Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor.

AIMS/HYPOTHESIS: Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. METHODS: We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. RESULTS: MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. CONCLUSIONS/INTERPRETATION: We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH.

Rab8a is involved in membrane trafficking of Kir6.2 in the MIN6 insulinoma cell line.

Although ATP-sensitive K+ (KATP) channels play an important role in the secretion of insulin by pancreatic beta cells, the mechanisms that regulate the intracellular transport of KATP channel subunit proteins (i.e., Kir6.2 and sulfonylurea receptor 1 (SUR1)) to the plasma membrane remain uncharacterized. We investigated the possibility that an interaction between KATP channel subunit proteins and Rab8a protein, a member of the RAS superfamily, may be involved in the membrane trafficking of KATP channels. Co-immunoprecipitation and immunostaining experiments using co-expression systems with fluorescent protein-tagged Kir6.2 were carried out to identify the coupling of KATP channels and Rab8a proteins in the insulin-secreting cell line, MIN6. Rab8a protein co-localized with Kir6.2 protein, a channel pore subunit (in a granular pattern), and with insulin. Knockdown of the Rab8a gene with RNA interference using small interfering RNA systems caused reductions in the amount of total KATP and plasma membrane surface KATP channels without decreasing the messenger RNA transcription of the KATP channel subunits. Rab8a gene knockdown also enhanced glucose-induced insulin secretion. These results suggest that Rab8a may be involved in membrane trafficking of KATP channels and the maintenance of normal insulin secretion in the MIN6 pancreatic beta cell line.

Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release From Macrophages and Impairs Insulin Secretion.

Pancreatic islet inflammation plays a crucial role in the etiology of type 2 diabetes (T2D). Macrophages residing in pancreatic islets have emerged as key players in islet inflammation. Macrophages express a plethora of innate immune receptors that bind to environmental and metabolic cues and integrate these signals to trigger an inflammatory response that contributes to the development of islet inflammation. One such receptor, Dectin-2, has been identified within pancreatic islets; however, its role in glucose metabolism remains largely unknown. Here we have demonstrated that mice lacking Dectin-2 exhibit local inflammation within islets, along with impaired insulin secretion and ß-cell dysfunction. Our findings indicate that these effects are mediated by proinflammatory cytokines, such as interleukin (IL)-1α and IL-6, which are secreted by macrophages that have acquired an inflammatory phenotype because of the loss of Dectin-2. This study provides novel insights into the mechanisms underlying the role of Dectin-2 in the development of islet inflammation.

[New estimation method for displacement characteristic of teeth with occlusal force records].

OBJECTIVES: To establish adequate occlusion for prostheses, evaluation of degree of displacement of teeth and implant is essential. However, it cannot be attained without complex equipment, which limits clinical application. To develop a new estimation method for displacement characteristic of teeth and implant by both occlusal force of individual teeth and existing data on degree of displacement, the relationship between increase of total occlusal force and occlusal force of individual teeth was evaluated. METHODS: Ten male subjects (mean age: 28.5, [range: 26-31] years) with clinically normal healthy dentition were selected. Electromyograms at maximum clenching at intercuspal position were recorded as 100 MVC. Then, clenching at 80, 60, 50, 30, 20, and 10 MVC was instructed with visual feedback. Occlusal forces were recorded with pressure-sensitive sheet (Dental-Prescale). The occlusal contacts were recorded by a silicone occlusal contact checking material (Black Silicone). The change of occlusal forces at first premolar was converted to displacement with existing data of degree of displacement (Goto et al). Then displacement characteristic of second premolar and first molar was calculated. RESULTS: The displacement characteristic of second premolar was similar to existing data on first premolar. Although the displacement characteristic of first molar was similar, the degree of displacement was small, which means occlusal force at the first molar increased more than at the first premolar as increase of the displacement. CONCLUSION: The results of this study, indicating similarity to past studies with complex equipment, suggest that the displacement characteristic of teeth could be estimated with both occlusal force of individual teeth and existing data on degree of displacement.

Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.

Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel ß-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other ß-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with ß1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with ß1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with ß1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with ß1-subunits. These results suggest that ß1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with ß1-subunits in a concentration-dependent manner.

ZD0947, a sulphonylurea receptor modulator, detects functional sulphonylurea receptor subunits in murine vascular smooth muscle ATP-sensitive K+ channels.

In order to identify functional sulphonylurea receptor (SUR.x) subunits of native ATP-sensitive K+ channels (KATP channels) in mouse portal vein, the effects of ZD0947, a SUR.x modulator, were investigated on spontaneous portal vein contractions, macroscopic membrane currents and unitary currents recorded (using patch-clamp techniques) in freshly dispersed mouse portal vein myocytes. Spontaneous contractions in mouse portal vein were reversibly reduced by ZD0947 in a concentration-dependent manner (Ki =293nM). The relaxation elicited by 3µM ZD0947 was antagonized by the additional application of glibenclamide (300nM), but not gliclazide (100-300nM). In the conventional whole-cell configuration, 100µM ZD0947 elicited inward glibenclamide-sensitive currents at a holding potential of -60mV that demonstrated selectivity for K+(i.e. KATP currents). The peak amplitude of the membrane current elicited by 30µM or 100µM ZD0947 was smaller than that elicited by 100µM pinacidil at -60mV. In the cell-attached mode, 100µM ZD0947 activated glibenclamide-sensitive K+ channels with a conductance (35 pS) similar to that of recombinant Kir6.1/SUR2B channels that were expressed in HEK293 cells and activated by 100µM ZD0947. These results demonstrate that ZD0947 caused a significant vascular relaxation through the activation of KATP channels and that SUR2B may be the major functional subunit of SUR.x in mouse portal vein KATP channels, based on its pharmacological selectivity.

A Retroperitoneal Isolated Enteric Duplication Cyst Mimicking a Teratoma: A Case Report and Literature Review.

Enteric duplication cysts lacking anatomic association with the gastrointestinal tract are called isolated enteric duplication cysts (IEDCs). We present an atypical case of a retroperitoneal IEDC with a tortuous tubular complex shape that enfolded the surrounding retroperitoneal fat and mimicked a retroperitoneal teratoma. Multiplanar reconstruction images should be used to evaluate such a lesion correctly. A tortuous tubular complex shape could be a key finding to differentiate from other retroperitoneal cysts.

Occlusal status of implant superstructures at mandibular first molar immediately after setting.

BACKGROUND: Occlusal contact on the implant superstructures is important for successful treatment. The purpose of this study was to investigate the occlusal contact of single implant superstructures at the mandibular first molar immediately after seating from weak to strong clenching. METHODS: Subjects were nine patients who had just been fitted with an implant prosthesis in the mandibular first molar region, with no missing teeth other than in the implant region. First, while masseter muscle activity was monitored, maximum clenching strength (100 % maximum voluntary contraction (MVC)) was determined with an electromyogram. Next, occlusal load and occlusal contact area were measured three times at clenching intensities of 40, 60, 80, and 100 % MVC by the use of pressure-sensitive film for occlusal force diagnostic and Occluzer for occlusal force measurement. Finally, the occlusal contact area was measured once each at 20, 40, and 60 % MVC using a silicone testing material and BiteEye for occlusal contact measurement. A two-way analysis of variance (ANOVA) was used to determine occlusal loading and occlusal area as dependent variables, and clenching strength and presence or absence of implant as between-subject factors. A multiple comparison test was performed using the Bonferroni method. RESULTS: The occlusal contact area and occlusal load of the implant prosthesis increased with clenching strength, and the increases in occlusal contact area and occlusal load of the implant prosthesis were less than those of the contralateral tooth at high clenching strength. However, significant difference was not observed when compared with both sides of the molar region regardless of clenching strength. CONCLUSIONS: The occlusal contact area of the implant had a tendency to be adjusted smaller than the natural tooth by a dental technician and a dentist. On the other hand, despite the small tissue displaceability of the implant, occlusal load on the implant prosthesis was smaller than on the natural tooth at high clenching strength.

Influence of occlusal loading force on occlusal contacts in natural dentition.

PURPOSE: Proper occlusal contact is important for the long-term success of prosthodontic therapy. We clarified the effects of occlusal loading force on occlusal contact in natural dentition by comparing measured values for occlusal loading and occlusal contact area. METHODS: Masseter muscle activity was measured in 10 subjects (2 male, 8 female; mean age, 27 years) with natural dentition using electromyography, with clenching at full strength with nothing interposed between the upper and lower teeth defined as 100% maximum voluntary contraction (MVC). Pressure-sensitive film (Occluzer) was used to examine occlusal contact points at 20, 40, 60, 80, 100 and 120% MVC. A material for checking accuracy of fit (BiteEye) was used to examine occlusal contact points at 20, 40, 60 and 80% MVC. ANOVA and the Bonferroni method were used to assess the results, with the level of significance set at 5%. Coefficients of variation (CV) were also calculated by dividing the standard deviation by the mean. RESULTS: Occlusal loading and occlusal contact area increased with clenching strength; however, CV showed differences between the methods at low and high MVC. CONCLUSIONS: With Occluzer, testing should be carried out at clenching strength ≥ 60% MVC. With BiteEye, testing should be carried out from light clenching strength at 20% MVC to moderate clenching strengths at 40-60% MVC. Occluzer and BiteEye (10 µm) gave similar occlusal contact areas at 60-80% MVC. These results suggest that combined use of Occluzer and BiteEye gives an accurate picture of occlusion from weak to strong clenching strength.