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Pancreatic preservation is an important part of diabetes management that may occur with improved peripheral insulin sensitivity and attenuated low-grade adipose tissue inflammation. The objective of the current study was to determine the response of obese, insulin-resistant fa/fa Zucker rats vs lean controls to dietary conjugated linoleic acid (CLA) supplementation with respect to pancreatic islet size, insulin resistance, and markers of inflammation and adipose glucose uptake. Six-week-old fa/fa and lean Zucker rats (n = 20 per genotype) were fed either a 1.5% CLA mixture or control diet for 8 weeks. Oral glucose tolerance testing was conducted at 7.5 weeks. Fasting serum haptoglobin, insulin, and C-peptide were assayed, and select messenger RNA (mRNA) and protein markers of inflammation and glucose metabolism were measured in adipose and liver tissues. CLA-fed fa/fa Zucker rats had smaller islet cell size, improved oral glucose tolerance and insulinemia, and attenuated serum haptoglobin levels compared with control-fed fa/fa Zucker rats, despite no differences in body weight and a slightly higher visceral adipose mass. CLA did not alter insulin sensitivity or islet size in lean Zucker rats. The CLA-fed fa/fa rats also had greater adipose glucose transporter-4 mRNA and less adipose tumor necrosis factor α mRNA and protein compared with control-fed fa/fa rats. In contrast, other markers of inflammation and glucose metabolism including adipose macrophage inflammatory factor, macrophage inflammatory protein-2, and liver pyruvate carboxylase and pyruvate dehydrogenase kinase 4 were not significantly changed. These results suggest that CLA supplementation preserved pancreatic function in conjunction with improved peripheral glucose use and reduced inflammation in fa/fa Zucker rats.

Hepatitis B virus (HBV) infection is a major cause of liver diseases. However, the mechanisms underlying HBV infection and pathogenesis remain largely unknown. The sex-determining region Y box 4 (Sox4) is a transcriptional factor, which preferentially regulates the development of various organs, tissues and cancers. But, the role of Sox4 in viral infection and pathogenesis has not been elucidated. Here, we demonstrated that Sox4 is up-regulated by HBV and revealed the mechanism by which HBV regulates Sox4 expression. First, HBV stimulates Sox4 expression through transcriptional factor Yin Yang 1 (YY1), which binds to Sox4 promoter to activate Sox4 transcriptional activity. Second, miR-335, miR-129-2 and miR-203 inhibit Sox4 expression by targeting its mRNA 3’UTR, while HBV suppresses the microRNAs expression, resulting in up-regulating Sox4 post-transcriptionally. Third, Sox4 protein is degraded by proteasome, while HBV surface protein (HBsAg) prevents Sox4 from degradation by directly interacting with the protein, thereby enhancing Sox4 production post-translationlly. More interestingly, HBV-activated Sox4 in turn facilitates HBV replication by direct binding to the viral genome via its HMG box. Thus, this study revealed a novel positive feedback mechanism by which Sox4 production and HBV replication are tightly correlated.

CLA has been shown to induce or suppress excess liver lipid accumulation in various animal models. Interestingly, the state of insulin resistance may be an important modulator of this effect. The objective of the current study was to determine how feeding a dietary CLA mixture would affect liver lipid accumulation in insulin-resistant/obese and lean rats in relation to liver function, lipidemia, liver TAG and phospholipid FA composition, and expression of hepatic markers of FA transport, oxidation, and synthesis. Six-week-old fa/fa and lean Zucker rats (n = 20/genotype) were fed either a 1.5% CLA mixture or a control diet for 8 wk. CLA supplementation reduced liver lipid concentration of fa/fa rats by 62% in concurrence with improved liver function (lower serum alanine aminotransferase and alkaline phosphatase) and favorable modification of the serum lipoprotein profile (reduced VLDL and LDL and elevated HDL) compared with controlfed fa/fa rats. The fa/fa genotype had two-thirds the amount of CLA (as % total FA) incorporated into liver TAG and phospholipids compared with the lean genotype. In both genotypes, CLA altered the hepatic FA profile (TAG greater than phospholipids) and these changes were explained by a desaturase enzyme index. Liver-FA-binding protein and acyl CoA oxidase, markers of FA transport and oxidation, respectively, were expressed at higher levels in CLA-fed fa/fa rats. In summary, these results illustrate a strong relationship between the state of insulin resistance and liver lipid metabolism and suggest that CLA acts to favorably modify lipid metabolism in fa/fa Zucker rats.

FIP3, isolated as a type 2 adenovirus E3–14.7-kDa interacting protein, is an essential component of the multimeric IκB-α kinase (IKK) complex and has been shown to interact with various components (Fas receptor-interacting protein, NF-κB-inducing kinase, IKKβ) of the NF-κB activation pathway. FIP3 has also been shown to repress basal and tumor necrosis factor (TNF) α-induced NF-κB activity as well as to induce cell death when overexpressed. The adenovirus E3–14.7-kDa protein (E3-14.7K) is an inhibitor of TNFα-induced cell death. In the current study, we generated deletion mutants to map the domains of FIP3, which are responsible for its various functions. The NF-κB inhibitory activity and the E3–14.7K binding domains were mapped at the carboxyl half of the FIP3 protein. We also found that the carboxyl-terminal half of FIP3 blocked TNFα-induced IκB-α phosphorylation and subsequent degradation, which suggests that the stabilization of the cytoplasmic inhibitor of NF-κB underlies the FIP3 inhibition of NF-κB activity. The amino-terminal 119 amino acids were responsible for the FIP3-IKKβ and FIP3-IKKα interaction, and the middle of the protein (amino acids 201–300) appeared to be both the FIP3 self-association domain as well as the FIP3-Fas receptor-interacting protein interaction domain. Thus, FIP3 might serve as a scaffold protein to organize the various components of the IκB-α kinase complex. Whereas the full-length protein is required for efficient cell death, the amino-terminal 200 amino acids are sufficient to cause rounding and detachment of the cells from the monolayer.

The predominant cause of death in diabetic patients is atherosclerotic coronary artery disease (CAD). Major gross cellular changes in the vascular wall of persons with CAD include endothelial injury and foam cell formation, as well as smooth muscle cell and fibroblast proliferation. This study examined the effects of glycated low density lipoprotein (glyLDL), a biochemical marker of diabetes, on cell viability, proliferation, and the expression of multiple growth factors in mouse embryo fibroblasts (MEF). The results demonstrated that exposure to ≥150 μg/mL of glyLDL for 24 h or 100 μg/mL of glyLDL for ≥48 h either significantly reduced cell viability or increased DNA fragmentation in MEF. GlyLDL treatment (25–100 μg/mL for up to 12 h) significantly increased the abundance of proliferating cell nuclear antigen (PCNA) and achieved a peak after 4 h exposure to glyLDL. Abundances of fibroblast growth factor-basic (FGF), transforming growth factor-β (TGF), and platelet-derived growth factor-A (PDGF) in MEF reached maximal levels after 2 h exposure to 50 μg/mL of glyLDL. The maximal increase of vascular endothelial growth factor (VEGF) was detected in MEF after 4 h of exposure to 50 μg/mL of glyLDL. Inhibitors for FGF (AZD4547), VEGF, or PDGF receptors (Axitinib), but not that for TGF receptor (LY364947), significantly decreased the abundance of (PCNA) in endothelial cells. The findings suggest that early exposure to a low dosage of glyLDL transiently increases the proliferation of MEF through the upregulation of FGF, VEGF, and (or) PDGF, and prolonged exposure to high concentrations of glyLDL reduced cell viability, which possibly accelerates atherogenesis under diabetic condition.

Impairments in mitochondrial function have been proposed to play an important role in the pathogenesis of diabetes. Atherosclerotic coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated low density lipoproteins (glyLDL) and oxidized LDL (oxLDL) were detected in patients with diabetes. Our previous studies demonstrated that oxLDL and glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). The present study examined the effects of glyLDL and oxLDL on mitochondrial respiration, membrane potential and the activities and proteins of key enzymes in mitochondrial electron transport chain (mETC) in cultured porcine aortic EC (PAEC). The results demonstrated that glyLDL or oxLDL significantly reduced oxygen consumption in Complex I, II/III and IV of mETC in PAEC compared to LDL or vehicle control using oxygraphy. Incubation with glyLDL or oxLDL significantly reduced mitochondrial membrane potential, the activities of mitochondrial ETC enzymes - NADH dehydrogenase (Complex I), succinate cytochrome c reductase (Complex II + III), ubiquinol cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV) in PAEC compared to LDL or control. Treatment with oxLDL or glyLDL reduced the abundance of subunits of Complex I, ND1 and ND6 in PAEC. However, the effects of oxLDL on mitochondrial activity and proteins were not significantly different from glyLDL. The findings suggest that the glyLDL or oxLDL impairs mitochondrial respiration, as a result from the reduction of the abundance of several key enzymes in mitochondria of vascular EC, which potentially may lead to oxidative stress in vascular EC, and the development of diabetic vascular complications.

Impairments in mitochondrial function have been proposed to play an important role in the pathogenesis of diabetes. Atherosclerotic coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated low density lipoproteins (glyLDL) and oxidized LDL (oxLDL) were detected in patients with diabetes. Our previous studies demonstrated that oxLDL and glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). The present study examined the effects of glyLDL and oxLDL on mitochondrial respiration, membrane potential and the activities and proteins of key enzymes in mitochondrial electron transport chain (mETC) in cultured porcine aortic EC (PAEC). The results demonstrated that glyLDL or oxLDL significantly reduced oxygen consumption in Complex I, II/III and IV of mETC in PAEC compared to LDL or vehicle control using oxygraphy. Incubation with glyLDL or oxLDL significantly reduced mitochondrial membrane potential, the activities of mitochondrial ETC enzymes - NADH dehydrogenase (Complex I), succinate cytochrome c reductase (Complex II + III), ubiquinol cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV) in PAEC compared to LDL or control. Treatment with oxLDL or glyLDL reduced the abundance of subunits of Complex I, ND1 and ND6 in PAEC. However, the effects of oxLDL on mitochondrial activity and proteins were not significantly different from glyLDL. The findings suggest that the glyLDL or oxLDL impairs mitochondrial respiration, as a result from the reduction of the abundance of several key enzymes in mitochondria of vascular EC, which potentially may lead to oxidative stress in vascular EC, and the development of diabetic vascular complications.

The isomer-specific effects of conjugated linoleic acid (CLA) on hepatic steatosis were assessed in fa/fa Zucker rats, a model for insulin resistance and the metabolic syndrome. Eight weeks of feeding trans-10,cis-12 CLA significantly improved glucose tolerance without changing body weight or visceral adipose mass. The trans-10,cis-12 isomer was also associated with reduced liver lipid content, improved liver function and reduced inflammation; these effects were not observed in rats fed the cis-9,trans-11 CLA isomer. Reduced liver lipid content did not correlate with activation of AMP-activated protein kinase or suppressed activation of sterol-regulatory element binding protein-1, two key regulators of hepatic lipid metabolism. Interestingly, rats fed cis-9,trans-11 CLA had fewer cytoplasmic lipid droplets in hepatocytes compared to rats fed control diet, but these droplets were larger in size. Conversely, fa/fa rats fed the trans-10,cis-12 CLA isomer had greater numbers of hepatic lipid droplets that were smaller in size, resulting in overall lower total lipid within these droplets. Changes in lipid droplets were associated with lower hepatic levels of PERILIPIN-2 (formerly known as adipophilin) in rats fed trans-10,cis-12 CLA, whereas amounts of other members of the PERILIPIN family of lipid droplet proteins were unaffected by dietary CLA. However, CLA isomers differentially affected the subcellular localization of these proteins. Treatment of H4IIE rat hepatoma cells with CLA isomers neither prevented nor reversed, but rather induced cytoplasmic lipid droplet formation, suggesting that the anti-steatotic effects of trans-10,cis-12 CLA are likely indirect and potentially mediated via increased lipid utilization by peripheral tissues.

Atherosclerotic cardiovascular disease is the leading cause of mortality in the Western world. Dysfunction of the mitochondrial respiratory chain and overproduction of reactive oxygen species (ROS) are associated with atherosclerosis and cardiovascular disease. Oxidation increases the atherogenecity of LDL. Oxidized LDL may be apoptotic or nonapoptotic for vascular endothelial cells (EC), depending on the intensity of oxidation. A previous study demonstrated that nonapoptotic oxidized LDL increased activity of mitochondrial complex I in human umbilical vein EC. The present study examined the impact of extensively oxidized LDL (eoLDL) on oxygen consumption and the activities of key enzymes in the mitochondrial respiratory chain of cultured porcine aortic EC. Oxygraphy detected that eoLDL significantly reduced oxygen consumption in various mitochondrial complexes. Treatment with eoLDL significantly decreased NADH-ubiquinone dehydrogenase (complex I), succinate cytochrome c reductase (complex II/III), ubiquinone cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV) activities and the NAD+-to-NADH ratio in EC compared with mildly oxidized LDL, LDL, or vehicle. Butylated hydroxytoluene, a potent antioxidant, normalized eoLDL-induced reductions in complex I and III enzyme activity in EC. Mitochondria-associated intracellular ROS and release of ROS from EC were significantly increased after eoLDL treatment. These findings suggest that eoLDL impairs enzyme activity in mitochondrial respiratory chain complexes and increases ROS generation from mitochondria of arterial EC. Collectively, these effects could contribute to vascular injury and atherogenesis under conditions of hypercholesterolemia and oxidative stress.