Otsuka Long-Evans Tokushima Fatty Rats Research Articles

NBF2, an Algal Fiber-Rich Formula, Reverses Diabetic Dyslipidemia and Hyperglycemia In Vivo.

Ulva prolifera, known as Aonori in Japan, is an edible alga species that is mass-cultivated in Japan. Supplementation with Aonori-derived biomaterials has been reported to enhance metabolic health in previous studies. This was an experimental study that evaluated the metabolic health effects of NBF2, a formula made of algal and junos Tanaka citrus-derived biomaterials, on obesity and type 2 diabetes (T2DM). We used 18 obese and hyperglycemic Otsuka Long-Evans Tokushima Fatty (OLETF) rats that were assigned randomly to three groups of six animals: a high-dose NBF2 drink (20 mg/kg) group, a low-dose (10 mg/kg) NBF2 drink group and the control group that received 2 mL of tap water daily for a total of six weeks. We also used eight LETO rats as the normal control group. In addition to the glucose tolerance test (OGTT), ELISA and real-time PCR assays were performed. High-dose and lowdose NBF2 improved insulin sensitivity, as well as glycemic and lipid profiles, as compared with control rats. The OGTT showed that both NBF2 groups and LETO rats had normalized glycemia by the 90-min time-point. NBF2 up-regulated PPARα/γ-mRNA and Sirt2-mRNA gene expressions in BAT and improved the blood pressure profile. These findings suggest that the NBF2 formula, which activates PPAR-α/γ mRNA and Sirt2-mRNA, may reverse dyslipidemia and hyperglycemia in T2DM.

PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats.

Bladder dysfunction associated with type 2 diabetes mellitus (T2DM) includes urine storage and voiding disorders. We examined pathological conditions of the bladder wall in a rat T2DM model and evaluated the effects of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats were used as the T2DM and control groups, respectively. Tadalafil was orally administered for 12 weeks. Micturition behavior was monitored using metabolic cages, and bladder function was evaluated by cystometry. Bladder blood flow was evaluated by laser speckle imaging, and an organ bath bladder distention test was used to measure adenosine triphosphate (ATP) release from the bladder urothelium. The expression levels of vesicular nucleotide transporter (VNUT), hypoxia markers, pro-inflammatory cytokines and growth factors in the bladder wall were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Bladder wall contractions in response to KCl and carbachol were monitored using bladder-strip tests. With aging, OLETF rats had higher micturition frequency and greater urine volume than LETO rats. Although bladder capacity was not significantly different, non-voiding bladder contraction occurred more frequently in OLETF rats than in LETO rats. Bladder blood flow was decreased and ATP release was increased with higher VNUT expression in OLETF rats than in LETO rats. These effects were suppressed by tadalafil administration, with accompanying decreased HIF-1α, 8-OHdG, IL-6, TNF-α, IGF-1, and bFGF expression. The impaired contractile responses of bladder strips to KCl and carbachol in OLETF rats with aging were restored by tadalafil administration. The T2DM rats had polyuria, increased ATP release induced by decreased bladder blood flow and impaired contractile function. PDE5 inhibition improved these changes and may prevent T2DM-associated urinary frequency and bladder storage and voiding dysfunctions.

The Effects of 1-Kestose on the Abundance of Inflammation-Related Gene mRNA in Adipose Tissue and the Gut Microbiota Composition in Rats Fed a High-Fat Diet.

Chronic inflammation in adipose tissue is thought to contribute to insulin resistance, which involves the gut microbiota. Our previous studies have demonstrated that ingestion of 1-kestose can alter the gut microbiota composition, increase cecal butyrate levels, and improve insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Additionally, we found that 1-kestose supplementation ameliorated insulin resistance in obese rat models fed a high-fat diet (HFD), although the effects of 1-kestose on the abundance of inflammation-related gene in adipose tissue and gut microbiota composition in these rats were not explored. This study aimed to investigate the impact of 1-kestose on these parameters in HFD-fed rats, compared to OLETF rats. Male Sprague-Dawley rats were divided into two dietary groups, control or HFD, for 19 wk. Each group was further subdivided to receive either tap water or tap water supplemented with 2% (w/v) 1-kestose throughout the study. We evaluated gene expression in adipose tissue, as well as short-chain fatty acids (SCFAs) levels and microbial composition in the cecum contents. 1-Kestose intake restored the increased relative abundance of tumor necrosis factor (Tnf) mRNA in adipose tissue and the reduced level of butyrate in the cecum contents of HFD-fed rats to those observed in control diet-fed rats. Additionally, 1-kestose consumption changed the composition of the gut microbiota, increasing Butyricicoccus spp., decreasing UGC-005 and Streptococcus spp., in the cecum contents of HFD-fed rats. Our findings suggest that 1-kestose supplementation reduces adipose tissue inflammation and increases butyrate levels in the gut of HFD-fed rats, associated with changes in the gut microbiota composition, distinct from those seen in OLETF rats.

The effects of resistance training on cardiovascular factors and anti-inflammation in diabetic rats

Diabetes induces a range of macrovascular and microvascular changes, which lead to significant clinical complications. Although many studies have tried to solve the diabetic problem using drugs, it remains unclear. In this study, we investigated whether resistance exercise affects cardiovascular factors and inflammatory markers in diabetes. The study subjected Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which have genetically induced diabetes mellitus, to a resistance exercise program for 12 weeks and assessed the levels of cardiovascular factors and inflammatory markers using western blotting analysis, ELISA, and immunohistochemistry. During the training period, OLETF + exercise (EX) group exhibited lower body weight and reduced glucose levels when compared with OLETF group. Western blotting analysis, ELISA, and immunohistochemistry revealed that the levels of PAI-1, VACM-1, ICAM-1, E-selectin, TGF-β, CRP, IL-6, and TNF-α were decreased in OLETF + EX group when compared with the OLETF group. Moreover, the anti-inflammatory markers, IL-4 and IL-10, were highly expressed after exercise. Therefore, these results indicate that exercise may influence the regulation of cardiovascular factors and inflammatory markers, as well as help patients with metabolic syndromes regulate inflammation and cardiovascular function.

Ezetimibe, Niemann-Pick C1 like 1 inhibitor, modulates hepatic phospholipid metabolism to alleviate fat accumulation.

Ezetimibe, which lowers cholesterol by blocking the intestinal cholesterol transporter Niemann-Pick C1 like 1, is reported to reduce hepatic steatosis in humans and animals. Here, we demonstrate the changes in hepatic metabolites and lipids and explain the underlying mechanism of ezetimibe in hepatic steatosis. We fed Otsuka Long-Evans Tokushima Fatty (OLETF) rats a high-fat diet (60kcal % fat) with or vehicle (control) or ezetimibe (10mgkg-1) via stomach gavage for 12 weeks and performed comprehensive metabolomic and lipidomic profiling of liver tissue. We used rat liver tissues, HepG2 hepatoma cell lines, and siRNA to explore the underlying mechanism. In OLETF rats on a high-fat diet, ezetimibe showed improvements in metabolic parameters and reduction in hepatic fat accumulation. The comprehensive metabolomic and lipidomic profiling revealed significant changes in phospholipids, particularly phosphatidylcholines (PC), and alterations in the fatty acyl-chain composition in hepatic PCs. Further analyses involving gene expression and triglyceride assessments in rat liver tissues, HepG2 hepatoma cell lines, and siRNA experiments unveiled that ezetimibe's mechanism involves the upregulation of key phospholipid biosynthesis genes, CTP:phosphocholine cytidylyltransferase alpha and phosphatidylethanolamine N-methyl-transferase, and the phospholipid remodeling gene lysophosphatidylcholine acyltransferase 3. This study demonstrate that ezetimibe improves metabolic parameters and reduces hepatic fat accumulation by influencing the composition and levels of phospholipids, specifically phosphatidylcholines, and by upregulating genes related to phospholipid biosynthesis and remodeling. These findings provide valuable insights into the molecular pathways through which ezetimibe mitigates hepatic fat accumulation, emphasizing the role of phospholipid metabolism.

Detraining after short-term exercise induces hyperphagia and obesity with fatty liver and brown adipose tissue whitening in young male OLETF rats.

This study examined the effects of exercise and detraining at a young age on fat accumulation in various organs. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were assigned to either the non-exercise sedentary (OLETF Sed) or exercise groups. The exercise group was subdivided into two groups: exercise between 4 and 12 weeks of age (OLETF Ex) and exercise between 4 and 6 weeks of age followed by non-exercise between 6 and 12 weeks of age (OLETF DT). Body weight was significantly lower in the OLETF Ex group than in the OLETF Sed group at 12 weeks of age. Fat accumulation in the epididymal white adipose tissue, liver, and brown adipose tissue was suppressed in the OLETF Ex group. During the exercise period, body weight and food intake in the OLETF DT group were significantly lower than those in the OLETF Sed group. However, food intake was significantly higher in the OLETF DT group than in the OLETF Sed group after exercise cessation, resulting in extreme obesity with fatty liver and brown adipose tissue whitening. Detraining after early-onset exercise promotes hyperphagia, causing extreme obesity. Overeating should be avoided during detraining periods in cases of exercise cessation at a young age.

Pseudocannabinoid H4CBD Reduces Cardiac Damage Indicator 4HNE in OLETF (Otsuka Long Evans Tokushima Fatty) Rats

Cardiovascular disease (CVD) is a leading cause of death globally and MetS is an associated precursor of CVD. Of the 6 known cluster factor conditions of MetS, obesity, and poor glucose tolerance are known to contribute to high lipid peroxidation, which can contribute to the generation of damaging free radical species and a pro-oxidant system. Cannabidiol (CBD) is a known antioxidant and a growing understanding of CBD analogs and pseudocannabinoids, like H4CBD, implicate similar antioxidant effects. However, little is known about the effects of CBD analogs on MetS-associated cardiac tissue damage. To better understand these effects during extreme metabolic dysfunction, 41-weeks-old animals were assigned into three groups, (n=8/group): (1) one-lean-strain control, Long Evans Tokushima Otsuka (LETO), (2) untreated OLETF, and (3) OLETF + H4CBD (200 mg/kg/day x 4 weeks). Our hypothesis focuses on the prospect that ongoing treatment with H4CBD in a MetS model using Otsuka Long-Evans Tokushima Fatty (OLETF) rats will reduce indicators of lipid peroxidation including 4-hydroxynonenal (4HNE), a toxic end product of lipid peroxidation. After the 4-week treatment, cardiac tissue was analyzed for 4-hydroxynonenal (4HNE) protein expression. We found that H4CBD decreased cardiac 4HNE protein expression in treated OLETF rats by 68% (p<0.05) compared to OLETF control, which indicates H4CBD attenuated cardiac lipid peroxidation overall. Although the 4-week treatment of H4CBD was not suffcient to ameliorate MetS-associated hypertension, the reduction of 4HNE suggests that the attenuation of cardiac damage is independent of arterial pressure. These data indicate that H4CBD and its derivatives hold promise for therapeutic use in managing CVD and related metabolic syndrome-associated pathologies. Undergraduate Research Training Initiative for Student Enhancement (U-RISE), Center for Medical Cannabis Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exogenous T4 Increased Cardiac Thioredoxin Protein Expression in Insulin Resistant OLETF Rats

Thioredoxin (Trx) is a protein that regulates cellular signaling and antioxidant defense. Cellular antioxidant mechanisms are designed to protect cells from oxidative damage caused by inappropriately elevated reactive oxygen species (ROS) and other free radicals. Oxidative damage can lead to cellular damage and impaired function. Diabetic cardiomyopathy (CDM) is associated with impaired redox balance and oxidative injury. Altered fuel metabolism is a key feature of CDM characterized by an imbalance in fatty acid and glucose utilization, favoring fatty acid overutilization. Excessive fatty acid beta-oxidation in the mitochondria leads to generation of free radicals including superoxide, which can contribute to oxidative injury. Thyroid hormones regulate substrate utilization and their dysregulation may contribute to oxidative injury. Therefore, we hypothesized that exogenous thyroxine (exoT4) upregulates Trx protein expression and ameliorate oxidative injury in the hearts of insulin resistant rats. Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exoT4 on heart Trx, oxidative damage, and glucose metabolism. Rats were assigned to four groups: (1) lean, control Long Evans Tokushima Otsuka (LETO; n=6), (2) LETO + T4 (8 μg/100g BM/d × 5 wks; n=7), (3) untreated, insulin resistant OLETF (n=6), and (4) OLETF + T4 (n=7). OLETF group displayed a 31% decreased in TRX compared to Leto. T4 increased Trx protein expression by 88% in OLTEF compared to untreated OLETF. This data suggests that exoT4 has the potential to upregulate cardiac Trx-dependent antioxidant mechanisms in insulin resistant OLETF rats to ameliorate oxidative injury and improve cardiac glucose metabolism. This work is supported by the American Heart Association and the Undergraduate Research Opportunity Center (UROC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alteration of Sweet and Bitter Taste Sensitivity with Development of Glucose Intolerance in Non-insulin-Dependent Diabetes Mellitus Model OLETF Rats.

Patients with diabetes exhibit altered taste sensitivity, but its details have not been clarified yet. Here, we examined alteration of sweet taste sensitivity with development of glucose intolerance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model of non-insulin-dependent diabetes mellitus. Compared to the cases of Long Evans Tokushima Otsuka (LETO) rats as a control, glucose tolerance of OLETF rats decreased with aging, resulting in development of diabetes at 36-weeks-old. In brief-access tests with a mixture of sucrose and quinine hydrochloride, OLETF rats at 25 or more-weeks-old seemed to exhibit lower sweet taste sensitivity than age-matched LETO ones, but the lick ratios of LETO, but not OLETF, rats for the mixture and quinine hydrochloride solutions decreased and increased, respectively, aging-dependently. Expression of sweet taste receptors, T1R2 and T1R3, in circumvallate papillae (CP) was almost the same in LETO and OLETF rats at 10- and 40-weeks-old, while expression levels of a bitter taste receptor, T2R16, were greater in 40-weeks-old rats than in 10-weeks-old ones in both strains. There was no apparent morphological alteration in taste buds in CP between 10- and 40-weeks-old LETO and OLETF rats. Metagenomic analysis of gut microbiota revealed strain- and aging-dependent alteration of mucus layer-regulatory microbiota. Collectively, we concluded that the apparent higher sweet taste sensitivity in 25 or more-weeks-old OLETF rats than in age-matched LETO rats was due to the aging-dependent increase of bitter taste sensitivity in LETO rats with alteration of the gut microbiota.

Cholesterol-Lowering and Antioxidant Effects of Pork-Liver Protein Hydrolysate in Otsuka Long-Evans Tokushima Fatty Rats.

In this study, we investigated the effects of a porcine liver protein hydrolysate (PLH) diet on lipid metabolism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type II diabetes. OLETF rats (20-wk-old males) were pair-fed with either a PLH diet containing 20% PLH or a casein diet for 14 wk. Dietary PLH significantly lowered serum cholesterol and phospholipid concentrations, mainly by decreasing low-density lipoprotein and high-density lipoprotein fractions. Fecal cholesterol was significantly increased in the PLH diet group; however, the total bile acid concentration in the feces was not significantly different between the groups. In addition, the PLH diet significantly decreased serum thiobarbituric acid reactive substance concentrations. These results suggest that dietary PLH exerts hyperlipidemic and antioxidant effects, indicating that it is a novel functional food ingredient.

Effect of Anagliptin on Vascular Injury in the Femoral Artery of Type 2 Diabetic Rats.

Patients with diabetes mellitus (DM) often experience complications such as peripheral arterial disease (PAD), which is thought to be caused by vascular damage resulting from increased oxidative stress. Dipeptidyl peptidase-4 inhibitors have been reported to reduce oxidative stress, although the exact mechanism remains unclear. This study aimed to investigate the impact of long-term (6 weeks) anagliptin treatment at a dose of 200 mg/kg/d against oxidative stress in the femoral artery of Otsuka Long-Evans Tokushima Fatty (OLETF) rats using a well-established animal model for type 2 DM. Serum toxic advanced glycation end-products concentrations and blood glucose levels after glucose loading were significantly elevated in OLETF rats compared to Long-Evans Tokushima Otsuka (LETO) rats but were significantly suppressed by anagliptin administration. Plasma glucagon-like peptide-1 concentrations after glucose loading were significantly increased in anagliptin-treated rats. Superoxide production and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in femoral arteries were significantly increased in OLETF rats compared to LETO rats but were significantly decreased by anagliptin administration. The expressions of NADPH oxidase components (p22phox in the intima region and p22phox and gp91phox in the media region) in the femoral artery were significantly increased in OLETF rats compared to LETO rats but were significantly suppressed by anagliptin administration. Furthermore, the femoral artery showed increased wall thickness in OLETF rats compared to LETO rats, but anagliptin administration reduced the thickening. This study suggests that long-term anagliptin administration can reduce oxidative stress in femoral arteries and improve vascular injury.

Human omentin-1 reduces vascular insulin resistance and hypertension in Otsuka Long-Evans Tokushima Fatty rats.

Hypertension is one of the major risk factors for renal failure and cardiovascular diseases, and is caused by various abnormalities including the contractility of blood vessels. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which mimic human type 2 diabetes, are frequently used to study obesity-induced insulin resistance (IR) and hypertension. Human omentin-1 is one of the recently identified adipocytokines. We previously demonstrated that human omentin-1 not only caused vasodilation in rat isolated blood vessels, but also prevented inflammatory responses, a possible mechanism relating IR, in human vascular endothelial cells. Taken together, we hypothesized that human omentin-1 may reduce obesity-induced IR and hypertension in OLETF rats. OLETF rats were intraperitoneally administered with human omentin-1 for 7days. Human omentin-1 had no influence on overweight, hyperglycemia, urinary glucose extraction, hyperinsulinemia, and systemic IR in OLETF rats. Human omentin-1 decreased systolic blood pressure in OLETF rats. The measurement of isometric contraction revealed that human omentin-1 had no influence on the agonist-induced contractile and relaxant responses in isolated thoracic aorta from OLETF rats. However, the relaxant response mediated by human insulin was converted into the contractile response in thoracic aorta from OLETF rats, which was prevented by human omentin-1. The Western blotting revealed that human omentin-1 improved the decrease in endothelial nitric oxide synthase activation in isolated thoracic aorta from OLETF rats. In summary, we for the first time revealed that human omentin-1 partly reduces vascular IR and thereby inhibits hypertension in OLETF rats.

Pseudocannabinoid H4CBD improves glucose response during advanced metabolic syndrome in OLETF rats independent of increase in insulin signaling proteins.

Cannabidiol (CBD) use has grown exponentially more popular in the last two decades, particularly among older adults (>55 yr), though very little is known about the effects of CBD use during age-associated metabolic dysfunction. In addition, synthetic analogues of CBD have generated great interest because they can offer a chemically pure product, which is free of plant-associated contaminants. To assess the effects of a synthetic analogue of CBD (H4CBD) on advanced metabolic dysfunction, a cohort of 41-wk-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were administered 200 mg H4CBD/kg by oral gavage for 4 wk. Animals were fed ad libitum and monitored alongside vehicle-treated OLETF and Long-Evans Tokushima Otsuka (LETO) rats, the lean-strain controls. An oral glucose-tolerance test (oGTT) was performed after 4 wk of treatment. When compared with vehicle-treated, OLETF rats, H4CBD decreased body mass (BM) by 15%, which was attributed to a significant loss in abdominal fat. H4CBD reduced glucose response (AUCglucose) by 29% (P < 0.001) and insulin resistance index (IRI) by 25% (P < 0.05) compared with OLETF rats. However, H4CBD did not statically reduce fasting blood glucose or plasma insulin, despite compensatory increases in skeletal muscle native insulin receptor (IR) protein expression (54%; P < 0.05). H4CBD reduced circulating adiponectin (40%; P < 0.05) and leptin (47%; P < 0.05) and increased ghrelin (75%; P < 0.01) compared with OLETF. Taken together, a chronic, high dose of H4CBD may improve glucose response, independent of static changes in insulin signaling, and these effects are likely a benefit of the profound loss of visceral adiposity.NEW & NOTEWORTHY Cannabis product use has grown in the last two decades despite the lack of research on Cannabidiol (CBD)-mediated effects on metabolism. Here, we provide seminal data on CBD effects during age-associated metabolic dysfunction. We gave 41-wk-old OLETF rats 200 mg H4CBD/kg by mouth for 4 wk and noted a high dose of H4CBD may improve glucose response, independent of static changes in insulin signaling, and these effects are likely a benefit of loss of visceral adiposity.

Reduced Striatal Dopamine Transporter Availability and Heightened Response to Natural and Pharmacological Stimulation in CCK-1R-Deficient Obese Rats.

Alterations in dopamine neurotransmission are associated with obesity and food preferences. Otsuka Long-Evans Tokushima Fatty (OLETF) rats that lack functional cholecystokinin receptor type-1 (CCK-1R), due to a natural mutation, exhibit impaired satiation, are hyperphagic, and become obese. In addition, compared to lean control Long-Evans Tokushima (LETO) rats, OLETF rats have pronounced avidity for over-consuming palatable sweet solutions, have greater dopamine release to psychostimulants, reduced dopamine 2 receptor (D2R) binding, and exhibit increased sensitivity to sucrose reward. This supports altered dopamine function in this strain and its general preference for palatable solutions such as sucrose. In this study, we examined the relationship between OLETF's hyperphagic behavior and striatal dopamine signaling by investigating basal and amphetamine stimulated motor activity in prediabetic OLETF rats before and after access to sucrose solution (0.3 M) compared to non-mutant control LETO rats, as well as availability of dopamine transporter (DAT) using autoradiography. In the sucrose tests, one group of OLETF rats received ad libitum access to sucrose while the other group received an amount of sucrose equal to that consumed by the LETO. OLETFs with ad libitum access consumed significantly more sucrose than LETOs. Sucrose exerted a biphasic effect on basal activity in both strains, i.e., reduced activity for 1 week followed by increased activity in weeks 2 and 3. Basal locomotor activity was reduced (-17%) in OLETFs prior to sucrose, compared to LETOs. Withdrawal of sucrose resulted in increased locomotor activity in both strains. The magnitude of this effect was greater in OLETFs and the activity was increased in restricted compared to ad-libitum-access OLETFs. Sucrose access augmented AMPH-responses in both strains with a greater sensitization to AMPH during week 1, an effect that was a function of the amount of sucrose consumed. One week of sucrose withdrawal sensitized AMPH-induced ambulatory activity in both strains. In OLETF with restricted access to sucrose, withdrawal resulted in no further sensitization to AMPH. DAT availability in the nucleus accumbens shell was significantly reduced in OLETF compared with aged-matched LETO. Together, these findings show that OLETF rats have reduced basal DA transmission and a heightened response to natural and pharmacological stimulation.

Adenosine monophosphate deaminase in the endoplasmic reticulum-mitochondria interface promotes mitochondrial Ca2+ overload in type 2 diabetes rat hearts.

We previously showed that upregulation of myocardial adenosine monophosphate deaminase (AMPD) is associated with pressure overload-induced diastolic dysfunction in type 2 diabetes hearts. Here, we examined involvement of AMPD localized in the endoplasmic reticulum-mitochondria interface in mitochondrial Ca2+ overload and its pathological significance. We used type 2 diabetes Otsuka Long-Evans Tokushima Fatty rats (OLETF) and non-diabetes Long-Evans Tokushima Otsuka Fatty rats (LETO) as well as AMPD3-overexpressing H9c2 cells and human embryonic kidney 293 cells. OLETF, but not LETO, showed diastolic dysfunction under the condition of phenylephrine-induced pressure overload. The levels of 90-kDa AMPD3 in outer mitochondrial membranes/endoplasmic reticulum and mitochondria-associated endoplasmic reticulum membrane (MAM) fractions were significantly higher in OLETF than in LETO. The area of the MAM quantified by electron microscopic analysis was 57% larger, mitochondrial Ca2+ level under the condition of pressure overload was 47% higher and Ca2+ retention capacity in MAM-containing crude mitochondria isolated before the pressure overloading was 21% lower in OLETF than in LETO (all P-values <0.05). Transfection of FLAG-AMPD3 in cells resulted in significant enlargement of the MAM area, and impairment in pyruvate/malate-driven adenosine triphosphate-stimulated and uncoupler-stimulated mitochondrial respiration compared with those in control cells. The findings suggest that 90-kDa AMPD3 localized in the endoplasmic reticulum-mitochondria interface promotes formation of the MAM, inducing mitochondrial Ca2+ overload and dysfunction in type 2 diabetes hearts.

Alcohol-induced increase in BMP levels promotes fatty liver disease in male prediabetic stage Otsuka Long-Evans Tokushima Fatty rats.

Alcohol consumption exacerbates liver abnormalities in animal models, but whether it increases the severity of liver disease in early diabetic or prediabetic rats is unclear. To investigate the molecular mechanisms underlying alcohol-induced liver steatosis or hepatitis, we used a prediabetic animal model. Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Fatty (LETO) rats were pair-fed with an ethanol-containing liquid diet for 6 weeks. Compared with controls, OLETF and LETO rats displayed more pronounced liver steatosis and higher plasma levels of serum glutamic oxaloacetic transaminase(SGOT)andserumglutamatepyruvatetransaminase(SPGT), indicating liver injury. Ethanol-fed LETO (Pd-L-E) rats showed mild liver steatosis and no inflammation compared with ethanol-fed OLETF (Pd-O-E) rats. Although precursor and active SREBP-1 levels in the liver of ethanol-fed OLETF rats significantly increased compared with control diet-fed OLETF rats (Pd-O-C), those of Pd-L-E rats did not. Bone morphogenetic protein (BMP) and TGF-β1 balance in Pd-O-E rats was significantly altered because BMP signaling was upregulated by inducing BMP2, BMP4, BMP7, BMP9, Smad1, and Smad4, whereas TGF-β1, Smad3, and Erk were downregulated. Activation of TGF-β/Smad signaling inhibited BMP2 and BMP9 expression and increased epithelial-mesenchymal transition (EMT) marker levels (Hepcidin, Snail, and Twist) in the liver of LETO rats. Livers of ethanol-fed OLETF rats showed increased levels of vimentin, FSP-1, α-SMA, MMP1, MMP13, and collagen III compared with rats of other groups, whereas EMT marker levels did not change. Thus, BMP exerted anti- and/or pro-fibrotic effects in ethanol-fed rats. Therefore, BMP and TGF-β, two key members of the TGF-β superfamily, play important but diverse roles in liver steatosis in young LETO and OLETF rats.

Downregulation of extramitochondrial BCKDH and its uncoupling from AMP deaminase in type 2 diabetic OLETF rat hearts.

Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

Mumefural prevents insulin resistance and amyloid-beta accumulation in the brain by improving lowered interstitial fluid pH in type 2 diabetes mellitus.

The present study tried to clarify if mumefural would prevent hyperglycemia, one of the typical symptoms of type 2 diabetes mellitus (T2DM), since mumefural is an extract from Japanese apricots preventing hyperglycemia. To clarify if mumefural would prevent T2DM pathogenesis, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats, T2DM model. Mumefural diminished hyperglycemia, HOMA-IR and plasma triglyceride concentration in OLETF rats under fasting conditions. In addition, mumefural elevated protein expression of sodium-coupled monocarboxylate transporter 1 (SMCT1) in the distal colon participating in absorption of weak organic acids, which behave as bases but not acids after absorption into the body. Mumefural also increased the interstitial fluid pH around the brain hippocampus lowered in OLETF rats compared with non-T2DM LETO rats used as control for OLETF rats. Amyloid-beta accumulation in the brain decreased in accordance with the pH elevation. On the one hand, mumefural didn't affect plasma concentrations of glucagon, GLP-1, GIP or PYY under fasting conditions. Taken together, these observations indicate that: 1) mumefural would be a useful functional food improving hyperglycemia, insulin resistance and the lowered interstitial fluid pH in T2DM; 2) the interstitial fluid pH would be one of key factors influencing the accumulation of amyloid-beta.

Effects of continuous thiamine intake on onset and progression of type 2 diabetes in leptin-receptor deficient mice

Aside from the COVID-19 pandemic, the obesity and diabetes pandemics have threatened global health. Patients with diabetes are more likely to experience serious complications from COVID-19; thus, preventing obesity-associated diabetes is of paramount important. Furthermore, the development of a method to prevent diabetes and elucidation of its pathology is a currently urgent issue. We previously reported that thiamine plays a key role in suppressing abnormal glycolipid metabolism in Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of obesity-associated diabetes. However, whether thiamine affects only OLETF rats or other animal models including a type 2 diabetes model with a different pathology requires elucidation. In this study, leptin-receptor deficiency mice were used as a model of type 2 diabetes with a different pathology to evaluate the efficacy of thiamine. The mice had free access to water containing 0.2% thiamine for 9 weeks, and the results showed that food and water consumption decreased in db/db-homo mice. Urine output, body weight gains and blood glucose levels decreased in mice that received thiamine. There were 5 mice and 1 mouse with a fasting glucose level of ≥ 300 mg/dL in the db/db-homo control group (n = 10) and db/db-homo thiamine group (n = 10), respectively, suggesting that thiamine intake may suppress an increase in blood glucose levels. The results of the present study suggest that demand for thiamine may exceed the normal range in in vivo mouse models of diabetes and continuous thiamine intake affects diabetes onset and progression.

PS-B03-8: ORAL CANNABIDIOL REDUCES ARTERIAL BLOOD PRESSURE DURING EARLY PHASE METABOLIC SYNDROME IN OLETF RATS

Objective: Cardiovascular disease (CVD) is the leading cause of death in the U.S. and a primary outcome of metabolic syndrome (MetS). MetS is diagnosed by the presentation of at least 3 of the 6 conditions including abdominal obesity, elevated arterial pressure, and glucose intolerance ± insulin resistance. Identifying the root cause of these conditions individually is difficult but developing new treatments, which can ameliorate the multiple conditions of MetS has greater potential to reduce later-onset CVD. The benefits of cannabidiol (CBD) is widely recognized for potent anti-inflammatory and antioxidative properties, which may be cardioprotective during isolated risk factor conditions. However, these effects have not been examined during MetS conditions. Design: A cohort of 14-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were administered 175 mg CBD/kg by oral gavage for 5 weeks (CBD; n = 8). Animals were fed ad libitum and monitored alongside vehicle-treated OLETF (OLETF; n = 8) and vehicle-treated Long-Evans Tokushima Otsuka (LETO) rats, the lean-strain controls (LETO; n = 8). Rats were implanted with biotelemeters to chronically measure arterial pressure [DSI; HD-S10]. An oral glucose tolerance test (oGTT) was performed after 5 weeks of treatment. Results: CBD reduced arterial pressure within the first week of dosing (6 ± 1.4 mmHg or 4.1%; p &lt; 0.05) and, after 5 weeks, by 9.7 ± 1.7 mmHg (6.5%; p &lt; 0.05) from baseline. CBD blunted body mass gain and reduced abdominal adiposity by 39% (p &lt; 0.0001) compared to untreated OLETF. Additionally, glucose absorption (AUCglucose 10 minutes after glucose bolus) was reduced 21% (p &lt; 0.01) and enhanced uptake (slope 60 minutes to 120 minutes after glucose bolus) by 160% (p &lt; 0.05). However, neither glucose response overall nor static fasting blood glucose were improved. On the other hand, insulin response (AUCinsulin) was reduced 77% (p &lt; 0.0001) compared to OLETF, which ultimately reduced calculated insulin resistance index (IRI) of 79% (p &lt; 0.0001). Moreover, CBD treatment reduced fasting plasma insulin by 76% compared to OLETF (p &lt; 0.001). CBD decreased adiposity by 59% (p &lt; 0.0001) associated with reduced plasma leptin. Conclusions: A chronic, high dose of CBD reduced the MetS-associated hypertension and other cluster factors. The renin-angiotensin-aldosterone system is a known driver of hypertension in humans with MetS and in the OLETF rat model and could be directly or indirectly modulated by CBD (analysis on-going). Collectively, CBD could be a novel and effective intervention strategy for early-stage hypertension and other metabolic risk factors associated with MetS.