Dynamic changes in fat oxidation in human primary myocytes mirror metabolic characteristics of the donor

Abstract

Metabolic flexibility of skeletal muscle, that is, the preference for fat oxidation (FOx) during fasting and for carbohydrate oxidation in response to insulin, is decreased during insulin resistance. The aim of this study was to test the hypothesis that the capacity of myotubes to oxidize fat in vitro reflects the donor's metabolic characteristics. Insulin sensitivity (IS) and metabolic flexibility of 16 healthy, young male subjects was determined by euglycemic hyperinsulinemic clamp. Muscle samples were obtained from vastus lateralis, cultured, and differentiated into myotubes. In human myotubes in vitro, we measured suppressibility (glucose suppression of FOx) and adaptability (an increase in FOx in the presence of high palmitate concentration). We termed these dynamic changes in FOx metabolic switching. In vivo, metabolic flexibility was positively correlated with IS and maximal oxygen uptake and inversely correlated with percent body fat. In vitro suppressibility was inversely correlated with IS and metabolic flexibility and positively correlated with body fat and fasting FFA levels. Adaptability was negatively associated with percent body fat and fasting insulin and positively correlated with IS and metabolic flexibility. The interindividual variability in metabolic phenotypes was preserved in human myotubes separated from their neuroendocrine environment, which supports the hypothesis that metabolic switching is an intrinsic property of skeletal muscle.

Figure 1

Characteristics of in vitro metabolic switching assays. (A) Dose response of FOx (measured by ¹⁴CO₂ production) to an increasing glucose concentration (suppressibility). (B) Dose response of FOx (measured by ¹⁴CO₂ production) to an increasing palmitate concentration (adaptability). Arrows indicate a dynamic change in FOx, representing an in vitro correlate to the clinical phenotype. Muscle cells were grown and differentiated into myotubes in 24-well plates. FOx assays were performed after 5 days of differentiation. Myotubes were incubated for 3 hours in serum-free media with increasing concentrations of glucose or palmitate. Assays were performed in duplicate, and data were normalized to protein content. Data are from 1 representative experiment.

Figure 2

In vitro suppressibility is an intrinsic characteristic of muscle cells. The potential of glucose to suppress FOx (CO₂) in vitro (% suppression of ¹⁴CO₂ = [1 – (FOx at 5 mM glucose / FOx at 0 mM glucose)] × 100) is correlated with in vivo metabolic flexibility [as indicated by ΔRQ (clamp), an insulin-stimulated change in RQ, measured by indirect calorimetry during the clamp] (A); in vivo IS represented by glucose disposal rate, measured by clamp (B); percent body fat, measured by dual energy X-ray absorptiometry (C); and in vivo fasting FFA levels (D). Muscle cells from 16 individuals were grown and differentiated into myotubes in 24-well plates. Myotubes were preincubated in glucose- and serum-free medium and incubated for 3 hours with 1 μCi/ml ¹⁴C-palmitate, without glucose (to measure maximal FOx) or with 5 mM glucose (to measure suppressed FOx). After incubation, ¹⁴CO₂ and ¹⁴C-intermediate metabolites of FOx were determined. Assays were performed in duplicates and data were normalized to protein content.

Figure 3

In vitro adaptability is an intrinsic characteristic of muscle cells. The capacity of muscle cells to increase FOx (measured by ¹⁴CO₂ production) in the presence of high palmitate concentration (fold increase in ¹⁴CO₂ = ¹⁴CO₂ at 100 μM palmitate / ¹⁴CO₂ at 0 μM palmitate) in vitro is correlated with flexible clinical phenotype [ΔRQ (clamp)] (A); insulin-sensitive clinical phenotype (IS is represented by the glucose disposal rate, measured by clamp) (B); VO2max in vivo (C); percent body fat, measured by dual energy X-ray absorptiometry (D); and fasting insulin levels on a standard diet (E). Muscle cells from 16 individuals were grown and differentiated into myotubes in 24-well plates. Myotubes were preincubated in glucose- and serum-free medium and incubated for 3 hours with 1 μCi/ml ¹⁴C-palmitate, in the presence or absence of 100 μm cold palmitate. After incubation, levels of ¹⁴CO₂ and ¹⁴C-intermediate metabolites of FOx were determined. Assays were performed in duplicate, and data were normalized to protein content. Data were adjusted for basal ¹⁴CO₂ production (¹⁴CO₂ production at 1 μCi/ml labeled palmitate and 0 μM cold palmitate).

Figure 4

Model of in vitro metabolic switching. Glucose suppressibility: In insulin-free medium, adaptable cells are able to maintain a relatively high rate of FOx in the presence of glucose compared with nonadaptable cells. According to our studies, in vivo insulin responsiveness decreases with increasing in vitro suppressibility, which indicates greater glucose suppression of FOx in insulin resistance and metabolic inflexibility. Metabolic adaptability: Adaptable cells possess a higher capacity to increase FOx when exposed to a high palmitate concentration compared with nonadaptable cells. According to our studies, in vivo insulin responsiveness increases with increasing in vitro adaptability, which indicates a greater capacity to increase FOx in subjects with higher IS.