Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans

Abstract

Background and aims: A diminution in skeletal muscle mitochondrial function due to ectopic lipid accumulation and excess nutrient intake is thought to contribute to insulin resistance and the development of type 2 diabetes. However, the functional integrity of mitochondria in insulin-resistant skeletal muscle remains highly controversial.

Methods: 19 healthy adults (age:28.4 ± 1.7 years; BMI:22.7 ± 0.3 kg/m2) received an overnight intravenous infusion of lipid (20% Intralipid) or saline followed by a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity using a randomized crossover design. Skeletal muscle biopsies were obtained after the overnight lipid infusion to evaluate activation of mitochondrial dynamics proteins, ex-vivo mitochondrial membrane potential, ex-vivo oxidative phosphorylation and electron transfer capacity, and mitochondrial ultrastructure.

Results: Overnight lipid infusion increased dynamin related protein 1 (DRP1) phosphorylation at serine 616 and PTEN-induced kinase 1 (PINK1) expression (P = 0.003 and P = 0.008, respectively) in skeletal muscle while reducing mitochondrial membrane potential (P = 0.042). The lipid infusion also increased mitochondrial-associated lipid droplet formation (P = 0.011), the number of dilated cristae, and the presence of autophagic vesicles without altering mitochondrial number or respiratory capacity. Additionally, lipid infusion suppressed peripheral glucose disposal (P = 0.004) and hepatic insulin sensitivity (P = 0.014).

Conclusions: These findings indicate that activation of mitochondrial fission and quality control occur early in the onset of insulin resistance in human skeletal muscle. Targeting mitochondrial dynamics and quality control represents a promising new pharmacological approach for treating insulin resistance and type 2 diabetes.

Clinical trial registration: NCT02697201, ClinicalTrials.gov.

Conflict of interest statement

Declaration of competing interest The authors declare that there are no conflicts of interest.

Copyright © 2021 Elsevier Inc. All rights reserved.

Figures

Figure 1.

CONSORT diagram depicting study enrollment, treatment allocation, and analysis.

Figure 2.

Schematic illustration of the experimental design.

Figure 3.

Expression of proteins regulating mitochondrial fission, fusion, and quality control. (A-C) Representative immunoblots of phosphorylated and total DRP1 and MFF, Mid49, Mid51, FIS1, MFN1, MFN2, OPA1, PINK1, Parkin, and Vinculin (loading control). (D-E) Densitometric quantification of protein expression relative to saline treatment. Data are shown as the mean ± SEM. *p<0.05. Comparisons of treatment were assessed by paired Students t-test.

Figure 4.

Mitochondrial membrane potential and ultrastructure. (A) Representative confocal micrographs of resting mitochondrial membrane potential (Δψm; 150X magnification). Micrographs are shown as TMRM alone (left) or the merge of TMRM, mitotracker deep red, and DAPI. (B) Transmission electron micrographs of mitochondrial ultrastructure and content (Scale bars (black) = 2 μm). Quantitation of (C) Δψm, (D) mitochondrial fragmentation, (E) mitochondrial content, and (F) mitochondrial associated lipid droplets. Differences are represented relative to saline (%). Data are shown as the mean ± SEM. *p<0.05. Comparisons of treatment were assessed by paired Students t-test.

Figure 5.

Oxidative phosphorylation and electron transfer capacity. (A-B) Assessment of leak respiration (L), OXPHOS (P) and ET (E) capacity in permeabilized skeletal muscle fibers. (C) Ratio of maximal ADP-stimulated O2 flux in the presence of pyruvate and malate to the leak rate in the absence in ADP. (D) Ratio of maximal ADP-stimulated O2 flux in the presence of palmitoylcarnitine to the leak rate in the absence in ADP. (E) Tissue concentrations of ATP. (F) Enzymatic activity of citrate synthase. (G) mtDNA content (COXII/18S). PM: pyruvate and malate, D: adenosine diphosphate, G: glutamate, S: succinate, F: FCCP, As: ascorbate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol. Data are shown as the mean ± SEM. Comparisons of treatment × time were assessed by two-way repeated measures ANOVA with Tukey’s multiple comparisons. Comparisons of treatment were assessed by paired Students t-test.

Figure 6.

Glucose homeostasis and insulin sensitivity. (A) Fasting glucose and clamp-derived euglycemia, (B) fasting insulin and clamp-derived hyperinsulinemia. (C) peripheral insulin sensitivity, (D) basal rate of HGP, and (E) suppression of HGP by insulin. Data are shown as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.01. Comparisons of treatment × time were assessed by two-way repeated measures ANOVA with Tukey’s multiple comparisons. Comparisons of treatment were assessed by paired Students t-test.

Figure 7.

Whole-body substrate metabolism. (A) Fasting and insulin stimulated FFAs and (B) suppression of FFA’s in response to insulin stimulation. Basal (C) carbohydrate, (D) protein, (E) and fat oxidation rates. (F) Change in the rate of fat oxidation from basal to insulin stimulation. Data are shown as the mean ± SEM with exception to panel C which is displayed as a box (mean) and whiskers (10–90% CI). *p<0.05, **p<0.01, ***p<0.01. Comparisons of treatment × time were assessed by two-way repeated measures ANOVA with Tukey’s multiple comparisons. Comparisons of treatment were assessed by paired Students t-test.