A high-fat diet supplemented with medium-chain triglycerides ameliorates hepatic steatosis by reducing ceramide and diacylglycerol accumulation in mice.

Non-alcoholic fatty liver disease (NAFLD) is projected to be the most common chronic liver disease worldwide and is closely linked to obesity, insulin resistance and type 2 diabetes. Currently, no pharmacological treatments are available to treat NAFLD, and lifestyle modification, including dietary interventions, is the only remedy. Therefore, we conducted a study to determine whether supplementation with medium-chain triglycerides (MCTs), containing a mixture of C8 and C10 (60/40), attenuates NAFLD in obese and insulin-resistant mice. To achieve that, we fed C57BL/6 male mice a high-fat diet (HFD) for 12 weeks to induce obesity and hepatic steatosis, after which obese mice were assigned randomly either to remain on the HFD or to transition to an HFD supplemented with MCTs (HFD + MCTs) or a low-fat diet (LFD) for 6 weeks as another dietary intervention model. Another group of mice was kept on an LFD throughout the study and used as a lean control group. Obese mice that transitioned to HFD + MCTs exhibited improvement in glucose and insulin tolerance tests, and the latter improvement was independent of changes in adiposity when compared with HFD-fed mice. Additionally, supplementation with MCTs significantly reduced hepatic steatosis, improved liver enzymes and decreased hepatic expression of inflammation-related genes to levels similar to those observed in obese mice transitioned to an LFD. Importantly, HFD + MCTs markedly lowered hepatic ceramide and diacylglycerol content and prevented protein kinase C-ε translocation to the plasma membrane. Our study demonstrated that supplementation with MCTs formulated mainly from C8 and C10 effectively ameliorated NAFLD in obese mice.

Sucrose-Enriched and Carbohydrate-Free High-Fat Diets Distinctly Affect Substrate Metabolism in Oxidative and Glycolytic Muscles of Rats.

Skeletal muscle substrate preference for fuel is largely influenced by dietary macronutrient availability. The abundance of dietary carbohydrates promotes the utilization of glucose as a substrate for energy production, whereas an abundant dietary fat supply elevates rates of fatty acid (FA) oxidation. The objective of this study was to determine whether an obesogenic, high-fat, sucrose-enriched (HFS) diet or a carbohydrate-free ketogenic diet (KD) exert distinct effects on fat, glucose, and ketone metabolism in oxidative and glycolytic skeletal muscles. Male Wistar rats were fed either a HFS diet or a KD for 16 weeks. Subsequently, the soleus (Sol), extensor digitorum longus (EDL), and epitrochlearis (Epit) muscles were extracted to measure palmitate oxidation, insulin-stimulated glucose metabolism, and markers of mitochondrial biogenesis, ketolytic capacity, and cataplerotic and anaplerotic machinery. Sol, EDL, and Epit muscles from KD-fed rats preserved their ability to elevate glycogen synthesis and lactate production in response to insulin, whereas all muscles from rats fed with the HFS diet displayed blunted responses to insulin. The maintenance of metabolic flexibility with the KD was accompanied by muscle-fiber-type-specific adaptive responses. This was characterized by the Sol muscle in KD-fed rats enhancing mitochondrial biogenesis and ketolytic capacity without elevating its rates of FA oxidation in comparison with that in HFS feeding. Conversely, in the Epit muscle, rates of FA oxidation were increased, whereas the ketolytic capacity was markedly reduced by the KD in comparison with that by HFS feeding. In the EDL muscle, the KD also increased rates of FA oxidation, although it did so without altering its ketolytic capacity when compared to HFS feeding. In conclusion, even though obesogenic and ketogenic diets have elevated contents of fat and alter whole-body substrate partitioning, these two dietary interventions are associated with opposite outcomes with respect to skeletal muscle metabolic flexibility.

The ketogenic diet promotes triacylglycerol recycling in white adipose tissue and uncoupled fat oxidation in brown adipose tissue, but does not reduce adiposity in rats.

The purpose of this study was to determine whether the weight-reducing and fat burning effects of the ketogenic diet (KD) could be attributed to alterations in the energy dissipating pathways of brown adipose tissue (BAT) uncoupled oxidation, and white adipose tissue (WAT) browning and triacylglycerol (TAG) recycling. To investigate this, male Wistar rats were fed one of the following three diets for either 8 or 16 weeks: a standard chow (SC), a high-fat, sucrose-enriched (HFS) obesogenic diet, or a KD. At the end of the intervention, subcutaneous inguinal (Sc Ing) and epididymal (Epid) fat, and interscapular and aortic BAT (iBAT and aBAT, respectively) were extracted. These tissues were used for the analysis of proteins involved in WAT browning and thermogenesis. Isolated adipocytes from WAT were assayed for basal and isoproterenol (Iso)-stimulated lipolysis and basal and insulin-stimulated lipogenesis, and BAT adipocytes were assayed for the determination of coupled and uncoupled glucose and palmitate oxidation. Adiposity similarly increased in HFS- and KD-fed rats at weeks 8 and 16. However, in HFS-fed animals insulin-stimulated lipogenesis and Iso-stimulated lipolysis were impaired in WAT adipocytes, whereas in KD-fed animals these pathways remained intact. The KD also significantly elevated WAT glycerol kinase levels, and favored TAG recycling under conditions of enhanced lipolysis. In BAT, the KD significantly increased uncoupling protein-1 levels and uncoupled fat oxidation. In summary, the KD preserved insulin sensitivity and lipolytic capacity in WAT and also upregulated energy-dissipating pathways in BAT, but it was not sufficient to prevent an increase in adiposity.

Insulin-resistant female rat skeletal muscles display diacylglycerol-mediated protein kinase C activation and inflammation without ceramide accumulation.

This study investigated the role of diacylglycerol (DAG)-mediated protein kinase C (PKC) activation, ceramide accumulation and inflammation in insulin-resistant female oxidative and glycolytic skeletal muscles induced by an obesogenic high-fat sucrose-enriched (HFS) diet. The HFS diet impaired insulin-stimulated AKTThr308 phosphorylation and glycogen synthesis, whereas rates of fatty acid oxidation and basal lactate production were significantly elevated in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. Insulin resistance was accompanied by increases in triacylglycerol (TAG) and DAG contents in Sol and EDL, whereas in Epit muscles only TAG content and markers of inflammation were associated with HFS diet-induced insulin resistance. Analysis of membrane-bound/cytoplasmic PKC fractions revealed that the HFS diet promoted activation/translocation of PKCδ and θ isoforms in Sol, EDL and Epit muscles. However, none of these muscles displayed alterations in ceramide content in response to HFS feeding. This could be explained by a significant increase in Dgat2 mRNA expression in Sol, EDL and Epit muscles, which likely diverted most of the intramyocellular acyl-CoAs toward TAG synthesis instead of ceramides. Overall, this study helps elucidate the molecular mechanisms underlying insulin resistance caused by diet-induced obesity in female skeletal muscles with distinct fibre type compositions. KEY POINTS: Feeding female Wistar rats a high-fat sucrose-enriched diet (HFS) led to diacylglycerol (DAG)-induced PKC activation and insulin resistance in oxidative and glycolytic skeletal muscles. HFS diet-induced toll-like receptor 4 (Tlr4) expression did not lead to increased ceramide content in female skeletal muscles. In highly glycolytic female muscles, elevated TAG content and markers of inflammation underlay HFS diet-induced insulin resistance. The HFS diet suppressed glucose oxidation and increased lactate production in oxidative and glycolytic female muscles. Increased Dgat2 mRNA expression likely diverted most of the intramyocellular acyl-CoAs toward TAG synthesis and prevented ceramide formation in skeletal muscles of HFS-fed female rats.

An obesogenic diet impairs uncoupled substrate oxidation and promotes whitening of the brown adipose tissue in rats.

Brown adipose tissue (BAT) is rich in mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass and its UCP1 content increase in rodents chronically fed a high-fat sucrose-enriched (HFS) diet, marked expansion of adiposity still occurs in these animals, suggesting insufficient BAT-mediated HFS diet-induced thermogenesis. Thus, the objective of this study was to investigate the metabolic and molecular mechanisms that regulate BAT thermogenesis in HFS-induced obesity. To accomplish this, rats were fed either a standard chow or HFS diet for 8 weeks. Subsequently, glucose and fatty acid metabolism and the molecular mechanisms underlying these processes were assessed in freshly isolated primary BAT adipocytes. Despite increasing BAT mass and its UCP1 content, the HFS diet reduced uncoupled glucose and palmitate oxidation in BAT adipocytes. It also markedly diminished tyrosine hydroxylase content and lipolysis in these cells. Conversely, glucose uptake, lactate production, glycerol incorporation into lipids, palmitate incorporation into triacylglycerol (TAG), phosphoenolpyruvate carboxykinase and glycerol kinase levels, and lipoprotein lipase and cluster of differentiation 36 gene expression were increased. In summary, a HFS diet enhanced glyceroneogenesis and shifted BAT metabolism toward TAG synthesis by impairing UCP1-mediated substrate oxidation and by enhancing fatty acid esterification in intact brown adipocytes. These adaptive metabolic responses to chronic HFS feeding attenuated BAT thermogenic capacity and favoured the development of obesity. KEY POINTS: Despite increasing brown adipose tissue (BAT) mass and levels of thermogenic proteins such as peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1B and uncoupling protein 1 (UCP1), an obesogenic high-fat sucrose-enriched (HFS) diet attenuated uncoupled glucose and fatty acid oxidation in brown adipocytes. Brown adipocytes diverted glycerol and fatty acids toward triacylglycerol (TAG) synthesis by elevating the cellular machinery that promotes fatty acid uptake along with phosphoenolpyruvate carboxykinase and glycerol kinase levels. The HFS diet increased glucose uptake that supported lactate production and provided substrate for glyceroneogenesis and TAG synthesis in brown adipocytes. Impaired UCP-1-mediated thermogenic capacity and enhanced TAG storage in BAT adipocytes were consistent with reduced adipose triglyceride lipase and tyrosine hydroxylase levels in HFS diet-fed animals.

Obesogenic versus ketogenic diets in the regulation of the renin-angiotensin system in rat white and brown adipose tissues.

OBJECTIVE: The ketogenic diet (KD) has been reported to reverse metabolic dysfunction in obesity. However, it remains unknown how the KD affects the balance between the classical and counterregulatory renin-angiotensin system (RAS) arms in adipose tissue, which carries important implications for metabolic function in adipocytes. The aim of this study was to compare the effects of the obesogenic diet and the KD on RAS balance in white and brown fat. METHODS: Nine male Wistar rats were fed a standard chow (SC), 11 fed a high-fat sucrose-enriched (HFS) obesogenic diet, and 12 a KD. At the end of the 8-wk feeding period, subcutaneous inguinal (Sc Ing), epididymal (Epid), and interscapular brown adipose tissue (iBAT) fat depots were extracted and subsequently used for the measurement of RAS proteins and MasR gene expression. RESULTS: In SC-fed rats, the Sc Ing fat displayed the highest levels of angiotensin-converting enzyme (ACE)1, but very low levels of angiotensin II types 1 and 2 receptors (AT1R and AT2R) and ACE2. Conversely, the highest levels of ACE2, AT1R, and AT2R were found in iBAT. The HFS diet increased AT1R protein in Sc Ing fat and iBAT, whereas the KD maintained low AT1R levels in these fat depots. However, in Sc Ing and Epid fat depots, the KD elevated AT2R levels and significantly reduced Epid ACE1 levels. CONCLUSION: Despite fat depot-specific differences in RAS components, the obesogenic diet promoted the classical RAS arm, whereas the KD attenuated it and enhanced the counterregulatory arm.

The ketogenic diet prevents steatosis and insulin resistance by reducing lipogenesis, diacylglycerol accumulation and protein kinase C activity in male rat liver.

Obesity-associated insulin resistance plays a major role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The accumulation of diacylglycerol (DAG), ceramides and inflammation are key factors that cause NAFLD. In recent years, the ketogenic diet (KD) has emerged as an effective non-pharmacological intervention for the treatment of NAFLD and other obesity-related metabolic disorders. What remains undetermined is how the KD affects DAG and ceramide content and insulin sensitivity in the liver. Thus, this research was designed to assess these variables, as well as glucose and fat metabolism and markers of inflammation in livers of rats exposed for 8 weeks to one of the following diets: standard chow (SC), obesogenic high-fat, sucrose-enriched diet (HFS) or a KD. Despite having a higher fat content than the HFS diet, the KD did not cause steatosis and preserved hepatic insulin signalling. The KD reduced DAG content and protein kinase C-ε activity, but markedly increased liver ceramide content. However, whereas the KD increased ceramide synthase 2 (CerS2) expression, it suppressed CerS6 expression, an effect that promoted the production of beneficial very long-chain ceramides instead of harmful long-chain ceramides. The KD also enhanced the liver expression of key genes involved in mitochondrial biogenesis and fatty acid oxidation (Pgc-1α and Fgf21), suppressed inflammatory genes (Tnfα, Nf-kb, Tlr4 and Il6), and shifted substrate away from de novo lipogenesis. Thus, through multiple mechanisms the KD exerted anti-steatogenic and insulin-sensitizing effects in the liver, which supports the use of this dietary intervention to treat NAFLD. KEY POINTS: The accumulation of diacylglycerol (DAG), ceramides and inflammation are key factors that cause insulin resistance and non-alcoholic fatty liver disease (NAFLD). This study provides evidence that a ketogenic diet (KD) rich in fat and devoid of carbohydrate reduced DAG content and preserved insulin signalling in the liver. The KD shifted metabolism away from lipogenesis by enhancing genes involved in mitochondrial biogenesis and fatty acid oxidations in the liver. The KD also promoted the production of beneficial very long-chain ceramides instead of potentially harmful long-chain ceramides. Through multiple mechanisms, the KD exerted anti-steatogenic and insulin-sensitizing effects in the liver, which supports the use of this dietary intervention to treat NAFLD.

Obesogenic and Ketogenic Diets Distinctly Regulate the SARS-CoV-2 Entry Proteins ACE2 and TMPRSS2 and the Renin-Angiotensin System in Rat Lung and Heart Tissues.

BACKGROUND: Obesity increases the severity of SARS-CoV-2 outcomes. Thus, this study tested whether obesogenic and ketogenic diets distinctly affect SARS-CoV-2 entry proteins and the renin-angiotensin system (RAS) in rat pulmonary and cardiac tissues. METHODS: Male Sprague-Dawley rats were fed either standard chow (SC), a high-fat sucrose-enriched diet (HFS), or a ketogenic diet (KD) for 16 weeks. Afterwards, levels of angiotensin converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), RAS components, and inflammatory genes were measured in the lungs and hearts of these animals. RESULTS: In the lungs, HFS elevated ACE2 and TMPRSS2 levels relative to SC diet, whereas the KD lowered the levels of these proteins and the gene expressions of toll-like receptor 4 and interleukin-6 receptor relative to HFS. The diets did not alter ACE2 and TMPRSS2 in the heart, although ACE2 was more abundant in heart than lung tissues. CONCLUSION: Diet-induced obesity increased the levels of viral entry proteins in the lungs, providing a mechanism whereby SARS-CoV-2 infectivity can be enhanced in obese individuals. Conversely, by maintaining low levels of ACE2 and TMPRSS2 and by exerting an anti-inflammatory effect, the KD can potentially attenuate the severity of infection and migration of SARS-CoV-2 to other ACE2-expressing tissues.

Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats.

This study investigated whether oxidative and glycolytic rat skeletal muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.

Cardiac Autophagy Deficiency Attenuates ANP Production and Disrupts Myocardial-Adipose Cross Talk, Leading to Increased Fat Accumulation and Metabolic Dysfunction.

Increased myocardial autophagy has been established as an important stress-induced cardioprotective response. Three weeks after generating cardiomyocyte-specific autophagy deficiency, via inducible deletion of autophagy-related protein 7 (Atg7), we found that these mice (AKO) had increased body weight and fat mass without altered food intake. Glucose and insulin tolerance tests indicated reduced insulin sensitivity in AKO mice. Metabolic cage analysis showed reduced ambulatory activity and oxygen consumption with a trend of elevated respiratory exchange ratio in AKO mice. Direct analysis of metabolism in subcutaneous and visceral adipocytes showed increased glucose oxidation and reduced ATGL expression and HSL phosphorylation with no change in lipid synthesis or fatty acid oxidation. Importantly, we found AKO mice had reduced myocardial and circulating levels of atrial natriuretic peptide (ANP), an established mediator of myocardial-adipose cross talk. When normal ANP levels were restored to AKO mice with use of osmotic pump, the metabolic dysfunction evident in AKO mice was corrected. We conclude that cardiac autophagy deficiency alters myocardial-adipose cross talk via decreased ANP levels with adverse metabolic consequences.

Effects of the adiponectin mimetic compound ALY688 on glucose and fat metabolism in visceral and subcutaneous rat adipocytes.

Adiponectin regulates white adipose tissue (WAT) metabolism and promotes insulin-sensitizing and anti-atherosclerotic effects in vivo. In this context, small molecule adiponectin receptor agonists have become of great therapeutic value for the treatment of metabolic diseases. Here, we investigated the effects of the adiponectin mimetic compound ALY688 on WAT metabolism. To accomplish this, rat epididymal (Epid) and subcutaneous inguinal (Sc Ing) adipocytes were isolated and incubated with ALY688. Subsequently, several parameters of glucose and fat metabolism were assessed. ALY688 promoted AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, enhanced glucose oxidation, and suppressed fat oxidation in adipocytes from both fat depots. ALY688 did not affect basal and insulin-stimulated rates of glucose uptake, glucose incorporation into lipids, and AKTSer473 and p38 mitogen-activated protein kinase (MAPK) phosphorylations in either Epid or Sc Ing adipocytes. ALY688 did not alter basal lipolysis in Epid and Sc Ing adipocytes, but it enhanced isoproterenol-induced lipolysis in Epid adipocytes. Adiponectin receptor 2 (AdipoR2) mRNA was the prevalent isoform expressed in all adipocytes, and Epid adipocytes displayed significantly higher AdipoR2 mRNA expression than Sc Ing adipocytes. In conclusion, ALY688 can regulate adiposity and affect glycaemic control by altering substrate portioning in the WAT in a fat depot-specific manner.

Low phosphatase activity of LiaS and strong LiaR-DNA affinity explain the unusual LiaS to LiaR in vivo stoichiometry.

BACKGROUND: LiaRS mediates Bacillus subtilis response to cell envelope perturbations. A third protein, LiaF, has an inhibitory role over LiaRS in the absence of stimulus. Together, LiaF and LiaRS form a three-component system characterized by an unusual stoichiometry, a 4:1 ratio between LiaS and LiaR, the significance of which in the signal transduction mechanism of LiaRS is not entirely understood. RESULTS: We measured, for the first time, the kinetics of the phosphorylation-dependent processes of LiaRS, the DNA-binding affinity of LiaR, and characterized the effect of phosphorylation on LiaR oligomerization state. Our study reveals that LiaS is less proficient as a phosphatase. Consequently, unspecific phosphorylation of LiaR by acetyl phosphate may be significant in vivo. This drawback is exacerbated by the strong interaction between LiaR and its own promoter, as it can drive LiaRS into losing grip over its own control in the absence of stimuli. These intrinsic, seemingly 'disadvantageous", attributes of LiaRS are likely overcome by the higher concentration of LiaS over LiaR in vivo, and a pro-phosphatase role of LiaF. CONCLUSIONS: Overall, our study shows that despite the conservative nature of two-component systems, they are, ultimately, tailored to meet specific cell needs by modulating the dynamics of interactions among their components and the kinetics of phosphorylation-mediated processes.

Cold acclimation enhances UCP1 content, lipolysis, and triacylglycerol resynthesis, but not mitochondrial uncoupling and fat oxidation, in rat white adipocytes.

The objective of this study was to investigate whether cold-induced browning of the subcutaneous (Sc) inguinal (Ing) white adipose tissue (WAT) increases the capacity of this tissue to oxidize fatty acids through uncoupling protein 1 (UCP1)-mediated thermogenesis. To accomplish that, rats were acclimated to cold (4°C for 7 days). Subsequently, interscapular and aortic brown adipose tissues (iBAT and aBAT, respectively), epididymal (Epid), and Sc Ing WAT were used for adipocyte isolation. In BAT adipocytes, cold acclimation increased UCP1 content and palmitate oxidation either in the absence or presence of oligomycin, whereas in Sc Ing adipocytes glucose and palmitate oxidation were not affected, although multilocular adipocytes were formed and UCP1 content increased upon cold acclimation in the WAT. Furthermore, isoproterenol-stimulated cold Sc Ing adipocytes exhibited significantly lower rates of palmitate oxidation than control cells when exposed to oligomycin. These findings provide evidence that, despite increasing UCP1 levels, cold acclimation essentially reduced mitochondrial uncoupling-mediated fat oxidation in Sc Ing adipocytes. Conversely, glycerol kinase and phosphoenolpyruvate carboxykinase levels, isoproterenol-induced lipolysis, as well as glycerol and palmitate incorporation into lipids significantly increased in these cells. Therefore, instead of UCP1-mediated mitochondrial uncoupling, cold acclimation increased the capacity of Sc Ing adipocytes to export fatty acids and enhanced key components of the triacylglycerol resynthesis pathway in the Sc Ing WAT.