Remodeling of oxidative energy metabolism by galactose improves glucose handling and metabolic switching in human skeletal muscle cells

Abstract

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [(14)C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [(14)C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.

Figure 1. Cellular handling of labeled galactose.

Myotubes were grown in DMEM-media with 5.5 mM glucose (A) or 5.5 mM galactose (B). For cellular uptake (n = 4) (C) and oxidation (CO₂-trapping) (n = 4) (D) the cells were exposed to [1-¹⁴C]galactose (1 µCi/ml, 200 µM) for 4 h before harvesting. For lipogenesis (n = 6) (E) the cells were exposed to [U-¹⁴C]glucose (1 µCi/ml, 200 µM) or [1-¹⁴C]galactose (1 µCi/ml, 200 µM) for 24 h before harvesting as described in Materials and Methods. Values represent nmol/mg cell protein given as means ± SEM. *P<0.05 vs. glucose pretreatment.

Figure 2. Effect of galactose treatment on oleic acid metabolism.

Myotubes were either grown in DMEM-media with 5.5 mM glucose or 5.5 mM galactose during the whole seeding period, or with 5.5 mM glucose during proliferation and 5.5 mM galactose during differentiation (Galactose_d). Thereafter, the cells were exposed to [1-¹⁴C]oleic acid (1 µCi/ml, 100 µM) for 4 h as described in Materials and Methods. The figures show cellular uptake (n = 6) (A), oxidation (n = 6) (B), % oxidized (CO₂/CA+CO₂) (n = 6) (C) and suppressibility (n = 3) (D) of [1-¹⁴C]oleic acid. Suppressibility, defined as the ability of the cells to decrease oleic acid oxidation by acutely added glucose, was calculated as: [(1-(oxidation of oleic acid at 5 mM glucose/oxidation of oleic acid at no glucose added))×100%]. Values represent means ± SEM. *P<0.05 vs. glucose.

Figure 3. Effect of galactose treatment on glucose metabolism.

Myotubes were grown in DMEM-media with 5.5 mM glucose or 5.5 mM galactose. Thereafter, the cells were exposed to [U-¹⁴C]glucose (1 µCi/ml, 100 µM) for 4 h as described in Materials and Methods. The figures show cellular uptake (n = 5–6) (A), oxidation (n = 5–6) (B) and reserve capacity (oxidation with FCCP (1 µM) – basal oxidation) (n = 3) (C) of [U-¹⁴C]glucose. Values represent means ± SEM. *P<0.05 vs. glucose.

Figure 4. Effect of chronic galactose treatment on gene and protein expressions.

Myotubes were grown in DMEM-media with 5.5 mM glucose or 5.5 mM galactose. Total RNA was isolated from the cells and analyzed by qPCR, while protein samples were harvested analyzed as described in Materials and Methods. Gene expressions were normalized to 36B4 and protein expressions to β-actin, except phosphorylated AMP-activated protein kinase (p-AMPK), which were normalized to total AMPK. Values in A and B represent fold change of genes/proteins in galactose-treated myotubes relative to glucose-treated myotubes, given as means ± SEM (n = 5). (A) Genes analyzed; CPT1b, carnitine palmitoyltransferase-1b; CYC1, cytochrome C; MCAD, acyl-coenzyme A-dehydrogenase; MYH2, myosin heavy chain 2, SLC2A1 and SLC2A4; glucose transporter 1 and 4, HKII; hexokinase II, GALK1 and 2; galactokinase 1 and 2, GALT; galactose-1-phosphate uridylyltransferase, PDK4; pyruvate dehydrogenase kinase 4. (B) Protein expression of myosin, ATP synthase subunit, slow muscle, complex II subunit, complex IV subunit II, complex I subunit NDUFB8, PDK4; pyruvate dehydrogenase 4, P-AMPK and AMPK. (C) Representative corresponding Western blots.

Figure 5. Effect of galactose treatment on mitochondrial and neutral lipid content.

Myotubes were grown in DMEM-media with 5.5 mM glucose or 5.5 mM galactose. The cells were stained for mitochondria, neutral lipid and nuclei as described in Materials and Methods. The figures show (A) pictures of stained myotubes with mitochondria (red), neutral lipids (green) and nuclei (blue), (B) mitochondrial content, (C) neutral lipid content. Results represent fold change relative to glucose given as means ± SEM, (n = 3) and the data are normalized to number of nuclei. *P<0.05 vs. glucose. Arbitrary units for mitochondrial content per nucleus and neutral lipid content were 552232±137107, 860881±218244, respectively for glucose and 942449±71302, 1299938±102390, respectively for galactose.