Mitochondrial dysfunction has divergent, cell type-dependent effects on insulin action

Abstract

The contribution of mitochondrial dysfunction to insulin resistance is a contentious issue in metabolic research. Recent evidence implicates mitochondrial dysfunction as contributing to multiple forms of insulin resistance. However, some models of mitochondrial dysfunction fail to induce insulin resistance, suggesting greater complexity describes mitochondrial regulation of insulin action. We report that mitochondrial dysfunction is not necessary for cellular models of insulin resistance. However, impairment of mitochondrial function is sufficient for insulin resistance in a cell type-dependent manner, with impaired mitochondrial function inducing insulin resistance in adipocytes, but having no effect, or insulin sensitising effects in hepatocytes. The mechanism of mitochondrial impairment was important in determining the impact on insulin action, but was independent of mitochondrial ROS production. These data can account for opposing findings on this issue and highlight the complexity of mitochondrial regulation of cell type-specific insulin action, which is not described by current reductionist paradigms.

Keywords: AMPK, AMP-activated protein kinase; AS160, Akt substrate of 160 kDa; Adipocyte; BSA, bovine serum albumin; ECAR, extracellular acidification rate; FoxO1, forkhead box protein O1; G.O., glucose oxidase; GLUT4, facilitative glucose transporter isoform 4; GP, glucose production; HI-FBS, heat-inactivated foetal bovine serum; Hepatocyte; IRS1, insulin receptor substrate 1; Insulin action; LDH, lactate dehydrogenase; MMP, mitochondrial membrane potential; Mitochondria; MnTBAP, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; Reactive oxygen species; SOD, superoxide dismutase; T2D, type 2 diabetes; TNFα, tumour necrosis factor alpha.

Figure 1

Diverse models of cell-autonomous insulin resistance in 3T3L1 adipocytes is not associated with a common perturbation in mitochondrial function. (A) Basal and insulin-stimulated (10 nM) 2-deoxyglucose uptake; (B) Insulin signalling; (C) LDH release; (D) Oxygen consumption due to basal respiration, ATP turnover and H⁺ leak; (E) Mitochondrial membrane potential (MMP); and (F) Mitochondrial ROS production, in vehicle (Veh), glucose oxidase (G.O.; 25 mU/mL), TNFα (10 ng/mL) and chronic insulin (10 nM) treated (24 h) cells. Data presented as mean ± SEM, n = 3–6 biological replicates. Denotes significantly different from; ^# vehicle/non-insulin-treated cells, * vehicle/insulin-treated cells; and ^† vehicle-treated cells (p < 0.05).

Figure 2

Impaired mitochondrial function induces insulin resistance in 3T3L1 adipocytes. (A) Basal respiration; (B) Mitochondrial function; (C) Extracellular acidification rate (ECAR); (D) ATP concentration; (E) Basal and insulin-stimulated (10 nM) 2-deoxyglucose uptake; (F) Insulin action; (G) Insulin signalling; and (H) Basal and insulin-stimulated (10 nM) 2-deoxyglucose uptake in the presence or absence of MnTBAP in vehicle, 50 nM or 100 nM oligomycin treated (24 h) 3T3L1 adipocytes. (I) Basal respiration; (J) Mitochondrial function; (K) ECAR; (L) ATP concentration; (M) Basal and insulin-stimulated (10 nM) 2-deoxyglucose uptake; (N) Insulin action; and (G) Insulin signalling in vehicle, 1 nM or 5 nM rotenone treated (24 h) 3T3L1 adipocytes. Data presented as mean ± SEM, n = 3–6 biological replicates. Denotes significantly different from; ^† vehicle-treated cells, ^# vehicle/non-insulin-treated cells, * vehicle/insulin-treated cells (p < 0.05).

Figure 3

Impaired mitochondrial function has no effect on insulin action in FAO hepatocytes. (A) Basal respiration; (B) Mitochondrial function; (C) Extracellular acidification rate (ECAR); (D) ATP concentration; (E) Basal and insulin-suppressed (0.1 nM) glucose production; (F) Insulin action; and (G) Insulin signalling in vehicle and 0.5 nM oligomycin treated (24 h) FAO hepatocytes. (H) Basal respiration; (I) Mitochondrial function; (J) ECAR; (K) ATP concentration; (L) Basal and insulin-suppressed (0.1 nM) glucose production; (M) Insulin action; (N) Insulin signalling; and (O) Basal and insulin-suppressed (0.1 nM) glucose production in the presence of absence of wortmannin (100 nM) in vehicle and 1 nM rotenone treated FAO hepatocytes. Data presented as mean ± SEM, n = 3–6 biological replicates. Denotes significantly different from; ^† vehicle-treated cells, ^# vehicle/non-insulin-treated cells, * vehicle/insulin-treated cells, Δ designated difference (p < 0.05).

Figure 4

Anti-diabetic agents rosiglitazone and phenformin induce mitochondrial dysfunction and have cell type-dependent effects on insulin action. (A) Mitochondrial function; (B) Basal and insulin-stimulated (10 nM) 2-deoxyglucose uptake; and (C) Insulin action in vehicle and 100μM rosiglitazone treated (24 h) 3T3L1 adipocytes. (D) Mitochondrial function; (E) Basal and insulin-stimulated (10 nM) 2-deoxyglucose uptake; and (F) Insulin action in the presence or absence of MnTBAP in vehicle and 100μM rosiglitazone treated (24 h) 3T3L1 adipocytes. (G) Mitochondrial function; (H) Basal and insulin-suppressed (0.1 nM) glucose production; (I) Insulin action; and (J) ATP levels in FAO hepatocytes treated (24 h) with increasing doses of rosiglitazone. (K) Mitochondrial function; (L) Basal and insulin-suppressed (0.1 nM) glucose production; (M) Insulin action; and (N) ATP levels in FAO hepatocytes treated (24 h) with increasing doses of phenformin. Data presented as mean ± SEM, n = 3–6 biological replicates. Denotes significantly different from; ^# vehicle/non-insulin-treated cells, * vehicle/insulin-treated cells, ^† vehicle-treated cells (p < 0.05). ND = not detected.

Figure 5

Rosiglitazone and phenformin do not impair mitochondrial ROS production in 3T3L1 adipocytes. (A) Basal and insulin-stimulated (10 nM) glucose uptake in 3T3L1 adipocytes treated with glucose oxidase (Gluc. Ox.; 25 mU/mL) and increasing doses of either rosiglitazone and phenformin; (B) Mitochondrial ROS production via mitosox fluorescence; and (C) Mitochondrial membrane potential (MMP) in 3T3L1 adipocytes treated (24 h) with glucose oxidase and rosiglitazone. (D) Mitochondrial ROS production via mitosox fluorescence; and (E) Mitochondrial membrane potential (MMP) in 3T3L1 adipocytes treated (24 h) with glucose oxidase and phenformin. Data presented as mean ± SEM, n = 3–6 biological replicates. Denotes significantly different from; * vehicle/insulin-treated cells, ^‡ glucose oxidase/insulin-treated cells, ^∫ glucose oxidase/vehicle-treated cells, ▪ corresponding treatment group/no glucose oxidase treated cells (p < 0.05).

Figure 6

Complexity linking impaired mitochondrial function to insulin action. Cell type- and impairment site-specific relationships between mitochondrial dysfunction and insulin action in adipocytes and hepatocytes.