Carnitine Palmitoyltransferase 1B Research Articles

Metabolic Reprogramming: A Byproduct or a Driver of Cardiomyocyte Proliferation?

Defining mechanisms of cardiomyocyte proliferation should guide the understanding of endogenous cardiac regeneration and could lead to novel treatments for diseases such as myocardial infarction. In the neonatal heart, energy metabolic reprogramming (phenotypic alteration of glucose, fatty acid, and amino acid metabolism) parallels cell cycle arrest of cardiomyocytes. The metabolic reprogramming occurring shortly after birth is associated with alterations in blood oxygen levels, metabolic substrate availability, hemodynamic stress, and hormone release. In the adult heart, myocardial infarction causes metabolic reprogramming but these changes cannot stimulate sufficient cardiomyocyte proliferation to replace those lost by the ischemic injury. Some putative pro-proliferative interventions can induce the metabolic reprogramming. Recent data show that altering the metabolic enzymes PKM2 [pyruvate kinase 2], LDHA [lactate dehydrogenase A], PDK4 [pyruvate dehydrogenase kinase 4], SDH [succinate dehydrogenase], CPT1b [carnitine palmitoyl transferase 1b], or HMGCS2 [3-hydroxy-3-methylglutaryl-CoA synthase 2] is sufficient to partially reverse metabolic reprogramming and promotes adult cardiomyocyte proliferation. How metabolic reprogramming regulates cardiomyocyte proliferation is not clearly defined. The possible mechanisms involve biosynthetic pathways from the glycolysis shunts and the epigenetic regulation induced by metabolic intermediates. Metabolic manipulation could represent a new approach to stimulate cardiac regeneration; however, the efficacy of these manipulations requires optimization, and novel molecular targets need to be defined. In this review, we summarize the features, triggers, and molecular regulatory networks responsible for metabolic reprogramming and discuss the current understanding of metabolic reprogramming as a critical determinant of cardiomyocyte proliferation.

Dietary supplementation with succinic acid improves growth performance and flesh quality of adult Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet

To evaluate the effects of dietary supplementation with succinic acid on growth performance, flesh quality, glucose, and lipid metabolism of Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet (HCD), five iso-nitrogenous and iso-lipidic diets were prepared as follows: HCD (control group) consisting of 55% corn starch and HCD supplemented with 0.5%, 1.0%, 2.0%, and 4.0% succinic acid, respectively. Tilapia with an initial body weight of 204.90±1.23 g randomly assigned to 15 tanks with 3 replicates per group and 10 fish per tank fed for 8 weeks. Increasing dietary succinic acid supplementation resulted in significant second-order polynomial relationship in the weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency rate (PER), viscerosomatic index, condition factor, and contents of muscular crude lipid and glycogen (P＜0.05). The hepatosomatic index, mesenteric fat index, liver glycogen content and crude lipid contents of the whole-body and liver demonstrated significantly linear and second-order polynomial relationship (P＜0.05). Quadratic curve model analysis based on WGR, SGR, PER, and FCR demonstrated that optimal supplementation with succinic acid in the HCD of Nile tilapia ranged from 1.83% to 2.43%. Fish fed with 1.0% succinic acid had higher muscular hardness (P＜0.05), increased the contents of alkali-soluble hydroxyproline in collagen, docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (n-3PUFA) in muscle (P＜0.05), and lower total fatty acid content in muscle (P＜0.05) compared with the control group. Compared to the control group, dietary supplementation with 1.0% succinic acid significantly increased the contents of total bounding amino acid (arginine, histidine, isoleucine, lysine, methionine, alanine, proline), total flavor amino acid content (free aspartic acid), the catalase (CAT) activity and total antioxidant capacity, and the mRNA relative expression levels of CAT, superoxide dismutase (SOD), and nuclearfactor erythroidderived 2-like 2 (Nrf2) in muscle (P＜0.05). Furthermore, succinic acid supplementation significantly up-regulated mRNA relative expression levels of glycolysis genes [hexokinase 2 (HK2), phosphofructokinase, muscle-A (PFKMA) and phosphofructokinase, muscle-B (PFKMB)], a key glycogen synthesis gene [glycogen synthase (GYS)], and lipid catabolism genes [carnitine palmitoyltransferase-1B (CPT1B), hormone sensitive lipase (HSL), and lipoprotein lipase (LPL)] (P<0.05), while down-regulating the mRNA relative expression level of fatty acid synthase (FASN) in muscle (P=0.004). In conclusion, dietary supplementation with 1.83% to 2.43% succinic acid improved muscle quality by increasing muscle antioxidant capacity and hardness, changing muscle amino acid and fatty acid composition, and regulating muscle glucose and lipid metabolism.

2-Methoxyestradiol ameliorates doxorubicin-induced cardiotoxicity by regulating the expression of GLUT4 and CPT-1B in female rats.

The clinical usage of doxorubicin (DOX) is hampered due to cardiomyopathy. Studies reveal that estrogen (E2) modulates DOX-induced cardiotoxicity. Yet, the exact mechanism is unclear. The objective of the current study is to evaluate the influence of E2 and more specifically its metabolite 2-methoxyestradiol (2ME) on cardiac remodeling and the reprogramming of cardiac metabolism in rats subjected to DOX cardiotoxicity. Seventy-two female rats were divided into groups. Cardiotoxicity was induced by administering DOX (2.5 mg/kg three times weekly for 2 weeks). In some groups, the effect of endogenous E2 was abolished by ovariectomy (OVX) or by using the estrogen receptor (ER) blocker Fulvestrant (FULV). The effect of administering exogenous E2 or 2ME in the OVX group was studied. Furthermore, the influence of entacapone (COMT inhibitor) on induced cardiotoxicity was investigated. The evaluated cardiac parameters included ECG, histopathology, cardiac-related enzymes (creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH)), and lipid profile markers (total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL)). The expression levels of key metabolic enzymes (glucose transporter-4 (GLUT4) and carnitine palmitoyltransferase-1B (CPT-1B)) were assessed. Our results displayed that co-treatment of E2 and/or 2ME with DOX significantly reduced DOX-induced cardiomyopathy and enhanced the metabolism of the heart through the maintenance of GLUT4 and CPT-1B enzymes. On the other hand, co-treatment of DOX with OVX, entacapone, or FULV increased the toxic effect of DOX by further reducing these important metabolic enzymes. E2 and 2ME abrogate DOX-induced cardiomyopathy partly through modulation of GLUT 4 and CPT-1B enzymes.

Effects of grape seed procyanidins on the lipid metabolism of growing-finishing pigs based on transcriptomics and metabolomics analyses

This study investigated how lipid metabolism in the longissimus thoracis is influenced by the diet supplemented with grape seed procyanidins (GSPs) in growing-finishing pigs. Forty-eight crossbred pigs were randomly assigned to four groups, each receiving a basal diet, or basal diet added with 150, 200, and 250 mg/kg GSPs. Transcriptomics and metabolomics were employed to explore differential gene and metabolite regulation. The expression of key lipid metabolism-related genes was tested via qRT-PCR, and the lipid and fatty acid composition of the longissimus thoracis were determined. Dietary GSPs at different concentrations upregulated lipoprotein lipase (LPL), which is involved in lipolysis, and significantly increased the mRNA expression levels of carnitine palmitoyltransferase-1B (CPT1B) and cluster of differentiation 36 (CD36), implicated in transmembrane transport of fatty acids. Dietary supplementation of GSPs at 200 or 250 mg/kg markedly reduced total cholesterol and triglyceride content in longissimus thoracis. Dietary GSPs significantly decreased the contents of low-density lipoprotein cholesterol and saturated fatty acids, while increasing unsaturated fatty acids. In conclusion, GSPs may regulate lipid metabolism, reducing cholesterol level, and improving fatty acid composition in the longissimus thoracis of growing-finishing pigs. Our findings provide evidence for the beneficial effects of GSPs as pig feed additives for improving lipid composition.

Effects of Dietary Eucommia ulmoides Leaf Extract Supplementation on Growth Performance, Meat Quality, Antioxidant Capacity, and Lipid Metabolism of Finishing Pigs.

This study aimed to investigate the effects of dietary Eucommia ulmoides leaf extract (ELE) on meat quality, antioxidant capacity, and lipid metabolism in finishing pigs. A total of 240 "Duroc × Landrace × Yorkshire" crossbred pigs with an initial weight of 74.70 ± 0.77 kg were randomly assigned to two groups: control group and 0.2% ELE group, with each group containing 10 replicates of 12 pigs per pen (half barrows and half gilts). The data showed dietary 0.2% ELE supplementation did not affect growth performance but tended to reduce the backfat thickness of the finishing pigs (p = 0.07). ELE diets increased pH value (p < 0.05) and meat color score (p = 0.01) and decreased 45 min L* value (p < 0.05), 24 h L* value (p = 0.01), pressurization loss (p = 0.01), and 24 h drip loss (p < 0.05) in longissimus dorsi (LD) muscle, accompanied by an increased (p < 0.05) proportion of monounsaturated fatty acids (MUFA) and decreased polyunsaturated fatty acids (PUFA) (p = 0.06) and n-6/n-3 PUFA ratio (p = 0.05) compared to controls. In addition, ELE supplementation increased inosine monophosphate (IMP) (p = 0.01), sweet amino acids (AAs) (p < 0.05), and total free AA content (p = 0.05) in LD. Meanwhile, increased activity of glutathione peroxidase (p < 0.05) and superoxide dismutase (p < 0.01) in both serum and LD muscle and decreased malondialdehyde content (p < 0.01) in LD muscle were detected with ELE treatment. Moreover, pigs fed ELE had a higher total protein (p < 0.01), albumin (p < 0.05), and high-density lipoprotein cholesterol (p < 0.05) and a lower total cholesterol (p < 0.01) and triacylglycerols (p = 0.06) in serum. Consistently, significant effects of dietary ELE were observed on the relative mRNA expression of lipid metabolism in the backfat and the LD muscle, respectively. ELE attenuated lipogenic processes in backfat, decreasing the relative expression of acetyl-CoA carboxylase and upregulating the relative expression of adipose triacyl glyceride lipase, carnitine palmitoyl transferase 1B, and fatty acid-binding protein 4 (p < 0.05). ELE also decreased the relative expression of CCAAT/enhancer-binding protein α (p < 0.05), fatty acid translocase (p < 0.05), carnitine palmitoyl transferase 1B (p < 0.01), and adipose triacyl glyceride lipase (p < 0.05) in LD muscle (p < 0.05). More specifically, lipogenesis appeared to be inhibited in both LD muscle and backfat, with the difference being that lipolysis was enhanced in backfat and inhibited in LD muscle. In conclusion, dietary ELE supplementation can potentially enhance carcass traits, sensory quality, and nutritional value of pork without negatively affecting intramuscular fat content. The underlying mechanism for these positive effects may be linked to the alterations in lipid metabolism and increased antioxidant capacity induced by ELE.

Effect of different levels of Lactobacillus added to diets on fat deposition and meat quality of Sunit lambs

In this study, we examined the impact of adding Lactobacillus to the diet on fat distribution and meat quality of Sunit lambs. For 90 days, twenty-four lambs (19.31 ± 0.47 kg) were fed diets that contained 0 (NP), 6 (P1), 12 (P2), or 24 (P3) g of Lactobacillus casei/d. The results suggested that dietary supplementation with Lactobacillus decreased serum triglyceride in Sunit lambs (P < 0.001). The loin muscle area displayed notable increases in the P1 group (P < 0.05). Meanwhile, tail and visceral fat deposition of lambs were reduced when Lactobacillus was added to the diet (P < 0.05). Compared with the NP group, the values of shear force and cooking loss of in the P1 group exhibited a significant reduction, and intramuscular fat content increased significantly (P < 0.05). Additionally, the P1 group showed an increase in polyunsaturated fatty acids and a decrease in saturated fatty acids in the longissimus thoracis and biceps femoris muscles (P < 0.05). The P1 group showed downregulation of protein kinase AMP-activated catalytic subunit alpha 2 (AMPKα2) and carnitine palmitoyltransferase 1B expression in the longissimus thoracis muscle (P < 0.05). However, there was an upregulation of acetyl-CoA carboxylase (ACC), sterol regulatory element binding transcription factor 1 (SREBF1), and fatty acid synthase (FASN) expression (P < 0.05). In conclusion, feeding Sunit lambs 6 g/d of Lactobacillus as a dietary supplement may be a valuable way to improve fat distribution and meat quality. The AMPK/ACC and AMPK/SREBF1/FASN signaling pathways may be involved in this outcome.

MiR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

Hypoxic exercise is an effective intervention for obesity, because it promotes weight loss by regulating fatty acid (FA) metabolism. The regulation of peroxisome proliferator-activated receptor β (PPARβ) by miR-122 may be involved in this process, but the detailed mechanisms are unknown. In order to address this issue, we probed how miR-122 affected the expression of factors associated with FA metabolism in skeletal muscle of obese rats undergoing hypoxic training. By injecting adeno-associated virus 9 containing miR-122 overexpression vector or miR-122 inhibitor into skeletal muscles of rats with a 4-week hypoxic exercise regimen, the miR-122 expression level can be regulated. Body composition and blood lipid levels were analyzed, and PPARβ, carnitine palmitoyltransferase 1b (CPT1b), acetylCoA carboxylase 2 (ACC2), and FA synthase (FAS) mRNA and protein levels were evaluated using quantitative reverse transcription quantitative PCR(RT-qPCR) and Western blot analysis. We found that miR-122 overexpression increased low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels and decreased PPARβ, ACC2, and FAS expression. Conversely, miR-122 inhibition decreased TG level, increased high-density lipoprotein cholesterol (HDL-C) level, and upregulated PPARβ, ACC2, FAS, and CPT1b. These data indicated that the negative regulation of PPARβ by miR-122 promotes FA metabolism by altering the levels of the factors related to FA metabolism in skeletal muscle of obese rat under hypoxic training, thus providing molecular-level insight into the beneficial effects of this intervention.

ABHD5-CPT1B: An Important Way of Regulating Placental Lipid Metabolism in Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is one of the most common metabolic disorders in pregnancy, and a novel association of maternal lipid profile has been suggested to play an important role. However, the molecular mechanism is not clear. Bio-analyzed combined with placental metabonomics and single-cell RNA-sequencing (scRNA-seq) successfully identified a potentially important molecule: α-β hydrolase domain-containing protein 5 (ABHD5). The syncytiotrophoblast (SCT) cell model was adopted as a fusion of BeWo cells in response to forskolin. On this basis, the high glucose-stimulated cell experiment was carried out. 15 women with GDM and 15 normal pregnant women were recruited for validation experiments. ABHD5 was mainly expressed in the trophoblast cells, especially in SCT cells, and significantly decreased in the GDM placenta. After stimulation by high glucose, the expression of ABHD5 was downregulated in a time-dependent manner in BeWo cells treated with forskolin. At the same time, lipid droplets (LDs) were increased in the SCT. LD storage was also increased in the SCT with siABHD5, while it was significantly reduced in SCT cells with high ABHD5 expression. However, this effect could be attenuated by downregulated carnitine palmitoyltransferase 1B (CPT1B). ABHD5-CPT1B is confirmed as an important regulator of placental lipid metabolism.

Exploiting the Metabolic Vulnerability of Leukemic Stem Cells (LSCs) in Inv(16) Acute Myeloid Leukemia (AML) with the Combination of Miristen, a Mir-126 Inhibitor, and Venetoclax

AML with inv(16)(p13q22) expresses the leukemogenic fusion gene CBFB-MYH11 and upregulates microRNA (miR)-126 that supports homeostasis, quiescence, and activity of LSCs. Genetic or pharmacologic depletion of miR-126 via miR-126 knock out (KO) or miRisten (a CpG-anti-miR-126 inhibitor) reduces LSCs in a Cbfb-MYH11 ( CM) knock-in (KI) AML model and inv(16) AML patient-derived xenografts (PDXs), respectively [PMID: 34686664]. However, the antileukemic underpinnings of miR-126 inhibition in inv(16) AML remain to be elucidated. LSCs reportedly rely on fatty acid metabolism/oxidative phosphorylation (OXPHOS) for their bioenergetic metabolic needs. To assess the role of miR-126 in metabolism, we performed metabolomic profiling for Lin-/c-Kit+ cells isolated from CM KI AML (miR-126 high) mice and CM with miR-126 KO mice ( CM/miR-126 Δ /Δ). Our data revealed that CM/miR-126 Δ /Δ had a significant decrease of short, medium as well as long-chain acyl carnitines and fatty acid (2 to 11 fold, p&amp;lt;0.05) compared to CM/miR-126 WT/ WT mice, suggesting that miR-126 deficit decreased fatty acid oxidation (FAO). Moreover, electron microscope imaging demonstrated mitochondrial fusion (i.e., larger and networked mitochondria) in CM Lin-/c-Kit+Sca1+ (LSK) cells vs syngeneic controls. In contrast, mitochondria fragmentation (fission) and decreased FAO/OXPHOS were observed in murine CM with miR-126 KO LSK. Accordingly, FAO and Seahorse assays demonstrated miRisten treatment led to mitochondria fragmentation and reduced FAO/OXPHOS and in human CD34 + CD38 ‒ inv(16) blasts (enriched for LSCs) vs normal CD34 + CD38 ‒ cells. Sprouty Related EVH1 Domain Containing 1 (SPRED1), a downstream target of miR-126, inhibits ERK/CREB activation and in turn increases antiapoptotic protein BCL-2 via phosphorylation and stabilization. In inv(16) AML cells, upregulated miR-126 downregulated SPRED1 and in turn increased ERK/CREB signaling and BCL-2 levels. This increase augments the ubiquitination of DRP1, a protein otherwise involved in mitochondrial fission, via NFE2-related factor 2 (NRF2), and increases Carnitine Palmitoyltransferase 1B (CPT1B), a rate-controlling protein for FAO and OXPHOS. In contrast, miR-126 KO or miR-126 inhibition by miRisten led to SPRED1 upregulation and decreased ERK/CREB signaling and BCL-2 levels, thereby resulting in increased Dynamin-related protein 1 (DRP1) and mitochondrial fission and a decrease in CPT1B, FAO, and OXPHOS. Thus, given the interplay of miR-126 with BCL-2 and its implication in mitochondrial fusion and increase of OXPHOS in inv(16) blasts and LSCs, we hypothesized a synergism between miRisten and the BCL-2 inhibitor venetoclax (VEN). In fact, combination of miRisten/VEN synergistically decreased FAO/OXPHOS, enhanced mitochondria fission and increased apoptosis of murine and human inv(16) blasts and LSCs [combination indices (CIs)&amp;lt;1, (ED50-ED95)]. Thus, we treated CM mice with scrambled oligonucleotides (sODN) (20 mg/kg i.v., daily for 3 weeks), miRisten (20 mg/kg i.v., daily for 3 weeks), VEN (100 mg/kg oral gavage, daily for 2 weeks), or miRisten/VEN (same dosages as single agents). Compared with sODN or single agents, miRisten/VEN significantly reduced bone marrow (BM) leukemic blast infiltration [miRisten/VEN 33.6±3.4% vs miRisten 49.8±2.6% (p=0.001), VEN 56.2±4.7% (p=0.001), or sODN 60.0±2.6% (p&amp;lt;0.0001)] and spleen weights [miRisten/VEN 0.228±0.038g vs miRisten 0.495±0.058g (p=0.001), VEN 0.479±0.06g (p=0.002), or sODN 0.853±0.039g (p&amp;lt;0.0001)] and prolonged survival of CM mice [median survival miRisten/VEN 125d vs miRisten 108d (p=0.005), VEN 106.5d (p=0.039), or sODN 91d (p&amp;lt;0.0001), respectively; secondary transplant median survival: miRisten/VEN 92d vs miRisten 70.5d (p=0.048), VEN 54d (p=0.006), or sODN 45d (p=0.0002)]. Similar results were also observed in inv(16) PDXs with significant reduction of BM huCD45+ leukemic blasts by miRisten/VEN compared to single agents or sODN [miRisten/VEN 13.7±1.9% vs miRisten 27.9±3.4% (p=0.003), VEN 25.3±4.6%(p=0.032), or sODN 68.6±5.3% (p&amp;lt;0.0001)]; survival analyses for treated PDX mice and secondary transplant recipients are in progress. In summary, our study provides the mechanism for combining miRisten with VEN in the treatment of inv(16) AML. While VEN is FDA approved for AML, miRisten has just undergone a successful toxicology evaluation and is underway to the clinic.

Effects of main active components of rosemary on growth performance, meat quality and lipid metabolism in finishing pigs

Rosemary extracts have been widely used as feed additives in recent years. This study aimed to investigate the effects of rosmarinic acid (RA) and ursolic acid (UA), the main active components of rosemary, on growth performance, meat quality and lipid metabolism in finishing pigs. A total of 72 finishing pigs (Landrace; initial age of 150 d) were randomly divided into 3 treatments with 8 replicates of 3 pigs each, and fed a basal diet or diet containing 500 mg/kg of RA or UA. The results showed that dietary supplementation of RA or UA had no significant effect on the growth performance and carcass traits of finishing pigs (P > 0.05). However, both RA and UA significantly increased the triglyceride (TG) level in soleus muscle (P < 0.001). Supplementation of RA increased the expression of genes related to lipogenesis and transport including fatty acid synthase (FAS) (P < 0.001), sterol regulatory element binding protein-1c (SREBP1c) (P < 0.001) and peroxisome proliferator-activated receptor γ (PPARγ) (P < 0.05), while UA increased the expression of fatty acid transport protein 1 (FATP1), a gene related to lipid uptake (P < 0.05). However, RA reduced the expression of adipogenesis-related gene acetyl-coenzyme A carboxylase α (ACCα) (P < 0.01). Characterization of cecal microbiota indicated that RA increased the microbial richness (chao 1, P < 0.001) and diversity (observed species, P < 0.01). Further analysis of the genera revealed that RA increased the relative abundance of Bacteroides and g-UCG-005 (P < 0.05), and UA enriched Prevotella (P < 0.001). Correlation analysis showed that g-UCG-005 was positively correlated with the expression of FAS, carnitine palmitoyl transferase 1B (CPT1B), SREBP1c and PPARγ (P < 0.01). In conclusion, dietary supplementation of RA or UA may increase fat deposition in muscle of finishing pigs by regulating lipid metabolism and gut microbiota.

Probiotics Increase Intramuscular Fat and Improve the Composition of Fatty Acids in Sunit Sheep through the Adenosine 5'-Monophosphate-Activated Protein Kinase (AMPK) Signaling Pathway.

This experiment aims to investigate the impact of probiotic feed on growth performance, carcass traits, plasma lipid biochemical parameters, intramuscular fat and triglyceride content, fatty acid composition, mRNA expression levels of genes related to lipid metabolism, and the activity of the enzyme in Sunit sheep. In this experiment, 12 of 96 randomly selected Sunit sheep were assigned to receive the basic diet or the basic diet supplemented with probiotics. The results showed that supplementation with probiotics significantly increased the loin eye area, and decreased plasma triglycerides and free fatty acids, increasing the content of intramuscular fat and triglycerides in the muscle and improving the composition of the fatty acids. The inclusion of probiotics in the diet reduced the expression of adenosine 5'-monophosphate-activated protein kinase alpha 2 (AMPKα2) mRNA and carnitine palmitoyltransferase 1B (CPT1B) mRNA, while increasing the expression of acetyl-CoA carboxylase alpha (ACCα) mRNA, sterol regulatory element-binding protein-1c (SREBP-1c) mRNA, fatty acid synthase mRNA, and stearoyl-CoA desaturase 1 mRNA. The results of this study indicate that supplementation with probiotics can regulate fat deposition and improves the composition of fatty acids in Sunit sheep through the signaling pathways AMPK-ACC-CPT1B and AMPK-SREBP-1c. This regulatory mechanism leads to an increase in intramuscular fat content, a restructuring of muscle composition of the fatty acids, and an enhancement of the nutritional value of meat. These findings contribute to a better understanding of the food science of animal resources and provide valuable references for the production of meat of higher nutritional value.

Fatty acid β-oxidation and mitochondrial fusion are involved in cardiac microvascular endothelial cell protection induced by glucagon receptor antagonism in diabetic mice.

The role of cardiac microvascular endothelial cells (CMECs) in diabetic cardiomyopathy is not fully understood. We aimed to investigate whether a glucagon receptor (GCGR) monoclonal antibody (mAb) ameliorated diabetic cardiomyopathy and clarify whether and how CMECs participated in the process. The db/db mice were treated with GCGR mAb or immunoglobulin G (as control) for 4 weeks. Echocardiography was performed to evaluate cardiac function. Immunofluorescent staining was used to determine microvascular density. The proteomic signature in isolated primary CMECs was analyzed by using tandem mass tag-based quantitative proteomic analysis. Some target proteins were verified by using western blot. Compared with db/m mice, cardiac microvascular density and left ventricular diastolic function were significantly reduced in db/db mice, and this reduction was attenuated by GCGR mAb treatment. A total of 199 differentially expressed proteins were upregulated in db/db mice versus db/m mice and downregulated in GCGR mAb-treated db/db mice versus db/db mice. The enrichment analysis demonstrated that fatty acid β-oxidation and mitochondrial fusion were the key pathways. The changes of the related proteins carnitine palmitoyltransferase 1B, optic atrophy type 1, and mitofusin-1 were further verified by using western blot. The levels of these three proteins were upregulated in db/db mice, whereas this upregulation was attenuated by GCGR mAb treatment. GCGR antagonism has a protective effect on CMECs and cardiac diastolic function in diabetic mice, and this beneficial effect may be mediated via inhibiting fatty acid β-oxidation and mitochondrial fusion in CMECs.

Transgenic mice producing the trans 10, cis 12-conjugated linoleic acid present reduced adiposity and increased thermogenesis and fibroblast growth factor 21 (FGF21)

Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) from ruminant-derived foodstuffs can induce body fat loss after oral administration. In the current study, a transgenic mouse that produced t10c12-CLA had been generated by inserting the Propionibacterium acnes isomerase (Pai) expression cassette into the Rosa26 locus, and its male offspring were used to elucidate the enduring influence of t10c12-CLA on overall health. Compared to their wild-type (wt) C57BL/6J littermates, both biallelic Pai/Pai and monoallelic Pai/wt mice exhibited reduced plasma triglycerides levels, and Pai/wt mice exclusively showed increased serum fibroblast growth factor 21. Further analysis of Pai/Pai mice found a decrease in white fat and an increase in brown fat, with more heat release and less physical activity. Analysis of Pai/Pai brown adipose tissues revealed that hyperthermia was associated with the over-expression of carnitine palmitoyltransferase 1B, uncoupling proteins 1 and 2. These findings suggest that the systemic and long-term impact of t10c12-CLA on obesity might be mediated through the pathway of fibroblast growth factor 21 when low doses are administered or through enhanced thermogenesis of brown adipose tissues when high doses are employed.

Intermittent Fasting Sustainably Improves Glucose Tolerance in Normal Weight Male Mice Through Histone Hyperacetylation

Abstract To explore the mechanism by which intermittent fasting (IF) exerts prolonged effects after discontinuation, we examined mice that had been subjected to 4 cycles of fasting for 72 hours and ad libitum feeding for 96 hours per week (72hIF), followed by 4 weeks of ad libitum feeding, focusing on expression of genes for lipid metabolism in the skeletal muscle and histone acetylation in the promoter region. The 72hIF regimen resulted in metabolic remodeling, characterized by enhanced lipid utilization and mitochondrial activation in the muscle. This long-term IF (72hIF) caused stronger metabolic effects than alternate day fasting (24hIF) wherein fasting and refeeding are repeated every 24 hours. Upregulation of lipid oxidation genes and an increase in oxygen utilization were sustained even at 4 weeks after discontinuation of 72hIF, associated with histone hyperacetylation of the promoter region of uncoupling protein 3 (Ucp3) and carnitine palmitoyl transferase 1b (Cpt1b) genes. An increase in leucine owing to fasting-induced muscle degradation was suggested to lead to the histone acetylation. These findings support the previously unappreciated notion that sustainable promotion of histone acetylation in lipid oxidation genes of the muscle and adipose tissues during and after IF may contribute to sustained metabolic effects of IF.

Role of SUMO-Specific Protease 2 in Leptin-Induced Fatty Acid Metabolism in White Adipocytes.

Leptin is a 16-kDa fat-derived hormone with a primary role in controlling adipose tissue levels. Leptin increases fatty acid oxidation (FAO) acutely through adenosine monophosphate-activated protein kinase (AMPK) and on delay through the SUMO-specific protease 2 (SENP2)-peroxisome proliferator-activated receptor δ/γ (PPARδ/γ) pathway in skeletal muscle. Leptin also directly increases FAO and decreases lipogenesis in adipocytes; however, the mechanism behind these effects remains unknown. Here, we investigated the role of SENP2 in the regulation of fatty acid metabolism by leptin in adipocytes and white adipose tissues. The effects of leptin mediated by SENP2 on fatty acid metabolism were tested by siRNA-mediated knockdown in 3T3-L1 adipocytes. The role of SENP2 was confirmed in vivo using adipocyte-specific Senp2 knockout (Senp2-aKO) mice. We revealed the molecular mechanism involved in the leptin-induced transcriptional regulation of carnitine palmitoyl transferase 1b (Cpt1b) and long-chain acyl-coenzyme A synthetase 1 (Acsl1) using transfection/reporter assays and chromatin immunoprecipitation. SENP2 mediated the increased expression of FAO-associated enzymes, CPT1b and ACSL1, which peaked 24 hours after leptin treatment in adipocytes. In contrast, leptin stimulated FAO through AMPK during the initial several hours after treatment. In white adipose tissues, FAO and mRNA levels of Cpt1b and Acsl1 were increased by 2-fold 24 hours after leptin injection in control mice but not in Senp2-aKO mice. Leptin increased PPARα binding to the Cpt1b and Acsl1 promoters in adipocytes through SENP2. These results suggest that the SENP2-PPARα pathway plays an important role in leptin-induced FAO in white adipocytes.

L-carnitine attenuated hyperuricemia-associated left ventricular remodeling through ameliorating cardiomyocytic lipid deposition.

Hyperuricemia (HUA) is associated with left ventricular remodeling (LVR) and thereby causes the initiation and development of a large number of cardiovascular diseases. LVR is typically accompanied by cardiomyocyte energy metabolic disorder. The energy supply of cardiomyocytes is provided by glucose and fatty acid (FA) metabolism. Currently, the effect of HUA on cardiomyocytic FA metabolism is unclear. In this study, we demonstrate that UA-induced cardiomyocyte injury is associated with cytoplasmic lipid deposition, which can be ameliorated by the FA metabolism-promoting drug L-carnitine (LC). UA suppresses carnitine palmitoyl transferase 1B (CPT1B), thereby inhibiting FA transport into the mitochondrial inner matrix for elimination. LC intervention can ameliorate HUA-associated left ventricular anterior wall thickening in mice. This study showed that FA transport dysfunction plays is a critical mechanism in both cardiomyocytic injury and HUA-associated LVR and promoting cytoplasmic FA transportation through pharmacological treatment by LC is a valid strategy to attenuate HUA-associated LVR.

Enriched functional milk fat ameliorates glucose intolerance and triacylglycerol accumulation in skeletal muscle of rats fed high-fat diets.

We examined the effect of a functional milk fat (FMF) on the glucose metabolism and its association with the intramuscular triacylglycerol (TAG) content in rats fed high-fat diets. Male Wistar rats were fed for 60days with S7 (soybean oil 7%), S30 (soybean oil 30%), MF30 (soybean oil 3% + milk fat 27%), or FMF30 (soybean oil 3% + FMF 27%) diets. An oral glucose tolerance test was performed. The levels of key metabolites in gastrocnemius muscle and mRNA levels of genes involved in glucose and lipid metabolism in muscle, epididymal white adipose tissue (EWAT), and serum were assessed. The S30 diet induced glucose intolerance and led to TAG, citrate, and glucose accumulation in muscle. Moreover, we observed a downregulation of uncoupling proteins (Ucp2 and Ucp3) and insulin receptor substrate-1 (Irs1) genes, lower carnitine palmitoyl transferase-1b (CPT-1b), and phosphofructokinase-1 (PFK1) activities in muscle and lower expression of adiponectin (Adipoq) in EWAT. The FMF30 diet ameliorated the glucose intolerance and normalized the glucose and TAG levels in muscle, preventing the accumulation of citrate and enhancing glucose utilization by the PFK1. The beneficial effects might also be related to the higher expression of Adipoq in EWAT, its receptor in muscle (Adipor1), and the expression of Ucp2, Ucp3, and Irs1 in muscle, restoring the alterations observed with the S30 diet. FMF30 modulated key genes involved in glucose and lipid metabolism in skeletal muscle, improving the glucose utilization and preventing TAG, glucose, and citrate accumulation.

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

Brown adipose tissue (BAT) is richin 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 8weeks. Subsequently, glucose and fatty acidmetabolism 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 inintact 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.

Ketogenic diet modulates cardiac metabolic dysregulation in streptozocin-induced diabetic rats

The ketogenic diet (KD) might improve cardiac function in diabetic cardiomyopathy, but the mechanisms remain unclear. This study investigated the effects of KD on myocardial fatty acid (FA), glucose, and ketone metabolism in diabetic cardiomyopathy. Echocardiograms, biochemistry, and micro-positron emission tomography were performed to evaluate cardiac function and glucose uptake in control rats and streptozotocin-induced diabetes mellitus (DM) rats with normal diet (ND) or KD for 6 weeks. Histopathology, adenosine triphosphate measurement, and Western blot were performed in the ventricular myocytes to analyze fibrosis, FA, ketone body, and glucose utilization. The ND-fed DM rats exhibited impaired left ventricular systolic function and increased chamber dilatation, whereas control and KD-fed DM rats did not. The KD reduced myocardial fibrosis and apoptosis in the DM rats. Myocardial glucose uptake in the micro-positron emission tomography was similar between ND-fed DM rats and KD-fed DM rats and was substantially lower than the control rats. Compared with the control rats, ND-fed DM rats had increased phosphorylation of acetyl CoA carboxylase and higher expressions of CD-36, carnitine palmitoyltransferase-1β, tumor necrosis factor-α, interleukin-1β, interleukin6, PERK, and e-IF2α as well as more myocardial fibrosis and apoptosis (assessed by Bcl-2, BAX, and caspase-3 expression); these increases were attenuated in the KD-fed DM rats. Moreover, ND-fed DM rats had significantly lower myocardial adenosine triphosphate, BHB, and OXCT1 levels than the control and KD-fed DM rats. The KD may improve the condition of diabetic cardiomyopathy by suppressing FA metabolism, increasing ketone utilization, and decreasing endoplasmic reticulum stress and inflammation.

Comparative proteomic analysis of hearts from mice with high-fat diet-induced metabolic cardiomyopathy

In patients with obesity or type 2 diabetes, the accumulation of lipotoxic by-products in cardiomyocytes leads to apoptosis and contractile dysfunction, eventually resulting in metabolic cardiomyopathy (MC). However, the underlying mechanisms remain unclear. Inb this study, a comparative proteome analysis was conducted to evaluate the differentially expressed proteins (DEPs) in the hearts of normal mice on standard diet (control group) and of high-fat diet (HFD)-induced MC mice (HFD group). We identified 90 DEPs unique to the control group and 18 DEPs unique to the HFD group. In 90 DEPs unique to the control group, only 74 DEPs were annotated in the gene ontology (GO) database. These annotated DEPs are involved in 114 biological processes, 68 molecular functions, and 174 cellular components. In 18 DEPs unique to the HFD group, only 14 DEPs were annotated in the GO database. These annotated DEPs are involved in 24 biological processes, 22 molecular functions, and six cellular components. Protein levels of two fatty acid metabolism-related enzymes, carnitine palmitoyltransferase 1B (CPT1B) and acetyl-CoA acyltransferase 2 (ACAA2), in the hearts of the mice in control group and HFD group were analyzed by immunostaining and Western blot. The results showed that the protein levels of CPT1B and ACAA2 were elevated in hearts of the mice in HFD group, which were consistent with the proteomic analysis. Our results reveal the differentially expressed proteome related to the progression of MC, providing a series of potential therapeutic targets for MC.