Chun-Lin Gao

Mitochondrial dysfunction is induced by high levels of glucose and free fatty acids in 3T3-L1 adipocytes.

Hyperglycemia and high free fatty acids (FFAs) are two well-known characteristics of type 2 diabetes, and are also implicated in the etiology of insulin resistance. However, their roles in mitochondrial dysfunction of white adipocytes are not well-studied. In this study, we investigated the effects of high glucose (25 mM), high free fatty acids (FFAs, 1mM), or a combination of both high glucose+high FFAs on mitochondrial function in differentiated 3T3-L1 adipocytes after 48 h of treatment. We found that high glucose, high FFAs, or high glucose+high FFAs reduced insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes. In addition, the mitochondria became smaller and more compact. Levels of the mitofusion protein mfn1 decreased and levels of the mitofission protein Drp1 increased as compared to controls. NRF1 was downregulated, and PGC-1 beta levels were diminished in the high glucose and high glucose+high FFAs conditions. Levels of PGC-1 alpha and mtTFA mRNA were greatly downregulated. No difference was found in the mitochondrial DNA (mtDNA) and intracellular ATP levels of treated cells compared to control cells. Cells treated with high glucose or high FFAs accumulated significant amounts of reactive oxygen species (ROS) and displayed a loss of the mitochondrial membrane potential. High glucose and high glucose+high FFAs led to similar decreases in intramitochondrial calcium concentration, although high FFAs had no effect. Therefore, high glucose and high FFAs can regulate insulin sensitivity, and mitochondrial dysfunction may occur in this process.

TNF-α induces mitochondrial dysfunction in 3T3-L1 adipocytes

TNF-alpha was the first proinflammatory cytokine identified linking obesity, insulin resistance and chronic inflammation. However, the mechanism of TNF-alpha in the etiology of insulin resistance is still far from clear. Because the mitochondria play an important role in energy metabolism, we investigated whether mitochondrial dysfunction is involved in pathogenesis of TNF-alpha-mediated insulin resistance. First, a fully differentiated insulin-resistant 3T3-L1 adipocyte model was established by incubating with 4 ng/ml TNF-alpha for 4 d, and then the mitochondrial morphology and functions were observed. TNF-alpha treatment induced pronounced morphological changes in the mitochondria, which became smaller and condensed, and some appeared hollow and absent of cristae. Mitochondrial dynamics changes were observed as increased mitofusion protein mfn1 and mitofission protein Drp1 levels compared with controls. No obvious effects on mitochondrial biogenesis were found. PGC-1alpha levels decreased, but no significant changes were found in mtTFA mRNA expression, NRF1mRNA expression and mitochondrial DNA (mtDNA). TNFalpha treatment also led to decreased mitochondrial membrane potential and reduced production of intracellular ATP, as well as accumulation of significant amounts of reactive oxygen species (ROS). Further research is required to determine if mitochondrial dysfunction is involved in the inflammatory mechanism of insulin resistance and may be a potential target for the treatment of insulin resistance.

IL-6 induces lipolysis and mitochondrial dysfunction, but does not affect insulin-mediated glucose transport in 3T3-L1 adipocytes

Interleukin-6 (IL-6) has emerged as an important cytokine involved in the regulation of metabolism. However, the role of IL-6 in the etiology of obesity and insulin resistance is not fully understood. Mitochondria are key organelles of energy metabolism, and there is growing evidence that mitochondrial dysfunction plays a crucial role in the pathogenesis of obesity-associated insulin resistance. In this study, we determined the direct effect of IL-6 on lipolysis in adipocytes, and the effects of IL-6 on mitochondrial function were investigated. We found that cells treated with IL-6 displayed fewer lipids and an elevated glycerol release rate. Further, IL-6 treatment led to decreased mitochondrial membrane potential, decreased cellular ATP production, and increased intracellular ROS levels. The mitochondria in IL-6-treated cells became swollen and hollow with reduced or missing cristae. However, insulin-stimulated glucose transport was unaltered. PGC-1α, NRF1, and mtTFA mRNA levels were markedly increased, and the mitochondrial contents were also increased. Our results demonstrate that IL-6 can exert a direct lipolytic effect and induce mitochondrial dysfunction. However, IL-6 did not affect insulin sensitivity in adipocytes in vitro. We deduce that in these cells, enhanced mitochondrial biogenesis might play a compensatory role in glucose transport.

Overexpression of NYGGF4 (PID1) induces mitochondrial impairment in 3T3-L1 adipocytes

NYGGF4 is a recently discovered gene that is involved in obesity-associated insulin resistance. The exact mechanism by which NYGGF4 induces insulin resistance has not yet been fully elucidated. In this study, we demonstrated that the overexpression of NYGGF4 in 3T3-L1 adipocytes decreased mitochondrial mass, mitochondrial DNA, and intracellular ATP synthesis. In addition, NYGGF4 overexpression also led to an imbalance of the mitochondrial dynamics and excess intracellular ROS production. Collectively, our results indicated that the overexpression of NYGGF4 caused mitochondrial dysfunction in adipocytes, which might be responsible for the development of NYGGF4-induced insulin resistance.

LYRM1, a novel gene promotes proliferation and inhibits apoptosis of preadipocytes

OBJECTIVE To characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects. METHODS AND RESULTS RT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1 expression is widely distributed, with the highest levels of expression occurring in adipose tissue. A fusion protein of LYRM1 and green fluorescent protein as well as western blot analysis were used to identify the subcellular localization of LYRM1 in the nucleus. Based on Oil red O staining and the expression profile of specific differentiation markers, ectopic LYRM1 expression was not found to significantly affect adipogenesis. MTT assays and cell cycle analysis showed that LYRM1 promotes preadipocyte proliferation, and data from annexin V-FITC and caspase-3 activity assays further determined that LYRM1 can inhibit apoptosis of preadipocytes. CONCLUSIONS By increasing cell proliferation and lowering the rate of apoptosis, LYRM1 has the potential to modulate the size of the preadipocyte pool and influence adipose tissue homeostasis.

Overexpression of LYRM1 induces mitochondrial impairment in 3T3-L1 adipocytes

Homo sapiens LYR motif containing 1 (LYRM1) is a recently discovered gene involved in adipose tissue homeostasis and obesity-associated insulin resistance. The exact mechanism by which LYRM1 induces insulin resistance has not yet been fully elucidated. In this study, we demonstrated that the overexpression of LYRM1 in 3T3-L1 adipocytes resulted in reduced insulin-stimulated glucose uptake, an abnormal mitochondrial morphology, and a decrease in intracellular ATP synthesis and mitochondrial membrane potential. In addition, LYRM1 overexpression led to excessive production of intracellular of reactive oxygen species. Collectively, our results indicated that the overexpression of LYRM1 caused mitochondrial dysfunction in adipocytes, which might be responsible for the development of LYRM1-induced insulin resistance.

Over-expression of LYRM1 inhibits glucose transport in rat skeletal muscles via attenuated phosphorylation of PI3K (p85) and Akt

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-d-[3H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.

Resistin induces rat insulinoma cell RINm5F apoptosis

Beta-cell apoptosis induced by adipokines may result in beta-cell dysfunction in type 2 diabetes. Resistin, an adipokine-linked obesity with type 2 diabetes, impairs glucose-stimulated insulin secretion (GSIS) in beta-cells. Presently, the effects of resistin on rat insulinoma cells RINm5F were examined. Treatment of RINm5F with resistin induced cell damage. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) protected resistin-mediated cytotoxicity in RINm5F. Incubation with resistin up-regulated caspase-3 activity and induced the formation of a DNA ladder. TIMP-1 attenuated these effects. The molecular mechanism of TIMP-1 inhibition of resistin-mediated cytotoxicity appeared to involve Akt phosphorylation and activation of IkB-α phosphorylation. Resistin treatment suppressed Akt phosphorylation and activated IkB-α phosphorylation, which could be attenuated by TIMP-1. We conclude that resistin can induce beta-cell apoptosis and that resistin-related beta-cell apoptosis can be prevented by TIMP-1.

The lin-4 Gene Controls Fat Accumulation and Longevity in Caenorhabditis elegans

Previous studies have determined that lin-4, which was the first miRNA to be discovered, controls the timing of cell fate determination and life span in Caenorhabditis elegans. However, the mechanism of lin-4 involvement in these processes remains poorly understood. Fat storage is an essential aspect of the life cycle of organisms, and the function of lin-4 in fat accumulation is not clear. In this study, we showed that the fat content is reduced remarkably in C. elegans lin-4 mutants. Quantitative RT-PCR analysis revealed a considerable decrease in the levels of SBP-1 and OGA-1 mRNA in lin-4 mutants. We also showed that lin-4 mutants have a significantly shorter life span than wild-type worms. DCF assay experiments showed that the reactive oxygen species (ROS) levels increased and mitochondrial DNA (mtDNA) copy number decreased in loss-of-function lin-4 mutants. These mutants also showed attenuation of locomotion. Taken together, our findings suggest that lin-4 may play an important role in regulating fat accumulation and locomotion and that lin-4 may control the life span of C. elegans by mediating ROS production.

Gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides by cDNA microarrays.

To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy.