2024 Volume 47 Issue 5 Pages 886-894
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.
Lifestyle-related diseases are caused by unhealthy lifestyle habits such as an unbalanced diet. Numerous lifestyle-related diseases develop and progress because of obesity and metabolic syndromes. Type 2 diabetes mellitus (T2DM) is a major lifestyle-related disease, and its incidence is increasing worldwide.1) Several treatments for T2DM have been developed; however, the QOL of patients with T2DM still tends to be lower than that of those without the disease. Metabolic dysfunction–associated steatotic liver disease (MASLD),2) formerly known as non-alcoholic fatty liver disease (NAFLD), is also a lifestyle-related disease and a hepatic manifestation of metabolic syndrome.3,4) Antioxidants and diabetes medicines are used to treat MASLD/NAFLD,5,6) and a sufficiently effective medicine against MASLD/NAFLD remains to be developed. Irrespective of the large number of patients with lifestyle-related diseases, few effective treatments are available for critically ill patients, and their QOL is reduced. Therefore, the development of innovative therapeutic methods that target lifestyle-related diseases is required.
Gene therapy has recently attracted attention as an advanced medical treatment. The Food and Drug Administration in the U.S. defines the purpose of gene therapy as follows: “Human gene therapy seeks to modify or manipulate the expression of a gene or to alter the biological properties of living cells for therapeutic use.” In 2019, the gene therapy products Tisagenlecleucel (Kymriah)—for the treatment of B-cell acute lymphoblastic leukemia7)—and Beperminogene perplasmid (Collategen)—for arteriosclerosis obliterans and Buerger’s disease8)—were approved in Japan. Clinical development and approval of various gene therapy products are underway. However, most target diseases for gene therapy are cancers and congenital genetic diseases, and there is limited research on lifestyle-related diseases.
In this review, we introduce studies on the development of vectors that are useful for gene therapy and basic research to develop new treatments for lifestyle-related diseases. The development of safe and high-performance vectors is essential for the practical application of gene therapy. A useful vector can be used for any therapeutic gene. Among the vectors, we focused on an adenovirus (Ad) vector due to its highly advantageous properties including high-titer production and highly efficient gene transduction into a wide spectrum of dividing and nondividing cells in vitro and in vivo.9,10) Subsequently, we conducted gene therapy studies for lifestyle-related diseases using the developed vectors.
Replication-incompetent Ad vectors are widely used in clinical gene therapy and basic research. Recently, Ad vectors have been used as vaccines against coronavirus disease 2019.11,12) Replication-incompetent Ad vectors have been used as gene delivery vehicles, enabling high transduction efficiencies, relatively large capacities for transgenes, and high-titer production. The replication-incompetent Ad vector lacks the E1 gene, which is crucial for the transcription of other Ad genes, and the target gene of interest is inserted into the E1-deleted region to express the transgene.9,10) Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector; however, they are expressed in the vector genome, resulting in the induction of cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause tissue damage and/or elimination of Ad vector-transduced cells, leading to short-lived transgene expression.13,14) We attempted to develop safe and functional Ad vectors by suppressing the leaky expression of Ad genes.
2.1. Development of Ad-E4-122aTLeaky expression of Ad genes occurs after transduction with a replication-incompetent Ad vector. However, no detailed analysis of the leaky expression of Ad gene profiles has been performed. To examine the expression profiles of Ad genes in cells following transduction with a replication-incompetent Ad vector, mRNA levels of the Ad genes, including the E2A, E2B, E4, hexon, penton base, fiber, and pIX genes, were determined using quantitative RT-PCR 2, 6, 12, 24, 48, 72, and 96 h after transduction. The E2A, E4, and pIX genes were found to be significantly expressed following Ad vector transduction.15) To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p, which exhibit liver- or spleen-specific expression, respectively, into the 3′-untranslated region of the E2A, E4, or pIX gene using a microRNA (miRNA)-regulated gene expression system.16) The liver is the main organ in which intravenously administered Ad vectors accumulate. The spleen is primarily involved in innate and acquired immune responses following Ad vector administration.
To examine whether in vivo leaky expression of Ad genes in organs was suppressed by the incorporation of miRNA-targeted sequences, Ad gene expression levels in the liver and spleen were determined using quantitative RT-PCR, following administration of Ad vectors. The leaky expression of these Ad vectors in the liver and spleen was significantly suppressed (2- to 100-fold) in an miRNA-dependent manner, compared to a conventional Ad vector, through the insertion of miRNA-targeted sequences. Among the Ad vectors developed, an Ad vector, i.e., Ad-E4-122aT, containing the miR-122a-targeted sequences in the 3′-untranslated region of the E4 gene expressed approximately 100-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector (Fig. 1).
Hepatotoxicity is a major adverse event following in vivo transduction of the Ad vector. To examine whether a reduction in the leaky expression of Ad genes by incorporating miRNA-targeted sequences leads to the suppression of hepatotoxicity associated with Ad vectors after intravenous administration, serum alanine aminotransferase (ALT) levels, which are enzymatic biomarkers of hepatotoxicity, were measured after the intravenous administration of Ad vectors. Ad-E4-122aT induced approximately 2- to 4-fold lower levels of ALT than the conventional Ad vector.
We attempted to elucidate the mechanism of hepatotoxicity suppression by insertion of miR-122a-targeted sequences into the E4 gene 3′-untranslated region. First, we hypothesized that hepatocytes transduced with Ad-E4-122aT may be less susceptible to Ad-specific cytotoxic T-lymphocyte (CTL) attack because of the lower levels of Ad antigen presentation compared to hepatocytes transduced with a conventional Ad vector. To test this hypothesis, Ad-specific CTL-mediated lysis of hepatocytes transduced with Ad vectors was determined using a lactate dehydrogenase assay. Hepatocytes transduced with a conventional Ad vector were more susceptible to Ad-specific CTL attacks than those transduced with Ad-E4-122aT. Second, we hypothesized that the suppression of leaky expression would result in reduction in Ad vector-induced immune-incompetent hepatotoxicity. Serum ALT levels were measured following intravenous administration of Ad vectors to Rag2/Il2rγ double-knockout mice, which have global defects in both cellular and humoral immunity due to the lack of T, B, and natural killer cells.17,18) Rag2/Il2rγ double-knockout mice exhibited a significant elevation in serum ALT levels following intravenous administration of a conventional Ad vector. In contrast, no increase in serum ALT levels was observed in Ad-E4-122aT-treated mice. These results indicate that miR-122a-mediated suppression of E4 gene expression in the liver significantly reduced the hepatotoxicity caused by the Ad vector through both adaptive and non-adaptive immune responses.
To examine whether the suppression of hepatotoxicity by Ad-E4-122aT improves transgene expression profiles, the murine secreted embryonic alkaline phosphatase (mSEAP) gene was inserted into the Ad vector genome as a reporter. mSEAP is a secreted form of murine embryonic alkaline phosphatase, which is a mouse endogenous protein that excludes the influence of immune responses to transgene products on the transgene expression profile. A conventional Ad vector carrying mSEAP (Ad-AHASEAP) and Ad-E4-122aT carrying mSEAP (Ad-E4-122aT-AHASEAP) exhibited mSEAP expression levels comparable to those on day 2 (Fig. 2). Ad-AHASEAP-mediated mSEAP expression levels gradually increased, plateauing 10 d after administration. Subsequently, mSEAP expression levels induced by Ad-AHASEAP gradually decreased. In contrast, the serum mSEAP levels induced by Ad-E4-122aT-AHASEAP were maintained for at least 149 d. In addition, the mSEAP expression levels induced by Ad-E4-122aT-AHASEAP were 1.5- to 34.1-fold higher than those induced by Ad-AHASEAP. These results indicate that the suppression of E4 gene expression in the liver leads to higher and longer-term transgene expression.
C57BL/6 mice were intravenously administered Ad vectors expressing the mSEAP gene. Blood samples were collected through retro-orbital bleeding on the indicated days after injection. mSEAP production in the serum was determined using the SEAP chemiluminescence assay. The data are expressed as the mean values ± standard deviation (S.D., n = 4). * p < 0.05 in comparison with Ad-AHASEAP. mSEAP expression in the phosphate-buffered saline (PBS)-treated mice was below the detectable level. This figure is reproduced from Shimizu et al., Mol. Ther. Methods Clin. Dev., 1, 14035 (2014).
Ad vector-mediated transduction can cause hepatotoxicity during two phases, approximately 2 and 10 d after administration. Early hepatotoxicity involves inflammatory cytokines such as interleukin-6 (IL-6) and interferons (IFNs); however, the exact mechanism remains to be clarified. Ad-E4-122aT transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT and a conventional Ad vector induced comparable levels of cytokines, including IL-6, IL-12β, IFN-β, and IFN-γ, in the spleen and liver. These data led us to hypothesize that Ad vector-induced inflammatory cytokine production alone does not cause hepatotoxicity during the early phase. We examined the mechanisms underlying Ad vector-induced hepatotoxicity during the early phase.19) To examine whether Ad vector-induced cytokines enhance hepatotoxicity caused by the leaky expression of Ad genes, primary mouse hepatocytes were transduced with either a conventional Ad vector or Ad-E4-122aT, in the presence or absence of IL-6. After transduction into hepatocytes, the conventional Ad vector induced significantly higher levels of cytotoxicity in the presence of IL-6 than in the absence of IL-6, whereas Ad-E4-122aT did not induce significant levels of cytotoxicity, irrespective of the presence or absence of IL-6. Moreover, addition of IL-6 significantly increased the leaky expression of Ad genes in conventional Ad vector-treated hepatocytes. Leaky expression of Ad genes and cytotoxicity in conventional Ad vector-treated hepatocytes in the presence of IL-6 were significantly suppressed upon the inhibition of IL-6 signaling pathways. Furthermore, Ad vector-mediated acute hepatotoxicity and leakage of Ad genes were significantly reduced in IL-6 knockout mice compared to wild-type mice. Thus, Ad vector-induced IL-6 causes the leaky expression of Ad genes in the liver, leading to hepatotoxicity during the early phase after systemic administration (Fig. 3). Therefore, Ad-E4-122aT is a promising framework for safe and effective gene therapy and basic research, including gene function analysis and elucidation of the mechanisms underlying Ad vector-mediated toxicity.
This figure is modified from Shimizu et al., J. Immunol., 206, 410–421 (2021).
The worldwide prevalence of T2DM and MASLD/NAFLD has increased in recent years. T2DM is characterized by a dysregulated glucose homeostasis. In T2DM, decreased insulin release from pancreatic β cells or suppression of insulin action leads to decreased glucose uptake by the peripheral tissues, resulting in increased blood glucose levels.20) Gluconeogenesis from the liver contributes more to glucose production in patients with T2DM than in normal individuals.21) MASLD/NAFLD, which is caused due to excessive accumulation of lipid in the liver, is the most common chronic diseases around the world. MASLD encompasses patients with hepatic steatosis and has at least one of five cardiometabolic risk factors, such as obesity.2) Abnormalities in glucose and lipid metabolism play a major role in the pathogenesis of T2DM and MASLD/NAFLD.
The liver is the primary organ responsible for glucose and lipid metabolism. We hypothesized that if genes with potential therapeutic value were highly expressed in the liver, abnormalities in glucose and lipid metabolism could be ameliorated, leading to the development of new treatments for lifestyle-related diseases such as T2DM. As systemic administration of Ad vectors results in liver-specific transgene expression, we decided to use Ad-E4-122aT for gene therapy studies for lifestyle-related diseases.
In this review, we present studies focusing on disease susceptibility genes and genes involved in the amount and type of lipids. In addition, we introduce research focusing on foods to advance research on the pathogenesis and treatment of lifestyle-related diseases.
3.1. Zinc Finger AN1-Type Domain 3 (ZFAND3)The pathology of T2DM differs between Japanese patients and patients of Westerners. Japanese patients with T2DM have less obesity, lower insulin secretion, and lower insulin resistance than Westerners patients with T2DM.22,23) Japanese patients with T2DM require treatment methods that are suitable for the Japanese population.
Genome-wide association studies have identified more than 300 loci associated with T2DM24); however, the mechanisms underlying their role in T2DM susceptibility remain largely unknown. ZFAND3, also known as the testis-expressed sequence 27, has been reported as a locus for T2DM in East Asian individuals.25) Little information is available regarding the physiological role of ZFAND3 in T2DM in vivo. To investigate the association between ZFAND3 and T2DM, we overexpressed ZFAND3 in the mouse livers using a ZFAND3-expressing Ad-E4-122a vector (Ad-ZFAND3).26)
To examine glucose metabolism, we performed glucose tolerance test. Glucose tolerance was improved in Ad-ZFAND3-treated mice compared to that in control Ad vector-treated mice. Insulin resistance in Ad-ZFAND3-treated mice also improved, as assessed using the insulin tolerance test. These results suggest that ZFAND3 overexpression in the liver improves glucose metabolism.
To investigate the effect of ZFAND3 on hepatic glucose metabolism in detail, primary mouse hepatocytes isolated from C57BL/6 mice were transduced with Ad-ZFAND3 or a control Ad vector. The expression levels of gluconeogenic genes (such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) were significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than in those transduced with the control Ad vector. The expression of hepatocyte nuclear factor 4α, a liver-specific transcription factor that induces the expression of gluconeogenesis genes,20,27) was significantly decreased in primary mouse hepatocytes transduced with Ad-ZFAND3. Furthermore, cAMP-induced glucose production was significantly lower in Ad-ZFAND3-transduced cells than in control Ad vector-transduced cells. These results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, thus serving as a potential novel therapeutic target for T2DM.
3.2. Lipoprotein Lipase (LPL)Adipose tissues store excess energy in the form of triglycerides and play an important role in energy homeostasis; however, adipose tissue dysfunction leads to the accumulation of triglycerides in nonadipose tissues as ectopic fat.28) Ectopic lipid accumulation in the liver is strongly associated with the development of hepatic insulin resistance, leading to abnormal glucose and lipid metabolism.29,30)
LPL has a major role in lipoprotein metabolism and hydrolyzes triglycerides in chylomicrons and very-low-density lipoproteins into glycerol and fatty acids.31,32) LPL is mainly expressed in adipose tissue and muscle, whereas it is barely expressed in the normal adult liver.32,33) We investigated whether liver-specific overexpression of LPL—using an LPL-expressing Ad-E4-122aT vector (Ad-LPL)—suppresses hepatic lipid accumulation and improves metabolism.34)
C57BL/6 mice were treated with Ad-LPL or control Ad vector and simultaneously fed a high-fat diet (HFD). Two weeks after the administration of Ad vectors, we performed a histopathological examination of the liver sections using hematoxylin–eosin (H&E) staining. Lipid droplet formation was considerably decreased in Ad-LPL-treated mice compared to that in control Ad vector-treated mice (Fig. 4A). Oil Red O staining of liver sections also revealed a lower number of lipid droplets in Ad-LPL-treated mice than in control Ad vector-treated mice (Fig. 4B). These results indicate that LPL overexpression in the liver suppresses hepatic lipid accumulation.
C57BL/6 mice were intravenously treated with Ad vectors through the tail vein and simultaneously fed HFD. PBS-treated mice were fed normal diet (ND) throughout the experimental period. Liver sections were obtained 2 weeks after administration of Ad-LPL, control Ad vector, or PBS and stained with (A) H&E or (B) Oil red O. Arrowheads indicate lipid droplets. Scale bar, 50 µm. (C) Fasting blood levels, (D) fasting insulin levels, and (E) HOMA-IR of mice 2 weeks after administration of Ad-LPL, control Ad vector, or PBS. The data are expressed as the mean ± standard error of the mean (S.E.M., n = 4–5). * p < 0.05 in comparison with control Ad vector. normal diet; HFD, high-fat diet. These figures are modified from Shimizu et al., PLOS ONE, 17, e0274297 (2022).
To determine the effect of LPL overexpression on glucose metabolism, we determined fasting blood glucose levels. Fasting blood glucose levels were considerably lower in Ad-LPL-treated mice than in control Ad vector-treated mice (Fig. 4C). Fasting serum insulin levels were considerably lower in Ad-LPL-treated mice than in the control Ad vector-treated mice (Fig. 4D). Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), an index of insulin resistance, was also considerably lower in mice administered Ad-LPL than in those administered the control Ad vector (Fig. 4E). These results suggest that LPL overexpression in the liver improves hepatic glucose metabolism and insulin resistance.
Hepatic LPL overexpression in HFD-fed mice was expected to alter gene expression in the liver. LPL hydrolyze triglyceride into fatty acids and glycerol. Fatty acids are degraded through oxidation. We focused on fatty acid oxidation-related genes in the mouse livers. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7–2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad vector-treated mouse livers. As mitochondria play a crucial role in fatty acid oxidation, we examined the mRNA and protein levels of citrate synthase, a marker of mitochondrial content. The levels of citrate synthase mRNA and protein in Ad-LPL-treated mouse livers were higher than those in control Ad vector-treated mouse livers. Transmission electron microscopy analysis, followed by subsequent measurement of the area of individual mitochondria, revealed a 2.3-fold smaller mitochondrial area in the control Ad vector-treated mouse livers than in the Ad-LPL-treated mouse livers. These results suggest that hepatic LPL overexpression partially maintains mitochondrial content. These findings may enable the development of new drugs for the treatment of lifestyle-related diseases such as T2DM and MASLD/NAFLD.
3.3. Lysophospholipid Acyltransferase 10 (LPLAT10)Recently, in addition to the amount of lipids, the types of lipids in the body have been investigated to elucidate the pathology of lifestyle-related diseases. Phospholipids, which are the major components of cellular membranes, consist of two fatty acids and one polar head group linked to a glycerol backbone. Differences in fatty acids, such as carbon chain length, double-bond numbers, and positions of double bonds, lead to different biological functions and diseases including T2DM.35,36) Fatty acids in phospholipids are cleaved by phospholipase A1/2 to form lysophospholipids, which are subsequently remodeled back to phospholipids by LPLATs37,38) (Fig. 5). LPLATs are involved in various biological processes and pathological conditions.39,40) LPLAT10, also called lysophosphatidylcholine acyltransferase 4 (LPCAT4) and lysophosphatidylethanolamine acyltransferase 2 (LPEAT2), plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. We investigated whether overexpression of LPLAT10 in the liver using an LPLAT10-expressing Ad-E4-122aT vector (Ad-LPLAT10) ameliorates abnormalities in glucose metabolism and hepatic lipid accumulation and improves metabolism.41)
To explore the effects of LPLAT10 on glucose metabolism, glucose tolerance assays were performed. Glucose levels were significantly lower in Ad-LPLAT10-treated mice than in the control Ad vector-treated mice. Insulin secretion levels after glucose loading in Ad-LPLAT10-treated mice were higher than those in the control Ad vector-treated mice. These results indicate that the liver-specific overexpression of LPLAT10 suppresses postprandial hyperglycemia by increasing postprandial insulin secretion.
Next, we attempted to elucidate the mechanism by which LPLAT10 overexpression in the liver increased postprandial insulin secretion from pancreatic β cells. We hypothesized that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) altered in the liver by LPLAT10 are secreted into the blood and act as humoral factors in the pancreas. First, PC and LPC species in the liver and serum were examined using LC-tandem mass spectrometry. Hepatic and serum levels of PC40 : 7, containing C18 : 1 and C22 : 6, increased in Ad-LPLAT10-treated mice. Then, we cultured MIN6 cells, a mouse insulinoma cell line, in a medium containing the serum of Ad-LPLAT10- or control Ad vector-treated mice and measured the amount of insulin secretion from MIN6 cells under high glucose stimulation. Sera from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion from mouse insulinoma MIN6 cells. MIN6 cells were cultured in a medium containing oleic acid (OA, C18 : 1) or docosahexaenoic acid (DHA, C22 : 6), a component of PC40 : 7, and the amount of insulin secreted from MIN6 cells was measured. Insulin secretion in MIN6 cells treated with OA did not significantly increase compared to that in control cells under both low- and high-glucose stimulation. DHA treatment significantly increased insulin secretion in MIN6 cells under high glucose stimulation. Finally, insulin secretion in primary mouse pancreatic islets treated with OA did not increase significantly compared to that in control cells, whereas insulin secretion in DHA-treated islets was 2-fold higher than that in control islets under high glucose conditions. These results indicate that changes in hepatic PC species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Thus, LPLAT10 is a potential novel therapeutic target for T2DM.
3.4. CranberryObesity is characterized by abnormal or excessive lipid accumulation, leading to the development of lifestyle-related diseases. Oxidative stress is increased in obesity42) and plays a critical role in the pathogenesis of lifestyle-related diseases; antioxidants are regarded as suitable agents for preventing metabolic syndrome.
Cranberries (Vaccinium oxycoccos L.) (Ericaceae) contain vitamins E and C and large amounts of phenolic polyphenols, including proanthocyanidins and anthocyanins, which exhibit antioxidative activity.43,44) Notably, the antioxidative activity of proanthocyanidins is considerably more potent than that of vitamin E or C,45) and cranberries contain markedly high levels of proanthocyanidins. We investigated the effects of polyphenol-rich cranberries on obesity-associated metabolic abnormalities over time in HFD-fed C57BL/6 mice.46)
To examine whether cranberry extract suppresses oxidative stress, liver sections were stained with an antibody against nitroguanosine, a marker of oxidative stress. We observed that oxidative stress was reduced in mice that were fed a HFD supplemented with 1 and 5% cranberry powder compared to HFD-fed control mice (Fig. 6A).
Mouse livers were collected at 8 weeks after beginning the diets. (A) Frozen sections were stained with anti-nitroguanosine antibody. (B) Liver sections were stained with H&E. Scale bar, 50 µm. HFD + CB1, HFD supplemented with 1% cranberry power; HFD + CB5, HFD supplemented with 5% cranberry power. These figures are modified from Shimizu et al., Biol. Bull. Biol., 42, 1295–1302 (2019).
We investigated the effects of cranberries on glucose and lipid metabolism. First, fasting glucose levels did not differ significantly between the HFD-fed and cranberry-supplemented-HFD-fed groups. Second, serum triglyceride levels were significantly lower in cranberry-supplemented-HFD-fed mice than in HFD-fed mice at 1 and 2 weeks after beginning the diet. Third, histological analysis revealed that adipocytes in epididymal adipose tissue in cranberry-supplemented-HFD-fed mice were markedly smaller than those in epididymal adipose tissue in HFD-fed mice 1 and 2 weeks after starting the diets. Fourth, histological analysis of the liver revealed that lipid droplet formation and hepatocyte ballooning, which are key MASLD/NAFLD characteristics, were both drastically decreased in cranberry-supplemented-HFD-fed mice relative to those in HFD-fed control mice (Fig. 6B). These results suggest that cranberry ameliorates HFD-induced metabolic disturbances and has considerable potential in preventing the progression of MASLD/NAFLD.
3.5. CalamondinThe citrus fruit Calamondin (Citrus mitis, Citrus microcarpa, or Citrus manurensis), a citrus cultivar, is a hybrid between Citrus reticulata Blanco and Fortunella spp.,47,48) and is widely grown in tropical and subtropical areas, including Taiwan, China, the Philippines, Vietnam, and Malaysia.49) Calamondin contains nobiletin and hesperidin, which are involved in the improvement of lipid metabolism,50–53) and vitamin C, which is an antioxidant.48,54,55) We investigated the effects of calamondin supplementation on obesity-associated metabolic abnormalities, including those in glucose and lipid metabolism, in HFD-fed C57BL/6 mice.56)
To examine glucose tolerance, we performed a glucose tolerance test four weeks after the initiation of the diet. The glucose tolerance of calomondin-supplemented HFD-fed mice improved compared to that of HFD-fed mice. Fasting insulin levels in the calamondin-fed mice were significantly lower than those in the HFD-fed mice. These results suggest that calamondin supplementation of HFD improves insulin sensitivity, leading to an improvement in glucose tolerance.
We evaluated the effects of calamondin on lipid metabolism. Serum triglyceride levels were significantly lower in calamondin-supplemented HFD-fed mice than in HFD-fed mice 2 weeks after initiating the diet. Histological analysis revealed lower lipid accumulation in the livers of calamondin-supplemented HFD-fed mice compared to that in the livers of HFD-fed mice. Hepatocyte ballooning and large lipid droplets were observed in HFD-fed mice after 4 weeks; however, these were nearly absent in calamondin-supplemented HFD-fed mice. The transcript-level expression of CD36, which plays a role in fatty acid uptake, was significantly lower in calamondin-supplemented HFD-fed mice than in untreated HFD-fed mice. These results suggest that calamondin suppresses hepatic lipid uptake, leading to reduced lipid accumulation in the liver.
The practical application of gene therapy for lifestyle-related diseases is expected to greatly improve the QOL of patients, not only through therapeutic effects but also by reducing the frequency of administration. Therefore, the development of gene therapies for lifestyle-related diseases is highly desirable. In a series of studies, we developed an improved Ad vector, Ad-E4-122aT, which exhibited higher and longer-term transgene expression and lower hepatotoxicity than a conventional Ad vector. We also elucidated the mechanisms of Ad vector-induced hepatotoxicity during the early phase using Ad-E4-122aT; Ad vector-induced IL-6 caused the leaky expression of Ad genes in the liver, leading to hepatotoxicity during the early phase after systemic administration. Next, we examined the therapeutic effects of ZFAND3, LPL, and LPLAT10 on lifestyle-related diseases using Ad-E4-122aT. Overexpression of ZFAND3, a susceptibility locus for T2DM, improves glucose tolerance and insulin resistance. Liver-specific overexpression of LPL and hydrolysis of triglycerides suppress lipid accumulation in the liver and improve glucose metabolism. Liver-specific overexpression of LPLAT10, which converts LPC into PC in the liver, suppresses postprandial hyperglycemia by increasing glucose-stimulated insulin secretion. In addition, we examined the effects of food consumption on lifestyle-related diseases for advancing the research on the pathophysiology and treatment of lifestyle-related diseases. Cranberries reduce oxidative stress in the liver and attenuate. MASLD/NAFLD progression Calamondin improves glucose tolerance and attenuates lipid accumulation. Our findings will lead to the development of new treatments, including gene therapy, for lifestyle-related diseases, such as T2DM and MASLD/NAFLD.
I would like to express my deepest gratitude to Dr. Toru Nishinaka, Dr. Tomoyuki Terada, and Dr. Koji Tomita of Osaka Ohtani University; Dr. Hiroyuki Mizuguchi and Dr. Fuminori Sakurai of Osaka University; and Dr. Hideo Shindou of National Center for Global Health and Medicine for their support and guidance. I am deeply grateful to Dr. Shotaro Michinaga of Meiji Pharmaceutical University and my students at Osaka Ohtani University for their assistance with the experiments. I would also like to thank my family members for their support.
This study was supported by JSPS KAKENHI (Grant Numbers: JP15K18939, JP18K14964, and 21K06680) and the Osaka Ohtani University Research Foundation. The author was a research fellow at the Japan Society for the Promotion of Science (12J01416).
The author declares no conflict of interest.
This review of the author’s work was written by the author upon receiving the 2023 Pharmaceutical Society of Japan Incentive Award for Women Scientists.
