Obese Zucker Rat Model Research Articles

Abstract 759: Soy protein diet changes metabolic profile in liver of 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary tumors in obese zucker rats

Abstract Obesity is associated with the risk of certain types of cancers development including breast cancer. Previously, we reported that obesity increased mammary tumor development using obese Zucker rat model. Several studies have shown the health benefit of soy protein consumption. However, the effects of soy protein diet on liver of mammary tumor for oxidative stress and DNA reaction is less known. We used DMBA induced-mammary tumor model to investigate the effects of soy protein diet on pro-oxidative environment and oxidative DNA modification in liver of obese DMBA mammary tumors rats. Female obese zucker rats (n=45) were either fed casein as control (OC; n=20) or soy protein isolate (OS; n=25), received DMBA (65mg/kg BW) at the age 50 days and sacrificed 20 weeks later. We used HPLC-ECD, HPLC-UV and LC-MS techniques for liver metabolic analysis. Comparing liver of OS vs. OC, soy protein 1) decreased the level of Methionine (P<0.02), increased 2) free reduced Homocysteine (P<0.02), 3) S-Adenosylmethionine (SAM, P<0.03), 4) S-Adenosyhomocysteine (SAH, P<0.001) and 5) 5methyl-Cytosine (5mC, P<0.03). Also, soy protein decreased (P<0.003) level of free oxidized Glutathione (GSSG), increased (P<0.02) GSH/GSSG oxidative stress ratio, without any changes a level of free reduced Glutathione (GSH), followed decrease (P<0.04) in liver DNA level of 8-OH-Guanosine (8-OH-G) and level (P<0.02) of 5hmethyl-Cytosine (5hmC). Our results shows soy protein diet capable change liver metabolism: a. Modify a methylation status by increasing levels of SAM (universal methyl group donor) following global liver DNA hypermethylation (5mC), and SAH (methylases inhibitor); b. Alleviate level of oxidative stress in liver by decreasing level of GSSG (not active glutathione), increasing GSH/GSSG ratio (antioxidative capacities), following lower ROS liver DNA damages by decrease levels of 8-OH-G and 5hmC. In conclusion, using obese DMBA-induced mammary tumor Zucker rat model, soy diet making a complex and significantly modification of metabolic conditions in liver affecting both methionine cycle and transsulfuration metabolic pathways. Citation Format: Reza Hakkak, Stepan Melnyk. Soy protein diet changes metabolic profile in liver of 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary tumors in obese zucker rats [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 759.

Effect of Metformin Treatment on Serum Metabolic Profile Changes in Lean and Obese Zucker Rat Model for Fatty Liver Disease.

Excessive weight and obesity are the leading risk factors for the development of chronic diseases, including diabetes. Metformin is capable of significantly improving coexisting complications of diabetes. We used a metabolomics approach to examine the effects of metformin administration on lean and obese (fa/fa) Zucker rats. After 1 week of acclimation, twenty-eight 5-week-old female lean and obese rats were randomly assigned to and maintained in the following four groups (seven rats/group) for 10 weeks: (1) lean control (LC); (2) obese control (OC); (3) lean metformin (LM); and (4) obese metformin (OM). At the end of 10 weeks, serum was collected and analyzed using HPLC with electrochemical detection, HPLC with UV detection, and liquid chromatography mass spectrometry. We selected 50 metabolites' peaks that were shared by all four groups of rats. Peak heights, as a defining factor, generally decreased in metformin-treated lean rats vs. untreated lean controls (3 LM:16 LC). Peak heights generally increased in metformin-treated obese rats vs. untreated obese controls (14 OM:5 OC). Overall, individual peaks were distributed as 11 that represented only lean rats, 11 that represented only obese rats, and 8 that were common among both lean and obese rats. In future studies, we will use a targeted metabolomics approach to identify those metabolites, map them to biochemical pathways and create a list of biomarkers. In summary, the current study contributed to a better understanding of the basic metabolic changes of lean and obese rats and demonstrated that both obesity and metformin make a significant impact on the metabolome of Zucker rats.

P31-022-23 Feeding Soy Protein Concentrate With Low and High Isoflavones Can Reverse Non-Alcoholic Fatty Liver Disease Caused by Obesity in Obese Zucker Rat Model

P31-022-23 Feeding Soy Protein Concentrate With Low and High Isoflavones Can Reverse Non-Alcoholic Fatty Liver Disease Caused by Obesity in Obese Zucker Rat Model

Feeding Soy Protein Concentrates with Low and High Isoflavones Alters 9 and 18 Weeks Serum Isoflavones and Inflammatory Protein Levels in Lean and Obese Zucker Rats

Soy's anti-inflammatory properties contribute to the health benefits of soy foods. This study was designed to investigate the bioavailability of soy isoflavones and whether the isoflavone content of soy protein concentrate diet would affect serum inflammatory proteins in an obese (fa/fa) Zucker rat model. Six-week-old male lean (L) and obese (O) Zucker rats were fed a casein control diet (C), soy protein concentrate with low isoflavones (SPC-LIF), or soy protein concentrate with high isoflavones (SPC-HIF) (7 rats/dietary group) before being killed at 9 and 18 weeks. Serum samples were analyzed for isoflavones and inflammatory proteins. At both time points, serum total (aglycone + conjugates) genistein, daidzein, and equol concentrations were significantly higher in L-SPC-HIF and O-SPC-HIF groups compared with L-SPC-LIF and O-SPC-LIF groups, respectively, and were not detectable in either L-C or O-C groups. At week 9, serum C-reactive protein (CRP) concentration was significantly lower in O-SPC-HIF group compared with O-C and O-SPC-LIF group, whereas proteins tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels did not differ between any groups. At week 18, serum CRP levels in both O-SPC-HIF and O-SPC-LIF groups were significantly lower compared with the O-C group. TNF-α level was higher in the O-SPC-LIF group compared with both O-C and O-SPC-HIF groups, whereas IL-6 levels were not different between any groups. Taken together, feeding Zucker rats SPC-LIF and SPC-HIF diets led to different serum isoflavone concentrations in both L and O Zucker rats and altered CRP and TNF-α levels in obese Zucker rats compared with controls.

Diet Containing Soy Protein Concentrate With Low and High Isoflavones for 9 Weeks Protects Against Non-alcoholic Fatty Liver Steatosis Using Obese Zucker Rats.

Non-alcoholic fatty liver disease (NAFLD), is one of the main liver diseases in the US and the world which often is related to obesity. Previously, we reported short- and long-term consumption of soy protein isolate diet with high isoflavones can reduce liver steatosis in the male and female obese Zucker rat model. However, the effects of high vs. low soy isoflavones on NAFLD is less known. The objectives of the present study were to examine the role of isoflavones levels in soy protein concentrate diets on protection against NAFLD in an obese rat model. Forty-two 6-week old lean (L, n = 21) and obese (O, n = 21) Zucker rats were randomly assigned to 1 of 3 dietary groups: casein diet (C = control), soy protein concentrate with low isoflavones (LIF), or soy protein concentrate with high isoflavones (HIF) for 9 weeks. Rats were weighed twice weekly. After 9 weeks, rats were sacrificed and samples of livers were taken for histopathological analysis. Serums were collected to measure ALT and AST levels. Results indicate that obese rats gained significantly more weight than lean rats for all three diet groups (P < 0.001). No significant difference in body weight between LC, LLIF and LHIF was noted. However, the OLIF and OHIF rats gained significantly more weight than OC rats (P < 0.001). Liver steatosis scores were significantly greater in obese rats compared to lean rats (P < 0.001). The OLIF and OHIF-fed rats had significantly reduced steatosis scores than OC rats (P = 0.013 and P < 0.001, respectively). The serum ALT levels were significantly greater in OC, OLIF and OHIF compared to LC, LLIF and LHIF, respectively (P < 0.001, P < 0.001, and P = 0.011). AST serum levels were greater in OC and OLIF compared to LC and LLIF, respectively (P = 0.001 and P = 0.022). In summary, we found that soy protein concentrate with isoflavones protects against liver steatosis and the protection is greater with a higher concentration of isoflavones.

Abstract 740: Obesity promotes liver pro-oxidative and DNA damage in mammary tumor of Zucker rats

Abstract Obesity is a major health problem in the US and world. Obesity is associated with the risk of cardiovascular disease, type 2 diabetes, and certain types of cancers such as breast cancer. Our laboratory reported that obesity increased mammary tumor model using obese Zucker rat model. However, the effects of obesity in the liver in regard to oxidative stress and DNA reaction is less known. In present study, we used a DMBA induced-mammary tumor model to further investigate the effects of obesity on creation of pro-oxidative environment in liver and oxidative DNA reaction. Lean and obese female rats were at 50 days of age received 7,12-dimethylbenz(α)anthracene (DMBA) and were sacrificed 20 weeks later. HPLC-ECD, HPLC-UV and LC-MS were used. Obesity capable significantly modify methylation status in liver caused higher (p &amp;lt; 0.001) SAM/SAH ratio (know as “methylation ratio”) and increased (p &amp;lt; 0.05) global DNA methylation. Also, obesity decrease GSH/GSSG ratio (known as “oxidative stress ratio”) with p &amp;lt; 0.0001 inducing higher oxidative stress and as results, increase presence both oxidized guanosine (8-OH-Guanosine) with p &amp;lt; 0.001 and oxidized 5metylCytosine (5hmCytosine) p&amp;lt;0.04 in liver DNA. 8-OH-Guanosine/5hmCytosine ratio was not significantly affected. Obesity caused significant oxidative stress in liver, causing oxidative DNA damage and change of DNA methylation pattern. These changes may contribute to the development of fatty liver in breast cancer models. Citation Format: Reza Hakkak, Stepan Melnyk, Soheila Korourian. Obesity promotes liver pro-oxidative and DNA damage in mammary tumor of Zucker rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 740.

Feeding Obese Zucker Rats With Soy Protein Concentrate With High Isoflavones Compared to Low Isoflavones Leads to a Significant Reduction of Serum Tumor Necrosis Factor-α

ObjectivesSoy isoflavones have been proposed to be the functional compounds that mediate the health benefits of soy-rich foods. This study was designed to investigate whether level of isoflavone content (high or low) of soy protein concentrate diet would affect serum TNF-α concentration in an obese Zucker rat model. Methods42 six-week old lean (L, n = 21) and obese (O, n = 21) Zucker rats were randomly assigned to one of 3 dietary groups after one week of acclimation: casein diet (C = control), soy protein with low isoflavones (LIF), or soy protein with high isoflavones (HIF) for 18 weeks (n = 7 rats/group). Rats were weighed twice weekly. After 18 weeks, rats were sacrificed and serum samples were collected. Serum TNF-α concentrations were determined by ELISA and compared between groups. ResultsAt the end of 18 weeks of dietary treatment, obese rats gained significantly more weight than lean rats in all three diet groups. In lean rats, there were no significant differences in body weight or serum TNF-α concentration between any diet groups (C, LIF, or HIF). In contrast, in obese rats, both LIF and HIF groups had significantly higher body weight compared to control group. While O-LIF and O-HIF groups did not differ in body weight, O-HIF group had significantly lower serum TNF-α concentration, suggesting a decreased level of systemic inflammation compared to O-LIF group. ConclusionsHIF soy protein diet significantly decreased serum TNF-α concentration in obese Zucker rats compared to LIF soy protein diet independent of body weight. Isoflavones may be responsible for the observed anti-inflammatory effect. Because systemic inflammation contributes to the etiology of a plethora of diseases, soy isoflavones may exert their many health benefits partially through downregulating systemic inflammation. Funding SourcesThis project is support USDA #12,889,791 to RH.

Short-Term Metformin Treatment Enriches Bacteroides dorei in an Obese Liver Steatosis Zucker Rat Model.

Obesity is the leading cause of health-related diseases in the United States and World. Previously, we reported that obesity can change gut microbiota using the Zucker rat model. Metformin is an oral anti-hyperglycemic agent approved by the FDA to treat type 2 diabetes (T2D) in adults and children older than 10 years of age. The correlation of short-term metformin treatment and specific alterations to the gut microbiota in obese models is less known. Short-term metformin has been shown to reduce liver steatosis. Here we investigate the effects of short-term metformin treatment on population of gut microbiota profile in an obese rat model. Five week old obese (n = 12) female Zucker rats after 1 week of acclimation, received AIN-93 G diet for 8 weeks and then rats were randomly assigned into two groups (6 rats/group): (1) obese without metformin (ObC), or (2) obese with metformin (ObMet). Metformin was mixed with AIN-93G diet at 1,000 mg/kg of diet. Rats were weighed twice per week. All rats were sacrificed at the end of metformin treatment at 10 weeks and fecal samples were collected and kept at −80°C. Total microbial DNA was collected directly from the fecal samples used for shotgun-metagenomics sequencing and subsequently analyzed using MetaPlAn and HUMAnN. After stringent data filtering and quality control we found significant differences (p = 0.0007) in beta diversity (Aitchison distances) between the ObC vs. ObMet groups. Supervised and unsupervised analysis of the log-ratios Bacteroides dorei and B. massiliensis vs. all other Bacteroides spp., revealed that B. dorei and B. massiliensis were enriched in the ObMet group, while the remaining Bacteroides spp. where enriched in the ObC group (p = 0.002). The contributional diversity of pathways is also significantly associated by treatment group (p = 0.008). In summary, in the obese Zucker rat model, short-term metformin treatment changes the gut microbiota profile, particularly altering the composition Bacteroides spp. between ObC and ObMet.

Metabolic Status of Lean and Obese Zucker Rats Based on Untargeted and Targeted Metabolomics Analysis of Serum.

Obesity is growing worldwide epidemic. Animal models can provide some clues about the etiology, development, prevention, and treatment of obesity. We examined and compared serum metabolites between seven lean (L) and seven obese (O) female Zucker rats to investigate the individual serum metabolic profile. A combination of HPLC-UV, HPLC-ECD, and LC-MS revealed more than 400 peaks. The 50 highest quality peaks were selected as the focus of our study. Untargeted metabolomics analysis showed significantly higher mean peak heights for 20 peaks in L rats, generally distributed randomly, except for a cluster (peaks 44–50) where L showed stable dominancy over O. Only eight peaks were significantly higher in O rats. Peak height ratios between pairs of L and O rats were significantly higher at 199 positions in L rats and at 123 positions in O rats. Targeted metabolomics analysis showed significantly higher levels of methionine, cysteine, tryptophan, kynurenic acid, and cysteine/cystine ratio in L rats and significantly higher levels of cystine and tyrosine in O rats. These results contribute to a better understanding of systemic metabolic perturbations in the obese Zucker rat model, emphasizing the value of both whole metabolome and individual metabolic profiles in the design and interpretation of studies using animal models.

Noninvasive estimation of local speed of sound by pulse-echo ultrasound in a rat model of nonalcoholic fatty liver

Objective. Speed of sound has previously been demonstrated to correlate with fat concentration in the liver. However, estimating speed of sound in the liver noninvasively can be biased by the speed of sound of the tissue layers overlying the liver. Here, we demonstrate a noninvasive local speed of sound estimator, which is based on a layered media assumption, that can accurately capture the speed of sound in the liver. We validate the estimator using an obese Zucker rat model of non-alcoholic fatty liver disease and correlate the local speed of sound with liver steatosis. Approach. We estimated the local and global average speed of sound noninvasively in 4 lean Zucker rats fed a normal diet and 16 obese Zucker rats fed a high fat diet for up to 8 weeks. The ground truth speed of sound and fat concentration were measured from the excised liver using established techniques. Main Results. The noninvasive, local speed of sound estimates of the livers were similar in value to their corresponding ‘ground truth’ measurements, having a slope ± standard error of the regression of 0.82 ± 0.15 (R 2 = 0.74 and p < 0.001). Measurement of the noninvasive global average speed of sound did not reliably capture the ‘ground truth’ speed of sound in the liver, having a slope of 0.35 ± 0.07 (R 2 = 0.74 and p < 0.001). Decreasing local speed of sound was observed with increasing hepatic fat accumulation (approximately −1.7 m s−1 per 1% increase in hepatic fat) and histopathology steatosis grading (approximately −10 to −13 m s−1 per unit increase in steatosis grade). Local speed of sound estimates were highly correlated with steatosis grade, having Pearson and Spearman correlation coefficients both ranging from −0.87 to −0.78. In addition, a lobe-dependent speed of sound in the liver was observed by the ex vivo measurements, with speed of sound differences of up to 25 m s−1 (p < 0.003) observed between lobes in the liver of the same animal. Significance. The findings of this study suggest that local speed of sound estimation has the potential to be used to predict or assist in the measurement of hepatic fat concentration and that the global average speed of sound should be avoided in hepatic fat estimation due to significant bias in the speed of sound estimate.

Effects of Short-Term Metformin Treatment on Gut Microbiota Profile Using Female Obese Zucker Rat Model

ObjectivesThe correlation of short-term metformin treatment and specific alterations to the gut microbiota in obese models is less known. So, the objectives of this experiment was to investigate the effects of short-term metformin treatment on population of gut microbiota profile in obese rat model. MethodsFive week old obese (n = 16) female Zucker rats after one week of acclimation, received AIN-93 G diet for 8 weeks and then rats were randomly assigned 8 rats/group): to 1) obese without metformin (ObC), or 2) obese with metformin (ObMet). Metformin were mixed with AIN-93G diet at 1000 mg/kg of diet. Rats were weighed twice per week. All rats were sacrificed 10 weeks post-metformin treatment and fecal samples were collected and kept at – 80c. Total microbial DNA were collected directly from the fecal samples using a PowerSoil® DNA isolation kit. Isolated DNA were used for shotgun-metagenomics data collection using Illumina NextSeq500 and analyzed using MetaPlAn and HUMAnN. DEICODE and Songbird used calculate log-ratios and differential ranks of taxa and functional pathways associated with metformin treatment respectively. The were then visualized using Qurro. ResultsThere was no significant difference between ObC vs. ObMet group body weight (P = 0.20). Overall microbial beta-diversity (DEICODE), showed significant separation between the obese control and metformin samples (P = 0.0007). Differential ranking (Songbird) of Bacteroides dorei and B. massiliensis vs. all other Bacteroides spp., revealed that B. dorei and B. massiliensis were enriched in the obese metformin group, while the remaining Bacteroides spp. where enriched in the obese control group (P = 0.002). The differential ranking of pathway diversity contributed by the Bacteroides were also associated with treatment group (P = 0.008). ConclusionsIn summary, in the obese zucker rat model, short-term metformin treatment changes the gut microbiota profile. Funding SourcesArkansas Biosciences Institute.

Effects of obesity and 10 weeks metformin treatment on liver steatosis.

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disease in adolescents and adults, and the risk of developing NAFLD increases with obesity. In the present study, it was shown that obesity increased fatty liver (steatosis) using an obese Zucker rat model. Metformin is an oral anti-hyperglycemic agent approved by the FDA for treatment of type 2 diabetes in adults and children >10 years of age. There is insufficient evidence regarding the effects of metformin on pediatric liver steatosis. Thus, in the present study, the effects of 10 weeks metformin treatment on liver steatosis and related serum markers for liver damage was assessed. Lean and obese (n=16 per group) 5-week old female Zucker rats were provided an AIN-93 G diet for 8 weeks to induce NAFLD, and then rats were randomly assigned to 4 groups (8 rats/group): i) lean without metformin (LC), ii) lean + metformin (LM), iii) obese without metformin (OC), and iv) obese + metformin (OM). Rats were provided ad libitum access to the diet containing metformin (1 g metformin per kg of food). Rats were weighed twice weekly and were sacrificed 10 weeks later. Serum was collected to measure the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), leptin and adiponectin. Livers were collected for histological analysis. The results showed that obese rats gained significantly more weight than lean rats in both the control and metformin treatment groups (P<0.001). OM treated rats exhibited a lower degree of liver steatosis compared with the OC rats (P<0.04). There were no significant differences in serum ALT levels between the groups. However, obesity significantly increased serum AST levels in both the control and metformin treatment groups (P=0.01). The ratio of leptin to adiponectin was increased in obese compared with the lean rats in both the control and metformin treatment groups (P<0.0001). There was no effect of metformin on serum biomarkers. In summary, short-term metformin treatment decreased liver steatosis but did not affect the serum markers of liver steatosis.

Long-Term Soy Protein Isolate Consumption Reduces Liver Steatosis Through Changes in Global Transcriptomics in Obese Zucker Rats.

To determine how soy protein isolate (SPI) ameliorated liver steatosis in male obese Zucker rats, we conducted global transcriptomic expression (RNAseq) analysis on liver samples of male rats fed either the SPI or a control casein (CAS)-based diet (n = 8 per group) for 16 weeks. Liver transcriptomics were analyzed using an Ilumina HiSeq system with 2 × 100 base pair paired-end reads method. Bioinformatics was conducted using Ingenuity Pathway Analysis (IPA) software (Qiagen, CA) with P < 0.05 and 1.3-fold differential expression cutoff values. Regression analysis between RNAseq data and targeted mRNA expression analysis of 12 top differentially expressed genes (from the IPA program) using quantitative PCR (qPCR) revealed a significant regression analysis (r2 = 0.69, P = 0.0008). In addition, all qPCR values had qualitatively similar direction of up- or down-regulation compared to the RNAseq transcriptomic data. Diseases and function analyses that were based on differentially expressed target molecules in the dataset predicted that lipid metabolism would be enhanced whereas inflammation was predicted to be inhibited in SPI-fed compared to CAS-fed rats at 16 weeks. Combining upstream regulator and regulator effects functions in IPA facilitates the prediction of upstream regulators (e.g., transcription regulators) that could play important roles in attenuating or promoting liver steatosis due to SPI or CAS diets. Upstream regulators that were predicted to be activated (from expression of down-stream targets) linked to increased conversion of lipid and transport of lipid in SPI-fed rats included hepatocyte nuclear factor 4 alpha (HNF4A) and aryl hydrocarbon receptor (AHR). Upstream regulators that were predicted to be activated in CAS-fed rats linked to activation of phagocytosis and neutrophil chemotaxis included colony stimulating factor 2 and tumor necrosis factor. The results provide clear indication that long-term SPI-fed rats exhibited diminished inflammatory response and increased lipid transport in liver compared to CAS-fed rats that likely would contribute to reduced liver steatosis in this obese Zucker rat model.

Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence.

Human stem cell therapy for type 2 diabetes/obesity (T2D/O) complications is performed with stem cell autografts, exposed to the noxious T2D/O milieu, often with suboptimal results. We showed in the Obese Zucker (OZ) rat model of T2D/O that when their muscle-derived stem cells (MDSC) were from long-term T2D/O male rats, their repair efficacy for erectile dysfunction was impaired and were imprinted with abnormal gene- and miR-global transcriptional signatures (GTS). The damage was reproduced in vitro by short-term exposure of normal MDSC to dyslipidemic serum, causing altered miR-GTS, fat infiltration, apoptosis, impaired scratch healing, and myostatin overexpression. Similar in vitro alterations occurred with their normal counterparts (ZF4-SC) from the T2D/O rat model for female stress urinary incontinence, and with ZL4-SC from non-T2D/O lean female rats. In the current work we studied the in vitro effects of cholesterol and Na palmitate as lipid factors on ZF4-SC and ZL4-SC. A damage partially resembling the one caused by the female dyslipidemic serum was found, but differing between both lipid factors, so that each one appears to contribute specifically to the stem cell damaging effects of dyslipidemic serum in vitro and T2D/O in vivo, irrespective of gender. These results also confirm the miR-GTS biomarker value for MDSC damage.

Effects of Obesity and Short-Term Metformin Treatment on Liver Steatosis in Female Zucker Rats

Abstract Objectives Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disease in adolescents in the US and World, and the risk has increased with the rise in obesity. We reported that obesity increases Fatty liver (steatosis) using an obese Zucker rat model. Metformin is an oral anti-hyperglycemic agent approved by the FDA to treat type 2 diabetes (T2D) in adults and children older than 10 years of age. There is insufficient evidence regarding the effects of metformin in pediatric liver steatosis. The objective of this study was to investigate the effects of short-term metformin treatment on liver steatosis and related serum markers for liver damage. Methods Five week old lean (n = 16) and obese (n = 16) female Zucker rats after one week of acclimation, received AIN-93 G diet for 8 weeks to induce NAFLD. After 8 weeks, lean and obese rats were randomly assigned to the following four groups (8 rats/group): 1) lean without metformin (LC), 2) lean with metformin (LMet), 3) obese without metformin (ObC), and 4) obese with metformin (ObMet). Metformin were mixed with AIN-93 G diet at 1000 mg/kg of diet. Rats were weighed twice per week. All rats were sacrificed 10 weeks post-metformin treatment and serum and livers were collected. Steatosis was semiquantitated as a score of 1 to 4 based upon the relative degree of steatosis within hepatocytes: 1) &amp;lt; 25%, 2) 25–50%, 3) 50–75%, and 4) &amp;gt;75%. Serums were collected to measure the levels of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) on a clinical analyzer. Results Obese rats gained significantly more weight (P &amp;lt; .001) than lean rats for both control and Metformin treatment groups and there was no significant difference between ObC vs. ObMet group (P = 0.20). The mean + SD liver steatosis scores for the LC, LMet, ObC and ObMet groups were 0.13 + o.3, 0.13 + o.3, 3.67 + 0.52 and 3.00 + 0.82. The ObMet treated rats had lower (P &amp;lt; 0.04) liver steatosis than ObC rats. There were no significant differences for the serum ALT and AST levels between groups. However, obesity increased significantly (P &amp;lt; 0.01) serum AST levels compared to LC but not in the metformin group. Conclusions In summary, in the obese zucker rat model, short-term metformin treatment decreased liver steatosis but did not impact serum markers of liver steatosis. Funding Sources Arkansas Children Research Institute/Arkansas Bioscience Institute.

Global Gene Expression and Pathway Analysis of Liver Obtained After 8 and 16 Weeks of Feeding Soy Isolate- or Casein-Based Diets in Male Obese Zucker Rats

Abstract Objectives To understand how soy protein isolate (SPI) reduced liver steatosis in male obese Zucker rats, we conducted global gene expression (RNAseq) analysis on liver samples of male rats fed either the SPI or a control casein (CAS)-based diet (n = 8 per group) for 8 or 16 weeks. Methods Data was analyzed using Ingenuity Pathway Analysis (IPA) software (Qiagen, CA) using a P &amp;lt; 0.05 and 1.3 fold differential expression cutoff values. Results Out of 12 top differentially expressed genes (both up- and down-regulated) that were listed at either 8 or 16 wks of feeding (24 total), only the expression of one gene exhibited a reversal (from down-regulated to up-regulated) between 8 and 16 wks indicating consistency in the liver steatosis model. An assessment of diseases and functions based on differentially expressed target molecules in the dataset revealed that lipid metabolism was predicted to be enhanced at 16 wks whereas inflammatory response was predicted to be inhibited in SPI-fed compared to CAS-fed rats at both 8 and 16 weeks. Using the upstream regulator analysis and regulator effects functions of the IPA program enables the prediction of a number of upstream regulators (e.g., transcription regulators) that could be playing important roles in attenuating or promoting liver steatosis due to SPI or CAS diets. Examples of upstream regulators that were predicted to be activated (based on expression of down-stream target molecules) that were linked to increased conversion of lipid and transport of lipid in SPI-fed rats included hepatocyte nuclear factor 4 alpha and aryl hydrocarbon receptor. Examples of upstream regulators that were predicated to be activated in CAS-fed rats that were linked to predicted activation of phagocytosis and neutrophil chemotaxis included colony stimulating factor 2 and tumor necrosis factor. Conclusions We believe these findings may shed light on mechanisms that SPI is able to reduce liver steatosis in this obese Zucker rat model. Funding Sources Arkansas Bioscience Institute and U of A Chancellor Innovative Fund.

Mitochondrial Respiration in Female Zucker Rats: Effects of Obesity and Short-Term Metformin Treatment

Abstract Objectives As childhood obesity rates climb, so has the incidence of type 2 diabetes among children and adolescents. Metformin, an FDA approved anti-hyperglycemic drug, is thought to target the mitochondria, but its effects on mitochondrial function in obesity have not been well studied. We used an obese Zucker rat model to investigate the effects of obesity and short-term metformin treatment on mitochondrial respiration. Methods Five-week old female Zucker rats (n = 16 lean, n = 16 obese) were fed AIN-93 G diet for 8 weeks before equally randomized to receive metformin (mixed in diet at 1 g/kg of feed); thus forming 4 groups with 8 rats each: lean +/– metformin and obese +/– metformin. Rats were sacrificed 10 weeks post-metformin treatment and spleens, perigonadal visceral adipose tissue (VAT) and skeletal muscle (SM; gracilis) collected. Mitochondrial respiration was measured in splenocytes by extracellular flux analysis and in VAT and SM fibers by high resolution respirometry. Results Effects of obesity on mitochondrial respiration were found in VAT and SM, but not splenocytes. In VAT, obese rats exhibited increased OXPHOS capacity over lean rats when octanoylcarnitine and malate were provided as substrates (obese vs lean: 1.33 vs 0.76 pmol O2/s/mg; SEdiff = 0.18, P = .005), and after subsequent additions of pyruvate (P = .012), glutamate (P = .009), and succinate (P = .045). In SM, OXPHOS capacity was increased when octanoylcarnitine and malate were provided as substrates (obese vs lean: 12.18 vs 5.45 pmol O2/s/mg; SEdiff = 2.31, P = .011) in obese vs lean rats. Metformin effects were observed only in splenocytes: coupling efficiency was decreased (metformin vs no metformin; 56.2% vs 69.8%; SEdiff = 4.1%, P = 0.005) and proton leak was increased (P &amp;lt; .001) in metformin treated rats as compared to rats not treated with metformin. Conclusions We found obesity was associated with increased mitochondrial respiration, particularly fatty acid oxidation, in VAT and SM. Short-term metformin treatment did not alter mitochondrial respiration in VAT or SM, but was found to increase proton leak and reduce coupling efficiency in splenocytes. Funding Sources Arkansas Children's Research Institute, Arkansas Biosciences Institute (R.H). S.R. and E.C. are also supported by the National Institute of General Medical Sciences of the National Institutes of Health.

Obesity and psychosocial stress influence the remodelling of pericardial adipose tissue

In obesity, fat is deposited at different sites around the body, but pericardial fat has been shown to have a particularly negative impact on the heart. Very little is known about the function of pericardial fat and how its phenotype is influenced by obesity. The aim of this study was to determine how obesity and stress affects the pericardial phenotype relative to visceral adipose tissue.Here we have used the obese Zucker rat (fa/fa) model induced by a mutation in the leptin receptor. Zucker rats develop hyperphagia with reduced energy expenditure, resulting in morbid obesity; impaired glucose and insulin sensitivity in a few weeks after birth. At 9 weeks of age lean (LZR) and obese (OZR) Zucker male rats were treated with or without unpredictable chronic mild stress (UCMS) for 8 weeks to mimic human clinical stress and depression. Adipose tissue can expand either through cellular hypertrophy or hyperplasia, with the former correlating with decreased metabolic health in obesity. To determine the phenotype for pericardial and visceral adipose tissue, tissue cellularity and gene expression were analysed. Tissue cellularity was determined following Haematoxylin and Eosin (H&amp;E) staining, whilst qPCR was used to examine gene expression.Data from these experiments showed that, as expected, the OZR group were considerably heavier than the lean controls (LZR). Total plasma corticosterone and glucose all increased in the obese group indicating that obesity in itself increases stress. Furthermore, UCMS stress conditions had an additive effect by increasing these parameters further in both the lean LZR and the obese OZR group indicating that stress had a negative metabolic impact.H&amp;E staining showed that there was greater heterogeneity in the cellularity of pericardial adipocytes compared with visceral adipose tissue, suggesting a mix of white, beige and brown‐like adipocytes. Analysis of tissue cellularity showed that obesity did not result in a significant alteration in adipocyte cell size in pericardial fat. However, when UCMS was combined with obesity there was a significant increase in adipocyte cell size relative to the lean group. This suggests that under non‐stressful conditions, pericardial adipose tissue does not expand predominantly through hypertrophy, unlike visceral adipocytes. However, stressful conditions combine with obesity to produce a more pathological pericardial adipose tissue phenotype.Gene expression analysis showed that UCMS stress caused an upregulation of leptin in the lean compared with the obese group. This suggests that leptin signalling is important for the stress‐dependent increase in adipose tissue leptin expression. Finally, the thermogenic marker UCP1 was upregulated in pericardial adipose tissue of obese rats relative to the lean controls but not in visceral tissue. These data strongly indicate that there is differential regulation of gene expression between pericardial and visceral adipose tissue which may have major implications for the heart in obesity.Support or Funding InformationUniversity of Reading, UK and the Petroleum Technology Development Fund, Nigeria

Effects of Obesity on Serum Calcium and Parathyroid Hormone in Zucker Rat Model (P08-032-19)

ObjectivesObesity has been epidemic in the United States and world for more than two decades. Obesity is associated with type 2 diabetes, cardiovascular disease and certain types of cancer. Primary Hyperparathyroidism (pHPT) is also more commonly seen in western societies and is also increasing. Previously, we have reported that obesity caused a significant reduction in serum Vitamin D using obese rat model. The role of obesity on serum Ca and PTH is less known. The main objective of this study was to investigate the effect of obesity on serum calcium (Ca) and parathyroid hormone (PTH) using obese Zucker rat model. Perhaps this could serve as an animal model for pHPT. MethodsFive-week-old female lean and obese Zucker rats were used. After one week of acclimation, lean and obese rats were maintained on AIN-93 G diet for 155 days. Rats were weighed twice per week. At the end of the experiment, rats were sacrificed and serum were collected to measure serum protein, Ca and PTH levels. Serum protein was measured by atomic absorbance spectrophotometry, serum calcium was measured by using with Arsenazo III method and serum PTH by a two-site enzyme-linked immunosorbent assay using a commercially available rat intact PTH ELISA kit. ResultsOur results shows that 1) obese rats gained significantly (P < 0.001) more weight than lean rats; 2) obesity caused a significant increase in serum protein (P < 0.05); 3) obesity increased significantly serum Ca (P < 0.05) and 4) increased a significant decrease PTH levels (P < 0.05). ConclusionsOur results show that obesity increased serum Ca and decreased PTH levels. This is a physiologic negative feedback relationship expected in a normal parathyroid system. If this had been a model for pHPT, the calcium and PTH levels would have been simultaneously elevated. It seems this animal model may not be model for pHPT. Funding SourcesArkansas Bioscience Institute.

Effects of Diet Containing Soy Protein Isolate on Liver Metabolic Methylation Status Using Obese Zucker Rat Model (P08-033-19)

ObjectivesThe obesity epidemic is continuing to grow in the United States and world for past two decades. There is a link between obesity and chronic diseases development such as diabetes, cardiovascular disease, certain types of cancer and liver diseases. Previously, we reported that obesity caused a significant increase liver steatosis and feeding soy protein isolate (SPI) reduced liver steatosis. The mechanism of SPI protection against liver steatosis is less known. We hypothesize that soy protein diet will reduce development of liver steatosis caused by obesity in part by changing methylation status. The objective of the present study was to investigate the effects of SPI feeding on liver metabolic methylation status using obese zucker rat model. MethodsAfter one week of acclimation, five weeks old female lean and obese Zucker rats (n = 8/group) were randomly fed AIN-93-G diet with either casein (CAS as control) or SPI as source of protein for 22 weeks. Rats were weighted twice per week. Liver sample metabolites concentrations were measured using HPLC with Electrochemical Detection and LC-MS. ResultsOur results shows that; 1) obesity increased body weight significantly (P < 0.001) for both CAS and SPI diets; 2) Obese SPI-fed rats significantly (P < 0.001) reduced liver steatosis compared to obese CAS-fed rats. Also, our results show that liver metabolic profile in lean SPI-fed rats significantly (P < 0.025) increased SAM/SAH ratio (methylation ratio) compare to CAS-fed rats. Obese SPI-fed rats significantly (P < 0.001) decrease level of Homocysteine in liver and increase significantly (P < 0.001) Methionine/Homocysteine ratio. ConclusionsIn summary we showed that SPI diet can reduce liver steatosis by changing methylation status and improved metabolism of Homocysteine, toxic intracellular compound, through remethylation to Methionine. Funding SourcesArkansas Children’s Research Institute’s University Medical Group Fund grant program and Arkansas.