Chow-fed Animals Research Articles

Assessment of Mitochondrial Function in a Mouse Model of Mild Chronic Kidney Disease Induced by High-Fat and High-Salt Diet

Intro: Kidneys are known for high energy demands involved in active transport and are thus highly oxidative organs, consuming the second-highest amount of oxygen per gram of tissue at rest (only exceeded by the heart). Chronic kidney disease (CKD) is a complex disorder that presents substantial challenges in understanding its pathophysiological mechanisms, including mitochondrial alterations. However, there are no established mouse models that recapitulate the CKD phenotype observed in humans.Hypothesis: We hypothesized that a high-fat, high-salt diet would induce a mild CKD phenotype and mitochondrial dysfunction in mice. Methods: We subjected C57Bl/6 mice to a high-fat (42%) and high-salt (8% NaCl) diet (HF/HNaCl) or standard chow diet for 16 weeks (n=4 per group). Additionally, we conducted comprehensive investigations into mitochondrial function, focusing on state 3 mitochondrial oxygen consumption (Oroboros O2K) and adenosine triphosphate (ATP) production (Horiba Fluoromax). Differences between groups were determined using Student’s t-tests. Results: HF/HNaCl feeding resulted in polyurea indicated by an increase in 24-hour urine output (Chow: 1.5 ± 0.2 mL; HF/HNaCl: 3.7 ± 0.8 mL; p=0.05) and a 30% reduction in glomerular filtration rate compared to chow-fed animals (Chow: 911.2 ± 107.8 μL/min/100g; Hf/HNaCl: 639.5 ± 25.9 μL/min/100g; p=0.08), indicative of the development of mild kidney disease. Although subtle changes were observed in mitochondrial oxygen consumption and ATP production, the alterations were not statistically significant. Discussion: Our findings suggest that the induced mild CKD state via the dietary regimen led to moderate renal impairment without substantial impacts on mitochondrial respiratory capacity and ATP synthesis. The establishment of this mouse model provides a valuable platform for further investigations into the complex interplay between renal dysfunction and mitochondrial dynamics in the context of mild CKD, offering insights that may pave the way for the development of targeted therapeutic interventions for this prevalent and debilitating condition. This project was supported by grants from the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 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.

Hepatic lipid droplet-associated proteome changes distinguish dietary-induced fatty liver from glucose tolerance in male mice.

Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared with high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD- and HStD-fed mice had more and larger LDs than Chow-fed animals. We observed striking differences in liver LD proteomes of HFD- and HStD-fed mice compared with Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver LD proteome consisting of proteins common in HFD- and HStD-fed mice, as well as a proteome associated with glucose tolerance that included proteins shared in Chow and HStD but not HFD-fed mice. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase to perilipin 5 in the LD proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.NEW & NOTEWORTHY This study identified a fatty liver lipid droplet proteome and one associated with glucose tolerance. Notably, glucose intolerance was linked with changes in the ratio of adipose triglyceride lipase to perilipin 5 that is indicative of dysregulation of neutral lipid homeostasis.

High fat diet is associated with gut microbiota dysbiosis and decreased gut microbial derived metabolites related to metabolic health in young Göttingen Minipigs.

The objectives were 1) to characterize a Göttingen Minipig model of metabolic syndrome regarding its colon microbiota and circulating microbial products, and 2) to assess whether ovariectomized female and castrated male minipigs show similar phenotypes. Twenty-four nine-week-old Göttingen Minipigs were allocated to four groups based on sex and diet: ovariectomized females and castrated males fed either chow or high-fat diet (HFD) for 12 weeks. At study end, body composition and plasma biomarkers were measured, and a mixed meal tolerance test (MMT) and an intravenous glucose tolerance test (IVGTT) were performed. The HFD groups had significantly higher weight gain, fat percentage, fasting plasma insulin and glucagon compared to the chow groups. Homeostatic model assessment of insulin resistance index (HOMA-IR) was increased and glucose effectiveness derived from the IVGTT and Matsuda´s insulin sensitivity index from the MMT were decreased in the HFD groups. The HFD groups displayed dyslipidemia, with significantly increased total-, LDL- and HDL-cholesterol, and decreased HDL/non-HDL cholesterol ratio. The colon microbiota of HFD minipigs clearly differed from the lean controls (GuniFrac distance matrix). The main bacteria families driving this separation were Clostridiaceae, Fibrobacteraceae, Flavobacteriaceae and Porphyromonadaceae. Moreover, the species richness was significantly decreased by HFD. In addition, HFD decreased the circulating level of short chain fatty acids and beneficial microbial metabolites hippuric acid, xanthine and trigonelline, while increasing the level of branched chain amino acids. Six and nine metabolically relevant genes were differentially expressed between chow-fed and HFD-fed animals in liver and omental adipose tissue, respectively. The HFD-fed pigs presented with metabolic syndrome, gut microbial dysbiosis and a marked decrease in healthy gut microbial products and thus displayed marked parallels to human obesity and insulin resistance. HFD-fed Göttingen Minipig therefore represents a relevant animal model for studying host-microbiota interactions. No significant differences between the castrated and ovariectomized minipigs were observed.

Diabetes exacerbates inflammatory bowel disease in mice with diet-induced obesity.

The increased prevalence of inflammatory bowel disease (IBD) among patients with obesity and type 2 diabetes suggests a causal link between these diseases, potentially involving the effect of hyperglycemia to disrupt intestinal barrier integrity. To investigate whether the deleterious impact of diabetes on the intestinal barrier is associated with increased IBD severity in a murine model of colitis in mice with and without diet-induced obesity. Mice were fed chow or a high-fat diet and subsequently received streptozotocin to induce diabetic-range hyperglycemia. Six weeks later, dextran sodium sulfate was given to induce colitis. In select experiments, a subset of diabetic mice was treated with the antidiabetic drug dapagliflozin prior to colitis onset. Endpoints included both clinical and histological measures of colitis activity as well as histochemical markers of colonic epithelial barrier integrity. In mice given a high-fat diet, but not chow-fed animals, diabetes was associated with significantly increased clinical colitis activity and histopathologic markers of disease severity. Diabetes was also associated with a decrease in key components that regulate colonic epithelial barrier integrity (colonic mucin layer content and epithelial tight junction proteins) in diet-induced obese mice. Each of these effects of diabetes in diet-induced obese mice was ameliorated by restoring normoglycemia. In obese mice, diabetes worsened clinical and pathologic outcomes of colitis via mechanisms that are reversible with treatment of hyperglycemia. Hyperglycemia-induced intestinal barrier dysfunction offers a plausible mechanism linking diabetes to increased colitis severity. These findings suggest that effective diabetes management may decrease the clinical severity of IBD.

Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy — A focus on energy expenditure

ObjectiveBariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study was to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model. MethodsDiet-induced obese male Sprague–Dawley rats were either sham-operated, underwent VSG surgery or were pair-fed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of chow-fed rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 14C-2-deoxy-d-glucose (14C-2DG). Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals. ResultsFollowing VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in chow-fed animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlighted a patent neural link between the gut and BAT that included groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus. ConclusionsCollectively, these data support a role for BAT in mediating the metabolic sequelae following VSG surgery, particularly the improvement in glucose regulation, and highlight the need to better understand the contribution from this tissue in human patients.

Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis.

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.

Exercise does not improve insulin resistance and mitochondrial characteristics together.

The aim of this study was to investigate the relationship between mitochondrial content and respiratory function and whole-body insulin resistance in high-fat diet (HFD) fed rats. Male Wistar rats were given either a chow diet or an HFD for 12 weeks. After 4 weeks of the dietary intervention, half of the rats in each group began 8 weeks of interval training. In vivo glucose and insulin tolerance were assessed. Mitochondrial respiratory function was assessed in permeabilised soleus and white gastrocnemius (WG) muscles. Mitochondrial content was determined by the measurement of citrate synthase (CS) activity and protein expression of components of the electron transport system (ETS). We found HFD rats had impaired glucose and insulin tolerance but increased mitochondrial respiratory function and increased protein expression of components of the ETS. This was accompanied by an increase in CS activity in WG. Exercise training improved glucose and insulin tolerance in the HFD rats. Mitochondrial respiratory function was increased with exercise training in the chow-fed animals in soleus muscle. This exercise effect was absent in the HFD animals. In conclusion, exercise training improved insulin resistance in HFD rats but without changes in mitochondrial respiratory function and content. The lack of an association between mitochondrial characteristics and whole-body insulin resistance was reinforced by the absence of strong correlations between these measures. Our results suggest that improvements in mitochondrial respiratory function and content are not responsible for improvements in whole-body insulin resistance in HFD rats.

Consumption of a high-fat-high-sucrose diet partly diminishes mechanical and structural adaptations of cardiac muscle following resistance training.

[Purpose]The purpose of this study was to investigate the effects of a high-fat high-sucrose (HFHS) diet on previously reported adaptations of cardiac morphological and contractile properties to resistance training.[Methods]Twelve-week-old rats participated in 12-weeks of resistance exercise training and consumed an HFHS diet. Echocardiography and skinned cardiac muscle fiber bundle testing were performed to determine the structural and mechanical adaptations.[Results]Compared to chow-fed sedentary animals, both HFHS- and chow-fed resistance-trained animals had thicker left ventricular walls. Isolated trabecular fiber bundles from chow-fed resistance-trained animals had greater force output, shortening velocities, and calcium sensitivities than those of chow-fed sedentary controls. However, trabeculae from the HFHS resistance-trained animals had greater force output but no change in unloaded shortening velocity or calcium sensitivity than those of the chow-fed sedentary group animals.[Conclusion]Resistance exercise training led to positive structural and mechanical adaptations of the heart, which were partly offset by the HFHS diet.

Human apolipoprotein C1 transgenesis reduces atherogenesis in hypercholesterolemic rabbits

Background and aimsApolipoprotein (apo) C1 is a 6.6 kDa protein associated with HDL and VLDL. ApoC1 alters triglyceride clearance, and it also favors cholesterol accumulation in HDL, especially by inhibiting CETP in human plasma. Apart from studies in mice, which lack CETP, the impact of apoC1 on atherosclerosis in animal models expressing CETP, like in humans, is not known. This study aimed at determining the net effect of human apoC1 on atherosclerosis in rabbits, a species with naturally high CETP activity but with endogenous apoC1 without CETP inhibitory potential. MethodsRabbits expressing a human apoC1 transgene (HuApoC1Tg) were generated and displayed significant amounts of human apoC1 in plasma. ResultsAfter cholesterol feeding, atherosclerosis lesions were significantly less extensive (−22%, p < 0.05) and HDL displayed a reduced ability to serve as CETP substrates (−25%, p < 0.05) in HuApoC1Tg rabbits than in WT littermates. It was associated with rises in plasma HDL cholesterol level and PON-1 activity, and a decrease in the plasma level of the lipid oxidation markers 12(S)-HODE and 8(S)HETE. In chow-fed animals, the level of HDL-cholesterol was also significantly higher in HuApoC1Tg than in WT animals (0.83 ± 0.11 versus 0.73 ± 0.11 mmol/L, respectively, p < 0.05), and it was associated with significantly lower CETP activity (cholesteryl ester transfer rate, −10%, p < 0.05; specific CETP activity, −14%, p < 0.05). ConclusionsConstitutive expression of fully functional human apoC1 in transgenic rabbit attenuates atherosclerosis. It was found to relate, at least in part, to the inhibition of plasma CETP activity and to alterations in plasma HDL.

The effect of acetylsalicylic acid and pentoxifylline in guinea pigs with non-alcoholic steatohepatitis.

Therapeutic options are urgently needed for non-alcoholic fatty liver disease (NAFLD), but development is time-consuming and costly. In contrast, drug repurposing offers the advantages of re-applying compounds that are already approved, thereby reducing cost. Acetylsalicylic acid (ASA) and pentoxifylline (PTX) have shown promise for treatment of NAFLD, but have not yet been tested in combination. Guinea pigs were fed a high-fat diet for 16weeks and then continued on the diet while being treated with ASA, PTX or ASA+PTX for 8weeks. Chow-fed animals served as healthy controls. Guinea pigs were CT scanned before intervention start and at intervention end. Animals without steatosis (ie NAFLD) at week 16 were excluded from the data analysis. ASA and PTX alone or in combination did not improve hepatic steatosis, ballooning, inflammation or fibrosis nor did the treatments affect liver enzymes (aminotransferases and alkaline phosphatase) or circulating lipids. Liver triglyceride levels, relative liver weight and hepatic mRNA expression of monocyte chemoattractant protein 1, interleukin 8 and platelet-derived growth factor b were nominally decreased. Thus, in the current study, treatment with ASA and PTX alone or in combination for 8weeks did not ameliorate NASH or hepatic fibrosis in guinea pigs.

Ad libitum high fat diet consumption during adolescence and adulthood fails to impact the affective properties of cocaine in male Sprague-Dawley rats.

Recent research from our laboratories has demonstrated that long-term and ad libitum high fat diet (HFD) consumption during adolescence and adulthood increases the intravenous self-administration (IVSA) of cocaine in adult male Sprague-Dawley rats. One possible interpretation of these findings is that this dietary history influences the affective properties of cocaine, that is, cocaine's rewarding and/or aversive effects. In this context, our research and others suggest that the overall affective response to a drug, and its potential for use and abuse, reflects a balance between these properties in which the rewarding effects of a drug maintain its use and the aversive effects limit it. Accordingly, long-term HFD consumption might increase the rewarding effects of cocaine and/or decrease its aversive effects, resulting in greater IVSA. To examine this possibility, male Sprague-Dawley rats were maintained on either a HFD (n = 32) or chow diet (n = 32) beginning on postnatal day (PND) 21 and underwent combined cocaine-induced place preference and taste avoidance conditioning from PNDs 78-102. Under these conditions, cocaine (18 and 32 mg/kg, intraperitoneally [IP]), but not vehicle, was effective in inducing both a place preference and a taste avoidance; however, HFD- and chow-fed animals did not differ on either of these behavioral indices. These data suggest that the ability of ad libitum HFD consumption during adolescence to increase cocaine IVSA is not likely due to changes in the affective properties of cocaine. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

1769-P: Microglial Activation and Inactivation via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) Alters Peripheral Glucose Homeostasis

Obesity, a condition affecting more than one in three American adults, is associated with hypothalamic neuronal injury, inflammation, and gliosis- a process characterized by accumulation of activated microglia. Experimental evidence from our lab suggests that microglial activation and inflammatory signaling play a functional role in obesity pathogenesis and, furthermore, that this inflammation, or the blockade of such, may affect whole-body glucose homeostasis. Microglial-specific NF-kB knock-out mice are protected from diet-induced obesity, but, despite their lean phenotype, develop glucose intolerance similar to wild type controls. We therefore used stimulatory (hM3Dq) and inhibitory (hM4Di) DREADDs to test the hypothesis that microglial activation or inactivation acutely affects systemic glucose homeostasis. We generated novel mouse models that selectively express the hM3Dq- and hM4Di-DREADD transgenes in microglia. Expression was verified via qRT-PCR, flow cytometry, and IHC. Cohorts of mice and littermate controls were maintained on either normal chow diet or high-fat diet (HFD). Glucose tolerance tests were performed on mice following administration of the DREADD receptor agonist clozapine-N-oxide (CNO). Acute microglial activation via CNO administration in hM3Dq-DREADD animals improved glucose tolerance under both chow and HFD conditions. Acute CNO-induced microglial inactivation in hM4Di-DREADD animals increased fasting glucose in chow-fed animals and worsened glucose tolerance in HFD-fed animals. These findings provide the first evidence that peripheral glucose homeostasis is affected by microglial activity and inflammatory signaling and can be manipulated on an acute timescale using DREADD technology. Disclosure K.M. Ness: None. J. Douglass: None. M. Valdearcos-Contreras: None. M.D. Dorfman: None. A. Niraula: None. S.K. Koliwad: Consultant; Self; AstraZeneca. Stock/Shareholder; Self; Suggestic, Yes Health. J. Thaler: None. Funding National Institutes of Health (T32DK007247, R01DK119754)

Obesogenic Memory Maintains Adipose Tissue Inflammation and Insulin Resistance.

Background:Obesity is characterized by visceral adipose tissue (AT) inflammation. Immunosuppressive regulatory T cells (Tregs), phagocytic M2-like macrophages, and innate lymphoid cells type 2 (ILC2) control lean AT inflammation to maintain systemic insulin sensitivity, while the loss of these cells in obesity leads to AT inflammation and insulin resistance (IR).Objective:The objective of this study was to determine if weight loss following obesity would correct AT inflammation and systemic metabolism.Results:After six months of high fat diet (HFD) in male C57/Bl6 mice, flow analyses of epidydimal AT stromal vascular fraction (SVF) revealed depleted Tregs by 50%, doubling of CD8+ T cells, tripling of pro-inflammatory M1-like macrophages, and an 80% drop in ILC2 cells associated with changes in pro-inflammatory adipocyte and macrophage gene expression. Despite normalization of body weight, fat, and adipocyte size, mice ingesting 3 months of high-fat diet (HFD) followed by 3 months of chow-diet remained more insulin resistant and glucose intolerant than chow-fed animals. Adipocytes, AT Tregs, CD8+ T cells, ILC2 cells, and M1-like macrophages all failed to normalize with weight loss.Conclusions:Persistent AT inflammation contributes to the maintenance of IR despite body weight and fat normalization in previously obese mice. These findings highlight the importance of obesity prevention to avoid the consequences of “obesogenic memory.”

Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection

A Westernized-diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice. We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen in the gut, and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low-fat (LF) or chow-fed animals. Prior to Listeria infection short-term consumption of HF diet elevated faecal levels of Firmicutes. During active infection with L. monocytogenes microbiota changes were further exacerbated but host inflammatory responses were significantly down-regulated relative to Listeria-infected LF or chow-fed groups, suggestive of a profound tampering of the host response influenced by infection in the context of a HF diet. Overall the results indicate that short-term consumption of a Westernized-diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.

ApoF knockdown increases cholesteryl ester transfer to LDL and impairs cholesterol clearance in fat-fed hamsters

Cholesteryl ester transfer protein (CETP) regulates intravascular lipoprotein metabolism. In vitro studies indicate that ApoF alters CETP function by inhibiting its activity with LDL. To explore in vivo the complexities driving ApoF's effects on CETP, we developed a siRNA-based hamster model of ApoF knockdown. In both male and female hamsters on chow- or fat-fed diets, we measured lipoprotein levels and composition, determined CETP-mediated transfer of cholesteryl esters (CEs) between lipoproteins, and quantified reverse cholesterol transport (RCT). We found that apoF knockdown in chow-fed hamsters had no effect on lipoprotein levels or composition, but these ApoF-deficient lipoproteins supported 50-100% higher LDL CETP activity in vitro. ApoF knockdown in fat-fed male hamsters created a phenotype in which endogenous CETP-mediated CE transfer from HDL to LDL increased up to 2-fold, LDL cholesterol increased 40%, HDL declined 25%, LDL and HDL lipid compositions were altered, and hepatic LDLR gene expression was decreased. Diet-induced hypercholesterolemia obscured this phenotype on occasion. In fat-fed female hamsters, ApoF knockdown caused similar but smaller changes in plasma CETP activity and LDL cholesterol. Notably, ApoF knockdown impaired HDL RCT in fat-fed hamsters but increased sterol excretion in chow-fed animals. These in vivo data validate in vitro findings that ApoF regulates lipid transfer to LDL. The consequences of ApoF knockdown on lipoproteins and sterol excretion depend on the underlying lipid status. By minimizing the transfer of HDL-derived CE to LDL, ApoF helps control LDL cholesterol levels when LDL clearance mechanisms are limiting.

Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity

Aims/hypothesisIn the context of diabetes, the health benefit of antioxidant treatment has been widely debated. In this study, we investigated the effect of antioxidant treatment during the development of insulin resistance and hyperphagia in obesity and partial lipodystrophy.MethodsWe studied the role of antioxidants in the regulation of insulin resistance using the tamoxifen-inducible fat-specific insulin receptor knockout (iFIRKO) mouse model, which allowed us to analyse the antioxidant’s effect in a time-resolved manner. In addition, leptin-deficient ob/ob mice were used as a hyperphagic, chronically obese and diabetic mouse model to validate the beneficial effect of antioxidants on metabolism.ResultsAcute induction of insulin receptor knockout in adipocytes changed the substrate preference to fat before induction of a diabetic phenotype including hyperinsulinaemia and hyperglycaemia. In healthy chow-fed animals as well as in morbidly obese mice, this diabetic phase could be reversed within a few weeks. Furthermore, after the induction of insulin receptor knockout in mature adipocytes, iFIRKO mice were protected from subsequent obesity development through high-fat diet feeding. By genetic tracing we show that the persistent fat mass loss in mice after insulin receptor knockout in adipocytes is not caused by the depletion of adipocytes. Treatment of iFIRKO mice with antioxidants postponed and reduced hyperglycaemia by increasing insulin sensitivity. In ob/ob mice, antioxidants rescued both hyperglycaemia and hyperphagia.Conclusions/interpretationWe conclude that fat mass reduction through insulin resistance in adipocytes is not reversible. Furthermore, it seems unlikely that adipocytes undergo apoptosis during the process of extreme lipolysis, as a consequence of insulin resistance. Antioxidants have a beneficial health effect not only during the acute phase of diabetes development, but also in a temporary fashion once chronic obesity and diabetes have been established.

Phenotypic effects of dietary stress in combination with a respiratory chain bypass in mice.

The alternative oxidase (AOX) from Ciona intestinalis was previously shown to be expressible in mice and to cause no physiological disturbance under unstressed conditions. Because AOX is known to become activated under some metabolic stress conditions, resulting in altered energy balance, we studied its effects in mice subjected to dietary stress. Wild‐type mice (Mus musculus, strain C57BL/6JOlaHsd) fed a high‐fat or ketogenic (high‐fat, low‐carbohydrate) diet show weight gain with increased fat mass, as well as loss of performance, compared with chow‐fed animals. Unexpectedly, AOX‐expressing mice fed on these metabolically stressful, fat‐rich diets showed almost indistinguishable patterns of weight gain and altered body composition as control animals. Cardiac performance was impaired to a similar extent by ketogenic diet in AOX mice as in nontransgenic littermates. AOX and control animals fed on ketogenic diet both showed wide variance in weight gain. Analysis of the gut microbiome in stool revealed a strong correlation with diet, rather than with genotype. The microbiome of the most and least obese outliers reared on the ketogenic diet showed no consistent trends compared with animals of normal body weight. We conclude that AOX expression in mice does not modify physiological responses to extreme diets.

Diet-Induced Obesity Disturbs Microglial Immunometabolism in a Time-of-Day Manner.

Background: Disturbance of immunometabolic signaling is a key process involved in the progression of obesity. Microglia—the resident immune cells in the brain, initiate local immune responses. It is known that hypercaloric diets lead to microglial activation. Previously, we observed that hypothalamic microglial cells from mice fed high-fat diet (HFD) lose their day/night rhythm and are constantly activated. However, little is known about daily rhythmicity in microglial circadian, immune and metabolic functions, either in lean or obese conditions. Therefore, we hypothesized that HFD disturbs microglial immunometabolism in a day/night-dependent manner.Methods: Obesity was induced in Wistar rats by feeding them HFD ad libitum for the duration of 8 weeks. Microglia were isolated from HFD- and chow-fed control animals at six time points during 24 h [every 4 h starting 2 h after lights on, i.e., Zeitgeber Time 2 (ZT2)]. Gene expression was evaluated using quantitative RT-PCR. JTK_Cycle software was used to estimate daily rhythmicity. Statistical analysis was performed with two-way ANOVA test.Results: Consumption of the obesogenic diet resulted in a 40 g significantly higher body weight gain in week 8, compared to chow diet (p < 0.0001), associated with increased adiposity. We observed significant rhythmicity of circadian clock genes in microglia under chow conditions, which was partially lost in diet-induced obesity (DIO). Microglial immune gene expression also showed time-of-day differences, which were disrupted in HFD-fed animals. Microglia responded to the obesogenic conditions by a shift of substrate utilization with decreased glutamate and glucose metabolism in the active period of the animals, and an overall increase of lipid metabolism, as indicated by gene expression evaluation. Additionally, data on mitochondria bioenergetics and dynamics suggested an increased energy production in microglia during the inactive period on HFD. Finally, evaluation of monocyte functional gene expression showed small or absent effect of HFD on peripheral myeloid cells, suggesting a cell-specific microglial inflammatory response in DIO.Conclusions: An obesogenic diet affects microglial immunometabolism in a time-of-day dependent manner. Given the central role of the brain in energy metabolism, a better knowledge of daily rhythms in microglial immunometabolism could lead to a better understanding of the pathogenesis of obesity.

Ablating The Rab-GTPase Activating Protein TBC1D1 Predisposes Rats To High Fat Diet-Induced Cardiomyopathy

While the pathogenesis of diabetic cardiomyopathy is poorly understood, impaired insulin signalling within the heart is thought to contribute to the development of this pathology. TBC1D1, a Rab-GTPase activating protein, is involved in glucose homeostasis and substrate metabolism within skeletal muscle, however, the function of TBC1D1 within the heart is relatively unknown. PURPOSE: To examine the role of TBC1D1 in overall cardiac morphology and substrate utilization using a rat knock-out (KO) model. METHODS: 7 weeks of high-fat feeding was provided as a metabolic perturbation to further elucidate the interaction between TBC1D1 and diet-induced cardiac contractile function. Experiments were conducted at 12 weeks of age, with the exception of cardiomyocyte isolation, which was conducted at 7 weeks of age. Animals were anaesthetized with 2.5% isoflurane before assessments of cardiac function, or surgical removal of the left ventricle. The left ventricle was immediately utilized for bioenergetics assessments, fixed for histology or immediately frozen in liquid nitrogen for Western blotting. RESULTS: In chow-fed animals, TBC1D1 ablation increased plasma membrane GLUT4 content and glucose uptake, as well as plasma membrane FABPpm content and palmitate oxidation, consistent with activating cellular trafficking through the ablation of TBC1D1. While echocardiograms suggested indices of cardiac function were unaltered in chow fed KO animals, when challenged with a 7 week high-fat diet, TBC1D1 KO rats displayed a 4-fold increase in fibrosis in association with attenuated stroke volume, cardiac output and end diastolic volume, suggesting a predisposition to diet-induced cardiomyopathy. Mitochondrial respiratory capacity and substrate sensitivity to pyruvate and ADP were not altered by diet or TBC1D1 ablation, nor were rates of mitochondrial hydrogen peroxide emission, or markers of oxidative stress. CONCLUSIONS: Altogether, ablation of TBC1D1 improves indices of cardiovascular function in rats fed a standard diet, but increases fibrosis and compromises indices of cardiac function in rats consuming a high-fat diet. Therefore, TBC1D1 may exert cardioprotective effects in the development of diabetic cardiomyopathy. This research is supported by NSERC funding.

Nrf2 Dysfunction in the RVLM is Associated with Obesity‐Induced Sympathoexcitation

Sympathetic overactivity contributes to the pathogenesis of obesity‐induced hypertension. The rostral ventrolateral medulla (RVLM) is an important brainstem region that regulates sympathetic nerve activity (SNA) to peripheral tissues. Oxidative stress and neuroinflammation in the RVLM are believed to be important contributors for augmented SNA in obesity, however, the molecular mechanisms that initiate and sustain the pro‐inflammatory milieu in the RVLM during obesity are not clear. Nuclear factor erythroid 2‐related factor (Nrf2) is a master transcriptional factor that regulates the expression of anti‐oxidant and anti‐inflammatory genes. We hypothesized that obesity impairs Nrf2 signaling to increase oxidative stress in the RVLM leading to increases in SNA and hypertension. To test this, 2 months‐old male C57BL/6J mice were fed a high‐fat diet (HFD; 60% fat) or normal chow diet (10% fat for 16 weeks and were instrumented with radiotelemeters in the last 2 weeks for measuring blood pressure and SNA. Heart rate variability and depressor response to ganglionic blockade were used to assess SNA. At the end of 16 weeks, animals were sacrificed, brainstem was isolated and sectioned. Superoxide levels were assessed in the frozen sections by dihydroethidium staining (DHE). RVLM was microdissected for gene expression analysis. Data were analyzed by students t‐test and p&lt;0.05 was considered statistically significant. HFD feeding resulted in a mild but significant increase in mean arterial pressure (103.7 ± 0.9 vs 108.6 ± 0.73 mmHg, normal chow diet vs HFD; P&lt;0.05; n=4) and systolic blood pressure (121.2 ± 1.2 vs 129 ± 0.67 mmHg, p&lt;0.05; n=4) compared to normal chow‐fed animals. Ganglionic blockade using hexamethonium showed greater depressor response (−0.21 ± 2.9 vs 13.13 ± 1.3 mmHg, normal chow diet vs HFD; p&lt;0.05; n=4) to HFD feeding. Furthermore, low‐frequency blood pressure variability and the ratio of low‐frequency to high‐frequency heart rate variability in HFD mice were significantly higher than in the control mice suggesting HFD upregulated SNA. In addition, obesity‐induced sympathoexcitation was associated with increased superoxide levels (DHE staining) and decreased mRNA levels of Nrf2 (1.05 ± 0.16 vs 0.48 ± 0.06 fold, normal diet vs HFD; p&lt;0.05) and its target genes (e.g. NQO1 1.08 ± 0.22 vs 0.51 ± 0.08 fold, p&lt;0.05 and SOD2 1.03 ± 0.13 vs 0.48 ± 0.05 fold; p&lt;0.05) in the RVLM of HFD mice. Collectively, our results show for the first time, evidence for obesity‐associated Nrf2 dysfunction in the RVLM. Future studies will investigate the mechanistic role of Nrf2 signaling in the RVLM in obesity‐induced sympathoexcitation.Support or Funding InformationRAC fund, CVHS, Oklahoma State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.