein acylations such as lysine acetylation and ß-hydroxybutyrylation. Here, to understand how BOHB systemically regulates acylations from an overarching perspective, we administered a ketogenic diet to mice to increase BOHB concentration and examined acylations. We found that global acetylation and ß-hydroxybutyrylation dramatically increase in various organs except for the brains, where the increase was much smaller than in the other organs. Interestingly, we observe no increase in histone acetylation in the organs where significant global protein acetylation occurs despite a substantial rise in histone ß-hydroxybutyrylation. Finally, we compared the transcriptome data of the mice's liver after the ketogenic diet to the public databases, showing that upregulated genes are enriched in those related to histone ß-hydroxybutyrylation in starvation. Our data indicate that a ketogenic diet induces diverse patterns of acylations depending on organs and protein localizations, suggesting that different mechanisms regulate acylations and that the ketogenic diet is associated with starvation in terms of protein modifications.

Genome-wide screening for regulators of degradation of insulin secretory granules with a fluorescent reporter.

Insulin is essential in controlling blood glucose levels, and its synthesis and secretion have been well investigated. In contrast, how insulin secretory granules (ISGs) are degraded in pancreatic beta cells remains largely unknown. To clarify the mechanism, we constructed a fluorescent reporter detecting ISG degradation, where EGFP and mCherry are tandemly conjugated to a cytoplasmic region of ZnT8, an ISG membrane-localized protein. Depletion of serum and amino acid stimulated lysosomal ISG degradation detected with the reporter. Next, with MIN6 cells expressing Cas9 and the reporter, we investigated the involvement of conventional Atg5/7-dependent autophagy to show that it is dispensable for the ISG degradation process. Finally, we performed genome-wide screening by enriching the cells lacking the ISG degradation and showed that pathways regulating autophagy are not identified. These results suggest that alternative degradation in lysosomes, instead of conventional autophagy, may be involved in ISG degradation.

A novel monoclonal antibody with improved FcγR blocking ability demonstrated non-inferior efficacy compared to IVIG in cynomolgus monkey ITP model at considerably lower dose.

Intravenous immunoglobulin (IVIG) is a well-established treatment for various autoimmune and inflammatory diseases. However, the standard dose prescribed for autoimmune diseases, including immune thrombocytopenic purpura (ITP), is 2 g/kg, which is markedly high and leads to a high treatment burden. In this study, we generated fragment crystallizable (Fc)-modified anti-haptoglobin (Hp) monoclonal antibodies with non-inferior efficacy compared to IVIG at considerably lower doses than IVIG, as shown by in vitro experiments. We evaluated binding activity of anti-Hp antibodies to Fc gamma receptors (FcγRs) with ELISA and inhibitory activity against the ADCC reaction. Furthermore, we successfully established a novel cynomolgus monkey ITP model and demonstrated that the anti-Hp antibody exerted its effect in this model with only a single dose. This Fc-modified anti-Hp monoclonal antibody could be a valuable therapeutic replacement for IVIG for the treatment of ITP.

Establishment of Pancreatic ß-Cell-Specific Gene Knockout System Based on CRISPR-Cas9 Technology With AAV8-Mediated gRNA Delivery.

The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic ß-cells associated with the pathogenesis of diabetes. However, it is expensive and time consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre expression mice. We establish a ßCas9 system with mice expressing Cas9 in pancreatic ß-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant ßCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in ß-cells. We also show significant ß-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to ßCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO ß-cells into glucagon-expressing cells. We successfully generated a convenient ß-cell-specific gene KO system with ßCas9 mice and AAV8-mediated gRNA delivery.

Monitoring autophagic flux in vivo revealed its physiological response and significance of heterogeneity in pancreatic beta cells.

Autophagy plays an essential role in preserving cellular homeostasis in pancreatic beta cells. However, the extent of autophagic flux in pancreatic islets induced in various physiological settings remains unclear. In this study, we generate transgenic mice expressing pHluorin-LC3-mCherry reporter for monitoring systemic autophagic flux by measuring the pHluorin/mCherry ratio, validating them in the starvation and insulin-deficient model. Our findings reveal that autophagic flux in pancreatic islets enhances after starvation, and suppression of the flux after short-term refeeding needs more prolonged re-starvation in islets than in the other insulin-targeted organs. Furthermore, heterogeneity of autophagic flux in pancreatic beta cells manifests under insulin resistance, and intracellular calcium influx by glucose stimulation increases more in high- than low-autophagic flux beta cells, with differential gene expression, including lipoprotein lipase. Our pHluorin-LC3-mCherry mice enable us to reveal biological insight into heterogeneity in autophagic flux in pancreatic beta cells.

Early effects of olanzapine on serum levels of ghrelin, adiponectin and leptin in patients with schizophrenia.

Although treatment with antipsychotics, particularly olanzapine and clozapine, has been implicated in weight gain and higher incidence of diabetes, the mechanism of these adverse reactions remains unclear. The purposes of this study were to explore the early effects of olanzapine on serum levels of ghrelin, adiponectin and leptin, three recently identified hormones that play crucial roles in the regulation of energy balance and glucose metabolism. Thirteen patients with schizophrenia who had not received any medication in the 4 weeks prior to this study were included. The patients received olanzapine at an average dose of 14.5mg/day. Serum levels of ghrelin, adiponectin, leptin and insulin, as well as weight and fasting glucose, were investigated at the baseline and at 4 weeks. Serum ghrelin levels had decreased (p 0.03) and leptin had increased (p 0.02), while adiponectin and insulin levels had not significantly changed at Week 4 (p 0.29 and p 0.25, respectively). Weight had increased (p 0.01), while fasting glucose had not significantly changed (p 0.46). These findings suggest that ghrelin levels decrease and leptin levels increase after initiation of olanzapine therapy. Weight gain is also considered to be an early change, while change in insulin sensitivity is not an early change of treatment with olanzapine. Further large-scale and longitudinal studies are warranted to elucidate metabolic changes involving ghrelin, adiponectin, leptin and insulin and their impact on weight and glucose metabolism during treatment with olanzapine and other antipsychotics.

Monitoring markers of disease activity for interstitial lung diseases with serum surfactant proteins A and D.

OBJECTIVES: Surfactant protein (SP) A and D are specific serum markers for interstitial lung diseases including idiopathic pulmonary fibrosis (IPF). The authors evaluated the critical roles of these markers on the prognoses of patients with IPF and the mechanisms of their elevation in sera. METHODOLOGY: The authors evaluated the relationship between prognosis and the serum markers in 82 IPF patients. The protein content and mRNA expression of the markers were evaluated using rats with interstitial pneumonia induced by bleomycin administration. RESULTS: Higher levels of serum SP-D at the time of the initial visit to the Sapporo Medical University Hospital were associated with poorer prognoses, while SP-A showed no significant affect on survival. Causes of the elevation in sera were due to the acceleration of, not only production in the lungs, leakage into the circulation. The elevation was associated with alveolitis but not fibrosis. CONCLUSIONS: SP-D is a good predictor of the prognosis in patients with IPF.

Gefitinib-induced interstitial lung disease showing improvement after cessation: disassociation of serum markers.

Gefitinib (ZD1839), a small-molecule epidermal growth factor receptor tyrosine kinase inhibitor, is an anticancer agent for patients with non-small cell lung carcinoma. Recently, however, as a result of accumulating evidence, it has been recognized that gefitinib can give rise to lethal lung toxicity. The authors report a case of interstitial lung disease (ILD) induced by gefitinib, which improved promptly following cessation of the administration of the agent. Clinical signs suggesting a good prognosis were noted, namely, findings similar to acute eosinophilic pneumonia on CT and a disassociation in the elevation of specific serum markers of ILD. At the time of onset of ILD, serum concentrations of surfactant protein (SP)-A and SP-D were significantly increased, whereas that of KL-6 was not increased. A previous study of three cases of lethal lung toxicity resulting from gefitinib administration revealed a significant and almost equal increase in KL-6, SP-A and SP-D. These results suggest that SP-A and SP-D may be indicators of gefitinib-induced ILD and that KL-6 is a predictor of outcome. Using a combination of these markers may help to establish a differential prognosis in patients with gefitinib-induced ILD.

Genetic structure of hybrid zones between Pinus pumila and P. parviflora var. pentaphylla (Pinaceae) revealed by molecular hybrid index analysis.

Pinus species have three differently inherited genomes: paternal chloroplast, maternal mitochondrial, and biparental nuclear. Our previous study on the hybrid zones between alpine Pinus pumila and montane to subalpine P. parviflora var. pentaphylla demonstrated contrasting patterns of introgression of two cytoplasmic genomes, i.e., the paternal cpDNA flowed from P. parviflora var. pentaphylla to P. pumila, and the maternal mtDNA flowed in the reverse direction. In the present study, we developed codominant nuclear DNA markers diagnostic or mostly diagnostic for each parental species by single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR) products, using expressed sequence tag (EST) primers of Pinus taeda. To describe the introgressive patterns of the nuclear genes, the molecular hybrid index (MHI) showing the overall proportion of alleles inferred to be derived from P. pumila was determined for each plant collected in hybrid zones on Mt. Asahidake and Mt. Higashiazuma, Japan. At Mt. Asahidake, the MHI values changed clinally according to the altitudes at which the plants were collected. However, at Mt. Higashiazuma, there was a gap in the MHI values between the plants above and below the Abies and Tsuga forest zone (alt. 1800-1900 m). This suggested that the zone plays a role in creating an effective barrier to gene flow in the hybrid zone.

Ubiquitin and ubiquitin-related proteins in neurons and dendrites of brains of atypical Pick's disease without Pick bodies.

Nine cases of atypical Pick's disease without Pick bodies were investigated immunohistochemically. Ubiquitin (ub)-positive and tau-negative structures were mainly found in the cerebral cortex and hippocampal dentate gyrus. In the cerebral cortex, most of the ub-positive structures had ub-positive dendrites in the neuropil, although some also showed diffuse ub-positive staining in the neuronal cytoplasm. These ub-positive structures were distributed throughout layers II-IIIab and layers V-VI. Granular cells of the dentate gyrus had ub-positive intraneuronal inclusions. When the numbers of ub-positive neurons and dendrites were evaluated in relation to the degree of neuronal loss in the cerebral cortex, the number of ub-positive neurons was significantly lower in regions showing very mild neuronal loss and higher in regions showing moderate neuronal loss. In contrast, ub-positive dendrites were detected even in cortical regions showing very mild neuronal loss. Immunoelectron-microscopically, ub-positive structures contained ub-positive ribosome-like granular components in the neuronal cytoplasm and dendrites, which were occasionally related to the rough endoplasmic reticulum and accompanied by a few filamentous components. Almost all ub-positive structures were positive for ub-binding protein p62 in double-immunostaining method. Some ub-positive or negative neurons in the cerebral cortex were positively immunolabeled with anti-ub ligase (Parkin) and anti-ub C-terminal hydrolase antibodies, whereas dendrites were not labeled by these antibodies. From the present study, it is suggested that in the cerebral cortex, these ubiquitinated proteins may firstly accumulate in the dendrites at the onset of neuronal degeneration, then appear in the neuronal cytoplasm before finally disappearing with neuronal loss.

Clinicopathological study of two subtypes of Pick's disease in Japan.

We examined the clinical and neuropathological findings in 3 cases of Pick's disease with Pick bodies (PiD) and 7 cases of atypical Pick's disease without Pick bodies (aPiD). PiD and aPiD cases corresponded clinically to frontotemporal dementia and semantic dementia, respectively, based on the clinical diagnostic criteria of frontotemporal lobar degeneration. Brain CT showed that cerebral atrophy was accentuated at an early stage of the illness in the anterior portion of the frontal lobes in PiD cases and in the anterior portion of the temporal lobes in aPiD cases. Neuropathologically, PiD cases showed more circumscribed lobar atrophy than aPiD cases. Both PiD and aPiD cases revealed moderate to severe degeneration with neuronal loss and gliosis in the affected cerebral cortex and subcortical nuclei, but only aPiD cases had pyramidal tract degeneration. Immunohistochemical analyses demonstrated that tauopathy with phosphorylated tau accumulation in the Pick bodies in PiD cases, while aPiD cases showed ubiquitinopathy with ubiquitin accumulation in the intraneuronal and dendritic inclusions. These findings suggested that two subtypes of Pick's disease in Japan can be distinguished not only neuropathologically but also clinically based on differences in pathogenesis.