Control Chow Research Articles

Lactobacillus plantarum Lp2 improved LPS-induced liver injury through the TLR-4/MAPK/NFκB and Nrf2-HO-1/CYP2E1 pathways in mice.

BackgroundInflammatory liver diseases present a significant public health problem. Probiotics are a kind of living microorganisms, which can improve the balance of host intestinal flora, promote the proliferation of intestinal beneficial bacteria, inhibit the growth of harmful bacteria, improve immunity, reduce blood lipids and so on. Probiotics in fermented foods have attracted considerable attention lately as treatment options for liver injury.ObjectiveThe aim of this study was selected probiotic strain with well probiotic properties from naturally fermented foods and investigated the underlying mechanisms of screened probiotic strain on lipopolysaccharide (LPS)-induced liver injury, which provided the theoretical foundation for the development of probiotics functional food.DesignThe probiotic characteristics of Lactobacillus plantarum Lp2 isolated from Chinese traditional fermented food were evaluated. Male KM mice were randomly assigned into three groups: normal chow (Control), LPS and LPS with L. plantarum Lp2. L. plantarum Lp2 were orally administered for 4 weeks before exposure to LPS. The liver injury of LPS-induced mice was observed through the evaluation of biochemical indexes, protein expression level and liver histopathology.Results and discussionsAfter treatment for 4 weeks, L. plantarum Lp2 administration significantly reduced the LPS-induced liver coefficient and the levels of serum or liver aspartate transaminase (AST), alanine aminotransferase (ALT), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and LPS, as well as decreasing the histological alterations and protein compared with the LPS group. Western-blotting results showed that L. plantarum Lp2 activated the signal pathway of TLR4/MAPK/NFκB/NRF2-HO-1/CYP2E1/Caspase-3 and regulated the expression of related proteins.ConclusionsIn summary, L. plantarum Lp2 suppressed the LPS-induced activation of inflammatory pathways, oxidative injury and apoptosis has the potential to be used to improve liver injury.

Oligofructose restores postprandial short-chain fatty acid levels during high-fat feeding.

Obesity is associated with consumption of a Western diet low in dietary fiber, while prebiotics reduce body weight. Fiber induces short-chain fatty acid (SCFA) production, and SCFA administration is beneficial to host metabolic homeostasis. However, the role of endogenous SCFA signaling in the development of obesity is contentious. Therefore, the primary objective of this study is to evaluate the postprandial time course of SCFA production and uptake in healthy (chow-fed), Western diet-fed (high-fat diet [HFD]) obese, and oligofructose-treated HFD-fed (HFD + OFS) rats. Male Sprague-Dawley rats were maintained on chow or HFD for 5 weeks, with or without supplementation of 10% OFS for 3 weeks. SCFAs were measured in the ileum, cecum, colon, portal vein, and vena cava at 0, 2, 4, 6, and 8 hours postprandially. Postprandial cecal and portal vein SCFAs were decreased in obese rats compared with lean chow controls, whereas no differences were observed in fasting SCFA concentrations. OFS supplementation increased SCFA levels in the cecum and portal vein during obesity. Butyrate levels were positively associated with portal glucagon-like peptide 1 and adiposity and with Roseburia relative abundance. The current study demonstrates that obesity is associated with reduced SCFA production, and that OFS supplementation increases SCFA levels. Additionally, postprandial butyrate production appears to be beneficial to host energy homeostasis.

CONSUMPTION OF Hibiscus sabdariffa DRIED CALYX ETHANOL EXTRACT IMPROVED REDOX IMBALANCE AND GLUCOSE PLASMA IN VITAMIN B12 RESTRICTION DIET IN RATS

This study aimed to evaluate the effect of Hibiscus sabdariffa L. dried calyx ethanolic (HSE) extract on the redox imbalance and glucose plasma of vitamin B12 restriction Sprague-Dawley rats. The rat was fed a Vitamin B12 Restriction Diet for 16 weeks and treated with HSE as the treatment group. One group was fed a restriction diet not treated with HSE as a positive control group, and another was fed a control chow diet not treated with HSE as a negative control group. Vitamin B12, MDA, SOD activity enzyme, and glucose levels were evaluated in this study. The extract had a significant antioxidant capacity in terms of reducing the production of free radical scavenging activity. The HSE can repair the skewed redox imbalance and oxidative damage generated by a vitamin B12-deficient diet in the rat liver. The result of fasting glucose plasma levels, in 16 weeks showed both significantly different in all groups. In Sprague-Dawley rats with vitamin B12 restriction treated with HSE, a decrease in the glucose plasma in week sixteen was observed, together with a reduction of hepatic redox imbalance. Treatment with HSE protected hepatocytes from oxidative damage caused by vitamin B12 deficiency.

Norbixin, a natural dye that improves serum lipid profile in rabbits and prevents LDL oxidation

We hypothesized that norbixin, which is a carotenoid used as an orange/red natural food coloring additive, has anti-atherogenic properties. An in vitro oxidation assay with human LDL and a rabbit model of atherosclerosis were used to test this hypothesis. Norbixin inhibited the oxidation of isolated human LDL in a concentration-dependent manner. In the in vivo assay, rabbits were fed with a regular chow (control) or an atherogenic diet (0.5% cholesterol) alone or supplemented with norbixin (10, 30 or 100 mg/kg b.w.) for 60 days. Norbixin supplementation (30 and 100 mg/kg b.w.) increased HDL levels and reduced triglyceride levels and the atherogenic index of rabbits. This effect was associated with the decrease of serum levels of oxidized LDL, oxidized LDL antibodies and aortic tissue levels of lipid and protein oxidation in the atherogenic rabbits supplemented with norbixin. Atherogenic diet increased enzymatic (superoxide dismutase, catalase, glutathione reductase, and thioredoxin reductase-1) and non-enzymatic (non-protein thiol groups content) antioxidant defense systems in the aortic tissue but reduced the activity of paraoxonase-1 in the serum. All these changes were prevented by norbixin supplementation (10, 30 and 100 mg/kg b.w.). These results suggest that norbixin has atheroprotective potential by improving serum lipid profile and preventing oxidative modifications of circulating LDL and aortic tissue. Norbixin may, therefore, be beneficial in the control of atherosclerosis risk factors and can be further investigated as a candidate to be used not only as a functional food ingredient but also for therapeutic applications and in the nutraceutical industry.

Abstract 254: Dietary fish oil and aspirin to augment anti-PD-1 immunotherapy in metastatic melanoma

Abstract Introduction: First line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI) induces objective responses in less than 50% of metastatic melanoma patients. Aspirin (ASA) use is associated with improved responsiveness to ICI, with inhibition of myeloid-induced immunosuppression in the tumor microenvironment (TME) a purported mechanism. Cyclooxygenase modulators ASA (pharmacologic inhibitor) and dietary fish oil (FO, substrate) shift myeloid and T cell populations to anti-inflammatory, pro-resolution phenotypes in immunity and cancer. Methods: C57-BL6/J mice were injected with YUMM 1.7 melanoma in the flank. Mice were fed control chow and water (CTL) or omega-3 rich (FO) chow (10% w/w, 30%kcal/kcal), given ASA in drinking water (600 ug/mL), or FO/ASA combined (COMBO) starting 12 days post tumor implantation, the same day as starting intraperitoneal αPD1 or IgG2a vehicle (q3-4 days). An alternative cohort received pre-αPD-1 treatment ASA and FO starting day 7. Tumors were assessed for growth, harvested, and characterized via flow cytometry on day 32. Results: When diets and water were started concurrent with αPD-1/IgG2a, FO decreased tumor volume vs. CTL for both vehicle (28%) and αPD-1 (27%) treatments; ASA and COMBO (p = 0.0501) did not. Compared to CTL, FO and COMBO increased monocytes (CD45+, CD11b+, Ly6C+, Ly6G-) and PD-L1+ monocytes in the TME with no effect on tumor associated macrophages (TAMS); ASA decreased the percent of Arginase 1+, CD206+ TAMs. ASA increased PD1+ CD8 T cells, while COMBO increased CD4 and CD8 T cells including LAG3+ and PD1+ CD8 T cells. When diets and water were started on day 7, FO + αPD-1 decreased tumor volume (21%) vs. αPD-1 controls; ASA and COMBO did not. Compared to CTL, FO and COMBO similarly altered immune populations as above, with FO additionally increasing total CD3+ T-cells, PD-L1+ CD4 T cells, and PD-1+ CD8 T cells; COMBO increased dendritic cells (CD11c+, MHCII+, F4/80-). TAMS were not assessed. Results significant (p< 0.05) by ANOVA or t-test unless specified. Food eaten, water imbibed, and weight did not vary between groups. Conclusions: Dietary fish oil impaired in vivo melanoma tumor growth with and without αPD-1 ICI despite increases in classically deleterious monocyte populations, while aspirin decreased M2-like macrophages in the TME without alterations in tumor growth. Modulation of the cyclooxygenase axis may be a cost-effective method to skew TME immune populations to favor αPD-1 immunotherapy responses in metastatic melanoma. Citation Format: Alexander C. Chacon, Shuyang S. Qin, Alexa D. Melucci, Katherine M. Jackson, Paul R. Burchard, Yatee A. Dave, Rachel Jewell, Brian A. Belt, William Tabayoyong, David C. Linehan, Peter A. Prieto. Dietary fish oil and aspirin to augment anti-PD-1 immunotherapy in metastatic melanoma [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 254.

Dietary fish oil augments the tumor immune microenvironment in anti-PD-1 treated melanoma.

e21564 Background: Less than half of metastatic melanoma patients experience objective responses to first line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI). Omega-3 fatty acids in dietary fish oil (FO) shift myeloid and T-cell populations to anti-inflammatory, pro-resolution phenotypes in immunity and cancer. We investigated whether dietary FO supplementation could augment tumor microenvironment (TME) immune profiles and improve ICI response in melanoma. Methods: C57-BL6/J mice were injected with YUMM 1.7 melanoma in the flank. Mice were fed control chow (CTL) or omega-3 rich (FO) chow (10% w/w, 30%kcal/kcal) starting 7 days post tumor implantation. Intraperitoneal αPD1 or IgG2a vehicle were injected q3-4 days starting day 12. To assess the temporal flux of TME immune populations, tumors from mice in analogous experiments were assessed for growth, harvested on day 12, day 22, or day 32, and characterized via flow cytometry. Results: In multiple models, FO decreased YUMM tumor growth vs. CTL by day 32 (> 21% for all models). FO halved tumor associated macrophage content, including M2 macrophages (Arg1+, CD206+, CD68+, F480+), at day 12. Conversely, FO increased M2 macrophages at day 22, leveling even with CTL at day 32. At all three timepoints, FO increased TME monocytes (Ly6C+, Ly6G-). In the T cell compartment, FO decreased TME CD4 and CD8 T cell content (including PD1+ CD4 and PD1+ CD8) at day 12, an effect which was reversed by day 22 (increased CD4 and CD8s vs CTL). FO increased PD-L1+ CD4 T cells and CD8+ T cells, including PD1+ CD8 T cells, on day 32. When fish oil diets were started on day 12 (concurrent with immunotherapy), FO decreased tumor volume, increased monocytes (and PD-L1+ monocytes), and increased CD4 and PD1+ CD8 T cells by day 32, all less significantly than when starting diets 5 days prior to initiation of immunotherapy. All results significant (p < 0.05) by 2-way ANOVA or t-test unless specified. Food eaten, water imbibed, and weight change did not vary between groups. Conclusions: Fish oil impairs in vivo murine melanoma tumor growth with and without αPD-1 ICI. Fish oil modulated myeloid and T cell lineages via delaying M2 macrophage and CD4 / CD8 T-cell appearance in the tumor microenvironment, with concomitant increases in monocytes and key T cell effectors involved in αPD-1 ICI responsiveness by the time decrements in tumor growth were noted. Dietary fish oil may benefit patients with advanced melanoma treated with αPD-1 immunotherapy; further study in humans is warranted.

1394-P: Peri-Islet Glial-Cell Expansion Occurs in Response to High-Fat Diet-Induced Obesity and Hyperglycemia

Peri-islet glial cells (PIGCs) are positioned to control neuronal-endocrine-vascular cell communication within the islet, but their role in islet cell function is not known. Hypothalamic glial cells become reactive in response to high fat diet (HFD) feeding and are implicated in the pathogenesis of obesity and type 2 diabetes. We hypothesize that metabolic stress such as diet-induced obesity and hyperglycemia promote the expansion of PIGCs, similar to what has been observed in the central nervous system. To test this hypothesis, we used the glial cell marker, glial fibrillary acidic protein (GFAP) , to identify PIGCs and compared GFAP immunoreactivity in pancreas from the following groups. First, pancreas was harvested from HFD- or chow fed control C57BL/6J male mice after 2 days, 2 weeks or 8 weeks on diet (n=6-8/group) . Second, pancreas was taken from 12-wk old db/db male mice and their control littermates with or without empagliflozin (EMPA) treatment (20mg/kg/d for 6 weeks; n=4-7/group) . Islet PIGC area was quantified as GFAP-positive area/total islet area x100%. Mann-Whitney test and one-way ANOVA were used for data analysis. We found that HFD feeding increased mouse pancreatic peri-islet GFAP expression relative to chow controls at 2 days (HFD 1.4±0.2%, chow 0.8±0.1%; p=0.02) , 2 weeks (HFD 3.5±0.4%, chow 1.2±0.1%; p<0.001) and 8 weeks (HFD 5.4±0.3%, chow 1.8±0.2%; p=0.002) in association with body weight gain and impaired glucose tolerance. Similar results were observed in obese and hyperglycemic db/db mouse vs. control littermates (db/db 5.0±1.4%, control 1.2±0.4%; p=0.004) . There was a trend for EMPA treatment to reduce islet PIGC area in the db/db mice (EMPA 2.0±0.5, VEH 5.0±1.4%, p=0.08) . In conclusion, these findings indicate that islet gliosis is observed in two obese and hyperglycemic mouse models and raise the possibility that pancreatic ‘gliosis’ may have a relevant role in islet-cell function regulation under metabolic challenge. Disclosure J.Niu: None. J.B.Moss: None. W.Rosario: Employee; Merck & Co., Inc. R.Arumugam: None. D.J.Hackney: None. R.L.Hull-meichle: Research Support; Casma Therapeutics. J.Tong: None. Funding National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097550) ;Department of Veterans Affairs (IBX004063)

MO261: Sparsentan, The Dual Endothelin Angiotensin Receptor Antagonist (DEARA), Attenuates Albuminuria and Protects from the Development of Renal Injury to a Greater Extent Than Losartan in the GDDY Mouse Model of IGA Nephropathy: A 16-Week Study

Abstract BACKGROUND AND AIMS gddY mice are an IgA nephropathy (IgAN)-prone mouse model that develops albuminuria by 8 weeks (wks) of age with glomerular IgA, IgG and C3 deposits and progressive mesangioproliferative glomerulonephritis. We have previously shown that sparsentan (SP), a novel, highly selective, first-in-class, single-molecule dual endothelin angiotensin receptor antagonist (DEARA) being developed for the treatment of focal segmental glomerulosclerosis and IgAN, protected gddY mice from the development of albuminuria and glomerulosclerosis in an 8-week study. Here we compare the impact of SP and losartan (LS), an angiotensin II subtype 1 receptor antagonist (AT1R), on albuminuria, % of sclerotic glomeruli (GS), podocyte number, and glycocalyx damage in gddY mice treated for 16 weeks. METHOD gddY mice at 4 weeks of age were treated for 8 or 16 weeks either with chow as a control (C) (n = 10) or containing SP at 900 ppm (SP900) (n = 12) or with control chow and LS in drinking water to deliver 30 mg/kg of LS (LS30) (n = 12). Systolic blood pressure (BP) was measured by tail-cuff plethysmography at 8, 12 and 18 weeks of age. Albuminuria/creatinine ratio (ACR) was assessed every 2 weeks until 20 weeks of age. Kidney biopsies (n = 4–5 per group) were taken from a subset of mice sacrificed at age 12 weeks and at the end of the study at 20 weeks of age (5–7 per group) and processed for determination of GS, the number of podocytes assessed by immunohistochemistry using an anti-WT-1 antibody (30 glomeruli per animal), and measurement of the glycocalyx area using FITC-labeled lectin (10 glomeruli per animal). RESULTS gddY mice treated with SP900 or LS30 had an equivalent and significant (P < .01) reduction in BP compared with C mice at 12 and 18 weeks of age (18 weeks of age mean BP ± SD mmHg: C, 147 ± 28; SP900, 100 ± 6; LS30, 101 ± 8). Despite similar systemic hemodynamic effects, SP900 treatment resulted in a greater and more rapid reduction in ACR from baseline compared with either mice treated with C or LS30 during the first 4 weeks of treatment (Fig. 1). Development of GS was significantly attenuated in mice at 20 weeks of age treated with SP900 (P < .001) compared with C mice, but not in those treated with LS30 (mean sclerosis score ± SD: C, 28 ± 17; SP900, 3 ± 1; LS30, 19 ± 21). Compared with C mice at 20 weeks of age, SP900 (P < .0001) and LS30 (P < .05) significantly prevented the reduction in WT-1 immunoreactivity (WT-1 positive cells/glomerulus, mean ± SD: C, 7.3 ± 0.5; SP900, 10.0 ± 0.5; LS30 8.1 ± 0.8). Notably, SP900-treated mice had significantly greater WT-1 staining (P < .0001) and glycocalyx area (Fig. 2) than LS30 mice. CONCLUSION gddY mice treated from 4–20 weeks of age with sparsentan demonstrated a more rapid attenuation of ACR and greater protection from GS than mice treated with losartan. Sparsentan protected the glomeruli from loss of podocytes and glycocalyx to a greater extent than losartan despite equivalent lowering of BP. These results suggest that the ability of sparsentan to protect gddY mice from progression of IgAN nephropathy may go beyond effects mediated through reduction in blood pressure and antagonism of AT1R alone and may be attributed to its dual mechanism of action. If translated to the clinic, these data may support sparsentan as a new approach to the treatment of IgAN.

MO615: The Lund Mets Rat, An Obese Type 2 Diabetic Preclinical Model, Develops Non-Alcoholic Steatohepatitis, Diabetic Nephropathy and Heart Failure with Preserved Ejection Fraction Under High Fat/Cholesterol/Fructose Diet

Abstract BACKGROUND AND AIMS Beyond the glucose control, anti-diabetic drugs need to demonstrate benefits on metabolic comorbidities. An animal model that recapitulates obesity and type 2 diabetes (T2D) comorbidities [i.e. non-alcoholic steato-hepatitis (NASH), nephropathy and heart failure with preserved ejection fraction (HFpEF)] is still needed for preclinical drug development. To overcome this limitation, we evaluated the effects of a high fat/cholesterol/fructose (HFCF) diet in the Lund MetS rat, a congenic BBDR.cg-lepr.cp model generated by introgression of the Koletsky leptin receptor mutation into the BioBreeding Diabetes Resistant (BBDR) rat. METHOD 17-week-old, male, lean control (ctrl) or Lund MetS obese T2D rats were fed a control chow (CC) diet or a HFCF diet, respectively for 8 weeks. Blood biochemistry was measured at 0, 4 and 8 weeks of diet. Kidney, heart and liver parameters were assessed at the end of the 8-week diet period. RESULTS Compared with ctrl, Lund MetS rats were obese (56% higher body weight) and diabetic with significantly higher %HbA1c (up to + 3%) and blood glucose levels (up to 3-fold higher) during the 8-week HFCF diet period. Significantly higher plasma insulin (up to 11-fold), total cholesterol (up to 5-fold), triglycerides (up to 8-fold), transaminases (up to 10-fold higher) levels were also observed in Lund MetS rats, as compared with ctrl. Hepatic total cholesterol, triglycerides and fatty acids levels were significantly higher in Lund Mets rats (14-, 7.3- and 6.7-fold higher as compared to ctrl, respectively). Liver histopathological scoring confirmed a NASH phenotype in Lund MetS rats fed the HFCF diet with strong liver steatosis, hepatic inflammation and portal to bridging fibrosis. Kidney function was substantially altered with a 60% decline in glomerular filtration rate and a 16-fold increase in urine albumin-to-creatinine ratio (both P < 0.0001 versus ctrl). Finally, echocardiography indicated diastolic dysfunction with significantly reduced E/A and greater E/e’ ratios, along with a preserved ejection fraction in Lund MetS rats, as compared with lean ctrl. CONCLUSION The present data demonstrate that the Lund MetS rat fed an HFCF diet that recapitulates the major metabolic comorbidities of obesity/T2D and may be a useful model for preclinical drug development.

High Fat Diet‐Induced Neuroplasticity in Cardiac‐Projecting Dorsal Motor Nucleus of the Vagus (DMV) Motoreurons

The DMV is critical in the maintenance of gastrointestinal (GI) function and metabolism through its axonal projections to subdiaphragmatic organs. However, there is a subpopulation of choline acetyltransferase (ChAT) positive DMV neurons that send projections to cardiac tissue. Given their anatomical apposition to nuclei regulating GI and metabolic function, it is likely that cardiac vagal motorneurons (CVNs) in the DMV are sensitive to nutrient signals such as high fat diet (HFD). We hypothesized that HFD exposure for 15 days modulates the electrophysiological properties of DMV CVNs, thus restraining their ability to discharge.Mice underwent retrograde tracing to identify DMV CVNs, were challenged with HFD (60%) or control chow, and were used for whole‐cell patch‐clamp electrophysiology. When comparing DMV CVNs from control and HFD‐fed animals under current clamp conditions, no significant differences in firing frequency, resting membrane potential, or input resistance were found under normal conditions. However, DMV CVNs from HFD‐fed mice exhibited a significant increase in evoked action potentials (APs) following blockade of inhibitory, gamma‐aminobutyric acid type‐A receptor (GABAAR) neurotransmission with bicuculline methiodide (BIC; 30 µM; n=10; 5.02±1.23 APs) compared to the previous artificial cerebrospinal fluid conditions (aCSF; 4.2±0.92 APs; p<0.0001). CVNs from controls demonstrated no significant differences between BIC and aCSF conditions (n=10; p=0.1194). Taken together, HFD increased CVN sensitivity to antagonism of GABAARs. Additional studies using voltage clamp configuration assessed differences in the two signaling modalities of GABA: phasic/synaptic and tonic/extrasynaptic. Spontaneous phasic inhibitory post‐synaptic currents were not different in frequency, amplitude, or decay time. However, DMV CVNs from HFD animals had a significant increase in tonic GABAAR current after the application of BIC (n=9; 1.35±0.19 pA/pF) compared to controls (n=7; 0.8±0.11 pA/pF; p=0.0361). Given that δ subunit‐containing GABAARs (GABAA(δ)Rs) are theorized to mediate tonic GABAergic current, follow‐up experiments assessed the application of the specific GABAA(δ)R agonist, THIP (3 µM) followed by BIC. Although BIC‐sensitive increases in tonic current remained in CVNs from HFD mice, no significant currents were induced in either group after THIP application, suggesting that all GABAA(δ)Rs are actively contributing to tonic currents under these conditions. Since no antagonists exist for GABAA(δ)Rs, we developed a transgenic mouse line where the δ subunit is knocked out of ChAT motorneurons (i.e. ChAT/GABAA(δ)R‐null mice) to determine whether the electrophysiological changes induced by HFD could be prevented. Preliminary findings indicate that ablation of GABAA(δ)Rs in ChAT positive DMV CVNs was enough to prevent HFD‐induced increases in evoked APs following BIC application (n=6; 4.58±1.05 APs) compared to aCSF (4.5±0.98 APs; p=0.9559). These data suggest that DMV CVNs express more GABAA(δ)Rs responsible for the generation of long‐term inhibition after HFD exposure, thus restraining their discharge and potentially reducing their capacity for cardiovascular regulation. These early changes in CVN electrophysiology could impact not only cardiovascular function, but lead to other comorbidities, including obesity, since exercise capacity is linked to vagal regulation from the DMV. Further studies are warranted to determine the mechanistic underpinnings of HFD‐induced CVN neuroplasticity.

Role of Proximal Tubule DPP4 in Inflammation and Kidney Disease Progression

Obesity and overweight leads to an entity called obesity related kidney disease (ORKD), which is a major risk factor for chronic kidney disease (CKD) progression to End Stage Renal Disease and/or dialysis. ORKD is characterized by proximal tubule brush border vacuolization and injury, basement membrane thickening, glomerular accumulation of fat globules and glomerulomegaly. We have developed a mouse model of Western diet (high fat and high sucrose)‐induced obesity that manifests all the features of ORKD. In addition, our lab observed that obese mice fed a Western diet, manifest early hyperfiltration (3 months into feeding). It is later followed by reduction in glomerular filtration rate (GFR, beginning at 18 months of age), and this pattern mimics human diabetic kidney disease progression. Our lab also observed that in ORKD, there is increased Dipeptidyl Peptidase 4 (DPP4) activity in the kidney, especially the proximal tubules and proximal tubule specific deletion of DPP4 improves the GFR. Lastly, our lab demonstrated that there is a major role for inflammation in the obese kidney and DPP4 inhibitors mitigate the inflammation. However, it is not known if and how proximal tubule DPP4 contributes to inflammation and pathophysiological changes in ORKD. Therefore, we hypothesized that proximal tubule DPP4 mediates inflammation and contributes to the pathophysiological changes in ORKD, thereby identifying DPP4 as a target to prevent CKD progression.MethodsProximal tubule specific DPP4fl/fl;SGLT2Cre+ (PT DPP4KO) and DPP4fl/fl;SGLT2Cre‐ (PT DPP4WT) mice were fed a WD and control chow (CD) for 6 mths, 15 mths and till sacrifice (18 to 27 mths). Inflammation was assessed both by flow cytometry assessment of kidney immune cells and expression of pro and anti‐inflammatory genes and proteins).ResultsThere were more pro‐inflammatory as well as DPP4 expressing macrophages in the WD‐fed WT group compared to the KO group (p<0.05). There was also activation of the T‐cells in particular the CD8s which was suppressed in the KO group (p<0.05). Deep sequencing of kidney mRNA revealed activation of pro‐inflammatory cytokines including Th1, Th17, M1 macrophage‐based cytokines and TLR4 and ICAM‐1. The KO group showed improvement in both TLR4 and ICAM‐1 (p<0.05). At the protein level, there was an increase in pro‐fibrotic (Desmin) and pro‐inflammatory (ERK) proteins in the WD‐fed WT groups and phosphorylation of ERK was suppressed in the KO group (p<0.05). When inflammation was assessed across the months it seemed that the earliest changes were in the growth pathways and beta‐oxidation of lipids, followed by the more inflammatory genes later in the course of the disease.ConclusionWD feeding led to hyperfiltration followed by a decline in GFR that was partly mitigated by PT deletion of DPP4 and this was associated with improved kidney histology and less fibrosis. WD feeding in WT mice was associated with increase in expression of several pro inflammatory genes and TLR4 and ICAM 1 were normalized in KO mice. DPP4 protein expression in kidney was associated with increased phosphorylation of ERK and this was suppressed in KO mice. Overall, these data suggest that suppression of early activation of DPP4 in ORKD may slow down the progression of CKD.

Role of Dipeptidyl Peptidase 4 and Effects of a Western Diet in Renal Sodium Transport and Tubular Injury

The protein dipeptidyl peptidase 4 (DPP4) is a target in the management of Type 2 diabetes mellitus (T2DM) and inhibition decreases albuminuria in humans with high cardiovascular risk such as subjects with T2DM, obesity/insulin resistance and hypertension. Our laboratory and others have shown that inhibition or genetic deletion of DPP4 has nephroprotective effects not only in rodent models of DM/insulin resistance but also obesity and hypertension. In addition, all three conditions with high cardiovascular risk are characterized by sodium retention. Recent data shows that DPP4 may affect sodium transport within the kidney via binding to sodium/hydrogen exchanger 3 (NHE3) and translocating to the villi in the brush border of the tubules. We have observed increased tubular damage and sodium uptake in a high fat/high sugar diet (Western diet) model of obesity in the setting of increased DPP4 activity. Therefore, we hypothesized that reduction/suppression of DPP4 expression/signaling and/or activity may lead to decreased sodium retention in the kidney in the obese mice.MethodsProximal tubule specific DPP4fl/fl;SGLT2Cre+ (PT DPP4KO) and DPP4fl/fl;SGLT2Cre‐ (PT DPP4WT) mice were fed a WD and control chow (CD) till sacrifice (18 to 27 mths). 24 hour urine collection and measurement of sodium excretion was done in these mice while on the diets. Gene and protein expression of transporters (sodium and water) was quantitated. Whole body DPP4‐/‐ (DPP4KO) and DPP4+/+ (DPP4WT) were on control chow diet and placed in metabolic cages for sodium balance studies. On days 3 and 4, mice were given 1% NaCl in drinking water. Daily mice weight, food and water consumption and urinary excretion of sodium and potassium and creatinine were measured.ResultsThe PT DPP4WT mice showed a much reduced 24 hour excretion of sodium compared to the PT DPP4KO mice (p<0.05) which was consistent with data from DPP4WT and DPP4KO mice on WD (unpublished) as well as data from DPP4 inhibitor treated mice (Nistala et al, Endocrinology 2014 Jun;155(6):2266‐76). Urinary sodium excretion was increased in both DPP4WT and DPP4KO mice on 1% NaCl in drinking water but the excretion was significantly increased in DPP4KO mice compared to DPP4WT mice. Excretion of glucose and other electrolytes reached a peak on the 3rd day and tapered off slightly on the 4th day. The increased excretion of sodium and water as well as slight reduction in food intake, led to gradual decrease in weight of the animals (~1gm) which was not significant between the WT and KO groups. The gene expression of many sodium and water transporters increased including NHE3, ENaCγ and AQP1/2 in the PT DPP4WT mice, while expression was reduced in the PT DPP4KO mice fed a Western Diet. The protein expression of NHE3 increased in WT mice and was suppressed in KO mice.ConclusionDPP4 expression was increased in PT DPP4WT mice fed a Western diet and reduced in the KO mice and this correlated with increased expression of NHE3 in the WT and reduction in KO mice. In the whole body DPP4KO mice, lack of DPP4 led to greater increase in sodium and water excretion. Taken together, proximal tubule DPP4 may be involved in regulating sodium and water homeostasis in CD and WD‐fed mice. Sodium excretion under conditions of low salt diet will help establish DPP4's role in sodium homeostasis.

Impact of Patterned Feeding of a Palatable Diet on Chronic Alcohol Drinking and Anxiety‐Like Behavior in Alcohol‐Preferring Rats

Previous studies from our lab have shown that patterned feeding of a palatable diet (PD; moderately high in fat) significantly reduced alcohol drinking in rats. In these studies, PD and alcohol were never presented concurrently, and alcohol testing occurred on non‐palatable diet access days (Mon, Wed, and Fri). Therefore, it is unclear if the patterned feeding of a PD would produce similar effects when alcohol is presented simultaneously in a chronic access paradigm. The present study examined the impact of patterned feeding of a PD on chronic alcohol drinking and anxiety‐like behavior in male and female alcohol‐preferring P‐rats. P‐rats (n=6/group) were allowed to drink alcohol 10% v/v in their home cages for six weeks, along with intermittent (24 hrs twice a week) PD access on Tuesdays and Thursdays. Control rats received chow on Tuesdays and Thursdays along with chronic alcohol access. Anxiety‐like behavior (Light/Dark box and Open Field testing) was assessed during acute alcohol withdrawal (24hr following alcohol access) on non‐PD access days. Water and chow were available ad libitum to all groups. Male and female data were analyzed together and separately. There were no significant between‐group differences in baseline alcohol drinking in chow and PD groups of rats. Overall, in cage alcohol drinking significantly (p<0.0001) increased with time, an effect evident in both male (p<0.001) and female (p<0.01) group of rats. Following initiation of patterned feeding, overall, a strong trend (p=0.07; main effect of treatment) towards reduced alcohol drinking was observed in rats receiving intermittent PD exposure compared to the chow controls. Further analysis revealed that alcohol drinking was only significantly reduced in males (p=0.0096), whereas alcohol drinking was identical in the female group of rats receiving PD compared to their chow controls. Similar effects were observed when alcohol drinking data on PD access days (Tuesdays and Thursdays) were analyzed, documenting reduced alcohol drinking when PD was concurrently available. Anxiety‐like behavior under acute alcohol deprivation conditions was not affected by patterned feeding of PD. Collectively, these data provide the effectiveness of patterned feeding in reducing chronic alcohol drinking and highlight striking sex differences in the ability of patterned feeding of PD in regulating these effects.

Diet-induced hypertension in rats is associated with increased renal vasoconstrictor response to angiotensin II after imitated endothelial dysfunction.

The mechanisms behind development of diet-induced hypertension remain unclear. The kidneys play a paramount role in blood volume and blood pressure regulation. Increases in renal vascular resistance lead to increased mean arterial blood pressure (MAP) due to reduced glomerular filtration rate and Na+ excretion. Renal vascular resistance may be increased by several factors, e.g. sympathetic output, increased activity in the renin-angiotensin system or endothelial dysfunction. We examined if a 14-week diet rich in fat, fructose or both led to increased renal vascular resistance and blood pressure. Sixty male Sprague-Dawley rats received normal chow (Control), high-fat chow (High Fat), high-fructose in drinking water (High Fructose), or a combination of high-fat and high-fructose diet (High Fat + Fruc) for 14weeks from age 4-weeks. Measurements included body weight (BW), telemetry blood pressures, renal blood flow in anesthetized rats, plasma concentrations of atrial natriuretic peptide and glucose, as well as vessel myography in renal segmental arteries. Body weight increased in both groups receiving high fat, whereas MAP increased only in the High Fat + Fruc group. Renal blood flow did not differ between groups showing that renal vascular resistance was not increased by the diets. After inhibiting nitric oxide and prostacyclin production, renal blood flow reductions to Angiotensin II infusions were exaggerated in the groups receiving high fructose. MAP correlated positively with heart rate in all rats tested. Our data suggest that diet-induced hypertension is not caused by an increase in renal vascular resistance. The pathophysiological mechanisms may include altered signaling in the renin-angiotensin system and increases in central sympathetic output in combination with reduced baroreceptor sensitivity leading to increased renal vasoconstrictor responses.

Abstract 224: Myeloid Cell PKM2 Deletion Enhances Efferocytosis And Reduces Atherosclerosis

Rationale: The glycolytic enzyme pyruvate kinase muscle 2 (PKM2) is upregulated in monocytes/macrophages of patients with atherosclerotic coronary artery disease. However, the role of cell type-specific PKM2 in the setting of atherosclerosis remains to be defined. Objective: We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis. Methods and Results: We generated novel myeloid cell-specific PKM2 -/- mice on Ldlr-deficient background (PKM2 mye-KO Ldlr -/- ). Controls were littermate PKM2 WT Ldlr -/- mice. To rule out sex-based differences, male and female mice were placed on a high-fat "Western" diet for 14 weeks, starting at eight weeks. PKM2 was upregulated in macrophages of Ldlr -/- mice fed the Western diet compared with a control chow diet. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2 -/- mice associated with decreased MCP-1 levels in plasma, reduced transmigration of macrophages in response to MCP-1, and impaired glycolytic rate. Macrophages isolated from myeloid-specific PKM2 -/- mice fed the Western diet exhibited reduced expression of pro-inflammatory genes, including MCP-1, IL-1β, and IL-12. Myeloid cell-specific PKM2 -/- mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP1 in macrophages in vitro and atherosclerotic lesions in vivo . Silencing LRP1 in PKM2-deficient macrophages restored inflammatory gene expression and reduced efferocytosis. As a therapeutic intervention, inhibiting PKM2 nuclear translocation using a small molecule reduced glycolytic rate, enhanced efferocytosis, and reduced atherosclerosis in Ldlr -/- mice. Conclusion: Genetic deletion or limiting PKM2 nuclear translocation in myeloid cells reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

Characterizing Hepatic Inflammation upon SGLT2 Inhibition in Diabetic TallyHo Mice

By 2030, Diabetes is estimated to become the 7th leading cause of death worldwide with roughly 90‐95% of all cases attributed to Type 2 diabetes (T2D). A common comorbidity of T2D is Non‐Alcoholic Fatty Liver Disease (NAFLD), characterized by the presence of lipid accumulation with or without hepatic steatosis (NASH). While the pathogenesis of NAFLD and NASH is not entirely understood, it is associated with insulin resistance, inflammation, and hyperlipidemia. Recently, sodium‐glucose co‐transporter 2 (SGLT2) inhibitors have emerged as a treatment option for patients with T2D. This drug effectively blocks renal glucose reabsorption and in turn, improves glycemic control. It is also associated with positive renal and cardiovascular outcomes. Given the attenuation of hyperglycemia, we sought to determine if SGLT2 inhibitors also offer protection from the development of NAFLD and NASH. To test this, spontaneously diabetic male and female TallyHo mice were maintained on either a control or high milk fat diet (60%) for 24‐weeks in the continued presence or absence of Empagliflozin, one of the most well‐characterized SGLT2 inhibitors on the market. As expected, at the conclusion of 24 weeks, blood glucose values were significantly lowered in the male mice treated with Empagliflozin. We also detected a reduction in hepatic lipid accumulation by both MRI and biochemical analysis. To determine if these protective effects correlated with a reduction in inflammation, we performed a large‐scale screen of more than 60 inflammatory markers on both plasma and livers collected from the high‐fat diet‐fed male mice. The data revealed that Empagliflozin attenuates systemic inflammation; we detected a downregulation in circulating pro‐inflammatory cytokines (CD30 ligand, IL‐1α, IL‐4, IL‐9, TIMP1, TPO) and an upregulation in MIP‐1γ, an anti‐inflammatory cytokine (p<0.05 for all cytokines). In contrast, we observed indications of increased inflammation in the Empagliflozin‐treated livers. Protein expression of cleaved caspase, the active form of the inflammatory‐driven protein, was elevated when compared to non‐treated milk fat diet‐fed mice (which was already elevated compared to normal chow controls). In addition, we noted heightened levels of five pro‐inflammatory cytokines (IL‐3, IL‐6, CCL2, M‐CSF, CCL1) while there was downregulation of another three (p40/p70, TNFSF8, CCL3; p<0.05). Efforts are currently underway to confirm the expression of these common inflammatory markers and to elucidate the mechanism behind the dysregulated hepatic inflammatory pathway upon SGLT2 inhibition.

The Effect of Dietary Fat and Sucrose on Cognitive Functioning in Mice Lacking Insulin Signaling in Neuropeptide Y Neurons.

Obesogenic diets can produce hippocampal insulin resistance and impairments to hippocampal-dependent cognition. This study investigated the effect of disrupted insulin signaling in Neuropeptide Y (NPY) neurons on diet-induced deficits in hippocampal-dependent memory. Wild-type mice and mice that had a targeted knockout of insulin receptors on NPY cells (IRlox/lox;NPYCre/+) were given ad libitum access to a high-fat diet (high fat; HF), 10% sucrose solution (high sugar; HS), both high-fat diet and sucrose solution (high fat, high sugar; HFHS), or a normal fat control chow for 12 weeks. Mice were tested in the Morris Water Maze (MWM), a hippocampal-dependent spatial memory task. Glucose homeostasis was assessed via a glucose tolerance test. Independent of genotype, consumption of HF, but not HS, diet increased energy intake, body weight, and plasma leptin, and impaired glucose tolerance. Disrupted insulin signaling in NPY cells and dietary interventions did not significantly affect the ability of mice to learn the location of the platform in the MWM. However, for IRlox/lox control mice, consumption of HF, but not HS, diet resulted in reduced time spent in the target quadrant during the probe trial, suggesting a hippocampal-dependent memory deficit. IRlox/lox;NPYCre/+ mice had poor performance in the probe trial regardless of diet, suggesting a floor effect. This study did not find adverse effects of chronic sucrose intake on metabolic outcomes or hippocampal-dependent memory. These data also suggest that the effects of HF diet on hippocampal-dependent memory may be dependent on insulin signaling in hippocampal NPY cells.

Catch‐up fat in male adults induces low testosterone and consequently promotes metabolic abnormalities and cognitive impairment

Catch-up fat in adults (CUFA) caused by rapid nutrition promotion after undernutrition plays an important role in the epidemic of insulin resistance (IR)-related diseases in developing societies. Insulin resistance is considered to be closely associated with reduced testosterone levels and cognitive function. However, the effects of CUFA on testosterone levels and cognitive function are unclear in males. To investigate the changes in testosterone levels and cognitive function in CUFA in male humans and rats, and explore their probable relationship and mechanisms in rats. The blood testosterone levels, fasting glucose, and blood insulin (FINS) were measured in subpopulation 1 (27 CUFA individuals, 61 controls without CUFA) aged 40-50 years to show the characteristics of sex hormone levels and the metabolic status in CUFA men. Cognitive Flexibility Inventory was conducted in subpopulation 2 (54 CUFA individuals, 214 controls) over 20 years to investigate the associations between sex hormone levels, cognitive function, and CUFA. Male rats (n = 27) were randomly allocated to the NC group (normal chow controls), RN group (CUFA, refeeding after caloric restriction), and RT group (RN with testosterone intramuscular injected while refeeding). The blood testosterone levels, intraperitoneal insulin tolerance test (IPITT), and FINS were measured, and the attentional set-shifting task test (ASST) for the assessment of cognitive function was performed in these animals. Insulin signaling pathway, N-methyl-d-aspartate receptors subtype 2A (NR2A) and 2B (NR2B) expression levels were determined in the rat cerebral cortex. The total testosterone levels decreased (medium [inter-quartile ranges], 13.43 [9.87-18.96] vs. 15.58 [13.37-24.96], p = 0.036), and HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) elevated (1.61 [1.08-2.33] vs. 1.24 [0.87-1.87], P = 0.037) in CUFA men in subpopulation 1. Additionally, cognitive impairment was observed in CUFA men in subpopulation 2. Moreover, our results indicated decreases in total and free testosterone levels, elevations in visceral lipid accumulation, FINS, HOMA-IR, blood glucose, and the area under the curve after IPITT, increases in the number of trials required to achieve the criterion of the first reversal of discrimination (R1) in ASST, and downregulation of IRS-1 mRNA expression, AKT phosphorylation, and the NR2A and NR2B expression in brain tissue in male CUFA rats. Notably, testosterone supplementation improved visceral lipid accumulation and IR-related metabolic disorders, cognitive dysfunction, decreases in IRS-1 mRNA expression, Akt phosphorylation, and NR2A and NR2B expression in brain tissue in male CUFA rodents. CUFA was characterized by reduced testosterone levels, metabolic abnormalities, and cognitive dysfunction in males, and testosterone supplementation attenuated these changes, as well as the alteration in insulin signaling and NR2A and NR2B expression in male CUFA rodents. Herein, we tentatively put forward that CUFA in males induces low testosterone, consequently promoting metabolic abnormalities and cognitive impairment probably mediated by defects in insulin signaling and NR2A, NR2B pathway in brain tissue.

Dietary fat promotes antibiotic-induced Clostridioides difficile mortality in mice

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea, and emerging evidence has linked dietary components with CDI pathogenesis, suggesting that dietary modulation may be an effective strategy for prevention. Here, we show that mice fed a high-fat/low-fiber “Western-type” diet (WD) had dramatically increased mortality in a murine model of antibiotic-induced CDI compared to a low-fat/low-fiber (LF/LF) diet and standard mouse chow controls. We found that the WD had a pro- C. difficile bile acid composition that was driven in part by higher levels of primary bile acids that are produced to digest fat, and a lower level of secondary bile acids that are produced by the gut microbiome. This lack of secondary bile acids was associated with a greater disturbance to the gut microbiome with antibiotics in both the WD and LF/LF diet compared to mouse chow. Mice fed the WD also had the highest level of toxin TcdA just prior to the onset of mortality, but not of TcdB or increased inflammation. These findings indicate that dietary intervention to decrease fat may complement previously proposed dietary intervention strategies to prevent CDI in high-risk individuals.

Hepatic glycogen participates in the regulation of hypothalamic pAkt/Akt ratio in high-sugar/high-fat diet-induced obesity

The hypothalamus is a major integrating centre that controls energy homeostasis and plays a major role in hepatic glycogen (HGlyc) turnover. Not only do hypothalamic and hepatic Akt levels influence glucose homeostasis and glycogen synthesis, but exposure to high-sugar/high-fat diets (HSHF) can also lead to hypothalamic inflammation and HGlyc accumulation. HSHF withdrawal overall restores energy and glucose homeostasis, but the actual relationship between hypothalamic inflammation and HGlyc after short-term HSHF withdrawal has not yet been fully elucidated. Here we investigated the short-term effects of HSHF withdrawal preceded by a 30-day HSHF intake on the liver-hypothalamus crosstalk and glucose homeostasis. Sixty-day old male Wistar rats were fed for 30 days a control chow (n = 10) (Ct), or an HSHF diet (n = 20). On the 30th day of dietary intervention, a random HSHF subset (n = 10) had their diets switched to control chow for 48h (Hw) whilst the remaining HSHF rats remained in the HSHF diet (n = 10) (Hd). All rats were anaesthetized and euthanized at the end of the protocol. We quantified HGlyc, Akt phosphorylation, inflammation and glucose homeostasis biomarkers. We also assessed the effect of propensity to obesity on those biomarkers, as detailed previously. Hd rats showed impaired glucose homeostasis, higher HGlyc and hypothalamic inflammation, and lower pAkt/Akt. Increased HGlyc was significantly associated with HSHF intake on pAkt/Akt lowered levels. We also found that HGlyc breakdown may have prevented a further pAkt/Akt drop after HSHF withdrawal. Propensity to obesity showed no apparent effect on hypothalamic inflammation or glucose homeostasis. Our findings suggest a comprehensive role of HGlyc as a structural and functional modulator of energy metabolism, and such roles may come into play relatively rapidly.