A Type 2 Ryanodine Receptor Variant in the Helical Domain 2 Associated with an Impairment of the Adrenergic Response.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is triggered by exercise or acute emotion in patients with normal resting electrocardiogram. The major disease-causing gene is RYR2, encoding the cardiac ryanodine receptor (RyR2). We report a novel RYR2 variant, p.Asp3291Val, outside the four CPVT mutation hotspots, in three CPVT families with numerous sudden deaths. This missense variant was first identified in a four-generation family, where eight sudden cardiac deaths occurred before the age of 30 in the context of adrenergic stress. All affected subjects harbored at least one copy of the RYR2 variant. Three affected sisters were homozygous for the variant. The same variant was found in two additional CPVT families. It is located in the helical domain 2 and changes a negatively charged amino acid widely conserved through evolution. Functional analysis of D3291V channels revealed a normal response to cytosolic Ca2+, a markedly reduced luminal Ca2+ sensitivity and, more importantly, an absence of normal response to 8-bromo-cAMP and forskolin stimulation in both transfected HEK293 and HL-1 cells. Our data support that the D3291V-RyR2 is a loss-of-function RyR2 variant responsible for an atypical form of CPVT inducing a mild dysfunction in basal conditions but leading potentially to fatal events through its unresponsiveness to adrenergic stimulation.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

[Figure: see text].

BHBA influences bovine hepatic lipid metabolism via AMPK signaling pathway.

ß-hydroxybutyric acid (BHBA), an important metabolite in ß-oxidation, is involved in the development of ketosis in dairy cows. It is known that AMP-activated protein kinase (AMPK) signaling pathway plays an important role in the regulation of lipid metabolism in hepatocytes. In the present study, bovine hepatocytes were treated with BHBA at variable concontrations and Compound C (Cpd C, an AMPK inhibitor) to investigate the effects of BHBA on the AMPK signaling pathway. The results showed that when the concentration of BHBA reached 1.2 mM, the AMPK signaling pathway was activated and the expression of sterol regulatory element binding protein-1c (SREBP-1c) as well as its target genes were significantly decreased. And these decreases were blocked by Cpd C. The binding activity and nucleus translocation of SREBP-1c showed a similar trend. The expression of peroxisome proliferator activated receptor-α (PPARα), carbohydrates response element binding protein (ChREBP) and their target genes were significantly increased while they were negatively suppressed by the Cpd C. The content of triglyceride (TG) had no obviously change in the BHBA and Cpd C-treated groups. These results indicate that BHBA can activate AMPK signaling pathway and regulate lipid synthesis and lipid oxidation genes of AMPK but showed no effect on TG in bovine hepatocytes.

SREBP-1c gene silencing can decrease lipid deposits in bovine hepatocytes cultured in vitro.

BACKGROUND: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c) is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. METHODS: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c) to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. RESULTS: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC) was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1), carnitine palmitoyltransferase І (CPT-І), carnitine palmitoyltransferase II (CPT- II), and ß-hydroxyacyl-CoA-DH (HADH) were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs), such as apolipoprotein B100 (ApoB), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTTP) were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR) was elevated. The concentrations of TG (triglyceride) and VLDL were also reduced. CONCLUSION: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

ß-Hydroxybutyrate activates the NF-κB signaling pathway to promote the expression of pro-inflammatory factors in calf hepatocytes.

BACKGROUND/AIMS: ß-hydroxybutyrate (BHBA) is the major component of ketone bodies in ketosis. Dairy cows with ketosis often undergo oxidative stress. BHBA is related to the inflammation involved in other diseases of dairy cattle. However, whether BHBA can induce inflammatory injury in dairy cow hepatocytes and the potential mechanism of this induction are not clear. The NF-κB pathway plays a vital role in the inflammatory response. METHODS: Therefore, this study evaluated the oxidative stress, pro-inflammatory factors and NF-κB pathway in cultured calf hepatocytes treated with different concentrations of BHBA, pyrrolidine dithiocarbamate (PDTC, an NF-κB pathway inhibitor) and N-acetylcysteine (NAC, antioxidant). RESULTS: The results showed that BHBA could significantly increase the levels of oxidation indicators (MDA, NO and iNOS), whereas the levels of antioxidation indicators (GSH-Px, CAT and SOD) were markedly decreased in hepatocytes. The IKKß activity and phospho-IκBα (p-IκBα) contents were increased in BHBA-treated hepatocytes. This increase was accompanied by the increased expression level and transcription activity of p65. The expression levels of NF-κB-regulated inflammatory cytokines, namely TNF-α, IL-6 and IL-1ß, were markedly increased after BHBA treatment, while significantly decreased after NAC treatment. However, the p-IκBα level and the expression and activity of p65 and its target genes were markedly decreased in the PDTC + BHBA group compared with the BHBA (1.8 mM) group. Moreover, immunocytofluorescence of p65 showed a similar trend. CONCLUSION: The present data indicate that higher concentrations of BHBA can induce cattle hepatocyte inflammatory injury through the NF-κB signaling pathway, which may be activated by oxidative stress.

Effects of nonesterified fatty acids on the synthesis and assembly of very low density lipoprotein in bovine hepatocytes in vitro.

High serum concentrations of nonesterified fatty acids (NEFA), which may affect the synthesis and assembly of very low density lipoproteins (VLDL), are associated with fatty liver during the early lactation period. Therefore, the objective of this study was to investigate the effects of NEFA on the synthesis and assembly of VLDL in bovine hepatocytes. Bovine hepatocytes were cultured and treated with different concentrations of NEFA. The mRNA expression of apolipoprotein B100 (ApoB100) and apolipoprotein E (ApoE) was significantly lower in the NEFA treatment groups than in the control group (0mM NEFA). The abundance of mRNA from microsomal triglyceride transfer protein (MTP) and the low density lipoprotein receptor (LDLR) was significantly lower in the medium- and high-dose NEFA treatment groups. The protein expression of ApoB100, ApoE, MTP, and LDLR was found to be significantly and dose-dependently decreased in groups of NEFA-treated hepatocytes. The VLDL content was also significantly decreased in the NEFA-treated hepatocytes. Large amounts of triglycerides accumulated in the hepatocytes. These results indicate that NEFA significantly inhibits the expression of ApoB100, ApoE, MTP, and LDLR, thereby decreasing the synthesis and assembly of VLDL and inducing TG accumulation in bovine hepatocytes.

Adiponectin activates the AMPK signaling pathway to regulate lipid metabolism in bovine hepatocytes.

Adiponectin (Ad) plays a crucial role in hepatic lipid metabolism. However, the regulating mechanism of hepatic lipid metabolism by Ad in dairy cows is unclear. Hepatocytes from a newborn female calf were cultured in vitro and treated with different concentrations of Ad and BML-275 (an AMPKα inhibitor). The results showed that Ad significantly increased the expression of two Ad receptors. Furthermore, the phosphorylation and activity of AMPKα, as well as the expression levels and transcriptional activity of peroxisome proliferator activated receptor-α (PPARα) and its target genes involved in lipid oxidation, showed a corresponding trend of upregulation. However, the expression levels and transcriptional activity of sterol regulatory element binding protein 1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP) decreased in a similar manner. When BML-275 was added, the p-AMPKα level as well as the expression and activity of PPARα and its target genes were significantly decreased. However, the expression levels of SREBP-1c, ChREBP and their target genes showed a trend of upregulation. Furthermore, the triglyceride (TG) content was significantly decreased in the Ad-treated groups. These results indicate that Ad activates the AMPK signaling pathway and mediates lipid metabolism in bovine hepatocytes cultured in vitro by promoting lipid oxidation, suppressing lipid synthesis and reducing hepatic lipid accumulation.

Acetic acid activates the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid) and BML-275 (an AMPKα inhibitor). Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

Effects of strontium on collagen content and expression of related genes in rat chondrocytes cultured in vitro.

Strontium stimulates cartilage matrix formation in vitro. However, the mechanisms governing these effects have not yet been extensively reported. In this study, chondrocytes were isolated from rat articular cartilage by enzymatic digestion and cultured for 24-72 h with 1-5 mM strontium. We investigated the effects of different concentrations of strontium on collagen content, type II collagen, insulin-like growth factor (IGF-1) and matrix metalloproteinase (MMP)-13 expression in rat cultured articular chondrocytes in vitro. The collagen content of the chondrocytes, determined as hydroxyproline, was measured by a colorimetry method. Type II collagen, IGF-1, and MMP-13 mRNA abundance and protein expression levels were determined by real-time polymerase chain reaction (real-time PCR) and western blot, respectively. The results showed that collagen content from the chondrocytes extracellular matrix increased with increasing strontium concentration. Moreover, 3 and 5 mM strontium strongly stimulated protein expression and mRNA levels of type II collagen and IGF-1. Conversely, MMP-13 expression in chondrocytes decreased dose-dependently with increasing strontium concentration. These results should provide insight into the ability of strontium to promote chondrocyte extracellular matrix synthesis. Strontium could promote collagen synthesis and suppress collagen degradation via the repression of MMP-13 expression.