The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes

Abstract

While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5 mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ∼1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose.

Ectopic brown adipose tissue formation within skeletal muscle after brown adipose progenitor cell transplant augments energy expenditure

“…1G, H), which is consistent with active thermogenesis in these adipocytes. The adipocytes differentiated from 3T3-L1 preadipocytes also had a considerable amount of uncoupled respiration, which is consistent with previous reports (26,27). iBAPC-derived adipocytes had comparable basal and uncoupled respiration to primary brown adipocytes, indicating that immortalization and extensive in vitro culture had no adverse effect on the thermogenic capacity of iBAPC-derived adipocytes (Fig.…”

FNDC5 overexpression and irisin ameliorate glucose/lipid metabolic derangements and enhance lipolysis in obesity

“…The media were replaced every 2 days thereafter until 85% of the cells contained lipid droplets. Eight days after the induction of differentiation, 3T3L1 adipocytes were ready to be used in experiments [9,10].…”

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization

“…After 3 days in culture, the cells were differentiated for 5 days as described above, and treated with 0, 10, or 50 M DMF for the last 24 h (n ϭ 6/group). The Seahorse extracellular flux analyzer XF-24 (Agilent Technologies, Inc., Santa Clara, CA), was used to measure the oxygen consumption rate (OCR), using XF Assay Medium (Cat # 102365-100, Agilent Technologies, Inc.) supplemented with 25 mM glucose (Cat # G8270, Sigma-Aldrich) (38). After measurement of basal respiration, oligomycin (5 g/ml, Cat # O4876, Sigma-Aldrich), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 M, Cat # C2920, Sigma-Aldrich), and rotenone (3 M, Cat # R8875, Sigma-Aldrich) ϩ antimycin A (4 M, Cat # A8674, Sigma-Aldrich) were added sequentially to determine ATP production/proton leak, spare respiratory capacity and nonmitochondrial respiration.…”