Fat Diet Feeding Research Articles

CRISPR/Cas9 based blockade of IL-10 signaling impairs lipid and tissue homeostasis to accelerate atherosclerosis.

Interleukin-10 (IL-10) is a widely recognized immunosuppressive factor. Although the concept that IL-10 executes an anti-inflammatory role is accepted, the relationship between IL-10 and atherosclerosis is still unclear, thus limiting the application of IL-10-based therapies for this disease. Emerging evidence suggests that IL-10 also plays a key role in energy metabolism and regulation of gut microbiota; however, whether IL-10 can affect atherosclerotic lesion development by integrating lipid and tissue homeostasis has not been investigated. In the present study, we developed a human-like hamster model deficient in IL-10 using CRISPR/Cas9 technology. Our results showed that loss of IL-10 changed the gut microbiota in hamsters on chow diet, leading to an increase in lipopolysaccharide (LPS) production and elevated concentration of LPS in plasma. These changes were associated with systemic inflammation, lipodystrophy, and dyslipidemia. Upon high cholesterol/high fat diet feeding, IL-10-deficient hamsters exhibited abnormal distribution of triglyceride and cholesterol in lipoprotein particles, impaired lipid transport in macrophages and aggravated atherosclerosis. These findings show that silencing IL-10 signaling in hamsters promotes atherosclerosis by affecting lipid and tissue homeostasis through a gut microbiota/adipose tissue/liver axis.

Abstract 2518: Elucidating the impact of obesity and weight loss on breast cancer tumor progression

Abstract Obesity is an established risk factor for breast cancer and worsened prognosis, however the mechanisms for this link have not been fully elucidated. Additionally, the consequence of weight loss (WL) prior to the diagnosis of cancer has not been fully explored. One possible link is via the immune system. Obesity is associated with metabolic dysfunction as well as inflammation of the adipose tissue. While WL is a great tool to combat metabolic dysfunction, we have shown that following WL the adipose tissue does not properly reprogram its immunophenotype and remains in a heightened inflammatory state. Accompanying the heightened inflammatory state and increased immune infiltration found in obesity, a novel subset of macrophages termed “lipid associated macrophages” (LAMs) with high expression of triggering receptor expressed on myeloid cells 2 (Trem2) have been discovered in adipose tissue of obese mice and humans. Data from our lab shows LAMs are found at low levels in the adipose tissue of lean mice and enhanced in weight gain (WG) and WL mice. While other investigators have shown Trem2-expressing tumor associated macrophages contribute to immunosuppression in the tumor microenvironment, the implications of enhanced Trem2+ LAMs found in obesity and WL have not been explored in the context of cancer. The purpose of this study is to elucidate the impact of obesity and WL on breast cancer tumor progression. Female mice were ovariectomized to model post-menopause and to enable greater weight gain. High fat diet (HFD) and low fat diet (LFD) feeding paradigms were used to produce three diet groups: lean, WG, and WL. Lean mice were maintained on LFD for the entirety of the study. WG mice were fed LFD for 12 weeks followed by HFD for 12 weeks. Mice in the WL group were fed HFD for 12 weeks to establish obesity and then fed LFD for 12 weeks to achieve WL. Four weeks prior to the end of the dietary paradigm, mice were injected with E0771 mammary cancer cells into the mammary fat pad. We confirmed that obesity drives accelerated tumor growth progression resulting in significantly increased tumor mass. We found that WL results in an intermediate phenotype between the lean and obese models, suggesting WL may provide some protection to tumor growth, but does not restore the tumor-bearing host to the same level of protection as a consistently lean model. We also demonstrated significantly increased gene expression of both Adgre1 (gene for F4/80) and Trem2 in obese ovarian and mammary adipose tissue compared to lean adipose tissue. Future studies will focus on understanding the mechanistic links between heightened inflammation in obese and WL adipose tissue and tumor progression. Additionally, we aim to elucidate the contribution of Trem2-expressing immune cells to tumor progression in the obese and WL contexts. Citation Format: Elysa M. Wolf, Barbara Fingleton, Alyssa H. Hasty. Elucidating the impact of obesity and weight loss on breast cancer tumor progression [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 2518.

1518-PUB: Role of PPARd in Regulating Autophagy Pathway in Improving Islet ß-Cell Function with GLP-1 Receptor Agonist

Aim: To investigate the effect and mechanism of glucagon-like peptide-1 receptor agonist (GLP-1 RA) on the function of islet β cells. Methods: Type 2 diabetes C57BL/6J mice were successfully induced by high fat diet (HFD) feeding for 12 weeks then were randomly divided into HFD+NaCl group (n=5) , HFD+Exenatide group (n=5, 24 nmol•kg-1­•d‑1) and NC group (n=5) . Tolerance tests and insulin measurement were performed after 8 weeks of drug therapy. Weight was measured weekly and fasting blood glucose was measured every 4 weeks. The pancreas were stained with LC3B Immunofluorescence at the end of the experiment. The PA induced primary islet β cells were treated with exendin-4 and PA induced Min6 cells were treated with exendin-4, GW501516 for 24h, which BSA as the control group, then detected the protein levels of PPARδ, LC3B-II and P62. At the same time, PA induced MIN6 cells were treated with GSIS. What's more, we silenced PPARδ transfected with siRNA to verify the role of PPARδ in GLP-1 receptor agonist activation of β-cell autophagy. Results: Compared with the normal control group, the body weight and fasting blood glucose increase and significant peripheral insulin resistance in the high fat group, while the above indexes are significantly improved in the high-fat fed mice after GLP-1 RA treatment. Immunofluorescence biopsies of pancreatic tissue show that LC3B expression decrease after high fat diet, while GLP-1 RA intervention significantly increase. In addition, GLP-1 RA not only can promote the expression of PPARδ and LC3B-II protein and inhibit the expression of P62 protein in primary islet β cells and MIN6 cells, but also significantly increase glucose stimulation insulin secretion in MIN6 cells. More importantly, GW501516 is found to be consistent with GLP-1 RA in MIN6 cells, while GLP-1 RA promote autophagy disappeare after PPARδ expression is silenced. Conclusion: GLP-1 RA can improve the function of islet β cells by activating the autophagy pathway through PPARδ. Disclosure Y.Su: None. B.Lin: None. Y.Chen: None. Z.Liu: None. L.Gan: None. W.Xu: None. Funding Science & Technology Project of Guangzhou（202102010175）

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)

Abstract P462: Bscl2/seipin Deficiency In Heart Causes Energy Deficit And Heart Failure Via Inducing Excessive Lipid Catabolism

Heart failure (HF) is one of the leading causes of death world-wide and is associated with cardiac metabolic perturbations. Human Type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease is caused by mutations in the BSCL2 gene. Global lipodystrophic Bscl2 –/– mice exhibit hypertrophic cardiomyopathy. Whether BSCL2 plays a direct role in regulating cardiac substrate metabolism and/or contractile function remains unknown. Here we show that mice with cardiac-specific deletion of Bscl2 ( Bscl2 cKO ) developed dilated HF. Myocardial BSCL2 deletion led to elevated ATGL expression and FA oxidation (FAO) along with reduced cardiac lipid contents. Cardiac dysfunction in Bscl2 cKO mice was independent of mitochondrial dysfunction and oxidative stress, but associated with decreased metabolic reserve and ATP levels. Importantly, heart failure in Bscl2 cKO mice could be partially reversed by pharmacological inhibition of FAO, or prevented by high fat diet (HFD) feeding. Lipidomic analysis further identified markedly reduced glycerolipids, glycerophospholipids, NEFA and acylcarnitines in Bscl2 cKO hearts, which were partially normalized by FAO inhibition or HFD. Our study reveals a new form of HF with excessive lipid catabolism, and identifies a crucial cardiomyocyte-specific role of BSCL2 in controlling cardiac lipid catabolism, energy state and contractile function. It also provides novel insights into metabolically treating energy-starved HF using FAO inhibitor or HFD.

Abstract P249: Angiotensin At1a Receptor Signaling In Agrp Neurons In Metabolism Control

The adverse cardiovascular responses to leptin are preserved, but beneficial metabolic effects, such asincreases in resting metabolic rate (RMR) are lost in obesity induced by high fat diet (HFD) feeding. Wepreviously reported angiotensin II (ANG) type 1a ( Agtr1a ; AT1A) receptors in a subset of AgRP neurons arecritically required for the integrative control of RMR. Understanding the mechanisms in this unique subset ofRMR controlling- AgRP neurons is the goal of this study and is critical to decipher the pathogenesis of obesity-associated hypertension. Male C57BL/6J mice were maintained on chow or HFD from 8-18 weeks of age. Cell-attached voltage clamp recording of AgRP neurons in ARC sections of Ai9(tdTomato) AgRP mice showed that ANGapplication results in three distinct cellular responses (n=65 neurons; 8 mice). In chow fed mice, ANG suppressedelectrical activity in 1/3 of AgRP neurons (ie. “Type 1” AgRP neurons) via a losartan-sensitive mechanism,indicating involvement of AT1 receptors (Firing rate; aCSF:1.23 ± 0.10 vs ANG:0.60 ± 0.08 vs ANG+LOS:1.37 ±0.11 Hz, p<0.05). ANG caused excitation in 1/3 of AgRP neurons (“Type 2” AgRP cells) which was mediatedthrough a PD-123,319-sensitive mechanism, implicating AT2 receptors (ANG:1.69 ± 0.12 vs ANG+PD:0.86 ±0.06 Hz, p<0.05). Finally, 1/3 of AgRP neurons did not respond to ANG (“Type 0” cells). Complementary to thesefindings, mice with AT1A deleted from AgRP cells (ie, Agrp -Cre x Agtr1a Flox x Ai9 mice) exhibited a completeloss of Type 1 responses but maintenance of Types 0 and 2 (n=23 cells; 4 mice). Further, pharmacologicaldissection of signaling cascades implicated a pertussis toxin-sensitive mechanism (Gαi cascade; ANG+PTX:1.00± 0.04 Hz, p<0.05) and multiple potassium + chloride channels in the ANG-mediated inhibition of Type 1 cells.Most intriguingly, the relative proportion of AgRP neurons exhibiting Type 1 (ANG -inhibited) vs Type 0 or 2responses was decreased with HFD (Type 1 cells proportion- chow: 35%,23 out of 65; HFD: 18%, 10 out of 56).These results establish a specific ANG-inhibited subtype of AgRP neuron and implicate the AT1A/Gαi signalingcascade in the inhibitory effect of ANG. Switch of Type 1 AgRP cells to Type 0 or 2 in response to HFD suggestsHFD-induced neural plasticity/adaptation.

1155-P: Neurocircuits Controlling Adaptive Thermogenesis Rely on Skin Detection of External Temperatures through Calcitonin Gene-Related Peptide Expressing Spinal Neurons

Heat-sensory neurons arising from the dorsal root ganglia (DRG) play a pivotal role in the detection of cutaneous temperature and transmission of external signals to the brain, ensuring the maintenance of thermoregulation. However, whether these thermoreceptor neurons contribute to adaptive thermogenesis has remained elusive. We show that genetic ablation of heat sensing Calcitonin Gene-Related Peptide α (CGRPα) neurons promotes resistance to weight gain upon high fat diet (HFD) feeding and increases energy expenditure in mice. Mechanistically, we find that loss of CGRPα-expressing sensory neurons is associated with reduced lipid deposition in adipose tissue, enhanced expression of fatty-acid oxidation genes, higher ex-vivo lipolysis in primary white adipocytes and enhanced mitochondrial respiration from iBAT. Remarkably, mice lacking CGRPα sensory neurons manifest increased tail cutaneous vasoconstriction at room temperature. This exacerbated cold perception was not associated with reduced core temperature, suggesting that heat production and heat conservation mechanisms are engaged. Specific denervation of CGRPα neurons in intrascapular BAT did not contribute to the increased metabolic rate observed upon global sensory denervation. Taken together, these findings highlight an important role for cutaneous thermoreceptors in regulating energy metabolism, by triggering counter-regulatory responses involving energy dissipation processes including lipid fuel utilization and cutaneous vasodilation. Disclosure C. Riera: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-15-INI-12 to C.R.)

Deletion of the nuclear receptor ROR\u03b1 in macrophages does not modify the development of obesity, insulin resistance and NASH

Retinoic acid receptor-related orphan receptor-alpha (RORα) is a transcription factor from the nuclear receptor family expressed by immune cells and involved in the development of obesity, insulin resistance (IR) and non-alcoholic steatohepatitis (NASH). It was recently reported that mice deficient for RORα in macrophages develop more severe NASH upon high fat diet (HFD) feeding due to altered Kupffer cell function. To better understand the role of RORα in obesity and IR, we independently generated a macrophage RORα-deficient mouse line. We report that RORα deletion in macrophages does not impact on HFD-induced obesity and IR. Surprisingly, we did not confirm an effect on NASH development upon HFD feeding nor in the more severe and obesity-independent choline-deficient, L-amino acid-defined diet model. Our results therefore show that RORα deletion in macrophages does not alter the development of obesity and IR and question its role in NASH.

211-OR: Hypothalamic Slug Couples Overnutrition States to Epigenetic Reprogramming of Leptin Neural Circuits, Obesity, and Type 2 Diabetes

Leptin protects against obesity and metabolic disease mainly through leptin neural circuits in the hypothalamus. Leptin binds to and activates hypothalamic leptin receptors (LepR), thereby suppressing food intake and increasing energy expenditure. Counterintuitively, obesity is associated with hyperleptinemia in rodents and humans. Moreover, leptin therapies fail to ameliorate obesity in humans. These observations support a notion that obese subjects develop leptin resistance. Multiple positive and negative regulators of LepR signaling have been identified to influence leptin sensitivity, including Sh2b1, SOCS3, and PTP1b. However, factors, which couple overnutrition states to leptin resistance, are not fully understood. Here, we observed that high fat diet (HFD) feeding markedly upregulates Slug, a nuclear epigenetic regulator, in the hypothalamus. Remarkably, LepR neuron-specific deletion of Slug (SlugΔLepR) reversed HFD-induced hyperleptinemia, energy imbalance, obesity, insulin resistance, glucose intolerance, and liver steatosis. Conversely, overexpression of Slug (MBHSlug) but not an epigenetic-defective Slug mutant in the mediobasal hypothalamus resulted in obesity, insulin resistance, glucose intolerance, and fatty liver disease in mice. We found that Slug physically binds to the LepR promoter and suppresses LepR promoter activity; accordingly, hypothalamic LepR expression was high in SlugΔLepR mice and low in MBHSlug mice. Furthermore, Slug increased the levels of LepR promoter H3K27 methylation, a repressive epigenetic mark, in the hypothalamus, presumably by recruiting H3K37 methytransferase Ezh2 to the LepR promoter. Indeed, Ezh2 inhibitor treatment blocked Slug suppression of LepR promoter activity. Collectively, we unravel a previously unrecognized Slug-elicited epigenetic reprograming of LepR pathways that couple overnutrition states to leptin resistance, obesity, and metabolic disease. Disclosure M. Kim: None. L. Jiang: None. M.G. Myers: Research Support; Self; AstraZeneca, Novo Nordisk Inc. L. Rui: None. Funding National Institutes of Health (F32DK120111)

1734-P: High Dietary Fat Intake Exacerbates Insulin Resistance in a Type 2 Diabetes Genetic Mouse Model

GLUT4, an insulin-sensitive glucose transporter, plays a major role of postprandial glucose disposal. We previously reported that human GLUT4 promoter-driven insulin receptor knockout (GIRKO) mice have reduced insulin signaling in central and peripheral tissues and develop hyperglycemia, albeit at a lower rate in an age-dependent manner on normal chow diet. Long-term high fat diet (HFD) feeding is well established for causing adiposity gain, increased inflammation, and insulin resistance. We hypothesized that HFD feeding will exacerbate the insulin resistance and result in rapid progression to overt diabetes in GIRKO mice. To test this, we examined the physiological response of euglycemic GIRKO mice to HFD in a time course study. We used indirect calorimetry analysis and found that GIRKO mice showed differences in energy partitioning shortly after HFD feeding started. The majority of GIRKO mice cohorts had 5-fold higher circulating insulin and 1.5-fold elevated blood glucose after 1 month of HFD feeding. In addition to the hyperglycemia and hyperinsulinemia, GIRKO mice also exhibited impaired glucose tolerance and islet hyperplasia, suggesting extreme insulin resistance exceeded the compensatory increase of insulin production. After 3 months on HFD, GIRKO mice had increased relative liver size, elevated hepatic triglycerides, and reduced hepatic glycogen, demonstrating impaired hepatic insulin signaling to stimulate glycogen synthesis and inhibit gluconeogenesis. We also confirmed the increase of key hepatic gluconeogenic gene expression, which likely contributed to hyperglycemia. The rapid progression of metabolic defects of GIRKO mice on HFD is not due to excessive weight or adiposity gain. In conclusion, our studies of HFD-fed GIRKO mouse will offer insights in the role of the dietary contribution to diabetes disease progression without the confounding factor of increased adiposity. Disclosure A. Reilly: None. S. Yan: None. A.J. Loncharich: None. H. Ren: None. Funding Indiana University Diabetes and Obesity Research Training Program (T32DK064466); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK120772, R00DK098294); Indiana Diabetes Center (P30DK097512); Indiana Clinical and Translational Sciences Institute (UL1TR002529)

301-OR: Lipin 1–Mediated Phosphatidic Acid Phosphatase Activity in Adipocytes Regulates Systemic Insulin Sensitivity on High-Fat Diet

Emerging evidence suggests that the capacity of adipocytes to appropriately store lipids and prevent their ectopic accumulation in liver and skeletal muscle may be an important regulator of systemic insulin sensitivity. Lipin 1 is a lipid phosphatase that is highly expressed in adipocytes and catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DAG); the penultimate step in triglyceride synthesis. Adipose tissue lipin 1 expression positively correlates with insulin sensitivity in obese humans and mouse models. To test the role of adipose tissue lipin 1 in systemic insulin sensitivity, we generated mice lacking lipin 1 in adipocytes (Adn-Lpin1-/- mice). On normal chow, loss of lipin 1 in adipocytes modestly reduced white and brown adipose tissue mass, but did not affect glucose tolerance or insulin sensitivity. However, in response to 2 weeks high fat diet (HFD; 60% fat) feeding, Adn-Lpin1-/- mice remained lean, but developed hyperglycemia compared to littermate wild type (WT) mice. Lipidomic analyses demonstrated an increase in liver lipids (PA, DAG, and triglyceride) in HFD-fed Adn-Lpin1-/- mice compared to WT controls, whereas only PA accumulated in skeletal muscle. Plasma triglyceride concentration was not different, but free fatty acids and glycerol were significantly reduced in Adn-Lpin1-/- vs. WT mice fed HFD, suggesting that lipolysis may be decreased. Plasma adiponectin and resistin were significantly reduced in HFD-fed Adn-Lpin1-/- vs. WT control mice while leptin concentrations were not different. Lastly, with 5 weeks of HFD feeding, despite being leaner, Adn-Lpin1-/- mice became markedly hyperglycemic and glucose intolerant compared to littermate WT controls. Collectively, these data suggest that loss of lipin 1 in adipocytes leads to insulin resistance on HFD, likely due to an impaired capacity to store lipids appropriately in adipose tissue. Disclosure A.J. Lutkewitte: None. B.N. Finck: Advisory Panel; Self; Cirius Therapeutics. Stock/Shareholder; Self; Cirius Therapeutics.

179-OR: Mitochondrially Targeted Tamoxifen Improves Diet-Induced Obesity and Diabetes and Reduces Adipose Tissue Senescence in Experimental Mice

Diabetes and ageing are mutually linked and senescent cells might be involved in the development of obesity and type 2 diabetes mellitus (T2DM). Recent studies have shown that senolytic agents may improve diabetes-related pathologies. To this end we have tested our novel anti-cancer agent with senolytic properties, mitochondrially targeted tamoxifen (MitoTam), in a mouse model of T2DM. Obesity and diabetic metabolic profile were induced by high fat diet (HFD) feeding in 20 C57BL/6J male mice for 6 months with another group fed a standard diet (SD) serving as controls. Subsequently, both groups were divided into 2 subgroups (n=10 each) treated either with MitoTam (2µg/1g of body weight dissolved in 4% ethanol in corn oil) or the excipient given i.p. twice a week for a period of 4 weeks. In HFD animals, MitoTam decreased body weight already after 2 weeks of treatment, with most pronounced reduction of visceral adipose tissue (VAT, P<0.001), whereas it had no effect on body weight in SD group. MitoTam also reduced fasting glucose (P<0.001) as well as postprandial glucose AUC (P<0.001) of HFD mice to levels comparable with SD group. This glucose-lowering effect was accompanied by a decrease in serum triglycerides (P<0.002), insulin (P<0.002), leptin (P<0.001) and glucose-dependent insulinotropic peptide (GIP, P<0.03) together with reduction of mRNA expression of leptin in epididymal adipose tissue (EAT, P<0.001), EAT adipocyte size and lipid accumulation in the liver (P<0.002). HFD-induced obesity and T2DM phenotype was associated with elevated levels of senescent markers p16 and β-gal in EAT (P<0.001 for p16 and P<0.03 for β-gal vs. SD), both of which were markedly reduced by MitoTam treatment (P<0.001 for p16 and P<0.002 for β-gal vs. HFD). In conclusion, our results suggest that MitoTam significantly improves HFD-induced obesity and diabetes in mice and that this effect might be in part mediated by alleviated senescence in adipose tissue. Disclosure J. Trnovska: None. E. Davidova: None. P. Svoboda: None. H. Kratochvilova: None. M. Mraz: None. S. Hubackova: None. J. Neuzil: None. M. Haluzik: Advisory Panel; Self; Lilly Diabetes, Sanofi. Consultant; Self; Ethicon US, LLC. Speaker’s Bureau; Self; AstraZeneca, Mundipharma International, Novartis AG, Novo Nordisk A/S. Funding Institute for Clinical and Experimental Medicine (00023001); Czech Science Foundation (GA 18-02550S)

2042-P: Loss of Apoptosis Repressor with Caspase Recruitment Domain Promotes High-Fat Diet Induced Hyperglycemia In Vivo

Apoptosis repressor with caspase recruitment domain (ARC) is an endogenous inhibitor of apoptosis signaling. We previously showed ARC is expressed in β cells and ameliorates amyloid-induced β-cell death in vitro. More recently, we found that wild type (WT) and whole-body ARC knockout (KO) mice on an FVB background fed a normal chow diet exhibit no differences in islet area, β-cell area, or α-cell area at 16 (n=5) or 35 weeks (n=7-8) of age. In the present study, we sought to determine whether ARC plays a role in maintaining glycemia in response to the challenge of high fat diet (HFD, 60% kcal from fat) feeding in vivo. At 12 weeks of age, just prior to starting 24 weeks of HFD, ARC KO mice displayed lower body weight than WT mice (0 wks: 26.2±0.6 vs. 30.6±0.8 g; p<0.01; n=13), and this weight difference persisted throughout the HFD study (24 wks: 44.7±2.5 vs. 51.9±1.0 g; p=0.04, n=4-9). Random plasma glucose (PG) was not different between genotypes prior to HFD feeding. With HFD challenge, PG increased significantly in ARC KO but not WT mice (see Table). This elevation in PG in ARC KO mice was observed after 4 weeks of HFD and remained throughout the study. These data indicate that loss of ARC promotes hyperglycemia in response to HFD feeding in vivo. Whether this hyperglycemia in ARC KO mice arises from impaired β-cell compensation due to reduced β-cell mass or function remains to be determined. Disclosure A.T. Templin: None. C.R. Schmidt: None. M.F. Hogan: None. N. Esser: None. R.N. Kitsis: None. R.L. Hull: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc. Funding U.S. Department of Veterans Affairs (I01-BX001060, IK2-BX004659); National Institutes of Health (P30DK017047)

Allograft inflammatory factor-1-like is not essential for age dependent weight gain or HFD-induced obesity and glucose insensitivity

The allograft inflammatory factor (AIF) gene family consists of two identified paralogs – AIF1 and AIF1-like (AIF1L). The encoded proteins, AIF1 and AIF1L, are 80% similar in sequence and show conserved tertiary structure. While studies in human populations suggest links between AIF1 and metabolic diseases such as obesity and diabetes, such associations with AIF1L have not been reported. Drawing parallels based on structural similarity, we postulated that AIF1L might contribute to metabolic disorders, and studied it using mouse models. Here we report that AIF1L is expressed in major adipose depots and kidney but was not detectable in liver or skeletal muscle; in notable contrast to AIF1, AIF1L was also not found in spleen. Studies of AIF1L deficient mice showed no obvious postnatal developmental phenotype. In response to high fat diet (HFD) feeding for 6 or 18 weeks, WT and AIF1L deficient mice gained weight similarly, showed no differences in fat or lean mass accumulation, and displayed no changes in energy expenditure or systemic glucose handling. These findings indicate that AIF1L is not essential for the development of obesity or impaired glucose handling due to HFD, and advance understanding of this little-studied gene and its place in the AIF gene family.

Abstract P3062: Single-nucleus Rna-sequencing Reveals Cell-specific Transcriptome Changes In The Hypothalamic Arcuate Nucleus In Response To Prolonged High-fat Diet

Selective leptin resistance (SLR) is a pathological state caused by prolonged high fat diet (HFD) feeding in which cardiovascular responses to leptin are preserved, but metabolic effects are attenuated. Thus, SLR is hypothesized to contribute to obesity-associated hypertension, but the mechanisms causing SLR are unclear. Desensitization of leptin signaling in cells of the arcuate nucleus (ARC) of the hypothalamus has been proposed as one of potential mechanisms contributing to SLR. Tanycytes, located along the walls of the third ventricle, play critical roles in the regulation of energy balance through the transport of blood-borne metabolic hormones such as leptin and ghrelin into the cerebrospinal fluid. Previous studies have reported that 10 weeks of 45% HFD (D12451) feeding is sufficient to cause SLR in wildtype C57BL/6J mice. To examine the transcriptomic changes induced by prolonged HFD exposure in tanycytes, we performed single-nucleus RNA-sequencing on nuclei isolated from ARC of ad libitum fed male C57BL/6J mice fed chow diet (7013; CD, n=7) or HFD (n=8) from 8 to 18 weeks of age. At 18 weeks, fat mass (+10.1±1.4 vs +3.2±0.4g, p<0.01) and plasma leptin (486±148 vs 119±43 pg/mL, p<0.05) were increased in mice fed HFD vs CD, energy expenditure was decreased (p<0.05), and caloric intake was normal after correction for body mass. After pooling replicates and filtering out nuclei with low quality reads, a total of 6,621 (CD) & 14,396 (HFD) nuclei remained. Unbiased clustering (Seurat) identified 23 major clusters of cell types within the arcuate. Ingenuity Pathway Analysis indicated enrichment (z-score +0.45, p<0.05) of the canonical “Leptin Signaling in Obesity” pathway in β2 tanycytes (which transport circulating leptin into the cerebrospinal fluid), but such pathways were de-enriched/inhibited in downstream neuronal clusters such as Agouti-related peptide ( Agrp ) neurons (z-score -1.6, p=0.009) and unchanged in neurons expressing proopiomelanocortin ( Pomc ) (p=0.2). Collectively, these results lead us to hypothesize that SLR is associated with normal transport of circulating leptin into the arcuate nucleus, while the primary dysfunction that causes SLR instead occurs within selected neuronal populations such as Agrp neurons.

171-OR: Metformin Prevents Microvascular Insulin Resistance and Preserves Endothelial Function in High-Fat Diet-Fed Rats

Endothelial dysfunction and vascular insulin resistance coexist and both contribute to the development of type 2 diabetes mellitus (T2DM) and increased cardiovascular risks. Insulin relaxes precapillary arterioles to increase insulin delivery to and action in muscle via recruiting muscle microvasculature and this action is impaired in the insulin resistant states. Metformin is the first line therapy for T2DM and has been shown to improve endothelial function beyond its hypoglycemic effects. Here we aim to examine whether metformin could restore vascular insulin responses in the setting of insulin resistance induced by high fat diet (HFD) feeding via the modulation of endothelial function. Adult, male Sprague-Dawley rats were fed on HFD with or without metformin treatment for 2 weeks. A chow fed group served as control. After dietary intervention, rats were fasted overnight and received a euglycemic-hyperinsulinemic clamp for 120 min. Insulin-mediated muscle microvascular recruitment was assessed before and during insulin infusion using contrast-enhanced ultrasound. Distal saphenous artery was isolated and used for determination of endothelial function and vascular insulin responses. Compared with controls, HFD feeding abolished insulin-induced muscle microvascular recruitment in muscle, decreased insulin-mediated whole body glucose disposal and blunted the vasodilatory response to insulin in small resistance vessel. Metformin treatment significantly enhanced insulin’s microvascular action, effectively rescued small arteriole endothelial function, but did not improve insulin-stimulated whole body glucose disposal in HFD rats. We conclude that metformin could reverse microvascular insulin resistance and attenuate endothelial dysfunction induced by HFD feeding. There findings may provide a novel mechanistic insight into metformin’s cardiovascular protective effect in patients with diabetes. Disclosure J. Liu: None. W. Chai: None. L. Gong: None. K.W. Aylor: None. Z. Liu: None. Funding American Diabetes Association (1-17-ICTS-059 to Z.L.); National Institutes of Health (R01HL094722, R01DK102359)

An adiponectin-S1P axis protects against lipid induced insulin resistance and cardiomyocyte cell death via reduction of oxidative stress

BackgroundAdiponectin exerts several beneficial cardiovascular effects, however their specific molecular mechanisms require additional understanding. This study investigated the mechanisms of adiponectin action in the heart during high fat diet (HFD) feeding or in palmitate (PA) treated H9c2 cardiomyoblasts.Methods6-week-old male adiponectin knock out (Ad-KO) mice were fed chow or 60% HFD for 6 weeks then received saline or recombinant adiponectin (3μg/g body weight) for an additional 2 weeks. After acute insulin stimulation (4 U/kg), tissue and serum samples were collected for analysis. H9c2 cardiomyocytes were treated ±0.1 mM PA, the adiponectin receptor agonist AdipoRon, or the antioxidant MnTBAP then assays to analyze reactive oxygen species (ROS) production and cell death were conducted. To specifically determine the mechanistic role of S1P, gain and loss of function studies were conducted with adding S1P to cells or the inhibitors THI and SKI-II, respectively.ResultsHFD feeding induced cardiac insulin resistance in Ad-KO mice, which was reversed following replenishment of normal circulating adiponectin levels. In addition, myocardial total triglyceride was elevated by HFD and lipidomic analysis showed increased levels of ceramides and sphingosine-1-phosphate (S1P), with only the latter being corrected by adiponectin administration. Similarly, treatment of H9C2 cardiomyoblasts with PA led to a significant increase of intracellular S1P but not in conditioned media whereas AdipoRon significantly increased S1P production and secretion from cells. AdipoRon or the antioxidant MnTBAP significantly reduced PA-induced cell death. Gain and loss of function studies suggested S1P secretion and autocrine receptor activation mediated the effect of AdipoRon to attenuate PA-induced ROS production and cell death.ConclusionOur data establish adiponectin signaling-mediated increase in S1P secretion as a mechanism via which HFD or PA induced cardiomyocyte lipotoxicity, leading to insulin resistance and cell death, is attenuated.

Bombax ceiba flowers extract ameliorates hepatosteatosis induced by ethanol and relatively moderate fat diet in rats.

Chronic excessive alcohol consumption could induce serious liver injury. In this study, therapeutic effect of aqueous methanol extract of Bombax ceiba L. flowers (BCE) (Family: Bombacaceae) was investigated against hepatic steatosis. This study included seven groups, and the research period was eight weeks. The first group served as control. The six remaining groups were divided into two categories, three groups in each. The first category was fed fat diet. The second category was fed fat diet and orally administrated ethanol, which was given in graduate doses from 2 g/kg/d to 6 g/kg/d. Then, one group from each category was orally treated with the standard drug fluvastatin (2 mg/Kg/d). Another group was orally treated with BCE (200 mg/kg/d). The third group left untreated. The results revealed that BCE significantly decrease both the body and liver weight. The treatment with BCE extract also ameliorates the effect of alcohol induced increase of liver enzyme activities. In addition, the extract was significantly increased hepatic liver antioxidants and decreased malondialdehyde (MDA) level. Also, serum lipid profiles: triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) were significantly decreased after BCE treatment. Histopathological study showed fatty changes induced by alcohol which were improved by BCE treatment. These data suggest that the BCE has anti-inflammatory, anti-oxidant and anti-steatosis potential properties against alcohol induced liver damage. This may be due to the presence of flavonoids and other phenol compounds.

Neuronal Deletion of the Mammalian Indy Homolog (Slc13a5) Increases Energy Expenditure in Mice

INDY (I’m Not Dead Yet) is a transporter of TCA cycle intermediates, mediating cellular citrate uptake and is highly expressed in liver and brain. Reduced expression of Indy in lower organisms extended lifespan, reduced whole body fat content and increased mitochondrial biogenesis. In mammals, whole body deletion of the mammalian Indy homolog (mIndy, Slc13a5) increased energy expenditure and protected mice from diet and aging induced obesity and insulin resistance. Here, we address the role of neuronal mIndy in energy homeostasis. We generated a neuronal mIndy knockout (NINKO) mouse model by crossing mIndy-floxed to NestinCre mice. Deletion of mIndy in neurons led to an increase in energy expenditure compared to NestinCre mice after 8 weeks of high fat diet (HFD) feeding (average 24h EE: NestinCre: 11.5±0.5 kcal/h/kg lean mass (lm); NINKO: 13.6±0.6 kcal/h/kg lm; p<0.001). The respiratory exchange ratio decreased from 0.78±0.01 in NestinCre mice to 0.77±0.01 in NINKO mice during the light phase (p<0.001), indicating augmented lipid oxidation in NINKO mice. Body temperature increased in NINKO compared to NestinCre mice (NestinCre: 37.0±0.1°C, NINKO: 37.3±0.1°C at 6pm; p<0.05). Fat mass was reduced and lean mass increased significantly in NINKO mice (NestinCre: 60±1% lm, 31±1% fat mass; NINKO: 63±1% lm, 27±1% fat mass after 8 weeks of HFD feeding; p<0.05). Hyperinsulinemic-euglycemic clamp studies showed improved insulin sensitivity in NINKO mice (steady state GINF NestinCre: 26±2 mg/kg/min; NINKO: 34.6±2 mg/kg/min; p<0.05). Together, these data suggest that neuronal mIndy is a critical regulator of energy and glucose homeostasis in mammals. Further studies will address the mechanisms involved in the effect. Disclosure A. Kurzbach: None. D.M. Willmes: None. T. Schumann: None. C. Henke: None. N. El-Agroudy: None. A. Kleinridders: None. A.L. Birkenfeld: None.

268: Expression of the fetal hepatic circadian gene Npas2 regulates weight gain following high fat diet (HFD) feeding

268: Expression of the fetal hepatic circadian gene Npas2 regulates weight gain following high fat diet (HFD) feeding