Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Development of an Improved Adenovirus Vector and Its Application to the Treatment of Lifestyle-Related Diseases.

The number of patients with lifestyle-related diseases such as type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has continued to increase worldwide. Therefore, development of innovative therapeutic methods targeting lifestyle-related diseases is required. Gene therapy has attracted considerable attention as an advanced medical treatment. Safe and high-performance vectors are essential for the practical application of gene therapy. Replication-incompetent adenovirus (Ad) vectors are widely used in clinical gene therapy and basic research. Here, we developed a novel Ad vector, named Ad-E4-122aT, exhibiting higher and longer-term transgene expression and lower hepatotoxicity than conventional Ad vectors. We also elucidated the mechanisms underlying Ad vector-induced hepatotoxicity during the early phase using Ad-E4-122aT. Next, we examined the therapeutic effects of the genes of interest, namely zinc finger AN1-type domain 3 (ZFAND3), lipoprotein lipase (LPL), and lysophospholipid acyltransferase 10 (LPLAT10), on lifestyle-related diseases using Ad-E4-122aT. We showed that the overexpression of ZFAND3 in the liver improved glucose tolerance and insulin resistance. Liver-specific LPL overexpression suppressed hepatic lipid accumulation and improved glucose metabolism. LPLAT10 overexpression in the liver suppressed postprandial hyperglycemia by increasing glucose-stimulated insulin secretion. Furthermore, we also focused on foods to advance research on the pathophysiology and treatment of lifestyle-related diseases. Cranberry and calamondin, which are promising functional foods, attenuated the progression of MASLD/NAFLD. Our findings will aid the development of new therapeutic methods, including gene therapy, for lifestyle-related diseases such as T2DM and MASLD/NAFLD.

Adenovirus Vector-Induced IL-6 Promotes Leaky Adenoviral Gene Expression, Leading to Acute Hepatotoxicity.

Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4, leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity.

Selective histamine H2 receptor agonists alleviate blood-brain barrier disruption by promoting the expression of vascular protective factors following traumatic brain injury in mice.

Histamine is a major neurotransmitter and alleviates neuronal damage after ischemic injury via H2 receptors. Herein, we investigated the effects of H2 receptor agonists on the blood-brain barrier (BBB) disruption after traumatic brain injury (TBI). Male ddY mice were used to generate the TBI model, in which a fluid percussion injury (FPI) was induced by a hydraulic impact. The BBB disruption was evaluated using Evans blue extravasation. H2 receptor agonists, amthamine and dimaprit, were administered into the lateral cerebroventricle (i.c.v.) or tail vein (i.v.) from 3 hours to 3 days after FPI. The i.c.v. or i.v. administration of amthamine and dimaprit reduced FPI-induced Evans blue extravasation and promoted mRNA expression of vascular protective factors, including angiopoietin-1 and sonic hedgehog. The co-administration of ranitidine, a H2 receptor antagonist, inhibited these effects. Expression of the H2 receptor was observed in astrocytes and brain microvascular endothelial cells (BMECs) in the injured cortex. Treatment with amthamine and dimaprit promoted mRNA expression of vascular protective factors in astrocytes and BMECs. These results suggest that H2 receptor agonists alleviate TBI-induced BBB disruption by increasing the expression of vascular protective factors in astrocytes and BMECs.

Pharmacological Inhibition of Transient Receptor Potential Vanilloid 4 Reduces Vasogenic Edema after Traumatic Brain Injury in Mice.

Vasogenic edema results from blood-brain barrier (BBB) disruption after traumatic brain injury (TBI), and although it can be fatal, no promising therapeutic drugs have been developed as yet. Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable channel that is sensitive to temperature and osmotic pressure. As TRPV4 is known to be responsible for various pathological conditions following brain injury, we investigated the effects of pharmacological TRPV4 antagonists on TBI-induced vasogenic edema in this study. A TBI model was established by inflicting fluid percussion injury (FPI) in the mouse cerebrum and cultured astrocytes. Vasogenic brain edema and BBB disruption were assessed based on brain water content and Evans blue (EB) extravasation into brain tissue, respectively. After FPI, brain water content and EB extravasation increased. Repeated intracerebroventricular administration of the specific TRPV4 antagonists HC-067047 and RN-1734 dose-dependently reduced brain water content and alleviated EB extravasation in FPI mice. Additionally, real-time PCR analysis indicated that administration of HC-067047 and RN-1734 reversed the FPI-induced increase in mRNA levels of endogenous causal factors for BBB disruption, including matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and endothelin-1 (ET-1). In astrocytes, TRPV4 level was observed to be higher than that in brain microvascular endothelial cells. Treatment with HC-067047 and RN-1734 inhibited the increase in mRNA levels of MMP-9, VEGF-A, and ET-1 in cultured astrocytes subjected to in vitro FPI. These results suggest that pharmacological inhibition of TRPV4 is expected to be a promising therapeutic strategy for treating TBI-induced vasogenic edema.

Transduction Properties of an Adenovirus Vector Containing Sequences Complementary to a Liver-Specific microRNA, miR-122a, in the 3'-Untranslated Region of the E4 Gene in Human Hepatocytes from Chimeric Mice with Humanized Liver.

Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3'-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.

Dietary calamondin supplementation slows the progression of non-alcoholic fatty liver disease in C57BL/6 mice fed a high-fat diet.

Obesity is associated with an increased risk of metabolic abnormalities. The citrus fruit calamondin contains nobiletin and hesperidin, which are involved in lipid metabolism, and vitamin C, which is an antioxidant. We investigated the metabolic profiles of C57BL/6 mice fed a normal diet, high-fat diet (HFD), HFD + 1% (w/w) calamondin puree (HFD + CL1), or HFD + 5% (w/w) calamondin puree (HFD + CL5). Glucose tolerance was significantly higher in HFD + CL than in HFD-fed mice. Histological analysis revealed less lipid accumulation in the livers of HFD + CL-fed mice than in those of HFD-fed control mice. Hepatocyte ballooning and large lipid droplets - key non-alcoholic fatty liver disease characteristics - were observed in HFD-fed mice after 4 weeks; however, they were nearly absent in HFD + CL-fed mice. The serum expression level of inflammation-associated Ccl2 was lower in HFD + CL-fed mice than in HFD-fed mice. Thus, calamondin may ameliorate HFD-induced metabolic disturbances, including the progression of non-alcoholic fatty liver disease.

Cranberry Attenuates Progression of Non-alcoholic Fatty Liver Disease Induced by High-Fat Diet in Mice.

Obesity is characterized by abnormal or excessive fat accumulation, which leads to the development of metabolic syndrome. Because oxidative stress is increased in obesity, antioxidants are regarded as suitable agents for preventing metabolic syndrome. Here, we examined the impact of cranberry, which contains various antioxidants, on metabolic profiles, including that during the progression of non-alcoholic fatty liver disease (NAFLD), in high-fat diet (HFD)-fed C57BL/6 mice. We observed that oxidative stress was diminished in mice that were fed HFD diets supplemented with 1 and 5% cranberry powder as compared with that in HFD-fed control mice. Notably, from 1 week after beginning the diets to the end of the study, the body weight of mice in the cranberry-treatment groups was significantly lower than that of mice in the HFD-fed control group; during the early treatment phase, cranberry suppressed the elevation of serum triglycerides; and adipocytes in the adipose tissues of cranberry-supplemented-HFD-fed mice were smaller than these cells in HFD-fed control mice. Lastly, we examined the effect of cranberry on NAFLD, which is one of the manifestations of metabolic syndrome in the liver. Histological analysis of the liver revealed that lipid-droplet formation and hepatocyte ballooning, which are key NAFLD characteristics, were both drastically decreased in cranberry-supplemented-HFD-fed mice relative to the levels in HFD-fed control mice. Our results suggest that cranberry ameliorates HFD-induced metabolic disturbances, particularly during the early treatment stage, and exhibits considerable potential for preventing the progression of NAFLD.

Transplacental delivery of factor IX Fc-fusion protein ameliorates bleeding phenotype of newborn hemophilia B mice.

Hemophilia B is an inherited hemorrhagic disorder characterized by a deficiency of blood coagulation factor IX (FIX) that results in abnormal blood coagulation. The blood coagulation is already evident in hemophiliacs at the fetal stage, and thus intracranial hemorrhage and other bleeding complications can occur at birth, leading to sequelae. Therefore, it is important to develop effective treatments for hemophiliacs in utero. In this study, in order to transplacentally deliver FIX from pregnant mice to their fetuses, an improved adenovirus (Ad) vector expressing human FIX fused with the IgG Fc domain (FIX Fc fusion protein), which plays a crucial role in neonatal Fc receptor (FcRn)-mediated transcytosis across the placenta, was intravenously administered to E13.5 pregnant mice. Significant levels of FIX Fc fusion protein were detected in 0-day-old newborn mice whose mothers were administered an Ad vector expressing FIX Fc fusion protein. Wild-type FIX overexpressed in the pregnant mice was not delivered to the fetuses. Plasma FIX levels in the newborn mice were relatively well correlated with those in their mothers, although transplacental delivery efficiencies of FIX Fc fusion protein were slightly reduced when the FIX Fc fusion protein was highly expressed in the mother mice. Plasma FIX levels in the newborn mice were about 3.6-6.4% of those in their mothers, Transplacental delivery of FIX Fc fusion protein to their fetuses successfully improved the blood clotting ability in the newborn mice.

Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.