Makio Kobayashi

Short-chain fatty acids and ketones directly regulate sympathetic nervous system via G protein-coupled receptor 41 (GPR41)

The maintenance of energy homeostasis is essential for life, and its dysregulation leads to a variety of metabolic disorders. Under a fed condition, mammals use glucose as the main metabolic fuel, and short-chain fatty acids (SCFAs) produced by the colonic bacterial fermentation of dietary fiber also contribute a significant proportion of daily energy requirement. Under ketogenic conditions such as starvation and diabetes, ketone bodies produced in the liver from fatty acids are used as the main energy sources. To balance energy intake, dietary excess and starvation trigger an increase or a decrease in energy expenditure, respectively, by regulating the activity of the sympathetic nervous system (SNS). The regulation of metabolic homeostasis by glucose is well recognized; however, the roles of SCFAs and ketone bodies in maintaining energy balance remain unclear. Here, we show that SCFAs and ketone bodies directly regulate SNS activity via GPR41, a Gi/o protein-coupled receptor for SCFAs, at the level of the sympathetic ganglion. GPR41 was most abundantly expressed in sympathetic ganglia in mouse and humans. SCFA propionate promoted sympathetic outflow via GPR41. On the other hand, a ketone body, β-hydroxybutyrate, produced during starvation or diabetes, suppressed SNS activity by antagonizing GPR41. Pharmacological and siRNA experiments indicated that GPR41-mediated activation of sympathetic neurons involves Gβγ-PLCβ-MAPK signaling. Sympathetic regulation by SCFAs and ketone bodies correlated well with their respective effects on energy consumption. These findings establish that SCFAs and ketone bodies directly regulate GPR41-mediated SNS activity and thereby control body energy expenditure in maintaining metabolic homeostasis.

Prognostic relevance of morphological types of intraductal papillary mucinous neoplasms of the pancreas

Objective The clinicopathological significance of four morphological types of intraductal papillary mucinous neoplasms of the pancreas (IPMNs; gastric, intestinal, pancreatobiliary and oncocytic) was assessed. Design Retrospective multicentre analysis of 283 surgically resected IPMNs. Results Of the 283 IPMNs, 139 were of the gastric type, 101 were intestinal, 19 were pancreatobiliary and 24 were oncocytic. These types were significantly associated with clinicopathological factors including sex (p=0.0032), age (p=0.00924), ectatic duct size (p=0.0245), detection of mural nodules (p=4.09×10−6), histological grade (p<2.20×10−16), macroscopic types with differential involvement of the pancreatic duct system (p=3.91×10−5), invasive phenotypes (p=3.34×10−12), stage (p<2.20×10−16) and recurrence (p=0.00574). Kaplan–Meier analysis showed significant differences in patient survival by morphological type (p=5.24×10−6). Survival rates at 5 and 10 years, respectively, were 0.937 (95% CI 0.892 to 0.984) for patients with gastric-type IPMNs; 0.886 (95% CI 0.813 to 0.965) and 0.685 (95% CI 0.553 to 0.849) for those with intestinal-type IPMNs; 0.839 (95% CI 0.684 to 1.000) and 0.734 (95% CI 0.526 to 1.000) for those with oncocytic-type IPMNs; and 0.520 (95% CI 0.298 to 0.909) and undetermined for those with pancreatobiliary-type IPMNs. Analysis by the Cox proportional hazards model comparing prognostic risks determined by stage and the morphological and macroscopic types indicated that staging was the most significant predictor of survival (p=3.68×10−8) followed by the morphological type (p=0.0435). Furthermore, the morphological type remained a significant predictor in a subcohort of invasive cases (p=0.0089). Conclusion In this multicentre retrospective analysis, the morphological type of IPMN appears to be an independent predictor of patient prognosis.

Intraductal Tubulopapillary Neoplasms of the Pancreas Distinct From Pancreatic Intraepithelial Neoplasia and Intraductal Papillary Mucinous Neoplasms

We have encountered cases of unusual intraductal pancreatic neoplasms with predominant tubulopapillary growth. We collected data on 10 similar cases of “intraductal tubulopapillary neoplasms (ITPNs)” and analyzed their clinicopathologic and molecular features. Tumor specimens were obtained from 5 men and 5 women with a mean age of 58 years. ITPNs were solid and nodular tumors obstructing dilated pancreatic ducts and did not contain any visible mucin. The tumor cells formed tubulopapillae and contained little cytoplasmic mucin. The tumors exhibited uniform high-grade atypia. Necrotic foci were frequently observed, and invasion was observed in some cases. The ITPNs were immunohistochemically positive for cytokeratin 7 and/or cytokeratin 19 and negative for trypsin, MUC2, MUC5AC, and fascin. Molecular studies revealed abnormal expressions of TP53 and SMAD4 in 1 case, but aberrant expression of β-catenin was not observed. No mutations in KRAS and BRAF were observed in the 8 cases that were examined. Eight patients are alive without recurrence, 1 patient died of liver metastases, and 1 patient is alive but had a recurrence and underwent additional pancreatectomy. The mitotic count and Ki-67 labeling index were significantly associated with invasion. All the features of ITPN were distinct from those of other known intraductal pancreatic neoplasms, including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and the intraductal variant of acinar cell carcinoma. Intraductal tubular carcinomas showed several features that were similar to those of ITPN, except for the tubulopapillary growth pattern. In conclusion, ITPNs can be considered to represent a new disease entity encompassing intraductal tubular carcinoma as a morphologic variant.

New insights into the pathogenesis of spinal muscular atrophy.

To clarify the pathomechanism of spinal muscular atrophy (SMA) with mutations in the gene for survival motor neuron (SMN) protein, postmortem neuropathological analyses were performed on spinal cords obtained at autopsy from 2 fetuses with SMA, 5 infants and a low teenager with SMA type 1, and a higher teenager with SMA type 2; the diagnosis of all of them was confirmed clinically and genetically. Histopathologically, it was noted that lower motor neurons (LMNs) in the SMA cases showed immature profiles characterized by fine Nissl bodies restricted to the periphery of small round somata with a few cell processes in the fetal period, and showed small-sized profiles in the postnatal period. LMNs began to reduce in size and number in the fetal period, ballooned neurons (BNs) appeared postnatally, and the remaining LMNs including BNs diminished with age. BNs were filled with phosphorylated neurofilament protein, and morphologically similar to but smaller than typical chromatolytic neurons as axonal reaction. The population of survived LMNs was relatively preserved in an SMA type 2 case, who lived to 17-year-old, as compared to SMA type 1 cases. Immunohistochemical analysis demonstrated expression of Bcl-2, Bax, activated caspase-3 and SMN in the LMNs prominent in the fetal cases. There was no significant difference in staining for these substances between the control and SMA cases. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed no significant signal in the control and SMA cases. Given that downregulation of SMN leads to a failure in neurite outgrowth and neuromuscular contact of LMNs, the present results suggest the involvement of a fetal developmental maturation error as well as a postnatal retrograde dying-back degeneration of LMNs in SMN-mutated SMA.

Morphological analysis of pancreatic endocrine cells in newborn animals delivered by experimental diabetic rats

Rats with mildly impaired glucose tolerance (MIGT) were prepared by the administration of streptozotocin and subsequent islet-isotransplantation. Of pups born to these rats, 65% exhibited macrosomia that weighed over 6.8 g (average weight of control pups was 5.96 g). The morphological changes in the endocrine pancreas in pups born of MIGT and normal control rats were studied using immunohistochemical methods and quantitative morphological analysis. In macrosomic pups, the percentages of pancreatic tissue devoted to islet areas and B cell areas were significantly higher than in control pups. The percentage of pancreas devoted to A cell areas was also higher in macrosomic than normal pups, while the ratio of A/B cell areas was in normal range. In non-macrosomic pups born to MIGT rats, the percentages of pancreas devoted to islet tissues, B cell, and A cell areas were each lower than these in macrosomic pups. Moreover, increase in the proportions of islet and B cell areas in the pancreatic tissue in pups born of MIGT mothers was linearly correlated with increase in birthright. There were no significant differences in D cell area as a proportion of pancreas among macrosomic, non-macrosomic and control pups. These findings suggest that insulin secreted from B cells play important roles in the control of fetal growth.

Roles of Fukutin, the Gene Responsible for Fukuyama-Type Congenital Muscular Dystrophy, in Neurons: Possible Involvement in Synaptic Function and Neuronal Migration

Fukutin is a gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), accompanying ocular and brain malformations represented by cobblestone lissencephaly. Fukutin is related to basement membrane formation via the glycosylation of α-dystoglycan (α-DG), and astrocytes play a crucial role in the pathogenesis of the brain lesion. On the other hand, its precise function in neurons is unknown. In this experiment, the roles of fukutin in mature and immature neurons were examined using brains from control subjects and FCMD patients and cultured neuronal cell lines. In quantitative PCR, the expression level of fukutin looked different depending on the region of the brain examined. A similar tendency in DG expression appears to indicate a relation between fukutin and α-DG in mature neurons. An increase of DG mRNA and core α-DG in the FCMD cerebrum also supports the relation. In immunohistochemistry, dot-like positive reactions for VIA4-1, one of the antibodies detecting the glycosylated α-DG, in Purkinje cells suggest that fukutin is related to at least a post-synaptic function via the glycosylation of α-DG. As for immature neurons, VIA4-1 was predominantly positive in cells before and during migration with expression of fukutin, which suggest a participation of fukutin in neuronal migration via the glycosylation of α-DG. Moreover, fukutin may prevent neuronal differentiation, because its expression was significantly lower in the adult cerebrum and in differentiated cultured cells. A knockdown of fukutin was considered to induce differentiation in cultured cells. Fukutin seems to be necessary to keep migrating neurons immature during migration, and also to support migration via α-DG.

Post-transcriptional regulation of fukutin in an astrocytoma cell line

Fukutin is the gene responsible for Fukuyama‐type congenital muscular dystrophy (FCMD), an autosomal recessive disease associated with central nervous system (CNS) and eye anomalies. Fukutin is involved in basement membrane formation via the glycosylation of α‐dystroglycan (α‐DG), and hypoglycosylation of α‐DG provokes the muscular, CNS and eye lesions of FCMD. Astrocytes play an important role in the pathogenesis of the CNS lesions, but the post‐transcriptional regulation of fukutin mRNA has not been elucidated. In this study, we investigated the characteristics of fukutin mRNA using an astrocytoma cell line that expresses fukutin and glycosylated α‐DG. The glycosylation of α‐DG was considered to be increased by over‐expression of fukutin and decreased by knockdown of fukutin. Knockdown of Musashi‐1, one of the RNA‐binding proteins involved in the regulation of neuronal differentiation, induced a decrease in fukutin mRNA. Immunoprecipitation and ELISA‐based RNA‐binding assay demonstrated possible binding between fukutin mRNA and Musashi‐1 protein. A relationship between fukutin mRNA and vimentin protein was also proposed. In situ hybridization for fukutin mRNA showed a positive cytoplasmic reaction including cytoplasmic processes. From these results, fukutin mRNA is suggested to be a localized mRNA up‐regulated by Musashi‐1 and to be a component of a mRNA‐protein complex which includes Musashi‐1 and (presumably) vimentin proteins.