Control Chow Research Articles

Abstract 18598: Contributions of Alpha-Ketoglutarate Dependent Oncometabolism to HFpEF in Two Mouse Models

Introduction: Switching from fatty acid oxidation to less efficient but more expeditious glycolysis is associated with oncometabolism and more recently cardiac hypertrophy. Hypothesis: We hypothesized that such a switch drives HFpEF in two diverse mouse models. Methods: WT/129J mice at age 4-weeks were subjected to biweekly p407 i.p. injections and a single i.v. injection of AAV9-cTnT-LDLR. A separate group of injected mice were fed a diet supplemented with alpha-ketoglutarate (2% αKG) at onset. 4-week-old Alport mice received either αKG diet or normal chow. Glucose uptake was measured by whole-body PET-CT imaging of 18F-FDG in four groups: (1) untreated (n=5), (2) LDLR/P407 at 1, 2, 4, 7 and 8-week treatment (n=3-5), (3) LDLR/P407 + αKG mice at 4-week treatment (n=5), and (4) 9-12-week Alport mice (n=5). Echocardiography was performed on all the groups (n=4-17). Mechanical and energetic measurements on skinned cardiac pupillary muscles were performed on late-stage Alport , 8-week-treated LDLR/P407 , 4-week-treated LDR/P407 + αKG mice, and untreated controls (n=4-9). Survival of LDLR/P407, LDLR/P407/αKG and control groups (n=8-13) were recorded. Results: PET-CT scans showed increased cardiac glucose uptake in Alport (FC=3, p< 0.05) and LDLR/P407 mice at 1, 2, 4 (FC = 2, p<0.05), and 8 weeks (FC=3.2, p<0.001). αKG diet increased cardiac glucose uptake in LDLR/P407 at 4 weeks compared to normal chow (FC=2.15, p<0.05). Doppler and echocardiography showed worse diastolic dysfunction (E/E’) (FC = 1.8, P<.05) in LDLR/P407 αKG mice at 4-weeks versus control chow and reduced heart size (volume) (FC = 0.41, P<0.05) and worse diastolic dysfunction in Alport mice fed αKG diet. Alport hearts favored a DRX state (FC= 1.52, p <.0001) while 8-week-LDLR/P407 hearts favored SRX (FC=1.1, p< 0.05). The SRX state was exacerbated by αKG diet in 4-week-LDLR/P407 relative to normal chow. Life spans of LDLR/P407 mice fed αKG diet were significantly shortened (p<0.001). Conclusions: The results support roles for oncometabolism in promoting HF and sudden death in Alport and LDLR/P407 mice, effects that were exacerbated by an αKG diet, and possibly caused by enhanced hypertrophic growth. The results suggest an αKGDH and phenotype-dependent redistribution of myosin energetic states

Abstract 19181: MicroRNA Regulation of Adipose Browning and Glucose Uptake Improves Cardiometabolic Disease

The prevalence of obesity is on the rise worldwide. Brown adipose tissue (BAT), a highly vascularized organ, is activated in response to cold temperatures to generate heat and modulate metabolic efficiency. MicroRNAs (miRNAs) are small, noncoding RNAs that are implicated in cardiometabolic disease. However, relevant miRNAs in the endothelium of the microvasculature of BAT, how they crosstalk to the surrounding adipose tissue in cardiometabolic disease is not fully understood. We profiled microRNAs from the endothelial cells (ECs) of brown adipose tissue (BAT) of mice with diet-induced obesity (DIO) using microRNA-Seq and identified microRNA-485-5p ( miR-485 ) as one of the most differentially regulated miRNAs compared to the chow control. MiR-485 expression was rapidly induced in ECs in response to glucose (2.2-fold) and palmitate (1.63-fold). Loss-of-function studies revealed that neutralization of miR-485 increased glucose uptake to 3T3-L1 adipocytes and human adipose organoids co-cultured with conditioned media from ECs deficient in miR-485 ; while also increasing the mRNA and protein expression of brown fat marker, UCP1. Multiplex ELISA analyses of conditioned media from ECs overexpressing miR-485 showed a significant decrease in the release of IL-25 from ECs (22.5%) while the inhibition of miR-485 in ECs increased IL-25 release by 36.3%. Interestingly, IL-25 increased glucose uptake to 3T3-L1 adipocytes. Systemic intravenous delivery of LNA-anti-miR-485 inhibitor increased expression of UCP1 (31.4%) in BAT by confocal immunofluorescence, improved BAT function as measured by multispectral optoacoustic tomography (MSOT), while improving glucose tolerance and insulin resistance by 10.2% and 14.2% respectively. Metabolic profiling revealed increased energy expenditure (25%), decreased respiratory exchange rate (6%), and increased left ventricular ejection fraction (LVEF) (9.5%). The global longitudinal strain was higher by 28% compared to control mice with DIO. Our findings indicate that miR-485 plays a key regulatory role in controlling adipose browning and metabolism in an EC-paracrine mediated crosstalk in DIO, an effect that could be exploited for therapeutic intervention in obesity-induced cardiometabolic disease.

Evaluation of GBT021601 As a Therapeutic Agent to Restore Bone Marrow Health and Effective Erythropoiesis in a Sickle Mouse Model

Introduction: Hypoxia-mediated damage resulting from vaso-occlusion in sickle cell disease (SCD) leads to abnormal angiogenesis, such as occurs with moyamoya disease and proliferative retinopathy. Abnormal angiogenesis in mouse models of SCD has been observed to impact the bone marrow niche, resulting in a structurally abnormal vascular network. A dysfunctional bone marrow environment results in ineffective erythropoiesis and can damage hematopoietic stem cells (HSCs). Healthy HSCs are essential to the maintenance of hemoglobin (Hb) levels regardless of the rapid turnover of red cells and because of their potential as material for gene therapy. Recently, red cell transfusions were shown to reverse pathogenic angiogenesis in a sickle cell mouse model. However, chronic red cell transfusions are difficult to sustain in individuals with SCD due to complications including iron overload, risk of alloimmunization, and the time and cost involved. GBT021601 is a potent sickle Hb polymerization inhibitor that improves the rheology of red blood cells (RBCs) and has been shown to increase total Hb by as much as 3 g/dL. Here, we explore the effectiveness of GBT021601 for restoring bone marrow health and decreasing proangiogenic factors while maintaining the stemness of marrow cells to preserve erythrocyte maturation. Understanding the impact of GBT021601 on aberrant angiogenesis and the function of HSCs will indicate if it has a role in improving the bone marrow niche and HSC fitness in an era of expanded cell-based therapies for SCD. Methods: Nine-week-old, humanized Townes HbSS SCD mice were split into 3 cohorts (n=8 per group: 4 male, 4 female) and for 12 weeks were fed either standard chow (control) or chow containing 0.1% or 0.2% of GBT021601. After 12 weeks, the mice were phlebotomized and euthanized. Spleens were collected and weighed. Whole bone marrow was collected via centrifugation from both tibias and one femur; the other femur was saved for subsequent 3D confocal microscopy. Bone marrow was analyzed using flow cytometry to measure erythroid cell maturation (Annexin V, TER119, and CD44) and HSC markers (Lin, Sca-1, c-kit). Immunoblot analysis was performed on peripheral plasma to measure hypoxia-induced signaling and markers of angiogenesis (VCAM-1, VEGF-A, ANG-1, and -2). Results: Hb increased by 3 g/dL on average with administration of GBT021601, and spleen weights were significantly reduced ( P=0.0023 and P=0.0015 for control vs 0.1% and 0.2%, respectively). Flow cytometry of the extracted bone marrow revealed a significant increase in mature RBCs and a decrease in erythroid progenitors at all stages ( P<0.001 for RBCs and basophils; P=0.04, P=0.002, and P=0.001 for pro-, poly-, and ortho-chromatic erythroblasts, respectively) (Figure 1). HSCs, defined as Lin-Sca-1 +c-kit +, were not significantly different between groups (control vs 0.1%, control vs 0.2%, and for 0.1% vs 0.2%). Immunoblot analysis revealed a reduction in VCAM-1 and ANG-1 markers with administration of GBT021601 relative to control. However, no difference between ANG-2 and VEGF-A markers was observed between treated and control mice (Figure 2). Conclusions: Treatment with GBT021601 was effective at reducing extramedullary hematopoiesis as determined by the reduction in spleen size compared with control mice. VCAM-1 and ANG-1 are increased in HbSS compared with HbAA mice; treatment with GBT021601 reduced factors that contribute to pathogenic angiogenesis. Treatment with GBT021601 did not change ANG-2 and VEGF-A expression; notably, VEGF-A levels are similar between HbSS and HbAA mice. Our findings support GBT021601 as a sustainable treatment to reduce ineffective erythropoiesis and abnormal angiogenesis-which can otherwise damage the bone marrow niche-thereby suggesting a potential benefit for individuals with SCD participating in future cell-based therapies with curative intent and improved outcomes. More work is planned to examine the ability of GBT021601 to normalize the bone marrow environment and reduce the risk of deleterious effects on allogenic or autologous edited HSCs.

Chronic sugar exposure increases daily intake of sugars but decreases avidity for sweeteners in mice

Little is known about how chronic sugar consumption impacts avidity for and daily intake of sugars. This issue is topical because modern humans exhibit high daily intakes of sugar. Here, we exposed sugar-naïve C57BL/6 mice (across two 28-day exposure periods, EP1 and EP2) to a control (chow and water) or experimental (chow, water and a 11 or 34% sugar solution) diet. The sugar solutions contained sucrose, glucose syrups, or high-fructose syrups. We used brief-access tests to measure appetitive responses to sucralose and sucrose solutions at three time points: baseline (before EP1), after EP1, and after EP2. We used lick rates to infer palatability, and number of trials initiated/test to infer motivation. Exposure to the control diet had no impact on lick rates or number of trials initiated for sucralose and sucrose. In contrast, exposure to the experimental diets reduced licking for the sweeteners to varying degrees. Lick rates were reduced by exposure to sugar solutions containing the 11% glucose syrups, 34% sucrose, 34% glucose syrups and 34% high-fructose syrups. The number of trials initiated was reduced by exposure to all of the sugar solutions. Despite the exposure-induced reductions in avidity for the sweetener solutions, daily intakes of virtually all of the sugar solutions increased across the exposure periods. We conclude that (i) chronic consumption of sugar solutions reduced avidity for the sweetened solutions, (ii) the extent of this effect depended on the concentration and type of sugar, and (iii) avidity for sweet-tasting solutions could not explain the persistently high daily intake of sugar solutions in mice.

Modulation of Small-Intestine Morphology in Mice by a Novel Supplement Containing Silybum marianum, Yeast β-Glucan, Prebiotics, and Minerals

AbstractSilymarin, derived from Silybum marianum, has recently demonstrated its potential to improve health in conditions such as obesity and metabolic disturbances. Understanding the impact of nutraceuticals on intestinal morphology is crucial for developing supplements that promote a higher quality of life. Therefore, this study aimed to investigate the effects of nutraceutical supplementation with silymarin on the morphology of the small intestine. Sixty-day-old adult male C57BL/6 mice were divided into two groups: one receiving a standard chow (control) and the other receiving a novel silymarin supplement (experimental). Following the experimental period, the animals were euthanized, and fragments of the small intestine were collected for histochemical analysis using Masson's trichrome and periodic acid-Schiff with Alcian blue staining techniques. Our results revealed an increase in the number of villi per analyzed field in the experimental group, accompanied by a decrease in basic mucin, crypt depth, mucosal thickness, and villus spacing. In conclusion, this novel nutraceutical supplementation may play a crucial role in modulating small intestine morphology and enhancing absorption capacity.

Quinoa (Chenopodium quinoa Willd.) supplemented cafeteria diet ameliorates glucose intolerance in rats.

Quinoa (Chenopodium quinoa Willd.) is a pseudocereal with rich nutritional composition, gluten free, and organoleptic. The primary aim of this study was to elucidate the possible protective roles of quinoa in glucose homeostasis in a model of cafeteria diet-induced obesity. Male Wistar rats (3 weeks of age) were randomly allocated to be fed by; control chow (CON; n = 6), quinoa (QUI; n = 6), cafeteria (CAF; n = 6), or quinoa and cafeteria (CAFQ; n = 6) for 15 weeks. CAFQ resulted in decreased saturated fat, sugar, and sodium intake in comparison with CAF. Compared to CON, CAF increased body weight gain, plasma insulin, plasma glucose, decreased liver IRS-1, AMPK mRNA expressions, and pancreatic β-cell insulin immunoreactivity, and developed hepatocyte degeneration and microvesicular steatosis. Compared to CAF, QUI lowered body weight, plasma glucose, and plasma insulin, increased liver IRS-1 and AMPK mRNA expressions, and pancreatic β-cell insulin immunoreactivity. Compared to CAF, CAFQ lowered plasma glucose, increased liver IRS-1 mRNA expressions, increased pancreatic β-cell insulin immunoreactivity, and lowered hepatocyte degeneration and microvesicular steatosis. Dietary treatments did not influence IRS-2, AKT2, and INSR mRNA expressions. HOMA-IR, HOMA-β, and QUICKI were also similar between groups. Restoration of insulin in CAFQ islets was as well as that of CON and QUI groups. In conclusion, as a functional food, quinoa may be useful in the prevention of obesity and associated metabolic outcomes such as glucose intolerance, disrupted pancreatic β-cell function, hepatic insulin resistance, and lipid accumulation.

Abstract P2087: Contributions Of A Glycolytic Switch To Hfpef In Two Mouse Models

Background: Switching from fatty acid oxidation to less efficient but more expeditious glycolysis is associated with oncometabolism and more recently cardiac hypertrophy. We hypothesized that such a switch drives HFpEF in two diverse mouse models. Method: WT/129J mice at age 4-weeks were subjected to biweekly p407 i.p. injections and a single i.v. injection of AAV9-cTnT-LDLR. A separate group of injected mice were fed a diet supplemented with alpha-ketoglutarate (2% aKG) at onset. 4-week-old Alport mice received either aKG diet or normal chow. Glucose uptake was measured by whole-body PET-CT imaging of 18F-FDG in four groups: (1) untreated mice (n=5), (2) LDLR/P407 mice at 1, 2, 4, 7 (n = 3) and 8 weeks treatment (n=5), (3) LDLR/P407 + aKG mice at 4-weeks treatment (n=5), and (4) 9-12-weeks Alport mice (n=5). Echocardiography was performed on untreated mice (n=14), 4-week LDLR/P407 treatment mice (n=17), and 4-week LDLR/P407 + aKG treatment mice (n=5). Mechanical and energetic measurements on skinned cardiac pupillary muscles were performed on late-stage Alport (n=8), 8-week-treated LDLR/P407 (n=9), 4-week-treated LDR/P407 + aKG mice (n=2), and untreated controls (n=8). Survival of LDLR/P407 (n=13), LDLR/P407/aKG (n=8) and control groups were recorded. Results: PET-CT scans showed increased cardiac glucose uptake in Alport (FC=3, p< 0.05) and LDLR/P407 mice at 1, 2, 4 (FC = 2, p<0.05), and 8 weeks (FC=3.2, p<0.001). aKG diet increased cardiac glucose uptake in LDLR/P407 at 4 weeks compared to normal chow (FC=2.15, p<0.05). Doppler and echocardiography showed worse diastolic dysfunction (E/E’) (FC = 1.8, P<.05) in LDLR/P407 αKG mice at 4-weeks versus control chow. Alport hearts favored a DRX state (FC= 1.52, p <.0001) while 8-week-LDLR/P407 hearts favored SRX (FC=1.1, p< 0.05). The SRX state was exacerbated by aKG diet in 4-week-LDLR/P407 relative to normal chow. aKG diet significantly shortened life spans of LDLR/P407 mice (p<0.001). Conclusion: The results support roles for oncometabolism in promoting HF and sudden death in Alport and LDLR/P407 mice, effects that were exacerbated by an aKG supplement, possibly via enhanced hypertrophic growth. Results suggest an αKGDH-dependent and phenotype-dependent redistribution of myosin energetic states.

Glycyrrhizic Acid Inhibits High-Mobility Group Box-1 and Homocysteine-Induced Vascular Dysfunction.

Hyperhomocysteinemia (HHcy) worsens cardiovascular outcomes by impairing vascular function and promoting chronic inflammation via release of danger-associated molecular patterns, such as high-mobility group box-1 (HMGB-1). Elevated levels of HMGB-1 have recently been reported in patients with HHcy. Therefore, targeting HMGB-1 may be a potential therapy to improve HHcy-induced cardiovascular pathologies. This study aimed to further elucidate HMGB-1's role during acute HHcy and HHcy-induced atherogenesis and to determine if inhibiting HMGB-1 with glycyrrhizic acid (Glyz) improved vascular function. Male New Zealand White rabbits (n = 25) were placed on either a standard control chow (CD; n = 15) or atherogenic diet (AD; n = 10) for 4 weeks. Rabbit serum and Krebs taken from organ bath studies were collected to quantify HMGB-1 levels. Isometric tension analysis was performed on abdominal aorta (AA) rings from CD and AD rabbits. Rings were incubated with homocysteine (Hcy) [3 mM] for 60 min to induce acute HHcy or rhHMGB-1 [100 nM]. Vascular function was assessed by relaxation to cumulative doses of acetylcholine. Markers of vascular dysfunction and inflammation were quantified in the endothelium, media, and adventitia of AA rings. HMGB-1 was significantly upregulated in serum (p < 0.0001) and Krebs (p < 0.0001) after Hcy exposure or an AD. Incubation with Hcy (p < 0.0001) or rhHMGB-1 (p < 0.0001) and an AD (p < 0.0001) significantly reduced relaxation to acetylcholine, which was markedly improved by Glyz. HMGB-1 expression was elevated (p < 0.0001) after Hcy exposure and AD (p < 0.0001) and was normalized after Glyz treatment. Moreover, markers of vascular function, cell stress and inflammation were also reduced after Glyz. These results demonstrate that HMGB-1 has a central role during HHcy-induced vascular dysfunction and inhibiting it with Glyz could be a potential treatment option for cardiovascular diseases.

Maternal age, obesity and hyperglycaemia are associated with a delay in preimplantation embryo development in mouse.

Fertility has decreased due to advanced maternal age and the rising prevalence of the metabolic syndrome. Using quantitative image analysis methods, we show that these factors are associated with delayed preimplantation embryo development in a mouse model. Delayed maternal age, obesity and diabetes are associated with reduced fertility. We investigated how age and obesity/metabolic syndrome impact fertility and hypothesized that its decrease is due to defects in preimplantation embryo development. Three groups of female C57Bl6 mice (12 weeks, 9 months and 1 year old) were fed either a high-fat diet for 8 weeks, to induce obesity and the metabolic syndrome, or a control chow diet. Body weight and composition, glucose tolerance and insulin resistance were assessed. Fecundity was evaluated by mating and pregnancy rates, as well as by the number of embryos. Embryo quality was assessed morphologically, and cell fate composition was analysed in preimplantation embryos by state-of-the-art single-cell quantitative confocal image analysis. The high-fat diet was associated with increased adiposity, glucose intolerance and insulin resistance, especially in the older mice. Fecundity was affected by age more than by the diet. Both age and high-fat diet were associated with reduced cell fate allocation, indicating a delay in the preimplantation embryo development, and with increased expression of GATA3, an inhibitor of placentation. These results support that age and the metabolic syndrome reduce fertility through mechanisms which are present at conception or very early in pregnancy.

Western diet-induced obesity interferes with the HPA axis-blunting effects of palatable food in male rats

Limited intermittent consumption of palatable food reduces HPA axis responses to stress in chow-fed rats, and this effect is dependent on the rewarding properties of the palatable food. However, obesity may be a state of reduced consummatory food reward, suggesting that palatable foods may be less effective at blunting HPA axis reactivity in the context of diet-induced obesity (DIO). To test this hypothesis, adult male Long-Evans rats were given unlimited access to Western (high-fat, high-sugar) diet (WD) vs. normal chow (controls). After 8 weeks of diet exposure, rats were given limited sucrose intake (LSI) consisting of additional twice-daily access to a small amount (4 ml) of either 3% or 30% sucrose drink, or water (controls) for 2 weeks. Rats then received an acute restraint stress challenge, with collection of tail blood samples for measurement of plasma corticosterone. WD-fed rats had increased caloric intake, body weight and adiposity, as expected. Rats offered LSI (3% or 30%) readily drank the maximal amount allowed (8 ml/day) and reduced their dietary intake to compensate for the sucrose calories, such that LSI did not alter body weight regardless of diet type. In chow-fed lean rats, LSI with either 3% or 30% sucrose reduced the plasma corticosterone response to restraint stress, but this effect was absent in WD-fed DIO rats. Together, these data support the hypothesis that obesity attenuates stress blunting by palatable foods and suggest the possibility that consequently, individuals with obesity may need to consume larger amounts of palatable food to obtain adequate stress relief.

1595-P: Empagliflozin Improves Nephropathy and Heart Failure with Preserved Ejection Fraction Despite Limited Effects on NASH in a Novel Obese Diabetic Hamster Model

Introduction: Sodium-glucose cotransporter 2 inhibitor empagliflozin (EMPA) has shown significant benefits for patients with diabetic nephropathy and heart failure with preserved ejection fraction (HFpEF), but the effects on nonalcoholic steatohepatitis (NASH) and liver fibrosis remain unclear. To further investigate the benefits of EMPA on these comorbidities, we established a novel diet-induced obese diabetic hamster model with human-like NASH and lipoprotein cholesterol metabolism, that also develops nephropathy and HFpEF. Methods: Obesity, NASH, and HFpEF were induced with a 20-week free choice diet, which presents hamsters with a choice between control chow or high fat/cholesterol diet, and normal water or 10% fructose water. After 20 weeks of diet, obese hamsters were intraperitoneally injected with streptozotocin (STZ) to induce diabetes and were then treated orally once daily for 5 weeks with vehicle or EMPA 30mg/kg. Results: Compared with vehicle, EMPA reduced hyperglycemia (vehicle: 373 +/- 43 mg/dL; EMPA: 182 +/- 30 mg/dL, p&amp;lt;0.01), increased pancreatic insulin content (+146%, p&amp;lt;0.05), significantly reduced plasma triglycerides and moderately raised LDL-cholesterol levels, as expected. However, EMPA did not improve NASH (including hepatocyte ballooning score) and liver fibrosis. EMPA reduced glomerular filtration rate and proteinuria (p=0.06 for both vs. vehicle), and significantly reduced urine albumin-to-creatinine ratio (-35%, p&amp;lt;0.01). In addition, EMPA showed significant improvement in HFpEF with reduced E/A and E’/E ratios with no alteration in ejection fraction, as measured by echocardiography. Conclusion: EMPA improves kidney parameters and HFpEF despite limited effects on NASH in obese diabetic hamsters. This preclinical model with multiple comorbidities will be useful to evaluate the combination of EMPA or other antidiabetic therapies with drugs targeting NASH. Disclosure F. Briand: Employee; PHYSIOGENEX. Stock/Shareholder; PHYSIOGENEX. C. Dubroca: Employee; CARDIOMEDEX. T. Sulpice: Employee; PHYSIOGENEX. Stock/Shareholder; PHYSIOGENEX. Employee; CARDIOMEDEX. Stock/Shareholder; CARDIOMEDEX.

97-OR: Antidiabetic TIX100 Improves NAFLD/NASH in Mice With and Without Diabetes and Obesity

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are often associated with diabetes, but are becoming an even bigger world-wide epidemic with over 1 billion people affected. Nevertheless, while some diabetes medications such as glucagon-like peptide 1 and peroxisome proliferator-activated receptor agonists have shown some beneficial effects in the context of NAFLD with diabetes, no approved therapy is currently available for NAFLD/NASH. Interestingly, the new antidiabetic small molecule thioredoxin-interacting protein (Txnip) inhibitor, TIX100, effectively protected obese and diabetic db/db mice against steatosis. TIX100 also significantly reduced the elevated expression levels of lipogenic genes found in db/db livers, many of which have also been shown to be elevated in human NAFLD and are being pursued as potential therapeutic targets. Importantly, the beneficial effects of TIX100 were not due to any weight loss as the severe obesity of the db/db mice was not affected by the treatment. In contrast, TIX100 did demonstrated strong antidiabetic effects and normalized the blood glucose of db/db mice to the levels observed in lean control animals. Therefore, to address the question of whether TIX100 would still be effective in the absence of diabetes, obesity and any genetic leptin-receptor deficiency, we next used a validated choline-deficient, amino acid-defined, high-fat diet to induce NASH. Indeed, compared to control chow, this diet significantly increased plasma liver enzymes indicating liver injury, led to elevated hepatic expression of inflammatory and fibrogenic markers and resulted in clear histological changes, demonstrating lipid accumulation and steatosis as well as pronounced fibrosis and upregulation of liver macrophages. Excitingly, TIX100 treatment blunted or improved all these pathological changes. Thus, TIX100 may not only be effective in diabetes, but may also provide an attractive novel approach for NAFLD/NASH. Disclosure S. Jo: None. J. Chen: None. G. Jing: None. K.M. Habegger: Research Support; Eli Lilly and Company, Novo Nordisk. Consultant; Glyscend Inc. Stock/Shareholder; Glyscend Inc. Research Support; Glyscend Inc. A. Shalev: Other Relationship; TIXiMED. Funding National Institutes of Health (R01DK078752); Human Islet Research Network (U01DK120379)

A 14-day pulse of PLX5622 modifies α-synucleinopathy in preformed fibril-infused aged mice of both sexes

Reactive microglia are observed with aging and in Lewy body disorders, including within the olfactory bulb of men with Parkinson's disease. However, the functional impact of microglia in these disorders is still debated. Resetting these reactive cells by a brief dietary pulse of the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 may hold therapeutic potential against Lewy-related pathologies. To our knowledge, withdrawal of PLX5622 after short-term exposure has not been tested in the preformed α-synuclein fibril (PFF) model, including in aged mice of both sexes. Compared to aged female mice, we report that aged males on the control diet showed higher numbers of phosphorylated α-synuclein+ inclusions in the limbic rhinencephalon after PFFs were injected in the posterior olfactory bulb. However, aged females displayed larger inclusion sizes compared to males. Short-term (14-day) dietary exposure to PLX5622 followed by control chow reduced inclusion numbers and levels of insoluble α-synuclein in aged males—but not females—and unexpectedly raised inclusion sizes in both sexes. Transient delivery of PLX5622 also improved spatial reference memory in PFF-infused aged mice, as evidenced by an increase in novel arm entries in a Y-maze. Superior memory was positively correlated with inclusion sizes but negatively correlated with inclusion numbers. Although we caution that PLX5622 delivery must be tested further in models of α-synucleinopathy, our data suggest that larger-sized—but fewer—α-synucleinopathic structures are associated with better neurological outcomes in PFF-infused aged mice.

Taurine supplementation reduces adiposity and hepatic lipid metabolic activity in adult offspring following maternal cafeteria diet

Maternal taurine supplementation has been shown to exert protective effects following a maternal obesogenic diet on offspring growth and metabolism. However, the long-term effects of maternal cafeteria diet on adiposity, metabolic profile, and hepatic gene expression patterns following supplementation of taurine in adult offspring remains unclear. In this study, we hypothesized that exposure to maternal taurine supplementation would modulate the effects of maternal cafeteria diet by reducing adiposity and hepatic gene expression patterns involved in lipid metabolism in adult offspring. Female Wistar rats were fed a control diet, control diet supplemented with 1.5% taurine in drinking water, cafeteria diet (CAF) or CAF supplemented with taurine (CAFT) from weaning. After 8 weeks, all animals were mated and maintained on the same diets during pregnancy and lactation. After weaning, all offspring were fed with control chow diet until the age of 20 weeks. Despite similar body weights, CAFT offspring had significantly lower fat deposition and body fat when compared with CAF offspring. Microarray analysis revealed that genes (Akr1c3, Cyp7a1, Hsd17b6, Cd36, Acsm3, and Aldh1b1) related to steroid hormone biosynthesis, cholesterol metabolism, peroxisome proliferator-activated receptor signaling pathway, butanoate metabolism, and fatty acid degradation were down-regulated in CAFT offspring. The current study shows that exposure to maternal cafeteria diet promoted adiposity and taurine supplementation reduced lipid deposition and in both male and female offspring and led to alterations in hepatic gene expression patterns, reducing the detrimental effects of maternal cafeteria diet.

Gut microbial metabolite delta - valerobetaine (VB) influences energy metabolism by altering fat utilization and satiety-related hormones in mice

There is mounting evidence that the gut microbiota exerts profound influences on systemic homeostasis and metabolism, including effects on host energy metabolism, fatty acid oxidation, and obesity. However, we know little about how the microbiota mechanistically influences these aspects of host biology. Previously, we used mass spectrometry-based metabolomics platforms to demonstrate remarkable differences in the metabolite composition in the hepatic mitochondria of germ-free (GF) and conventional mice and identified Δ-valerobetaine (VB) as a key discriminative molecule. VB was undetectable in GF mice but present in conventional mice. We showed that VB is produced by specific bacterial species and inhibited mitochondrial fatty acid oxidation by suppression of the carnitine shuttle. VB administration to GF and conventional mice increased visceral fat mass and exacerbated hepatic steatosis with a Western-style diet but not a chow control diet and can thus function as a microbiota-derived obesogen. Here we show that VB impacts the physiological aspects of energy metabolism. VB reduces the Oxygen consumption rate (OCR) in HEPG2 cells and alters mitochondria-related protein markers (PGC1α, AMPKα, TFAM) suggesting changes in VB can alter the function of mitochondria. To evaluate whole animal consequences, we utilized the CLAMS-HC metabolic cage system to measure the respiratory exchange ratio (RER) in conventional mice. We found that western diet-fed animals treated with VB (IP 50mg/kg) for a week significantly increased RER compared to control groups on the same diet, whereas there was no impact of VB on regular chow diet-fed animals. Higher RER indicates the usage of glucose rather than the usage of fat, thus shifting the resources to produce energy and enhancing the accumulation/retention of adipose tissue. We also found a significant increase in food intake among VB-treated animals regardless of diet. We further investigated the impact of VB on energy-regulating hormones from serum and found a significant alteration in leptin and ghrelin levels. These data suggest VB alters energy utilization in a diet-dependent fashion possibly through regulating energy satiety hormones. Manipulation of VB levels by changing the intake of dietary precursors, or with VB-producing bacterial taxa, may provide therapeutic approaches to disorders of energy/lipid metabolism. This project has been funded by U.S. NIH R01 AI64462. This is the full abstract presented at the American Physiology Summit 2023 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.

Soda intake influences phenotype, antioxidants and inflammatory status in high protein-fed wistar rats

An increasing population of people, especially young adults who exercise, consume high protein diets along with carbonated drinks. While there are numerous studies on the effect of high protein diets, there is a need to understand how protein diets in combination with carbonated drinks impact physiology. In order to assess these effects on wistar rats’ phenotype, antioxidants and inflammatory profiles, 64 wistar rats were divided into dietary groups of 8 male and 8 female animals each. The animals were fed standard diet as control (chow), chow and carbonated soda, a high protein diet (48.1% energy from protein) and a high protein diet with carbonated soda according to their groups.Body measurements, blood glucose levels, serum insulin levels, lipid peroxidation, antioxidant activity, adipokines and inflammatory markers concentrations were all determined. At the end of the study, body measurements, inflammatory markers and adipokine concentration were increased in animals fed the high protein diet and high protein-soda diet. There was a decrease in antioxidant and lipid peroxidation levels in protein fed male and female animals but those fed protein in combination with soda had increased lipid peroxidation levels.In conclusion, high protein diet in combination with carbonated soda impacts physiology differently from a high protein diet alone, and may stimulate weight gain, oxidative stress and HPD-related inflammation in Wistar rats.

Effects of GBT1118, a voxelotor analog, on intestinal pathophysiology in sickle cell disease.

Voxelotor is an allosteric haemoglobin (Hb) modulator that binds covalently and reversibly to Hb alpha chain to facilitate improved Hb-O2 affinity and arterial oxygen. It, therefore, reduces the susceptibility of erythrocytes carrying Haemoglobin S to sickle. In this study, we have used GBT1118, an analog of voxelotor, to treat male Townes sickle cell disease (SCD) mice to investigate whether the Hb modulator could attenuate the intestinal pathophysiologic changes associated with SCD. Compared with mice fed with control chow, GBT1118-treated mice showed improvement in the intestinal pathophysiology. These mice exhibited improved small intestinal barrier functions, reduced intestinal microbial density, reduced enterocyte injury, lower serum lipopolysaccharides and smaller spleens. These improvements were observed after only 3 weeks of GBT1118 treatment. Benefits were also observed after experimentally-induced vaso-occlusive crisis (VOC). Recovery from the VOC-induced changes was faster in mice that were treated with GBT1118. The improved small intestinal barrier function was associated with higher expression of genes encoding enterocyte E-cadherin, JAM-A, ZO-1, MUC-2 and occludin while the lower intestinal microbial density associated with higher expression of genes encoding the antimicrobial peptides defensin-α 1 and defensin-α 4. Our findings provide the evidence to support the beneficial effects of GBT1118 in SCD-related intestinal pathophysiology.

Cardiac disturbances and changes in tissue cytokine levels in mice fed with a high-refined carbohydrate diet

AimsThe consumption of highly refined carbohydrates increases systemic inflammatory markers, but its potential to exert direct myocardial inflammation is uncertain. Herein, we addressed the impact of a high-refined carbohydrate (HC) diet on mice heart and local inflammation over time. Main methodsBALB/c mice were fed with a standard chow (control) or an isocaloric HC diet for 2, 4, or 8 weeks (HC groups), in which the morphometry of heart sections and contractile analyses by invasive catheterization and Langendorff-perfused hearts were assessed. Cytokines levels by ELISA, matrix metalloproteinase (MMP) activity by zymography, in situ reactive oxygen species (ROS) staining and lipid peroxidation-induced TBARS levels, were also determined. Key findingsHC diet fed mice displayed left ventricular hypertrophy and interstitial fibrosis in all times analyzed, which was confirmed by echocardiographic analyses of 8HC group. Impaired contractility indices of HC groups were observed by left ventricular catheterization, whereas ex vivo and in vitro indices of contraction under isoprenaline-stimulation were higher in HC-fed mice compared with controls. Peak levels of TNF-α, TGF-β, ROS, TBARS, and MMP-2 occur independently of HC diet time. However, a long-lasting local reduction of the anti-inflammatory cytokine IL-10 was found, which was linearly correlated to the decline of systolic function in vivo. SignificanceAltogether, the results indicate that short-term consumption of HC diet negatively impacts the balance of anti-inflammatory defenses and proinflammatory/profibrotic mediators in the heart, which can contribute to HC diet-induced morphofunctional cardiac alterations.

Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis.

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.

Fenofibrate mildly stimulates browning-associated expression in white adipose tissues of young but not old male rats.

The aim of this study was to examine the effects of the hypolipemic drug fenofibrate (FF) and aging on the expression of factors/enzymes involved in brown adipose tissue (BAT) function and browning of white adipose tissue epididymal (eWAT) and subcutaneous (sWAT) depots. Young-adult and old male Wistar rats were fed standard chow (control) or supplemented with 0.1% or 0.5% FF for 30 days. Tissue samples were analysed for gene expression and protein content, and stained with Oil Red O or hematoxylin and eosin. In BAT of young rats, 0.5% FF increased only Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1 (CITED1) protein content and Fgf21 and Gpr109A mRNA expression. The expression of oxidative metabolism related genes (Pgc1α, Cpt1b, Mcad) decreased after 0.5% FF. In BAT of old rats, FF did not affect UCP1 and CITED1 content and had little effect on gene expression. Oil Red O staining of BAT revealed no changes in lipid droplet area upon treatment in either age group. In eWAT of young rats, 0.1FF elevated UCP1 protein content and Ucp1, Pgc-1α, and Mcad expression, whereas 0.5% FF increased PPARα content and Pgc-1α, Cpt1b, Mcad, and Gpr109A levels. In eWAT of old rats, only 0.1FF increased Pgc1α and Mcad expression. In both age groups median cell area of eWAT adipocytes was reduced after 0.5% FF. In sWAT Ucp1 gene expression was very low and UCP1 protein was undetectable. FF upregulated Ucp1, Cited1, Eva1, and Cpt1b expression in sWAT of young rats, with diminished effects in old rats. In both age groups 0.5% FF increased Fgf21 expression in sWAT. Median cell area of sWAT adipocytes decreased only in young rats treated with 0.5% FF. Our results reveal that fenofibrate differentially affects gene expression in BAT, with diminished effects in old compared to young rats. In WAT of young rats FF modestly stimulates the expression of factors/enzymes involved in lipid oxidative metabolism and browning. Aging reduces both these effects. Gpr109A may present a novel gene target upregulated by FF in BAT and eWAT.