Normal Chow Research Articles

Abstract 1058: Effects Of Soluble Guanylate Cyclase Activation On Progression Of Fibrocalcific Aortic Valve Stenosis (avs), Its Molecular Drivers, And Downstream Pathological Cardiac Electrical Remodeling

Objective: We recently discovered that activation of oxidized soluble guanylate cyclase with ataciguat (ATA) slows valve calcification and dysfunction in mice and humans with moderate aortic valve stenosis (AVS). We examined if ATA would attenuate multiple mechanisms contributing to AVS and end organ dysfunction associated with AVS, and hypothesized: 1) ATA attenuates osteogenic signaling in valve tissue from mice with AVS, thereby attenuating calcification and valve dysfunction, 2) AVS increases rates of sudden cardiac death in our model, and 3) treatment with ATA prevents QT prolongation and deleterious electrical remodeling commonly observed in AVS. Methods: Functional, molecular, and histopathological changes in valve tissue along with QT interval duration were measured in 3 independent groups of mice: non-AVS, AVS, and AVS+ATA (WD for 6 months then WD+ATA for 3 months (AVS+ATA). We gave 5 mg/kg isoproterenol (ISO, i.p.) to ldlr -/- /ApoB100-only mice fed normal chow (non-AVS) or western diet (AVS, fed WD for 9 months) to assess risk for malignant arrhythmias. Retrospective analysis of QT interval was also done on data from our recent Phase 1 clinical trial. Results: AVS+ATA mice had less valve calcification and slower progression of valve dysfunction through attenuation of canonical BMP signaling and reductions in Runx2 and SPP1 compared to AVS mice. QT prolongation was evident in AVS mice, and was significantly attenuated by treatment with ATA (p= 0.001) and coarsely associated with severity of valvular or ventricular function. While all non-AVS mice survived for 24 hours after ISO, 40% of mice with AVS reached a moribund equivalent within 4 hours. Retrospective analyses also showed reductions in QT interval duration in patients treated with 200 mg/day ATA for 14 days (p < 0.05). Conclusion: Collectively, this is the first study reporting increased propensity for malignant arrhythmias in our mouse model of AVS, QT prolongation as a likely underlying mechanism, and restoration of cardiac conduction dynamics with targeted restoration of nitric oxide signaling. Future studies focused on the role of sGC in the evolution and prevention of AVS and consequent deleterious electrical remodeling/malignant arrhythmia risk is warranted.

Advanced PamGene Coregulator Interactome Analysis of PPARgamma Reveals that Diet Influences Regulatory Protein Interactions and Signaling Mechanisms

Studies show a strong correlation with type 2 diabetes mellitus (T2DM) in obese patients developing congestive heart failure (CHF) with rosiglitazone treatment. Rosiglitazone, a thiazolidinedione, treats T2DM by improving insulin resistance. Its use has been halted due to a subset of patients developing CHF through unknown mechanisms. Rosiglitazone targets PPARγ, a nuclear receptor highly expressed in adipose tissue and, to a lesser extent, in cardiac tissue. An important mechanism of PPARγ regulation is through coregulators that can alter transcriptional activity through protein-protein interaction to drive PPARγ toward specific pathways. We hypothesized coregulator interaction with PPARγ may regulate its activity differently in adiposity, inducing CHF through adipose-derived peripheral signaling. To address this, we fed 8-week-old C57BL/6 mice a normal chow (NCD) or high-fat (HFD) diet for 16 weeks. Mice were then injected with rosiglitazone every 48 hours for 4 weeks. In NCD and HFD Rosi mice, we found significantly increased heart weights (p-value<0.05) and various weight changes in five adipose depots. To understand the roles of the PPARγ coregulator interactome in adiposity, we used our state-of-the-art PamGene PamStation Nuclear Hormone Receptor chip to measure the interaction of PPARγ with 155 coregulators in five adipose depots and the liver. In adipose depots increasing in weight due to Rosi treatment, such as retroperitoneal white adipose tissue (WAT) and mesenteric WAT, more coregulators associated with the PPARγ complex. Conversely, depots that did not change in weight with Rosi treatment, such as epididymal WAT and inguinal WAT, showed a reduction of coregulators leaving the PPARγ complex, indicating differential PPARγ regulation. Our data suggests that the coregulator interactome of PPARγ in adipose depots represents potential therapeutic targets, and understanding how PPARγ regulation is different in adiposity is crucial to understanding the negative side effects of rosiglitazone treatment. R01DK121797 (T.D.H.) R01DA058933 (T.D.H.). 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.

Deletion of Impdh2 in adipocyte precursors limits the expansion of white adipose tissue and enhances metabolic health with overnutrition

The equilibrium between the hypertrophic growth of existing adipocytes and adipogenesis is vital in managing metabolic stability in white adipocytes when faced with overnutrition. Adipogenesis has been established as a key player in combating metabolic irregularities caused by various factors. However, the benefits of increasing adipogenesis-mediated white adipose tissue (WAT) expansion for metabolic health regulation remain uncertain. Our findings reveal an increase in Impdh2 expression during the adipogenesis phase, both in vivo and in vitro. Xmp enhances adipogenic potential by fostering mitotic clonal expansion (MCE). The conditional knockout of Impdh2 in adipocyte progenitor cells(APCs) in adult and aged mice effectively curbs white adipose tissue expansion, ameliorates glucose tolerance, and augments energy expenditure under high-fat diet (HFD). However, no significant difference is observed under normal chow diet (NCD). Concurrently, the knockout of Impdh2 in APCs significantly reduces the count of new adipocytes induced by HFD, without affecting adipocyte size. Mechanistically, Impdh2 regulates the proliferation of APCs during the MCE phase via Xmp. Exogenous Xmp can significantly offset the reduction in adipogenic abilities of APCs due to Impdh2 deficiency. In summary, we discovered that adipogenesis-mediated WAT expansion, induced by overnutrition, also contributes to metabolic abnormalities. Moreover, the pivotal role of Impdh2 in regulating adipogenesis in APCs offers a novel therapeutic approach to combat obesity.

Hairless (Hr) Deficiency Mitigates High-Fat Diet-Induced Obesity and Insulin Resistance in Mice.

Obesity is a significant global health concern linked to excessive dietary energy intake. This research focuses on the mammalian hairless protein (HR), known for its role in skin and hair function, and its impact on metabolism. Examining male wild-type (Hr+/+) and Hr null (Hr-/-) mice over a 14-week normal chow diet (NCD) or high-fat diet (HFD) intervention. This study reveals that HR deficiency exhibited a protective effect against HFD-induced obesity and insulin resistance. This protective effect is attributed to increased energy expenditure in Hr-/- mice. Moreover, the brown adipose tissue (BAT) of Hr-/- mice displays elevated levels of the thermogenic protein, uncoupling protein 1 (Ucp1), and its key transcriptional regulators (PPARγ and PGC1α), compared to Hr+/+ mice. In summary, the findings underscore the protective role of HR deficiency in countering HFD-induced adiposity by enhancing insulin sensitivity, raising energy expenditure, and augmenting thermogenic factors in BAT. Further exploration of HR metabolic regulation holds promise for potential therapeutic targets in addressing obesity-related metabolic disorders.

Probucol mitigates high-fat diet-induced cognitive and social impairments by regulating brain redox and insulin resistance.

Probucol has been utilized as a cholesterol-lowering drug with antioxidative properties. However, the impact and fundamental mechanisms of probucol in obesity-related cognitive decline are unclear. In this study, male C57BL/6J mice were allocated to a normal chow diet (NCD) group or a high-fat diet (HFD) group, followed by administration of probucol to half of the mice on the HFD regimen. Subsequently, the mice were subjected to a series of behavioral assessments, alongside the measurement of metabolic and redox parameters. Notably, probucol treatment effectively alleviates cognitive and social impairments induced by HFD in mice, while exhibiting no discernible influence on mood-related behaviors. Notably, the beneficial effects of probucol arise independently of rectifying obesity or restoring systemic glucose and lipid homeostasis, as evidenced by the lack of changes in body weight, serum cholesterol levels, blood glucose, hyperinsulinemia, systemic insulin resistance, and oxidative stress. Instead, probucol could regulate the levels of nitric oxide and superoxide-generating proteins, and it could specifically alleviate HFD-induced hippocampal insulin resistance. These findings shed light on the potential role of probucol in modulating obesity-related cognitive decline and urge reevaluation of the underlying mechanisms by which probucol exerts its beneficial effects.

Intermittent fasting, exercise, and dietary modification induce unique transcriptomic signatures of multiple tissues governing metabolic homeostasis during weight loss and rebound weight gain

Obesity and its related metabolic diseases bring great challenges to public health. In-depth understanding on the efficacy of weight-loss interventions is critical for long-term weight control. Our study demonstrated the comparable efficacy of exercise (EX), intermittent fasting (IF), or the change of daily diet from an unhealthy to a normal chow (DR) for weight reduction, but largely divergently affected metabolic status and transcriptome of subcutaneous fat, scapular brown fat, skeletal muscles and liver in high-fat-high-fructose diet (HFHF) induced obese mice. EX and IF reduced systematic inflammation, improved glucose and lipid metabolism in liver and muscle, and amino acid metabolism and thermogenesis in adipose tissues. EX exhibited broad regulatory effects on TCA cycle, carbon metabolism, thermogenesis, propanoate-, fatty acid and amino acid metabolism across multiple tissues. IF prominently affected genes involved in mitophagy and autophagy in adipose tissues and core genes involved in butanoate metabolism in liver. DR, however, failed to improve metabolic homeostasis and biological dysfunctions in obese mice. Notably, by exploring potential inter-organ communication, we identified an obesity-resistant-like gene profile that were strongly correlated with HFHF induced metabolic derangements and could predict the degree of weight regain induced by the follow-up HFHF diet. Among them, 12 genes (e.g., Gdf15, Tfrc, Cdv3, Map2k4, and Nqo1) were causally associated with human metabolic traits, i.e., BMI, body fat mass, HbA1C, fasting glucose, and cholesterol. Our findings provide critical groundwork for improved understanding of the impacts of weight-loss interventions on host metabolism. The identified genes predicting weight regain may be considered regulatory targets for improving long-term weight control.

Resveratrol relieves HFD-induced insulin resistance in skeletal muscle tissue through antioxidant capacity enhancement and the Nrf2-Keap1 signaling pathway.

Resveratrol has received much attention due to its beneficial effects including antioxidant activity. The purpose of this study was to investigate the therapeutic effects of resveratrol treatment on oxidative stress and insulin resistance in the skeletal muscle of high-fat diet (HFD)-fed animals. A total of 30 six-week-old C57BL/6J mice were randomly allocated to three groups (10 animals in each group): The control group in which mice were fed a normal chow diet (NCD); the HFD group in which mice were fed an HFD for 26 weeks; and the HFD-resveratrol group in which HFD was replaced by a resveratrol supplemented-HFD (400mg/kg diet) after 10 weeks of HFD feeding. At the end of this period, gastrocnemius muscle samples were examined to determine insulin resistance and the oxidative status in the presence of HFD and resveratrol. Resveratrol supplementation in HFD-fed mice reduced body and adipose tissue weight, improved insulin sensitivity, and decreased oxidative stress as indicated by lower malonaldehyde (MDA) levels and higher total antioxidant capacity. The supplement also increased the expression and activity of antioxidative enzymes in gastrocnemius muscle and modulated Nrf2 and Keap1 expression levels. These results suggest that resveratrol is effective in improving the antioxidant defense system of the skeletal muscle in HFD-fed mice, indicating its therapeutic potential to combat diseases associated with insulin resistance and oxidative stress.

Effect of Aqueous Extract of Irvingia wombolu Seeds on Lipid Profile and Atherogenic Indices in Hyperlipidemic Wistar Rats

The effect of aqueous extract of Irvingia wombolu seeds on lipid profile and atherogenic indices in hyperlipidemic wistar rats was evaluated. Forty five (45) wistar rats were grouped into five groups of nine rats each. The animals were allowed seven days acclimatization period. Group one was the control group and it received normal rat chow and water throughout the study. Groups 2 to 5 were given high fat diet for 14 days after which they were fed with normal rat chow till the end of the study. At the end of the 14 days, group 2 was not treated while group 3-5 were treated with 250, 500 and 1000mg/kg body weight aqueous extract of Irvingia wombolu seed respectively for 28 days. The lipid profile of animals was assayed three times: first after 14 days induction period (phase 1) i.e day 0 of treatment, second was taken 14 days after treatment (phase 2), third was taken 28 days after treatment (phase 3).The study lasted for 49 days and distilled water was used as a vehicle for the extract.In phase 1, there was a significant decrease (p<0.05) in HDL (high density lipoprotein) level in all groups (2-5) compared to the control.there was a significant increase (p<0.05) in LDL ( low density lipoprotein), Total cholesterol, Triglyceride, VLDL (very low density lipoprotein), Non HDL, Cardic risk factor, Atherogenic coefficient, Atherogenic index of plasma levels in all groups (2-5) compared to the control. All doses of aqueous extract of Irvingia wombolu seed were able to increase HDL level and decrease other lipid profile parameters and atherogenic indices in hyperlipidemic rats. Thus, aqueous extract of Irvingia wombolu seed has antidyslipidemic potential.

Investigating the Effects of Diet-Induced Prediabetes on Skeletal Muscle Strength in Male Sprague Dawley Rats.

Type 2 diabetes mellitus, a condition preceded by prediabetes, is documented to compromise skeletal muscle health, consequently affecting skeletal muscle structure, strength, and glucose homeostasis. A disturbance in skeletal muscle functional capacity has been demonstrated to induce insulin resistance and hyperglycemia. However, the modifications in skeletal muscle function in the prediabetic state are not well elucidated. Hence, this study investigated the effects of diet-induced prediabetes on skeletal muscle strength in a prediabetic model. Male Sprague Dawley rats were randomly assigned to one of the two groups (n = 6 per group; six prediabetic (PD) and six non-pre-diabetic (NPD)). The PD group (n = 6) was induced with prediabetes for 20 weeks. The diet that was used to induce prediabetes consisted of fats (30% Kcal/g), proteins (15% Kcal/g), and carbohydrates (55% Kcal/g). In addition to the diet, the experimental animals (n = 6) were supplied with drinking water that was supplemented with 15% fructose. The control group (n = 6) was allowed access to normal rat chow, consisting of 35% carbohydrates, 30% protein, 15% fats, and 20% other components, as well as ordinary tap water. At the end of week 20, the experimental animals were diagnosed with prediabetes using the American Diabetes Association (ADA) prediabetes impaired fasting blood glucose criteria (5.6-6.9 mmol/L). Upon prediabetes diagnosis, the animals were subjected to a four-limb grip strength test to assess skeletal muscle strength at week 20. After the grip strength test was conducted, the animals were euthanized for blood and tissue collection to analyze glycated hemoglobin (HbA1c), plasma insulin, and insulin resistance using the homeostatic model of insulin resistance (HOMA-IR) index and malondialdehyde (MDA) concentration. Correlation analysis was performed to examine the associations of skeletal muscle strength with HOMA-IR, plasma glucose, HbA1c, and MDA concentration. The results demonstrated increased HbA1c, FBG, insulin, HOMA-IR, and MDA concentrations in the PD group compared to the NPD group. Grip strength was reduced in the PD group compared to the NPD group. Grip strength was negatively correlated with HbA1c, plasma glucose, HOMA-IR, and MDA concentration in the PD group. These observations suggest that diet-induced prediabetes compromises muscle function, which may contribute to increased levels of sedentary behavior during prediabetes progression, and this may contribute to the development of hyperglycemia in T2DM.

Role of HNF4α-cMyc Interaction in CDE Diet–Induced Liver Injury and Regeneration

Hepatocyte nuclear factor 4 alpha (HNF4α) is a nuclear factor essential for liver function that regulates the expression of cMyc and plays an important role during liver regeneration. This study investigated the role of the HNF4α-cMyc interaction in regulating liver injury and regeneration using the choline-deficient and ethionine-supplemented (CDE) diet model. Wild-type (WT), hepatocyte-specific HNF4α-knockout (KO), cMyc-KO, and HNF4α-cMyc double KO (DKO) mice were fed a CDE diet for 1 week to induce subacute liver injury. To study regeneration, normal chow diet was fed for 1 week after CDE diet. WT mice exhibited significant liver injury and decreased HNF4α mRNA and protein expression after CDE diet. HNF4α deletion resulted in significantly higher injury with increased inflammation, fibrosis, proliferation, and hepatic progenitor cell activation compared with WT mice after CDE diet but indicated similar recovery. Deletion of cMyc lowered liver injury with activation of inflammatory genes compared with WT and HNF4α-KO mice after CDE diet. DKO mice had a phenotype comparable to that of the HNF4α-KO mice after CDE diet and a complete recovery. DKO mice exhibited a significant increase in hepatic progenitor cell markers both after injury and recovery phase. Taken together, these data show that HNF4α protects against inflammatory and fibrotic changes after CDE diet-induced injury, which is driven by cMyc.

LRP1 facilitates hepatic glycogenesis by improving the insulin signaling pathway in HFD-fed mice.

LDL receptor-related protein-1 (LRP1) is a cell-surface receptor that functions in diverse physiological pathways. We previously demonstrated that hepatocyte-specific LRP1 deficiency (hLRP1KO) promotes diet-induced insulin resistance and increases hepatic gluconeogenesis in mice. However, it remains unclear whether LRP1 regulates hepatic glycogenesis. Insulin signaling, glycogenic gene expression, and glycogen content were assessed in mice and HepG2 cells. The pcDNA 3.1 plasmid and adeno-associated virus serotype 8 vector (AAV8) were used to overexpress the truncated β-chain (β∆) of LRP1 both invitro and invivo. On a normal chow diet, hLRP1KO mice exhibited impaired insulin signaling and decreased glycogen content. Moreover, LRP1 expression in HepG2 cells was significantly repressed by palmitate in a dose- and time-dependent manner. Both LRP1 knockdown and palmitate treatment led to reduced phosphorylation of Akt and GSK3β, increased levels of phosphorylated glycogen synthase (GYS), and diminished glycogen synthesis in insulin-stimulated HepG2 cells, which was restored by exogenous expression of the β∆-chain. By contrast, AAV8-mediated hepatic β∆-chain overexpression significantly improved the insulin signaling pathway, thus activating glycogenesis and enhancing glycogen storage in the livers of high-fat diet (HFD)-fed mice. Our data revealed that LRP1, especially its β-chain, facilitates hepatic glycogenesis by improving the insulin signaling pathway, suggesting a new therapeutic strategy for hepatic insulin resistance-related diseases.

Glycogen synthesis is required for adaptive thermogenesis in beige adipose tissue and affects diet-induced obesity.

Glycogen is a form of energy storage for glucose in different tissues such as liver and skeletal muscle. It remains incompletely understood how glycogen impacts on adipose tissue functionality. Cold exposure elevated the expression of Gys1 that encodes glycogen synthase 1 in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT). The in vivo function of Gys1 was analyzed using a mouse model in which Gys1 was deleted specifically in adipose tissues. Under normal chow conditions, Gys1 deletion caused little changes to body weight and glucose metabolism. Deletion of Gys1 abrogated upregulation of UCP1 and other thermogenesis-related genes in iWAT upon prolonged cold exposure or treatment with β3-adrenergic receptor agonist CL-316,243. Stimulation of UCP1 by CL-316,243 in adipose-derived stromal cells (stromal vascular fractions, SVFs) was also reduced by Gys1 deletion. Both the basal glycogen content and CL-316,243-stimulated glycogen accumulation in adipose tissues were reduced by Gys1 deletion. High-fat diet-induced obesity and insulin resistance were aggravated in Gys1-deleted mice. The loss of body weight upon CL-316,243 treatment was also abrogated by the loss of Gys1. In conclusion, our results underscore the pivotal role of glycogen synthesis in adaptive thermogenesis in beige adipose tissue and its impact on diet-induced obesity in mice.NEW & NOTEWORTHY Glycogen is one of major types of fuel reserve in the body and its classical function is to maintain blood glucose level. This study uncovers that glycogen synthesis is required for beige fat tissue to generate heat upon cold exposure. Such a function of glycogen is linked to development of high-fat diet-induced obesity, thus extending our understanding about the physiological functions of glycogen.

Long-Term Sodium Deficiency Reduces Sodium Excretion but Impairs Renal Function and Increases Stone Formation in Hyperoxaluric Calcium Oxalate Rats.

Excessive sodium intake is associated with nephrolithiasis, but the impact of sodium-deficient (SD) diets is unknown. Hence, we investigated the effects of short- and long-term SD diets on the expression of renal aquaporins and sodium transporters, and thus calcium oxalate (CaOx) crystal formation in hyperoxaluria rats. In a short-term sodium balance study, six male rats received drinking water and six received 0.75% ethylene glycol (EG) to induce hyperoxaluria. After a 30-day period of feeding on normal chow, both groups were treated with a normal-sodium diet for 5 days, followed by a sodium-free diet for the next 5 days. In a long-term SD study (42 days), four groups, induced with EG or not, were treated with normal-sodium water and sodium-free drinking water, alternately. Short-term sodium restriction in EG rats reversed the daily positive sodium balance, but progressively caused a negative cumulative water balance. In the long-term study, the abundant levels of of Na/H exchanger, thiazide-sensitive Na-Cl cotransporter, Na-K-ATPase, and aquaporins-1 from SD + EG rats were markedly reduced, corresponding to a decrease in Uosm, as compared to SD rats. Increased urine calcium, AP(CaOx)index, and renal CaOx deposition were also noted in SD + EG rats. Although the SD treatment reduced sodium excretion, it also increased urinary calcium and impaired renal function, ultimately causing the formation of more CaOx crystals.

544 Interleukin-6 protects renal dysfunction in mouse models of hypertension and salt-sensitive hypertension

OBJECTIVES/GOALS: We are investigating the role of IL-6 in regulating renal function by measuring mean arterial pressure (MAP), renal plasma flow (RPF) and glomerular filtration rate (GFR) in wild type (WT) and IL-6-knockout (KO) mice in established mouse models of angiotensin II (AII)-dependent- hypertension and -salt-sensitive hypertension. METHODS/STUDY POPULATION: Twelve-week-old male WT and KO mice on the C57BL6 background strain were infused with vehicle (V; saline) or angiotensin II (AII; 200 ng/kg/min) for 12-14 days. Half of the AII-treatment groups were maintained on a high salt (HS; 6% NaCl) diet for the duration of the experiment, while the other half of the AII treatment groups and both vehicle groups were fed normal rat chow. MAP was continuously measured by a fluid filled catheter in conscious mice for the duration of the experiment. RPF and GFR were measured on days 12-14 in anesthetized mice by the para-aminohippurate, and fluorescein isothiocynate-Inulin techniques, respectively. All data were analyzed by 2-way ANOVA; *p<0.05 vs. WT, same treatment; #p<0.05 vs.V, same genotype; ^p<0.05, AII vs. AII+HS, same genotype. RESULTS/ANTICIPATED RESULTS: MAP was 31% lower in KO vs WT mice. AII increased MAP (1.2-fold) in WT but not KO mice. HS diet magnified AII-induced increases in MAP in WT and moderately increased MAP in AII-KO mice: [MAP (mmHg): WT+V, 130±7.0; KO+V, 91.0+4.0*; WT+AII, 153±5.0#; KO+AII, 83.0±4.0*; WT+AII+HS, 150±11#; KO+AII+HS, 93.0±4.0#]. AII infusion reduced RPF in the KO but not WT mice. Addition of HS reduced RPF in WT and exacerbated AII-induced reductions in RPF in KO mice [RPF (ml/min/g): WT+V, 1.82±0.23; KO+V, 1.91+0.40; WT+AII, 3.16±0.75#; KO+AII, 1.65±0.42*; WT+AII+HS, 1.10±0.31#^; KO+AII+HS, 1.13±0.XX#^]. The HS diet reduced GFR in AII-infused KO but not WT mice [GFR (µl/min/g): WT+V, 756±XX; KO+V, 788±XX; WT+AII, 1010±63*#; KO+AII, 756±23*; WT+AII+HS, 1100±150#; KO+AII+HS: 540±210*#^]. DISCUSSION/SIGNIFICANCE: The absence of IL-6 in male mice attenuated AII- and/or AII+HS-induced increases in MAP; however, it exacerbated HS-induced reductions in RPF and GFR. These findings suggest inhibiting IL-6 has therapeutic potential as an antihypertensive but not as a renal protective agent in hypertension and salt-sensitive hypertension disease states.

A combination of gliclazide and metformin attenuates obesity-induced polycystic ovary syndrome in female Wistar rats

Presently, it is known that the progression of obesity concomitantly leads to polycystic ovary syndrome and infertility. This study aimed to evaluate the potential effects of metformin (M; insulin secretagogues) and gliclazide (G; insulin sensitizer) alone and their combination at different doses to treat obesity-induced PCOS. High high-fat diet was given to all female Wistar rats for nine weeks to induce obesity except for the normal control group which received a normal chow diet. Estradiol valerate (0.8 mg/kg) was also given to all obese rats to induce polycystic ovarian syndrome. After the induction, M (100, 300 mg/kg) and G (5, 10 mg/kg) were given orally either individually or in combination for 28 days. The notable (p < 0.0001) reduction in body weight and blood glucose level was observed in treatment groups in contrast to disease control (DCG). The marked (p < 0.05–0.0001) decrease in hemocylated hemoglobin, serum insulin, cholesterol, triglycerides, and testosterone was observed in treated groups, notably in combination groups (M100+G10 mg/kg) in contrast to DCG. There was a considerable (p < 0.01–0.0001) increase in progesterone E2, estradiol, luteinizing, and follicle-stimulating hormones in treated groups as compared to DCG. Treatment with M and G treated groups also exhibited marked (p < 0.05–0.0001) increases in SOD, CAT, and GSH while decreased in NO and MDA levels in ovary tissue as evidenced by the histological study of the ovary. Treatment with M and G alone and in combination significantly (p < 0.0001) restored the serum IL-6, NrF2, and NF-κB levels as compared to DCG. The results inveterate that the M and G combination (M100+G10, and M300+G10) was useful in treating obesity-induced infertility due to antioxidant properties, hypolipidemic effects, and modulation of inflammatory markers.

Loss of ceramide synthase 5 inhibits the development of experimentally induced aortic valve stenosis.

Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high-fat/high-cholesterol diet (Western diet, WD). We used wild-type (WT) and CerS5-/- mice on WD or normal chow in a wire injury model. We measured the peak velocity to determine AVS development and performed histological analysis of the aortic valve area, immune cell infiltration (CD68 staining), and calcification (von Kossa). Invitro experiments involved measuring the calcification of human aortic valvular interstitial cells (VICs) and evaluating cytokine release from THP-1 cells, a human leukemia monocytic-like cell line, following CerS5 knockdown. CerS5-/- mice showed a reduced peak velocity compared to WT only in the experiment with WD. Likewise, we observed reduced immune cell infiltration and calcification in the aortic valve of CerS5-/- mice, but only on WD. Invitro, calcification was reduced after knockdown of CerS5 in VICs, while THP-1 cells exhibited a decreased inflammatory response following CerS5 knockdown. We conclude that CerS5 is an important mediator for the development of AVS in mice on WD and regulates critical pathophysiological hallmarks of AVS formation. CerS5 is therefore an interesting target for pharmacological therapy and merits further investigation.

Elevation of hypothalamic ketone bodies induces a decrease in energy expenditures and an increase risk of metabolic disorder

ObjectiveKetone bodies (such as β-hydroxybutyrate or BHB) have been recently proposed as signals involved in brain regulation of energy homeostasis and obesity development. However, the precise role of ketone bodies sensing by the brain, and its impact on metabolic disorder development remains unclear. Nevertheless, partial deletion of the ubiquitous ketone bodies transporter MCT1 in mice (HE mice) results in diet-induced obesity resistance, while there is no alteration under normal chow diet. These results suggest that ketone bodies produced during the high fat diet would be important signals involved in obesity onset. MethodsIn the present study we used a specific BHB infusion of the hypothalamus and analyzed the energy homeostasis of WT or HE mice fed a normal chow diet. ResultsOur results indicate that high BHB levels sensed by the hypothalamus disrupt the brain regulation of energy homeostasis. This brain control dysregulation leads to peripheral alterations of energy expenditure mechanisms. ConclusionsAltogether, the changes induced by high ketone bodies levels sensed by the brain increase the risk of obesity onset in mice.

Protective Effect of Pemafibrate Treatment against Diabetic Retinopathy in Spontaneously Diabetic Torii Fatty Rats.

Diabetic retinopathy (DR) can cause visual impairment and blindness, and the increasing global prevalence of diabetes underscores the need for effective therapies to prevent and treat DR. Therefore, this study aimed to evaluate the protective effect of pemafibrate treatment against DR, using a Spontaneously Diabetic Torii (SDT) fatty rat model of obese type 2 diabetes. SDT fatty rats were fed either a diet supplemented with pemafibrate (0.3 mg/kg/day) for 16 weeks, starting at 8 weeks of age (Pf SDT fatty: study group), or normal chow (SDT fatty: controls). Normal chow was provided to Sprague-Dawley (SD) rats (SD: normal controls). Electroretinography (ERG) was performed at 8 and 24 weeks of age to evaluate the retinal neural function. After sacrifice, retinal thickness, number of retinal folds, and choroidal thickness were evaluated, and immunostaining was performed for aquaporin-4 (AQP4). No significant differences were noted in food consumption, body weight, or blood glucose level after pemafibrate administration. Triglyceride levels were reduced, and high-density lipoprotein cholesterol levels were increased. Extension of oscillatory potential (OP)1 and OP3 waves on ERG was suppressed in the Pf SDT fatty group. Retinal thickness at 1,500 microns from the optic disc improved in the Pf SDT fatty group. No significant improvements were noted in choroidal thickness or number of retinal folds. Quantitative analyses showed that AQP4-positive regions in the retinas were significantly larger in the Pf SDT fatty group than in the SDT fatty group. The findings suggest that pemafibrate treatment can exert protective effects against DR.

Liver ChREBP deficiency inhibits fructose-induced insulin resistance in pregnant mice and female offspring

High fructose intake during pregnancy increases insulin resistance (IR) and gestational diabetes mellitus (GDM) risk. IR during pregnancy primarily results from elevated hormone levels. We aim to determine the role of liver carbohydrate response element binding protein (ChREBP) in insulin sensitivity and lipid metabolism in pregnant mice and their offspring. Pregnant C57BL/6J wild-type mice and hepatocyte-specific ChREBP-deficient mice were fed with a high-fructose diet (HFrD) or normal chow diet (NC) pre-delivery. We found that the combination of HFrD with pregnancy excessively activates hepatic ChREBP, stimulating progesterone synthesis by increasing MTTP expression, which exacerbates IR. Increased progesterone levels upregulated hepatic ChREBP via the progesterone-PPARγ axis. Placental progesterone activated the progesterone-ChREBP loop in female offspring, contributing to IR and lipid accumulation. In normal dietary conditions, hepatic ChREBP modestly affected progesterone production and influenced IR during pregnancy. Our findings reveal the role of hepatic ChREBP in regulating insulin sensitivity and lipid homeostasis in both pregnant mice consuming an HFrD and female offspring, and suggest it as a potential target for managing gestational metabolic disorders, including GDM.

Abstract 3974: Macronutrient composition dictates MASH-associated HCC immunosurveillance through microbiota alteration and metabolic adaptation

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with an estimated incidence of over one million cases by 2025. The advances in vaccines against HBV and HCV have led to a decrease in the number of virus-related cases; however, the obesity epidemic has contributed to a drastic increase in metabolic dysfunction-associated steatohepatitis (MASH) related HCC cases (e.g., obesity and type 2 diabetes induced HCC). Considering this trend, we aim to understand how lifestyle choices, like food composition, in combination with alcohol consumption at a “social drinking level” impacts host metabolism, and subsequently the development of MASH-induced HCC. We hypothesize that the impact of dietary composition on dysbiosis and immune cells, particularly adaptive immune cell metabolic status, will drive the phenotype. We have developed a MASH-induced HCC murine model, with long-term dietary intervention including seven different customized diets: normal chow (NC), ketogenic, solid high fat diet (sHFD), or isocaloric liquid Lieber DeCarli Diets (LDC): high fat/low carb (HF), high fat/low carb with ethanol (HF-EtOH), low fat/high carb (LF), and low fat/high carb with ethanol (LF-EtOH). Mice began special diet in late adolescences and disease progression was monitored over the course of a year, where mice developed HCC. Our model allowed for a comprehensive understanding of MASH-induced HCC where disease initiation could be studied beyond known contributors like obesity or body mass index (BMI). The animal model was analyzed comprehensively with high-parameter flow cytometry (FC), 16S sequencing, bulk RNA-seq, scRNA/ATAC-seq, and WGS. Long-term diet intervention led to macronutrient specific microbiota dysregulation, alterations in systemic metabolism and suppression of adaptive immune response, consequently impacting the progression of HCC. Customized dietary interventions showed a specific phenotype regarding HCC development and progression with distinct mutational genotype and particular response to immunotherapy. T and B cell subpopulation deficiency had a significant impact on MASH-induced HCC development in a macronutrient composition dependent manner. In summary, each dietary intervention showed a distinct pattern and effect on dysbiosis, systemic metabolic reprograming, cellular transcriptomic and epigenetic alteration, and particularly immune cell effector function. Additionally, some compositions supported the immuno-escape mechanism of malignant cells through regulation of their MHC machinery. Collectively, these accumulated data construct an atlas for understanding the underlying molecular mechanisms contributing to lifestyle-induced HCC providing the knowledge needed to advance effective strategies for treatment and prevention. Citation Format: Nicolas T. Ryujin, Jian Huang, Albert Nguyen, Jared Edwards, Yuhe Cheng, Jessica Wen, Ashish Damania, Nadim Ajami, Spencer Rosario, Mark Long, Ludmil Alexandrov, Shabnam Shalapour. Macronutrient composition dictates MASH-associated HCC immunosurveillance through microbiota alteration and metabolic adaptation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3974.