Weeks Of High-fat Feeding Research Articles

7943 In Obese C57Bl6 Male Mice, Weight Loss due to Caloric Restriction Reduced Bone Morphology but Improved Components of Systemic Metabolism

Abstract Disclosure: C. Chlebek: None. C. McAndrews: None. S.N. Costa: None. C.J. Rosen: None. Obesity and calorie restriction individually alter systemic cellular metabolism and affect musculoskeletal tissue health. Obesity reduces trabecular and cortical bone quality. Clinically, obese patients are advised to lose weight, despite the negative effects of calorie restriction on bone in nonobese preclinical models. Little is known about bone quality following weight loss in obese preclinical models. We hypothesized that in obese models, caloric restriction would further worsen bone quality. To induce obesity, male C57Bl6 mice (8 weeks old) received 60% high fat diet for 12 weeks (high fat feeding phase). Following obesity induction, the HFD-CR group was acclimated to control low fat diet for 2 wks and then underwent 30% caloric restriction for 8 wks (caloric restriction feeding phase). The HFD group received 60% kcal high fat diet from ages 8 to 30 wks. LFD mice received control low fat 10% kcal diet for the study duration. Mice were euthanized at 30 wks of age. Body weight, body composition, and bone mineral density were recorded at baseline and at the end of each diet. Glucose and insulin tolerance were recorded at the end of the high fat and caloric restriction feeding phases. At euthanasia, cortical and trabecular bone morphology (µCT) were assessed.Adverse metabolic parameters worsened with high fat feeding but improved by caloric restriction. Compared to LFD animals, HFD and HFD-CR mice gained body weight, fat mass, and lean mass during the high fat feeding phase. After the caloric restriction feeding phase, body weight, fat mass, and lean mass were reduced in HFD-CR compared to both HFD and LFD controls (p < 0.0001). After 12 weeks of high fat feeding, glucose and insulin tolerance were reduced in HFD and HFD-CR mice compared to LFD (p < 0.0001). Caloric restriction improved both glucose and insulin tolerance in HFD-CR mice compared to both HFD and LFD animals (p < 0.0001). Caloric restriction improved areal bone mineral density, but at euthanasia, HFD-CR mice had worse bone morphology compared to LFD or HFD animals. Although areal bone mineral density (aBMD) was reduced by the high fat feeding phase, HFD-CR and LFD aBMD was not different at the end of the study (p < 0.0001). At euthanasia, HFD-CR mice had reduced cortical area and cortical thickness (p < 0.05). Marrow area and moment of inertia were not different between diets. Compared to LFD, both HFD and HFD-CR mice had reduced tibial bone volume fraction (p < 0.05). HFD-CR animals had smaller trabecular thickness (p < 0.05) and greater trabecular number (p < 0.01) compared to HFD mice. Despite improvements in components of systemic metabolism, caloric restriction in obese preclinical models reduced bone morphology in both trabecular and cortical compartments. This work will improve our understanding of the musculoskeletal system following caloric restriction in obese models and will aid in nutritional counseling for obese patients. Presentation: 6/1/2024

Total Sn-2 Palmitic Triacylglycerols and the Ratio of OPL to OPO in Human Milk Fat Substitute Modulated Bile Acid Metabolism and Intestinal Microbiota Composition in Rats

In this study, the impact of sn-2 palmitic triacyclglycerols (TAGs) in combination with their ratio of two major TAGs (1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) to 1,3-dioleoyl-2-palmitoylglycerol (OPO)) in human milk fat substitute (HMFS) on bile acid (BA) metabolism and intestinal microbiota composition was investigated in newly-weaned Sprague–Dawley rats after four weeks of high-fat feeding. Compared to those of control group rats, HMFS-fed rats had significantly increased contents of six hepatic primary BAs (CDCA, αMCA, βMCA, TCDCA, TαMCA and TβMCA), four ileal primary BAs (UDCA, TCA, TCDCA and TUDCA) and three secondary BAs (DCA, LCA and ωMCA), especially for the HMFS with the highest sn-2 palmitic acid TAGs of 57.9% and OPL to OPO ratio of 1.4. Meanwhile, the inhibition of ileal FXR-FGF15 and activation of TGR5-GLP-1 signaling pathways in HMFS-fed rats were accompanied by the increased levels of enzymes involved in BA synthesis (CYP7A1, CYP27A1 and CYP7B1) in the liver and two key thermogenic proteins (PGC1α and UCP1) in perirenal adipose tissue, respectively. Moreover, increasing sn-2 palmitic TAGs and OPL to OPO ratio in HMFS also altered the microbiota composition both on the phylum and genus level in rats, predominantly microbes associated with bile-salt hydrolase activity, short-chain fatty acid production and reduced obesity risk, which suggested a beneficial effect on host microbial ecosystem. These observations provided important nutritional evidence for developing new HMFS products for infants.

Intervention effect of Chuanxiong-Chishao herb pair on circRNA/lncRNA expression profile in a myocardial infarction-atherosclerosis model

This study aimed to explore the intervention effect of Chuanxiong-Chishao herb pair(CX-CS) on a myocardial infarction-atherosclerosis(MI-AS) mouse model and investigate its effect on the expression profile of circular RNAs(circRNAs)/long non-coding RNAs(lncRNAs) in ischemic myocardium and aorta. Sixty male ApoE~(-/-) mice were randomly assigned to a model group, high-, medium-, and low-dose CX-CS groups(7.8, 3.9, and 1.95 g·kg~(-1)), and a positive drug group(metoprolol 26 mg·kg~(-1) and simvastatin 5.2 mg·kg~(-1)), with 12 mice in each group. Male C57BL/6J mice were assigned to the sham group. The mice in the model group and the groups with drug intervention were fed on a high-fat diet for 10 weeks, followed by anterior descending coronary artery ligation. After that, the mice were fed on a high-fat diet for another two weeks to induce the MI-AS model. The mice in the sham group received normal feed, followed by sham surgery without coronary artery ligation. Mice in the groups with drug intervention received CX-CS or positive drug by gavage for four weeks from the 9th week of high-fat feeding, and those in the model group and the sham group received an equal volume of normal saline. Whole transcriptome sequencing was performed on the heart and aorta tissues of the medium-dose CX-CS group, the model group, and the sham group after administration. The results showed that the medium-and high-dose CX-CS groups showed improved cardiac function and reduced myocardial fibrosis area, and the medium-dose CX-CS group showed significantly reduced plaque area. CX-CS treatment could reverse the expression of circRNA＿07227 and circRNA＿11464 in the aorta of AS model and circRNA expression(such as circRNA＿11505) in the heart of the MI model. Differentially expressed circRNAs between the CX-CS-treated mice and the model mice were mainly enriched in lipid synthesis, lipid metabolism, lipid transport, inflammation, and angiogenesis in the aorta, and in angiogenesis, blood pressure regulation, and other processes in the heart. CX-CS treatment could reverse the expression of lncRNAs such as ENSMUST00000162209 in the aorta of the AS model and TCONS＿00002123 in the heart of the MI model. Differentially expressed lncRNAs between the CX-CS-treated mice and model mice were mainly enriched in lipid metabolism, angiogenesis, autophagy, apoptosis, and iron death in the aorta, and in angiogenesis, autophagy, and iron death in the heart. In summary, CX-CS can regulate the expression of a variety of circRNAs and lncRNAs, and its intervention mechanism in coronary heart disease may be related to the regulation of angiogenesis and inflammation in ischemic myocardium, as well as lipid metabolism, lipid transport, inflammation, angiogenesis in AS aorta.

1670-P: Sex-Dependent Effects of PERK Deletion in Thermogenic Adipocytes

Brown and beige adipocytes dissipate chemical energy as heat through thermogenic respiration, thereby playing important roles in adaptive thermogenesis and energy homeostasis in mice. The Protein Kinase R (PKR)-Like Endoplasmic Reticulum Kinase (PERK) has been shown to be required for mitochondrial thermogenesis in cultured brown adipocytes, and in mice lacking PERK in white and brown adipose tissues. Here, we tested the hypothesis that PERK expression specifically in thermogenic adipocytes is required for adaptive thermogenesis and to regulate energy homeostasis in mice, by selectively deleting PERK in Ucp1-expressing cells (brown and beige adipocytes) of male and female mice. At baseline conditions, mitochondria oxygen consumption rates (OCR) were significantly reduced in brown adipocytes isolated from male, but not from female KO mice. After being housed at thermoneutrality for 7 days (30 °C), wild type (WT) and KO mice were exposed to 4 °C for 3 days. Surprisingly, both male and female KO mice were able to properly adapt to cold exposure, as demonstrated by preserved core body temperatures relative to WT mice. Interestingly, age-induced weight gain, fat mass expansion and impairments in glucose homeostasis were all attenuated in KO males, while weight gain was exacerbated in KO females. This correlated with increased energy expenditure (EE) in male mice, while EE was reduced in female mice. We, next, tested the hypothesis that male KO mice would be resistant to diet-induced obesity. After 12 weeks of high-fat feeding (60% calories from fat), male KO mice became as obese, glucose intolerant and insulin resistant as WT mice. In conclusion, our data suggest that PERK expression in thermogenic adipocytes reduces mitochondrial OCR in male mice, but is dispensable for thermoregulation after short-term cold exposure in both male and female mice. Furthermore, our data reveal a sex-dependent effect of PERK in thermogenic adipocytes in the regulation of energy homeostasis, which is dependent on changes in EE. Disclosure J.Jena: None. L.M.García-peña: None. R.Pereira: None. Funding National Institutes of Health (DK125405, 2T32DK112751-06)

The triacylglycerol structure and composition of a human milk fat substitute affect the absorption of fatty acids and calcium, lipid metabolism and bile acid metabolism in newly-weaned Sprague-Dawley rats.

In this study, the effect of sn-2 palmitic triacylglycerols (sn-2 palmitic TAGs) and the ratio between the two major sn-2 palmitic TAGs (OPL to OPO ratio) in a human milk fat substitute (HMFS) on growth, fatty acid and calcium absorptions, and lipid and bile acid metabolic alterations was investigated in Sprague-Dawley rats. After 4 weeks of high-fat feeding, rats fed with the HMFS containing a sn-2 palmitic acid content of 57.87% and an OPL to OPO ratio of 1.4 showed the lowest TAG accumulation in their livers and hypertrophy of perirenal adipocytes, compared to the groups fed with fats containing a lower sn-2 palmitic acid content or a lower OPL to OPO ratio. Meanwhile, synergistically improved absorption of fatty acids and calcium and increased levels of total bile acids (BAs), especially for the tauro-conjugated BAs (TCDCA, TUDCA, TαMCA, TβMCA, TDCA and TωMCA), were observed in rats by both increasing the sn-2 palmitic acid content and the OPL to OPO ratio in HMFS. In addition, the levels of total BAs and tauro-conjugated BAs were negatively correlated with serum TAG, TC, and LDL-c levels and positively correlated with HDL-c levels according to Spearman's correlation analysis (P < 0.05). Collectively, these findings present new nutritional evidence for the potential effects of the TAG structure and composition of a human milk fat substitute on the growth and lipid and bile acid metabolism of the host in infancy.

ODP005 Molecular Characterization of Early Adipose Tissue Remodeling and Fibrosis in a Murine Model of Diet-Induced Obesity.

Abstract One of the many pathophysiological changes associated with obesity is the build up of structural and physiochemical extracellular matrix components in adipose depots, known as adipose tissue fibrosis. Like liver fibrosis, adipose tissue fibrosis occurs as a response to chronic injury and inflammation and can perpetuate inflammation and impede proper tissue functioning. Key adipose tissue fibrosis markers have been identified in obese adipose tissue, including collagen VI and its cleavage product endotrophin, however, adipose tissue fibrosis remains poorly characterized, particularly in its initiating phases. We have developed and characterized a murine model of diet-induced obesity (12 weeks of high fat feeding), insulin resistance, and early fibrotic development in both sexes. While diagnosable fibrosis was absent, as determined by picrosirius red staining, we detected elevated collagen VI, TGF-β, TNF-α, and TIMP-1 mRNA in gonadal white adipose of both sexes. TIMP-4 was decreased in males but elevated in females. Immunohistochemical analysis revealed pericellular build up of collagen VI immunoreactivity in males only, co-localized with immunoreactivity of its protease MMP-14, which is known to generate the pro-fibrotic fragment endotrophin. This work identifies key sex differences and fibrosis markers present during the initiation of adipose tissue fibrosis, earlier than diagnosable fibrosis can be detected, highlighting their potential as therapeutic targets for prevention or reversal of fibrosis in the treatment of obesity and insulin resistance. Presentation: No date and time listed

Abstract 108: Altered Erk / Klf4-mediated Control Of Agrp And Metabolic Rate During Obesity

Resting metabolic rate (RMR) adaptation contributes to the maintenance of human obesity, yet mechanisms controlling RMR in lean vs obese states remain poorly defined. Leptin contributes to RMR control in part through suppression of inhibitory Agouti-related peptide (AgRP) neurotransmission in the hypothalamic arcuate nucleus (ARC). Within AgRP neurons, leptin activates the extracellular signal-regulated kinase (ERK) cascade, which is thought to interrupt Agrp transcription through interference with the Krüppel-like factor 4 (KLF4) transcription factor, and our published data support altered ARC ERK signaling during obesity. We therefore hypothesized a role for ERK/KLF4 signaling within AgRP neurons for the suppression of AgRP transcription and thereby stimulation of RMR in the lean state, and changes in this pathway during obesity. In the GT1-7 cell model of AgRP neurons, leptin increased the phosphorylated ERK (pERK)/ERK ratio (n=4, 1 uM, 15 min +89% p&lt;0.05) and reduced Agrp mRNA (n=4, 1 uM, 1 hr -43.0% p&lt;0.05); similarly, siRNA knockdown of Klf4 reduced Agrp mRNA (n=4, -21.6% p&lt;0.05). In lean adult C57BL/6J mice, leptin increased the ARC pERK/ERK ratio (n=8, 1 ug/g ip 16.5-fold p&lt;0.05) and stimulated RMR (n=11, 2 ug/g ip 30 min +43.8% p&lt;0.05). Lean transgenic mice with conditional deletion of KLF4 from AgRP neurons ( Agrp -Cre x Klf4 flox ) exhibited increased RMR (n=13 KO +13.48% vs controls n=22 p&lt;0.05). These findings support the model that ERK inhibition of KLF4 within AgRP neurons mechanistically contributes to leptin-mediated control of Agrp and RMR in the lean state. Ten weeks of high fat feeding strongly increases endogenous plasma leptin in C57BL/6J mice, and this treatment increased the ARC pERK/ERK ratio (n=5 +54.4% p&lt;0.05), yet ARC KLF4 was increased (n=6 +74.6% p&lt;0.05) and Agrp mRNA was not suppressed (n=4 +1.6-fold, p=ns). Therefore, these data also support the concept that loss of ERK-mediated suppression of KLF4 within the AgRP neuron during obesity may contribute to the pathogenesis of RMR adaptation.

Abstract 114: Metabolic Adaptation During Obesity Is Associated With G Protein Switching By The Angiotensin At1a Receptor In Agrp Neurons

Obesity represents the single greatest impediment to continued improvements in hypertension and cardiovascular disease. Prolonged obesity is associated with “Resting Metabolic Rate (RMR) adaptation” (ie, reduced RMR relative to body composition), and RMR adaptation is proposed to explain resistance to weight loss / weight maintenance after obesity is established. Previously we showed that angiotensin II (ANG) Agtr1a (AT1A) receptors expressed in Agouti-related peptide (AgRP) neurons of the hypothalamic arcuate nucleus (ARC) are required for integrative control of RMR. We hypothesize that changes in AT1A signaling in AgRP cells underlie the development of RMR adaptation. ARC slices from mice expressing tdTomato in AgRP cells ( Agrp -Cre x Ai9) were used for cell-attached voltage clamp recording of AgRP neurons. ANG inhibited ~33% of AgRP neurons via an AT1A-dependent mechanism (deemed a “Type 1i” response, which was inhibitable by losartan (LOS) or genetic deletion of AT1A; Firing rate; aCSF:0.85 ± 0.03 vs ANG:0.20 ± 0.04 vs ANG+LOS:0.91 ± 0.11 Hz, p&lt;0.05) that was also blocked by the Gαi inhibitor, pertussis toxin (PTX), but not by the Gαq inhibitor, BIM-46187 (Δ Firing rate: PTX: -0.39 ± 0.09 Hz, p&lt;0.05 vs BIM: 0.10 ± 0.09 Hz). After 10 weeks of high-fat feeding, roughly half of Type 1 cells exhibited a spontaneous switch from the ANG/AT1A-mediated inhibitory (Type 1i) response to an ANG/AT1A-mediated stimulatory response (“Type 1s”; Firing rate; aCSF: 0.86 ± 0.03 vs ANG:1.10 ± 0.12 vs ANG+LOS:0.82 ± 0.03 Hz, p&lt;0.05), which was blocked by BIM-46187, but not by PTX (Δ Firing rate: PTX: 0.07 ± 0.06 Hz vs BIM: 0.49 ± 0.06 Hz, p&lt;0.05). AAV delivery of Cre-inducible DREADDs into the ARC of lean Agtr1a -Cre mice was then used to study the effects of Gαi vs Gαq signaling in Type 1 AgRP cells upon RMR control. Activation of the Gαi-coupled hM4Di DREADD in AT1A-expressing cells of the ARC was sufficient to increase RMR (+25% from baseline, n=8-10, p&lt;0.05), but activation of a Gαq-coupled DREADD had no effect. We conclude (i) that the AT1A receptor is expressed by a unique subtype of AgRP neuron; (ii) that inhibitory AT1A-Gαi signaling in the Type 1 AgRP cell stimulates RMR; and (iii) that loss of AT1A-Gαi coupling in these cells is sufficient to explain obesity-associated RMR adaptation.

ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.

1381-P: GDF15 Deficiency in Brown Adipose Tissue Does Not Affect Thermogenesis, but Exacerbates Diet-Induced Obesity

Growth and differentiation factor 15 (GDF15) is induced in brown adipocytes in response to thermogenic stimuli, as well as high-fat feeding in mice. However, whether brown adipose tissue (BAT) -derived GDF15 is required to regulate BAT thermogenesis or energy homeostasis is unclear. In the present study, we sought to test the hypothesis that BAT-derived GDF15 is necessary to mediate thermogenesis and systemic adaptations to diet-induced obesity (DIO) . We generated mice lacking GDF15 specifically in BAT by crossing Gdf15 floxed mice with mice harboring the Cre recombinase under the control of the Ucp1 promoter (GDF15 BAT KO) . GDF15 BAT KO mice were treated with the beta adrenoreceptor agonist CL316,243 (CL) for 5 days to induce thermogenesis, or fed a high-fat diet (60% fat content) for 12 weeks to induce DIO. CL treatment promoted a significant induction in Gdf15 mRNA levels in the BAT of WT mice, which, as expected, was prevented in KO mice. Nonetheless, serum GDF15 levels were similarly induced by CL in both WT and KO mice. CL-induced activation of thermogenic genes in BAT, such as Ucp1 and Dio2, was comparable between genotypes, and core body temperature was unchanged between WT and KO mice. After 12 weeks of high-fat feeding, KO mice had significantly increased body weight and fat mass relative to WT mice. However, food intake, locomotor activity and energy expenditure were unchanged between groups. Respiratory exchange ratio was significantly higher in KO mice, indicating a relative preference for carbohydrate as fuel. Although glucose tolerance was unchanged between genotypes, insulin sensitivity was impaired in KO mice. Taken together, our data indicates that BAT-derived GDF15 does not contribute to GDF15 circulating levels in response to CL treatment and is largely dispensable for CL-induced BAT thermogenesis. However, BAT-derived GDF15 seems to be required to regulate diet-induced weight gain and insulin sensitivity. Disclosure J.Jena: None. K.Kato: None. A.A.Marti: None. R.Pereira: None. Funding NIHDK125405

9-LB: Increased Islet Expression of Somatostatin and Glucagon in Male Rats with Insulin-Dependent Type 2 Diabetes

Background: Pancreatic islet remodelling in type 1 diabetes increases somatostatin (SST) expression, which inhibits glucagon counterregulation to hypoglycemia. Here, we characterized islet morphology and hormone composition in a rodent model of insulin-deficient type 2 diabetes (T2D) . Methods: Hypoinsulinemic T2D was induced in 8-9-week-old, male, Sprague-Dawley rats (n=4) via 3 weeks of high-fat feeding and a low-dose injection of streptozotocin (STZ; 35 mg/kg) . After 2 weeks of basal insulin treatment, pancreas tissue was extracted from T2D and healthy control (n=5) rats for immunohistochemical staining of representative islets. Results: Fasted plasma C-peptide levels of hyperglycemic (24.8±3.3 mmol/l) T2D rats were reduced by ∼44% (624±64 vs. 1107±121 pmol/l; p&amp;lt;0.001) , while glucagon was unchanged (75±31 vs. 98±39 pg/ml) . Fractional islet area of glucagon and SST were elevated by 40% (21±15 vs. 15±10%; p&amp;lt;0.001) and 83% (11±8 vs. 6±5%; p&amp;lt;0.001) , respectively, as compared to control rats (Figure) , which was most pronounced in small and medium-sized islets. Conclusions: Fractional area expression of glucagon and SST increased per islet in insulin-treated rats with advanced T2D. These findings may help account for the increase in SST signaling that underscores defective glucagon counterregulation to hypoglycemia in insulin-deficient diabetic states. Disclosure E. G. Hoffman: None. S. C. Atherley: None. N. Akbarian: None. N. Dsouza: None. R. Liggins: Employee; Zucara Therapeutics, Stock/Shareholder; Zucara Therapeutics. M. Riddell: Advisory Panel; Zealand Pharma A/S, Zucara Therapeutics, Consultant; Eli Lilly and Company, Jaeb Center for Health Research, Speaker’s Bureau; Dexcom, Inc., Eli Lilly and Company, Novo Nordisk. Funding GlycoNet

The microsomal triglyceride transfer protein inhibitor lomitapide improves vascular function in mice with obesity.

In this study, the effect of lomitapide, a microsomal triglyceride transfer protein inhibitor, on the cardiovascular function in obesity was investigated. Eight-week-old C57BL/6 mice were fed with high-fat diet for 12 weeks in the presence and absence of lomitapide. Lomitapide was administered by gavage (1 mg/kg/d) during the last 2 weeks of high-fat feeding. Body weight, blood glucose, body composition, and lipid profile were determined. Vascular function and endothelial function markers were studied in the aorta and mesenteric resistance arteries. Lomitapide treatment reduced body weight in mice with obesity. Blood glucose, percentage of fat mass, total cholesterol, and low-density lipoprotein levels were significantly reduced, and the percentage of lean mass was significantly increased after lomitapide treatment. The vascular response to sodium nitroprusside in the aorta and mesenteric arteries was similar among groups. However, the vascular response to acetylcholine was improved in the treated group. This was associated with decreased levels of vascular endoplasmic reticulum stress, inflammation, and oxidative stress. Treatment with lomitapide attenuated the increase in body weight in mice with obesity and restored the lipid profile and vascular function. These effects were accompanied by a decrease in inflammation and oxidative stress.

A Cycle of Inflammatory Adipocyte Death and Regeneration in Murine Adipose Tissue.

Adipose tissue (AT) expands by a combination of two fundamental cellular mechanisms: hypertrophic growth of existing adipocytes or through generation of new adipocytes, also known as hyperplastic growth. Multiple lines of evidence suggest a limited capacity for hyperplastic growth of AT in adulthood and that adipocyte number is relatively stable, even with fluctuations in AT mass. If the adipocyte number is stable in adulthood, despite well-documented birth and death of adipocytes, then this would suggest that birth may be coupled to death in a regenerative cycle. To test this hypothesis, we examined the dynamics of birth of new fat cells in relationship to adipocyte death by using high-fidelity stable isotope tracer methods in C57Bl6 mice. We discovered birth of new adipocytes at higher frequency in histological proximity to dead adipocytes. In diet-induced obesity, adipogenesis surged after an adipocyte death peak beyond 8 weeks of high-fat feeding. Through transcriptional analyses of AT and fractionated adipocytes, we found that the dominant cell death signals were inflammasome related. Proinflammatory signals were particularly evident in hypertrophied adipocytes or with deletion of a constitutive oxygen sensor and inhibitor of hypoxia-inducible factor, Egln1. We leveraged the potential role for the inflammasome in adipocyte death to test the adipocyte death-birth hypothesis, finding that caspase 1 loss of function attenuated adipocyte death and birth in murine visceral AT. These data collectively point to a regenerative cycle of adipocyte death and birth as a driver of adipogenesis in adult murine AT.

High fat diet-induced obesity leads to depressive and anxiety-like behaviors in mice via AMPK/mTOR-mediated autophagy

Depression is one of the most common mental illnesses in modern society. In recent years, several studies show that there are disturbances in lipid metabolism in depressed patients. High-fat diet may lead to anxiety and depression, but the mechanisms involved remain unclear. In our study, we found that 8 weeks of high-fat feeding effectively induced metabolic disorders, including obesity and hyperlipidemia in mice. Interestingly, the mice also showed depressive and anxiety-like behaviors. We further found activated microglia and astrocyte, increased neuroinflammation, decreased autophagy and BDNF levels in mice after high-fat feeding. Besides, high-fat feeding can also inhibit AMPK phosphorylation and induce mTOR phosphorylation. After treating with the mTOR inhibitor rapamycin, autophagy and BDNF levels were elevated. The number of activated microglia and astrocyte, and pro-inflammation levels were reduced. Besides, rapamycin can also reduce the body weight and serum lipid level in high fat feeding mice. Depressive and anxiety-like behaviors were also ameliorated to some extent after rapamycin treatment. In summary, these results suggest that high-fat diet-induced obesity may lead to depressive and anxiety-like behaviors in mice by inhibiting AMPK phosphorylation and promoting mTOR shift to phosphorylation to inhibit autophagy. Therefore, improving lipid metabolism or enhancing autophagy through the AMPK/mTOR pathway could be potential targets for the treatment of obesity depression.

Fetuin-A excess expression amplifies lipid induced apoptosis and β-cell damage.

Fetuin-A, a hepato-adipokine, is associated with lipid-mediated islet inflammation and inflicts β-cell death but the underlying mechanisms are still unclear. In an earlier report, it was shownthat fetuin-A promotes lipid-induced insulin resistance by acting as an endogenous ligand of toll like receptor 4. Recently, we have also reported that β-cells secrete fetuin-A on stimulation by palmitate causing β-cell dysfunction. The aim of this study was twofold: (a) screening the role of fetuin-A in survival of murine β-cells, and (b) to validate the effect of fetuin-A release and lipid induced apoptosis in mouse insulinoma cell line MIN6. Excess of lipid and fetuin-A in circulation induced significant deterioration of islet histoarchitecture and impeded insulin secretion by 2.7 ± 0.5-folds in 20 weeks high fat diet mice. Administration of fetuin-A (0.7 mg/g) along with 4 weeks of HFD produced similar results as20 weeks of high fat feeding. Treating high doses of palmitate alone (0.50 mM) as well as in combination with fetuin-A (100 µg/ml) for 24 h inflicted apoptosis in MIN6 through the mitochondrial pathway. Knockdown of fetuin-A gene partially inhibited palmitate inflicted apoptosis in MIN6 by 1.83 ± 0.25 times, however, fetuin-A when added in the medium caused re-emergence of apoptosis. Notably, apoptosis induced by palmitate conditioned media from MIN6, 3T3L1, and HepG2, was partially inhibited in fetuin-A KD MIN6. These results confirmed the critical role of circulatory fetuin-A and β-cell secreted fetuin-A in β-cell dysfunction and apoptosis under hyperlipidemic conditions.

199-LB: ATF4 Deletion in Brown Adipose Tissue Impairs Adaptive Thermogenesis but Attenuates Diet-Induced Insulin Resistance

Short-term cold exposure induces the integrated stress response (ISR) and its main effector, the activating transcription factor 4 (ATF4), in brown adipose tissue (BAT). Furthermore, induction of ATF4 in BAT in response to mitochondrial stress is required for systemic metabolic adaptations mediated by BATokine secretion. To test the hypothesis that ATF4 in BAT is required for cold-induced thermogenesis and to promote BATokine secretion, we selectively deleted ATF4 in BAT by crossing Atf4 floxed mice with mice harboring the Cre recombinase under the control of the Ucp1 promoter (ATF4 BAT KO). KO mice experienced a significant reduction in core body temperature after 3 days of cold exposure relative to wild type (WT) mice. Impaired thermogenesis correlated with reduced BAT mass, and decreased UCP1 and tyrosine hydroxylase protein levels. In addition, KO mice had impaired cold-induced browning of white adipose tissue (WAT) and reduced expression of fibroblast growth factor 21 (Fgf21) and growth and differentiation factor 15 (Gdf15), BATokines shown to be induced in response to BAT thermogenic activation. Surprisingly, although KO mice had equivalent weight gain and fat mass relative to WT mice after 12 weeks of high-fat feeding, glucose and insulin intolerance were ameliorated, and fasting insulin levels were significantly reduced, indicating increased insulin sensitivity. Improved glucose clearance correlated with increased levels of glucose transporters 1 and 4 (GLUT1 and GLUT4) in BAT, but not in WAT or skeletal muscle. Taken together, our data indicate that Atf4 expression is required for full thermogenic activation of BAT and to regulate FGF21 and GDF15 release as BATokines, likely coordinating BAT thermogenesis with systemic adaptations. Finally, our data suggest that in diet-induced obesity, ATF4 downregulation may increase glucose utilization in BAT, contributing to improved glucose homeostasis and insulin sensitivity. Disclosure S. Bjorkman: None. A. A. Marti: None. E. T. Weatherford: None. L. M. García-peña: None. M. J. Potthoff: None. E. D. Abel: Board Member; Self; MyoKardia, Other Relationship; Self; AstraZeneca. R. Pereira: None. Funding National Institutes of Health (R01DK125405)

Neuronatin Promotes SERCA Uncoupling and Its Expression is Inversely Associated with High Fat Diet Induced Weight Gain in vivo

Neuronatin (NNAT) is a paternally imprinted gene involved in many aspects of metabolism but the underlying cellular mechanism remains unknown. Its sequence homology with phospholamban (PLN) and sarcolipin (SLN) suggest that NNAT has a putative role in regulating the enzymatic activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in striated muscles. Both PLN and SLN physically interact and functionally inhibit SERCA by lowering its affinity for Ca2+, but only SLN has the unique ability to uncouple SERCA-mediated Ca2+ transport from ATP hydrolysis in mouse soleus muscles. Uncoupling the SERCA pump increases energy expenditure and may be beneficial in combatting energy imbalances, such as obesity. The objective of this study was to test our hypothesis that like SLN, NNAT would also promote SERCA uncoupling. In addition, we also examined whether NNAT expression would be altered in soleus muscles obtained from C57BL/6 mice fed a high fat diet (HFD). Using human embryonic kidney (HEK) cells co-transfected with SERCA and NNAT cDNA, we measured SERCA coupling ratio using an Indo-1 based fluorometric Ca2+ uptake assay and an enzyme-linked spectrophotometric ATPase assay. NNAT promoted uncoupling of both SERCA1a and SERCA2a isoforms in HEK cells in a dose-dependent manner with SERCA1a showing increased sensitivity when compared to SERCA2a. Western blotting was used to confirm HEK cell transfection as well as to assess NNAT and SERCA content in soleus muscles from mice fed a low (LFD) or HFD (60% kcal) for 12 weeks. A ~40% reduction in SERCA1a and a ~50% increase in SERCA2a protein content was observed in the soleus muscles of mice fed a HFD compared to LFD. Total NNAT content and its levels relative to SERCA (SERCA1a + SERCA2a) were reduced by ~40% and ~45%, respectively in HFD mice compared to LFD mice. Plotting muscle NNAT content (relative to SERCA) against total weight gained after 12 weeks of high fat feeding also revealed a significant inverse relationship (r2=0.49, p=0.01), suggesting that higher NNAT content (relative to SERCA) is related to reduced weight gained from a HFD in vivo. This is the first study to demonstrate NNAT as a SERCA uncoupler and that its expression in muscle is negatively associated with HFD induced weight gain. These findings suggest that NNAT may regulate whole-body metabolism and energy balance via SERCA uncoupling.

Exploring the role of ATP10A in diet‐induced obesity, insulin resistance, and type 2 diabetes

Type IV P-type ATPases (P4-ATPases) are enzymes that catalyze the translocation of lipids across plasma membranes. Our lab recently discovered that Atp10a, a mammalian P4-ATPase, can translocate glucosylceramide, a sphingolipid. Both Atp10a and glucosylceramide have been independently implicated in metabolic dysfunction. To explore the role of Atp10a in metabolic dysfunction and sphingolipid metabolism, we created a novel Atp10a knockout (KO) mouse model. Atp10a KO mice display a female-specific excess weight gain on standard chow that is exacerbated by 12 weeks of high fat feeding and this is attributable to increased adiposity. Additionally, they display an accumulation of neutral lipids and altered ceramide metabolism in their livers. After a 5 hour fast, these mice exhibit elevated blood glucose levels that are not coupled to a proportional elevation in insulin levels. Atp10a KO mice also exhibit elevated plasma free fatty acids, cholesterol, and triglycerides. Thus far, our studies have shown that knocking out Atp10a in a mouse results in sex-specific perturbations to body weight and composition, glucose homeostasis, plasma lipid levels, and liver metabolism.

Heat Treatment Improves Hepatic Mitochondrial Respiratory Efficiency via Mitochondrial Remodeling.

Nonacholic fatty liver disease, or hepatic steatosis, is the most common liver disorder affecting the western world and currently has no pharmacologic cure. Thus, many investigations have focused on alternative strategies to treat or prevent hepatic steatosis. Our laboratory has shown that chronic heat treatment (HT) mitigates glucose intolerance, insulin resistance, and hepatic steatosis in rodent models of obesity. Here, we investigate the direct bioenergetic mechanism(s) surrounding the metabolic effects of HT on hepatic mitochondria. Utilizing mitochondrial proteomics and respiratory function assays, we show that one bout of acute HT (42°C for 20 min) in male C57Bl/6J mice (n = 6/group) triggers a hepatic mitochondrial heat shock response resulting in acute reductions in respiratory capacity, degradation of key mitochondrial enzymes, and induction of mitophagy via mitochondrial ubiquitination. We also show that chronic bouts of HT and recurrent activation of the heat shock response enhances mitochondrial quality and respiratory function via compensatory adaptations in mitochondrial organization, gene expression, and transport even during 4 weeks of high-fat feeding (n = 6/group). Finally, utilizing a liver-specific heat shock protein 72 (HSP72) knockout model, we are the first to show that HSP72, a protein putatively driving the HT metabolic response, does not play a significant role in the hepatic mitochondrial adaptation to acute or chronic HT. However, HSP72 is required for the reductions in blood glucose observed with chronic HT. Our data are the first to suggest that chronic HT (1) improves hepatic mitochondrial respiratory efficiency via mitochondrial remodeling and (2) reduces blood glucose in a hepatic HSP72-dependent manner.

Impact of short-term high-fat feeding on lipid droplet content in mouse oocytes.

Mature mammalian oocytes contain lipid droplets (LDs), which are neutral lipid storage organelles critically important for energy metabolism. In mice, maternal obesity, induced by long-term (> 3 months) high-fat feeding, contributes to the accumulation of LDs in mature oocytes. However, few studies have investigated the influence of short-term high-fat feeding on LD content. In this study, we demonstrated that 3 weeks of high-fat feeding is sufficient to increase LD content and intracellular triacylglycerol levels. Using a two-step centrifugation technique to release LDs into the perivitelline space, we found that short-term high-fat feeding increased the level of LDs in MII oocytes and that 3 days of high-fat feeding were sufficient to increase efficiency of LD release. Collectively, our study suggests that short-term high fat feeding can have a higher impact on lipid metabolism during oocyte maturation.