TallyHo Mice Research Articles

154 - Improved bladder contraction after CrisprCas9-mediated deletion of matrix metalloproteinase-9 in female prediabetic TallyHo mice

154 - Improved bladder contraction after CrisprCas9-mediated deletion of matrix metalloproteinase-9 in female prediabetic TallyHo mice

Allogenic Vertebral Body Adherent Mesenchymal Stromal Cells Promote Muscle Recovery in Diabetic Mouse Model of Limb Ischemia

Chronic limb threatening ischemia (CLTI) carries a significant risk for amputation especially in diabetic patients with poor options for revascularization. Phase I trials have demonstrated efficacy of allogeneic mesenchymal stromal cells (MSC) in treating diabetic CLTI. Vertebral bone adherent mesenchymal stromal cells (vBA-MSC) are derived from vertebral bodies of deceased organ donors which offer the distinct advantage of providing a 1,000x greater yield compared to that of living donor bone aspiration. This study describes the effects of intramuscular injection of allogenic vBA-MSC in promoting limb perfusion and muscle recovery in a diabetic CLTI mouse model. A CLTI mouse model was created through unilateral ligation of the femoral artery in male polygenic diabetic TALLYHO mice. Treated mice were injected with vBA-MSC into the gracilis muscle of the ischemic limb 7 days post ligation. Gastrocnemius or tibialis muscle was assessed post-mortem for fibrosis by collagen staining, capillary density via immunohistochemistry, and mRNA by quantitative real time PCR. Laser Doppler perfusion imaging and plantar flexion muscle testing were performed to quantify changes in limb perfusion and muscle function. Compared to vehicle control, treated mice demonstrated indicators of muscle recovery including decreased fibrosis, increased perfusion, muscle torque, and angiogenesis. PCR analysis of muscle obtained 7- and 30-days post vBA-MSC injection showed an upregulation in expression of MyoD1 (p = 0.03) and MyH3 (p = 0.008) mRNA representing muscle regeneration, VEGF-A (p = 0.002 ; p = 0.004) signifying angiogenesis as well as IL-10 (p < 0.001), T regulatory cell marker Foxp3 (p = 0.04), and M2-biased macrophage marker Mrc1 (CD206) (p = 0.02). These findings indicate human allogeneic vBA-MSC ameliorate ischemic muscle damage and rescue muscle function. These results in a murine model will enable further studies to develop potential therapies for diabetic CLTI patients.

SGLT2 inhibition leads to a restoration of hepatic and circulating metabolites involved in the folate cycle and pyrimidine biosynthesis.

Inhibition of sodium-glucose cotransporter 2 (SGLT2) by empagliflozin (EMPA) and other "flozins" can improve glycemic control under conditions of diabetes and kidney disease. Though they act on the kidney, they also offer cardiovascular and liver protection. Previously, we found that EMPA decreased circulating triglycerides and hepatic lipid and cholesterol esters in male TallyHo mice fed a high-milk-fat diet (HMFD). The goal of this study was to determine whether the liver protection is associated with a change in metabolic function by characterizing the hepatic and circulating metabolic and lipidomic profiles using targeted LC-MS. In both male and female mice, HMFD feeding significantly altered the circulating and hepatic metabolome compared with low-fat diet (LFD). Addition of EMPA resulted in the restoration of circulating orotate (intermediate in pyrimidine biosynthesis) and hepatic dihydrofolate (intermediate in the folate and methionine cycles) levels in males and acylcarnitines in females. These changes were partially explained by altered expression of rate-limiting enzymes in these pathways. This metabolic signature was not detected when EMPA was incorporated into an LFD, suggesting that the restoration requires the metabolic shift that accompanies the HMFD. Notably, the HMFD increased expression of 18 of 20 circulating amino acids in males and 11 of 20 in females, and this pattern was reversed by EMPA. Finally, we confirmed that SGLT2 inhibition upregulates ketone bodies including β-hydroxybutyrate. Collectively, this study highlights the metabolic changes that occur with EMPA treatment, and sheds light on the possible mechanisms by which this drug offers liver and systemic protection.NEW & NOTEWORTHY Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, have emerged as a new treatment option for individuals with type 2 diabetes that have positive impacts on kidney and cardiovascular disease. However, less is known about their impact on other tissues, including the liver. Here, we report that empagliflozin reduces hepatic steatosis that is associated with restoring metabolic intermediates in the folate and pyrimidine biosynthesis pathways. These changes may lead to new approaches to treat nonalcoholic fatty liver disease.

Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells Promote Muscle Regeneration in a Diabetic Mouse Model of Critical Limb Threatening Ischemia

Critical limb threatening ischemia (CLTI), the end stage of peripheral arterial disease (PAD), is diagnosed in 500,000 patients each year, often results in amputation, and has a ~50% 5-year mortality rate. Diabetic CLTI patients experience especially high morbidity and mortality, and no effective non-surgical therapy exists for this population. Our Phase II MOBILE trial demonstrated that autologous bone marrow nucleated cells were unable to prevent amputations in diabetic patients; however, data from a Phase I trial shows that allogeneic bone marrow-derived mesenchymal stromal cells (BMD-MSC) stimulated angiogenesis in ischemic muscle, including diabetics. While allogeneic MSC may be an effective cell preparation to treat diabetic CLTI, passaging-related cell senescence prevents generation of sufficient cell numbers for therapeutic use. The development of induced pluripotent stem cell (iPSC)-derived MSC overcomes cell senescence issues and offers the possibility of genetic modifications to enhance cell function. The current study was designed to determine potential mechanisms by which iPSC-MSC stimulate muscle regeneration in a rodent CLTI model.&#x0D; The CLTI mouse model was created by ligation/excision of the femoral artery in male polygenic diabetic TallyHo mice. Mice with intramuscular administration of iPSC-MSC displayed positive indicators of muscle regeneration compared to vehicle control mice. Real-time PCR performed with gastrocnemius muscle obtained 7- or 30-days post iPSC-MSC injection showed an increase in mRNA expression for MyH3, MyoD1, Mrc1, FoxP3, and VEGF-A vs. vehicle treated muscle, indicating promotion of muscle regeneration, M2-biased macrophage expression, T regulatory cell (Treg) expansion, and vascular proliferation. Downregulation of the NADPHoxidase subunit p47phox indicated a decrease in oxidative stress in the treated mice. The results are consistent with iPSC-MSC promotion of muscle regeneration via a Treg mediated stimulation of the M1-M2 macrophage phenotypic shift. Thus, human iPSC-MSC could be an effective treatment to stimulate muscle regeneration and ameliorate CLTI in diabetic patients.

Increased tissue modulus and hardness in the TallyHO mouse model of early onset type 2 diabetes mellitus.

Individuals with type 2 diabetes mellitus (T2DM) have a higher fracture risk compared to those without T2DM despite having higher bone mineral density (BMD). Thus, T2DM may alter other aspects of resistance to fracture beyond BMD such as bone geometry, microarchitecture, and tissue material properties. We characterized the skeletal phenotype and assessed the effects of hyperglycemia on bone tissue mechanical and compositional properties in the TallyHO mouse model of early-onset T2DM using nanoindentation and Raman spectroscopy. Femurs and tibias were harvested from male TallyHO and C57Bl/6J mice at 26 weeks of age. The minimum moment of inertia assessed by micro-computed tomography was smaller (-26%) and cortical porosity was greater (+490%) in TallyHO femora compared to controls. In three-point bending tests to failure, the femoral ultimate moment and stiffness did not differ but post-yield displacement was lower (-35%) in the TallyHO mice relative to that in C57Bl/6J age-matched controls after adjusting for body mass. The cortical bone in the tibia of TallyHO mice was stiffer and harder, as indicated by greater mean tissue nanoindentation modulus (+22%) and hardness (+22%) compared to controls. Raman spectroscopic mineral:matrix ratio and crystallinity were greater in TallyHO tibiae than in C57Bl/6J tibiae (mineral:matrix +10%, p < 0.05; crystallinity +0.41%, p < 0.10). Our regression model indicated that greater values of crystallinity and collagen maturity were associated with reduced ductility observed in the femora of the TallyHO mice. The maintenance of structural stiffness and strength of TallyHO mouse femora despite reduced geometric resistance to bending could potentially be explained by increased tissue modulus and hardness, as observed at the tibia. Finally, with worsening glycemic control, tissue hardness and crystallinity increased, and bone ductility decreased in TallyHO mice. Our study suggests that these material factors may be sentinels of bone embrittlement in adolescents with T2DM.

Characterizing the impact of SGLT2 inhibition on hepatic inflammation and cellular response in a type 2 diabetic mouse model

Type 2 diabetes (T2D) is associated with several comorbidities including nonalcoholic fatty liver disease (NAFLD). NAFLD is characterized by the presence of steatosis with or without hepatic fibrosis and is associated with insulin resistance, inflammation, and hyperlipidemia. Recently, sodium-glucose co-transport 2 inhibitors (SGLT2i) such as Empagliflozin (Empa), have emerged as a treatment option for patients with T2D by blocking renal glucose reabsorption and improving glycemic control. Whether these inhibitors may attenuate T2D-associated NAFLD is still under investigation. To explore this possibility, we challenged TallyHo (TH) mice with a 60% high-milk fat diet (HMFD) with or without Empa (10mg/kg BW) for 12 weeks. Like humans, TH mice possess several quantitative trace loci associated with diabetic/obese phenotypes. In this study, we found that Empa treatment decreased hepatic steatosis in both male and female TH mice. However, the 12-week dietary challenge was not sufficient to induce fibrosis and inflammation (Kurtz et al. 2022). Thus, we have extended the trial to 24-weeks with the hypothesis that Empa promotes anti-inflammatory responses through macrophage infiltration and cellular signaling. Protein expression of the cGAS-STING pathway (STING, pTBK1, TBK1, pIRF3, IRF3), inflammasome pathway (AIM2, ASC/TMS1) and apoptosis signaling pathway (Caspase-3) showed no significant changes between diet or treatment. Additionally, activity of p50 and p65 of NF-κB (measured through nuclear expression) was not changed between treatment groups.While Empa does not appear to alter cellular-mediated inflammatory pathways, we did note changes in cytokine expression. Using a large-scale screen to probe for more than 60 inflammatory markers, we observed elevated levels of three pro-inflammatory cytokines (IL-3, CCL2, M-CSF; p&lt;0.05) in livers from Empa-treated mice while there was downregulation of p40/p70 (p&lt;0.01). The screen of plasma samples identified elevated levels of MIP-1γ (p&lt;0.01) and identified decreased levels of three cytokines (IL-9, TIMP1, TPO; p&lt;0.05) in Empa-treated mice. To further understand the role of macrophage infiltration as a response to increased inflammation, immunofluorescence on liver section with F4/80, a macrophage marker, was performed. There were no significant differences in the density or localization of Kupffer cells between treatments and diets for both sexes. Thus, it is hypothesized that Empa promotes the polarization of macrophages from M1 (pro-inflammatory) to M2 (anti-inflammatory) to promote tissue regeneration and repair. Staining of M1 and M2 macrophage markers are currently underway. Additionally, it has been determined that the upregulation of TIMP1 leads to matrix deposition. Efforts are currently underway to quantify portal inflammation using QuPath. Collectively, the data shows that Empa treatment for 24-weeks does not alter cellular signaling, and potentially alters macrophage signaling to combat NAFLD. NIDDK This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SGLT2 Inhibition Alters the Hepatic and Circulating Metabolome of Mice Under a High-Milk-Fat Diet

Recent studies have shown that the inhibition of Sodium-glucose cotransporter 2 (SGLT2), which reabsorbs glucose in the renal proximal tubule, can aid in glycemic control under conditions of type 2 diabetes (T2D). Though it acts directly on the kidney, these inhibitors also lead to great improvement in liver function. Previously, we challenged TallyHo mice, an inbred polygenetic mice model that recapitulates T2D, with a high milk fat diet (HMFD) for 12 weeks in the continued absence or presence of Empagliflozin (EMPA), an FDA-approved SGLT2 inhibitor. Male mice showed a decrease in circulating triglycerides, hyperglycemia, and hepatic lipid and cholesterol esters (Kurtz et al 2022). However, the mechanisms that underlie this improved liver phenotype is unknown. Thus, we sought to characterize the hepatic and circulating metabolic and lipidomic profiles that are altered and/or restored in the presence of EMPA using targeted and untargeted LC-MS. Control, HMFD, and HMFD + EMPA liver tissues and plasma samples were dissolved in a methanol/water 50/50 extraction buffer that contained debrisoquine (DBQ) for the positive mode standard and 4-nitrobenzoic acid for the negative mode standard. For the untargeted liver and plasma samples, a UPLC-QTOF analysis was performed. MetaboAnalyst was used to quantify metabolite changes with inclusion criteria set to a fold change of 2 and a p-value &lt;0.05. Of note, previous studies have shown that EMPA promotes the upregulation of ketone bodies including β-Hydroxybutyrate (BHB). We noted a similar upregulation of BHB in our metabolomic analysis and confirmed that EMPA led to a ~1.5-fold increase (p &lt; 0.022) in this circulating ketone body. In addition, of those metabolites that met the inclusion criteria, we identified 12 that were downregulated and 9 that were upregulated +EMPA in the male samples. Out of these, orotate, sarcosine, tartrate, and dihydrofolate levels were restored to those of control levels. In the female samples, 6 were downregulated and 3 were upregulated +EMPA. Indoxyl sulfate, butylcarnitine, and valerylcarnetine were all restored to control levels. Of particular interest is the finding that EMPA treatment restores levels of key metabolites that are involved in the folate and pyrimidine biosynthesis pathways. Previous studies have determined that folate deficiency can promote insulin resistance and the urea cycle, a pathway that is integrated with pyrimidine biosynthesis, is upregulated under conditions of T2D and non-alcoholic fatty liver disease. Future studies are aimed at identifying the role of EMPA's hepatic protection in these metabolic pathways. NIDDK This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Designed Host Defense Peptide for the Topical Treatment of MRSA-Infected Diabetic Wounds.

Diabetes mellitus is a chronic disease characterized by metabolic dysregulation which is frequently associated with diabetic foot ulcers that result from a severely compromised innate immune system. The high levels of blood glucose characteristic of diabetes cause an increase in circulating inflammatory mediators, which accelerate cellular senescence and dampen antimicrobial activity within dermal tissue. In diabetic wounds, bacteria and fungi proliferate in a protective biofilm forming a structure that a compromised host defense system cannot easily penetrate, often resulting in chronic infections that require antimicrobial intervention to promote the healing process. The designed host defense peptide (dHDP) RP557 is a synthesized peptide whose sequence has been derived from naturally occurring antimicrobial peptides (AMPs) that provide the first line of defense against invading pathogens. AMPs possess an amphipathic α-helix or β-sheet structure and a net positive charge that enables them to incorporate into pathogen membranes and perturb the barrier function of Gram-positive and Gram-negative bacteria along with fungi. The capacity of skin to resist infections is largely dependent upon the activity of endogenous AMPs that provided the basis for the design and testing of RP557 for the resolution of wound infections. In the current study, the topical application of RP557 stopped bacterial growth in the biofilm of methicillin-resistant Staphylococcus aureus (MRSA) USA300 infected wounds on the flanks of clinically relevant diabetic TALLYHO mice. Topical application of RP557 reduced bacterial load and accelerated wound closure, while wound size in control diabetic mice continued to expand. These studies demonstrate that RP557 reduces or eliminates an infection in its biofilm and restores wound-healing capacity.

Renal Metabolome in Obese Mice Treated with Empagliflozin Suggests a Reduction in Cellular Respiration.

Sodium glucose cotransporter, type 2 inhibitors, such as Empagliflozin, are protective of the kidneys by unclear mechanisms. Our aim was to determine how Empagliflozin affected kidney cortical metabolome and lipidome in mice. Adult male TALLYHO mice (prone to obesity) were treated with a high-milk-fat diet, or this diet containing Empagliflozin (0.01%), for 8 weeks. Targeted and untargeted metabolomics and lipidomics were conducted on kidney cortex by liquid chromatography followed by tandem mass-spectroscopy. Metabolites were statistically analyzed by MetaboAnalyst 5.0, LipidSig (lipid species only) and/or CEU Mass Mediator (untargeted annotation). In general, volcano plotting revealed oppositely skewed patterns for targeted metabolites (primarily hydrophilic) and lipids (hydrophobic) in that polar metabolites showed a larger number of decreased species, while non-polar (lipids) had a greater number of increased species (>20% changed and/or raw p-value < 0.05). The top three pathways regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism, with an amino acid network being highly affected, with 14 of 20 classic amino acids down-regulated. Out of 75 changed polar metabolites, only three were up-regulated, i.e., flavin mononucleotide (FMN), uridine, and ureidosuccinic acid. Both FMN and uridine have been shown to be protective of the kidney. Scrutiny of metabolites of glycolysis/gluconeogenesis/Krebs cycle revealed a 20–45% reduction in several species, including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, although overall lipid quantity was not higher, several lipid species were increased by EMPA, including those of the classes, phosphatidic acids, phosphatidylcholines, and carnitines. Overall, these analyses suggest a protection from extensive metabolic load and the corresponding oxidative stress with EMPA in kidney. This may be in response to reduced energy demands of the proximal tubule as a result of inhibition of transport and/or differences in metabolic pools available for metabolism.

Empagliflozin Treatment Attenuates Hepatic Steatosis by Promoting White Adipose Expansion in Obese TallyHo Mice.

Sodium-glucose co-transporters (SGLTs) serve to reabsorb glucose in the kidney. Recently, these transporters, mainly SGLT2, have emerged as new therapeutic targets for patients with diabetes and kidney disease; by inhibiting glucose reabsorption, they promote glycosuria, weight loss, and improve glucose tolerance. They have also been linked to cardiac protection and mitigation of liver injury. However, to date, the mechanism(s) by which SGLT2 inhibition promotes systemic improvements is not fully appreciated. Using an obese TallyHo mouse model which recapitulates the human condition of diabetes and nonalcoholic fatty liver disease (NAFLD), we sought to determine how modulation of renal glucose handling impacts liver structure and function. Apart from an attenuation of hyperglycemia, Empagliflozin was found to decrease circulating triglycerides and lipid accumulation in the liver in male TallyHo mice. This correlated with lowered hepatic cholesterol esters. Using in vivo MRI analysis, we further determined that the reduction in hepatic steatosis in male TallyHo mice was associated with an increase in nuchal white fat indicative of “healthy adipose expansion”. Notably, this whitening of the adipose came at the expense of brown adipose tissue. Collectively, these data indicate that the modulation of renal glucose handling has systemic effects and may be useful as a treatment option for NAFLD and steatohepatitis.

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&lt;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&lt;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.

Characterizing Murine Diabetic Glucose Metabolism Under Conditions of Attenuated Renal Glucose Reabsorption

While type 1 (T1DM) and type 2 (T2DM) diabetes mellitus differ in etiology, both are metabolic disorders characterized by chronic hyperglycemia; if left untreated, this can lead to serious complications including renal failure. Glucoregulation is critical to meeting tissues’ energy needs without exceeding adaptive glucose levels and involves several metabolic pathways including gluconeogenesis (occurring in the kidney and liver) and glycolysis, which are primarily regulated through certain rate‐limiting enzymes involved in the committed steps of each pathway. Beyond its metabolic contributions, the kidney also mediates glucoregulation through the process of glucose reabsorption in the proximal tubules (PT); crucial symporters known as sodium glucose cotransporters (SGLTs) move glucose into PT epithelial cells to facilitate its return to the bloodstream rather than excretion in the urine. Inhibiting renal glucose reabsorption via the modulation of SGLTs has been linked to improved diabetic outcomes, yet the impacts of this intervention on whole body glucose metabolism have not been fully characterized. Thus, we aimed to determine if SGLT modulation alters expression of rate‐limiting glucometabolic enzymes under conditions of T1DM and T2DM. Firstly, we compared renal and hepatic tissues isolated from nondiabetic and type 1 diabetic Olfactory Receptor 1393 (Olfr1393) knockout (KO) and wild type mice; Olfr1393 is a GPCR that has been previously demonstrated to impact localization of glucose transporters, and Olfr1393 KO mice exhibit improved diabetic phenotypes (Shepard et al., 2019; Schiazza et al., 2021). Secondly, hepatic tissues from T2DM TallyHo (TH) mice fed a milk fat diet with or without the SGLT2 inhibitor Empagliflozin were analyzed. Initial analysis of qPCR and Western blot data from each condition reveals diabetic differences in glucometabolic enzyme expression within each group; under conditions of T1DM, gluconeogenesis (via expression of glucose 6‐phosphatase) appears to be decreased in both the kidney and liver. In addition, we noted reciprocal regulation of glycolytic enzymes PKM 1 and 2; in the kidney, T1DM decreases PKM1 expression but upregulates PKM2. However, the data collectively suggest that SGLT modulation does not alter renal or hepatic glucose metabolism. Further in vivo exploration may elucidate patterns of similar or differential glucoregulation that will contribute to our current understanding of the body’s dynamic glucometabolic profile in the context of diabetes and the efficacy and whole body impacts of applying SGLT inhibition in the treatment of these disorders.

Protective Effects of Chaya against Mitochondrial and Synaptic Toxicities in the Type 2 Diabetes Mouse Model TallyHO.

The purpose of our study is to determine the protective effects of the chaya leaf against mitochondrial abnormalities and synaptic damage in the Type 2 diabetes (T2D) mouse model, TallyHO (TH). The TH mouse is a naturally occurring polygenic mouse model of diabetes that mimics many characteristics of human Type 2 diabetes. Only male TH mice develop hyperglycemia and moderate obesity. Female mice display moderate obesity but do not manifest overt diabetes. In this study, we evaluated three groups of mice over a period of 11 weeks: (1) the experimental group of TH diabetic mice fed with chaya chow; (2) a diabetic control group of TH diabetic mice fed with regular chow; and (3) a non-diabetic control group of SWR/J mice fed with regular chow. Body mass and fasting blood glucose were assessed weekly. Brain and other peripheral tissues were collected. Using qRT-PCR and immunoblotting analyses, we measured the mRNA abundance and protein levels of mitochondrial biogenesis, mitochondrial dynamics, autophagy/mitophagy, and synaptic genes. Using immunofluorescence analysis, we measured the regional immunoreactivities of mitochondrial and synaptic proteins. Using biochemical methods, we assessed mitochondrial function. We found increased body mass and fasting glucose levels in the TH diabetic mice relative to the non-diabetic control SWRJ mice. In chaya chow-fed TH diabetic mice, we found significantly reduced body mass and fasting glucose levels. Mitochondrial fission genes were increased and fusion, biogenesis, autophagy/mitophagy, and synaptic genes were reduced in the TH mice; however, in the chaya chow-fed TH diabetic mice, mitochondrial fission genes were reduced and fusion, biogenesis, autophagy/mitophagy, and synaptic genes were increased. Mitochondrial function was defective in the diabetic TH mice; however, it was rescued in the chaya chow-fed TH mice. These observations strongly suggest that chaya chow reduces the diabetic properties, mitochondrial abnormalities, and synaptic pathology in diabetic, TH male mice. Our data strongly indicates that chaya can be used as natural supplemental diet for prediabetic and diabetic subjects and individuals with metabolic disorders.

Candidate Gene Test for Quantitative Trait Loci Conferring Hypercholesterolemia on Mouse Chromosome 1

ObjectivesThe TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a subcongenic mouse with TH donor segment, ∼25 Mb, on chromosome (Chr) 1 in a C57BL/6J (B6) background that harbors quantitative trait loci (QTL) conferring hypercholesterolemia, named Tchol1 (Tallyho Associated Cholesterol 1). The subcongenic mouse developed hypercholesterolemia compared to B6 mice demonstrating that distal segment of Chr 1 from TH genome is necessary to cause the hypercholesterolemia. In this study, we tested the candidacy of the apolipoprotein A2 (Apoa2) gene for Tachol1 by the quantitative complementation test. Apoa2, known regulator of cholesterol metabolism, maps to the Tchol1 locus. MethodsTo carry out the quantitative complementation test, both TH-homozygous Tachol1 subcongenic and B6-homozygous (B6) mice were mated to the Apoa2 knockout heterozygous [wild-type (wt)/null] mice to produce four types of animals; TH/wt, TH/null, B6/wt, and B6/null. Both male and female mice were weaned onto standard rodent chow and maintained. Blood was collected when animals were euthanized at 16 weeks of age. Total plasma cholesterol levels were determined using colorimetric assays. A two-way ANOVA was used to evaluate Apoa2 (null vs. wt) and Tachol1 (TH vs. B6) interaction effects for dependent variables, followed by the multiple comparison post test with Tukey correction using GraphPad Prism 8. ResultsTotal plasma cholesterol levels were: 137 ± 5 (TH/wt), 119 ± 8 (TH/null), 103 ± 8 (B6/wt), and 80 ± 4 (B6/null) for males, and 149 ± 8 (TH/wt), 130 ± 9 (TH/null), 98 ± 3 (B6/wt), and 103 ± 6 (B6/null) for females [mean ± s.e.m; mg/dl]. Two-way ANOVA revealed no significant interaction between Tchol1 and Apoa2 knockout alleles for total plasma cholesterol levels in both males and females. However, there were significant main effects of Tchol1 and Apoa2 knockout alleles on total plasma cholesterol levels in males, while significant main effects of Tchol1 on them in females. ConclusionsNo significant interaction effect between knockout and QTL alleles is interpreted as evidence that the knockout locus is not equal to the QTL. Our results suggest that the Apoa2 gene is not identical to the Tchol1 QTL. Funding SourcesAHA 18AIREA33960437, NIH 1 R15 DK113604-01A1, the WV-INBRE grant (P20GM103434), and the COBRE ACCORD grant (1P20GM121299).

Glucagon-like peptide-1 receptor agonist inhibits aeroallergen-induced activation of ILC2 and neutrophilic airway inflammation in obese mice.

BackgroundObesity is a risk factor for the development of asthma. However, pharmacologic therapeutic strategies that specifically target obese asthmatics have not been identified. We hypothesize that glucagon‐like peptide‐1 receptor agonist (GLP‐1RA) treatment inhibits aeroallergen‐induced early innate airway inflammation in a mouse model of asthma in the setting of obesity.MethodsSWR (lean) and TALLYHO (obese) mice were challenged intranasally with Alternaria alternata extract (Alt‐Ext) or PBS for 4 consecutive days concurrent with GLP‐1RA or vehicle treatment.ResultsTALLYHO mice had greater Alt‐Ext‐induced airway neutrophilia and lung protein expression of IL‐5, IL‐13, CCL11, CXCL1, and CXCL5, in addition to ICAM‐1 expression on lung epithelial cells compared with SWR mice, and all endpoints were reduced by GLP‐1RA treatment. Alt‐Ext significantly increased BALF IL‐33 in both TALLYHO and SWR mice compared to PBS challenge, but there was no difference in the BALF IL‐33 levels between these two strains. However, TALLYHO, but not SWR, mice had significantly higher airway TSLP in BALF following Alt‐Ext challenge compared to PBS, and BALF TSLP was significantly greater in TALLYHO mice compared to SWR mice following airway Alt‐Ext challenge. GLP‐1RA treatment significantly decreased the Alt‐Ext‐induced TSLP and IL‐33 release in TALLYHO mice. While TSLP or ST2 inhibition with a neutralizing antibody decreased airway eosinophils, they did not reduce airway neutrophils in TALLYHO mice.ConclusionsThese results suggest that GLP‐1RA treatment may be a novel pharmacologic therapeutic strategy for obese persons with asthma by inhibiting aeroallergen‐induced neutrophilia, a feature not seen with either TSLP or ST2 inhibition.

19 DIET-INDUCED OBESITY UNIQUELY REGULATES GLUCOSE AND NACL ABSORPTION IN SPRAGUE DAWLEY RAT SMALL INTESTINAL EPITHELIAL CELLS

Background Traditional coupled NaCl absorption occurs via the dual operation of Na:H (NHE3) and Cl:HCO3 (DRA/PAT1) exchange in the brush border membrane of villus cells in the mammalian small intestine. In genetic models of obesity (Zucker rat and Tallyho mouse) and humans, coupled NaCl absorption was not affected. However, in these animal models, a novel coupling of Na-glucose co-transport (SGLT1) and Cl:HCO3 exchange was increased, potentially important in the pathogenesis of diabetes and hypertension associated with obesity. Human obesity is both genetic and environmental (e.g. diet mediated). But it is not known whether this novel coupled NaCl absorptive pathway is affected in diet-induced obesity (DIO). Hypothesis Novel coupled NaCl absorption mediated by SGLT1 and DRA/PAT1 is stimulated in DIO. Aim Determine the mechanisms of regulation of SGLT1 and DRA/PAT1 in DIO. Methods Sprague Dawley rats were fed with a high-fat diet (HFD; 60% fat calories) from 12-18 wks of age to induce obesity. Control rats were fed with standard chow. BBM vesicles (BBMV) were prepared from villus cells by Mg precipitation. Phlorizine sensitive, 3H-OMG uptake for SGLT1; amiloride-sensitive, 22Na uptake for NHE3 and DIDS sensitive 36Cl uptake for Cl:HCO3 exchange was performed in villus cell BBMV. Na/KATPase activity was determined as inorganic phosphate release. Western blot studies were performed using rat specific antibodies. Results In intact rat villus cells, SGLT1 uptake was significantly increased in HFD rats (2.4±0.2 nmol/mg protein×2 min in control and 6.4±0.6 in HFD; n=4, p Conclusions In DIO, BBM SGLT1 activity was stimulated in villus cells from HFD rats. This is not due to the altered Na extruding capacity of the cell. Cl:HCO3 exchange was also stimulated in DIO while NHE3 remained unaffected. These data indicate that in DIO, while the traditional coupled NaCl absorption is unaffected, a novel coupled NaCl absorptive pathway, via SGLT1 and DRA/PAT1 is stimulated resulting in enhanced glucose and NaCl absorption which is likely important in the pathophysiology of diabetes and hypertension of obesity.

Canagliflozin, an SGLT2 inhibitor, corrects glycemic dysregulation in TallyHO model of T2D but only partially prevents bone deficits.

Higher fracture risk in type 2 diabetes (T2D) is attributed to disease-specific deficits in micro-structural and material properties of bone, although the primary cause is not yet established. The TallyHO (TH) mouse is a polygenic model of early-onset T2D and obesity analogous to adolescent-onset T2D in humans. Due to incomplete penetrance of the phenotype, ~25% of male TH mice never develop hyperglycemia, providing a strain-matched, non-diabetic control. Utilizing this model of T2D, we examined the impact of glucose-lowering therapy with canagliflozin (CANA) on diabetic bone. Male TH mice with or without hyperglycemia (High BG, Low BG) were monitored from ~8 to 20weeks of age, and compared to age-matched, male, TH mice treated with CANA from ~8 to 20weeks of age. At 20weeks, untreated TH mice with high BG [High BG: 687±106mg/dL] exhibited lower body mass, decrements in cortical bone of the femur (decreased cross-sectional area and thickness; increased porosity) and in trabecular bone of the femur metaphysis and L6 vertebra (decreased bone volume fraction, thickness, and tissue mineral density), as well as decrements in cortical and vertebral bone strength (decreased yield force and ultimate force) when compared to untreated TH mice with low BG [Low BG: 290±98mg/dL; p<0.0001]. CANA treatment was metabolically advantageous, normalizing body mass, BG and HbA1c to values comparable to the Low BG group. With drug-induced glycemic improvement, cortical area and thickness were significantly higher in the CANA than in the High BG group, but deficits in strength persisted with lower yield force and yield stress (partially independent of bone geometry) in the CANA group. Additionally, CANA only partially prevented the T2D-related loss in trabecular bone volume fraction. Taken together, these findings suggest that the ability of CANA to lower glucose and normalized glycemic control ameliorates diabetic bone disease but not fully.

124-LB: Praliciguat, a Clinical-Stage Soluble Guanylate Cyclase (sGC) Stimulator, Improved Oral Glucose and Lipid Handling and Slowed Diabetic Weight Loss in TALLYHO Mice

Background: Praliciguat (IW-1973) is an sGC stimulator in clinical development as a potential treatment for diabetic kidney disease. In animal models, praliciguat distributes broadly to tissues and elicits hemodynamic, anti-inflammatory, anti-fibrotic, and metabolic effects. Here, we assessed the metabolic effects of praliciguat in diabetic TALLYHO mice housed at thermoneutrality in order to minimize any confounding contribution of brown fat thermogenesis on metabolism, which is rare in humans but common in mice. Methods: Six- to eight-week-old male TALLYHO/JngJ mice were maintained on low-fat diet (10% kcal from fat, LFD). Due to incomplete penetrance of the diabetic phenotype in this model, diabetic mice were identified prior to study initiation using HbA1c and urinary glucose/creatinine ratio as criteria. At 10-12 weeks of age, a diabetic control group (n=6) was maintained on LFD, while another group (n=6) was switched to LFD formulated with praliciguat to achieve a Cmax drug exposure similar to a 20 mg oral clinical dose. Plasma was collected at baseline and after 4 weeks of treatment. Oral glucose tolerance tests (OGTT) and oral lipid tolerance tests (LTT) were performed after 5 and 6 weeks of treatment, respectively. Results: At week 4, praliciguat treatment attenuated the HbA1c increase in TALLYHO diabetic mice (+0.40 vs. +0.95%, in praliciguat and control groups, respectively P&amp;lt;0.05). At week 5, fasting glucose (561 ± 33 vs. 650 ± 46 mg/dl; -14%, P&amp;lt;0.01) and OGTT glucose AUC (-12%, P&amp;lt;0.01) were lower in praliciguat-treated mice than in untreated controls. At week 6, fasting plasma triglycerides (721 ± 97 vs. 1134 ± 190 mg/dl; -36%, P&amp;lt;0.05) and LTT triglyceride AUC (-23%, P&amp;lt;0.01) were lower in praliciguat-treated mice than in untreated controls. Body weight loss and food intake were lower in praliciguat-treated mice than in untreated mice. Conclusion: Praliciguat improved nutrient handling in diabetic TALLYHO mice. Disclosure C. Schwartzkopf: None. A. Carvalho: Stock/Shareholder; Self; Cyclerion Therapeutics. J. Hadcock: None. M. Currie: Employee; Self; Cyclerion Therapeutics. Stock/Shareholder; Self; Cyclerion Therapeutics. J.E. Jones: None.

Subcongenic Analysis of Quantitative Trait Loci Conferring Obesity and Hypercholesterolemia on Mouse Chromosome 1

Abstract Objectives The TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a congenic mouse strain with TH donor segment on chromosome (Chr) 1 in a C57BL/6 J (B6) background that harbors quantitative trait loci (QTL) conferring obesity and hypercholesterolemia (The B6.TH-Chr1–128Mb). The congenic mouse developed increased adiposity and hypercholesterolemia and demonstrated that distal segment of Chr 1 from TH genome is necessary to cause both phenotypes. With the goal to identify the underlying genes regulating body fat and plasma cholesterol levels on mouse Chr 1, in this study we generated and characterized subcongenic lines with smaller TH donor segments. Methods Using the original B6.TH-Chr1–128Mb congenic mouse, we fixed three overlapping subcongenic lines over the distal region of Chr 1; one containing the full distal region of TH donor genome (39Mb in size) (B6.TH-Chr1–39Mb) and two nested overlapping lines (B6.TH-Chr1–30Mb and B6.TH-Chr1–20Mb). For mapping, all three lines of subcongenic mice and control B6 mice were weaned onto either chow or high fat (HF) diets at 4 weeks of age, and maintained on the respective diets. At 14 weeks of age, body composition including fat and lean mass was measured using EchoMRI-100 whole body composition analyzer, and plasma total cholesterol levels were determined using colorimetric assays. Data were analyzed by ANOVA using GraphPad Prism 8. Results On chow, body fat mass was not significantly different among all subcongenic mice and B6 mice. On HF diets, on the other hand, all three subcongenic lines of mice had significantly larger fat mass than B6 mice without genotype differences. These results suggest that the obesity candidate region is most likely located within the unique segment retained in B6.TH-Chr1–20Mb mice, delimited from the markers D1Mit105 and D1Mit361. Plasma total cholesterol levels were also significantly higher in B6.TH-Chr1–20Mb mice than them in B6 mice both on chow and HF diets. Conclusions We have created subcongenic lines, with smaller TH donor segments from the Chr 1 congenic interval, which develop detectable phenotypes, thus narrowing target intervals for positional cloning. Funding Sources This work was supported, in part, AHA 18AIREA33960437, NIH 1 R15 DK113604–01A1.