The Mitochondrial Mechanism Analysis Of High Level Of Glucose,High FFAs And UCP4 Impacting The Insulin Sensitivity Of 3T3-L1 Adipocytes

Obesity has been a global public health problem, and the situation in children and adolescents is not optimistic. The obesity incidence rate of US childhood has increased nearly 3 times during the past three decades, and 12% of the Chinese children and adolescents are overweight and obese. Obesity has been showed to affect the children's mental growth, and would lead to develop the obesity related metabolism syndrome( type 2 diabetes included). Recently, the lower age tendency of childhood type 2 diabetes is becoming obvious. The key link of type 2 diabetes and metabolism syndrome is insulin resistance, but the mechanism of which is not fully clarified untill now, so a systematic study for the pathogenesis of insulin resistance is needed.Hyperglycaemia and high level of free fatty acids (FFAs) in blood circulation are the most important and common sceneries. The concept of glucose toxicity and lipid toxicity was raised early in 1990s, which meant that high level of glucose or FFAs would lead toβ-Cell failure and loss of glucose-stimulated insulin secretion (GSIS). There were growing body of evidences demonstrated that not onlyβ-Cell, but other insulin targeted tissues, such as skeletal muscle, hepatic cell and vascular endothelial cell et al, were involved in the development of high glucose and high FFAs caused insulin resistance.As one of the important periphery insulin targeted tissues, the adipose tissue was not merely as an energy storage place, once it became insulin resistance, it would turn to release FFAs and secrete inflammatory factors (TNF-α, for example), the both conditions would exacerbate systematic insulin resistance, today it has been conceived as an important player of systematic insulin resistance. The adipose tissue contained plentiful of mitochondria, on the other hand, the mitochondria mechanism has been implicated closely in insulin resistance, but there is little message for the effect of high level of glucose and FFAs on mitochondrial function and adipocyte insulin sensitivity.UCP4, located in the inner mitochondrial membrane, was closely related to energy metabolism. We previously showed that UCP4 was significently upregulated in omental adipose tissue in diet-induced obese rats using suppression subtractive hybridization (SSH). Ectopic expression of UCP4 can promote proliferation and inhibit differentiation and lipid accumulation as well as apoptosis in 3T3-L1 preadipocytes. The insulin-stimulated glucose uptake and the expression of glucose transporters GLUT1 and GLUT4 in differentiated 3T3-L1 adipocytes were downregulated upon UCP4 overexpression. Whether mitochondrial mechanism is involved in the down regulated insulin sensitivity of UCP4 in adipocytes deserves futhur investigation.The aim of this study was to determine the effect of high glucose,high FFAs and UCP4 on adipocytes insulin sensitivity, and to detemine whether mitochondrial dysfunction plays a role. In the first part, the differentiated 3T3-L1 adipocytes were incubated with high glucose or/and high FFAs, adipocytes insulin sensitivity was observed, the mitochondrial function was analyzed after the treatment, the relationship of mitochondrial function and insulin sensitivity was investigated; In the second part, we examined the effect of UCP4 on adipocytes insulin sensitivity and mitochondrial function in vitro by establishing a stable preadipocyte cell line overexpressing UCP4, we preliminary assessed the possible role of mitochondrial dysfunction in the process. Part I: The mitochondrial mechanism analysis of high level of glucose,high FFAs impacting the insulin sensitivity of 3T3-L1 adipocytesObjective: To investigate the relationship and the effects of high glucose and high FFAs on insulin sensitivity and mitochondrial function in differentiated 3T3-L1 adipocytes.Methods: Differentiated 3T3-L1 adipocytes were treated with high glucose (25 mM) or high FFAs (1 mM) or both for 48 hours, 5 mmol/L glucose as control. Insulin sentivity was determined by insulin-stimulated 2-Deoxy-D-[3H] glucose uptake in differentiated 3T3-L1 adipocytes, mitochondria ultramicrostructure was displayed by electromicrograph morphometry, mitochondrial biogenesis related proteins were examined by western blot, and the mitochondrial DNA copynumber and mRNA expression of PGC-1αwas detected by Realtime PCR. The ATP content of the adipocytes was measured with ATP lite-glo, a luciferase-based luminescence assay kit. Mitochondrial membrane potential and intramitochondrial calcium as well as intracellular ROS was detemined by fluorescent molecular probes and FACS.Results: We found:①High glucose,high FFAs,or high glucose+high FFAs reduced insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes;②The treatment of high glucose, high FFAs, or high glucose+high FFAs induced smaller and more compact mitochondria, the mitochondrial matrix condensed, cristae collapsed and confused arranage;③Levels of the mitofusion protein mfn1 decreased in high FFAs group and high glucose+high FFAs group, and levels of the mitofission protein Drp1 increased in high FFAs group;④Levels of the mitochondrial biogenesis key factors PGC-1α,PGC-1β,NRF-1,mtTFA were downregulated in the treated groups ;⑤Intracellular ROS was significently increased while mitochondrial membrane potential was decreased in the treated groups, but there was no difference was detected when it came to mtDNA copy number and intracellular ATP content;⑥High glucose and high glucose+high FFAs treatment induced significent decreased intramitochondrial calcium, but high FFAs had no effect.Conclusion:High glucose and high FFAs could down regulate insulin sensitivity and cause mitochondrial dysfunction of adipocytes, mitochondrial dysfunction mightbe one of the mechanisms of adipocytes insulin resistance.Part II: The mitochondrial mechanism analysis of UCP4 overexpression affecting the insulin sensitivity of 3T3-L1 adipocytesObjective: To explore the effect of UCP4 on adipocytes insulin sensitivity, and to analyze the relationship of UCP4 overexpression induced mitochondrial function changes and insulin sensitivity in differentiated 3T3-L1 adipocytes.Methods: UCP4 eukaryotic expression vector was constructed and was stably transfected into 3T3-L1 preadipocytes. UCP4 expression was confirmed by Real Time PCR and Western-Blot analysis. The transfected cells with an empty expression vector (pcDNA3.1Myc/His B)(as control) or an UCP4 expression vector were induced to differentiated adipocytes; Insulin sentivity was determined by insulin-stimulated 2-Deoxy-D-[3H] glucose uptake in differentiated 3T3-L1 adipocytes, mitochondria ultramicrostructure was displayed by electromicrograph morphometry, mitochondrial biogenesis related proteins were examined by western blot, and the mitochondrial DNA copynumber and mRNA expression of PGC-1αwas detected by Realtime PCR. The ATP content of the adipocytes was measured with ATP lite-glo, a luciferase-based luminescence assay kit. Mitochondrial membrane potential and intramitochondrial calcium as well as intracellular ROS was detemined by FACS. Results:①Adipocytes insulin sensitivity was down regulated upon UCP4 overexpression;②Transmission electron microscopy (TEM) showed that adipocytes overexpressing UCP4 displayed different size and condensed mitochondria with collapsed and unclear cristae;③UCP4 overexpression impaired mitochondrial fusion and fission, as indicated by decreased mitofusin mfn1, and mitofission DRP1;④The adipocytes overexpressing UCP4 also showed decreased mRNA expression of key factors in mitochondrial biogenesis, including PGC-1αand mtTFA. NRF-1 and ERRβlevels were downregulated, while NRF-2 levels were upregulated and no change for SIRT1;⑤The adipocytes overexpressing UCP4 also showed decreased mitochondrial copy number (mtDNA) and intracellular ATP content, higher production of intracellular ROS and diminished levels of intramitochondrial calcium and mitochondrial membrane potential.Conclusion: UCP4 overexpression induced decreased insulin sensitivity and mitochondrial dysfunction in adipocytes, and mitochondrial dysfunction might be one of the mechanisms of adipocytes insulin resistance.