Metabolic remodeling of human skeletal myocytes by cocultured adipocytes depends on the lipolytic state of the system - PubMed (original) (raw)

. 2011 Jul;60(7):1882-93.

doi: 10.2337/db10-0427. Epub 2011 May 20.

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Metabolic remodeling of human skeletal myocytes by cocultured adipocytes depends on the lipolytic state of the system

Jean-Paul Kovalik et al. Diabetes. 2011 Jul.

Abstract

Objective: Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism.

Research design and methods: Interactions between these two tissues were modeled using a coculture system composed of primary human adipocytes and human skeletal myotubes derived from lean or obese donors. Metabolic analysis of myocytes was performed after coculture with lipolytically silent or activated adipocytes and included transcript and metabolite profiling along with assessment of substrate selection and insulin action.

Results: Cocultured adipocytes increased myotube mRNA expression of genes involved in oxidative metabolism, regardless of the donor and degree of lipolytic activity. Adipocytes in the basal state sequestered free fatty acids, thereby forcing neighboring myotubes to rely more heavily on glucose fuel. Under this condition, insulin action was enhanced in myotubes from lean but not obese donors. In contrast, when exposed to lipolytically active adipocytes, cocultured myotubes shifted substrate use in favor of fatty acids, which was accompanied by intracellular accumulation of triacylglycerol and even-chain acylcarnitines, decreased glucose oxidation, and modest attenuation of insulin signaling.

Conclusions: The effects of cocultured adipocytes on myocyte substrate selection and insulin action depended on the metabolic state of the system. These findings are relevant to understanding the metabolic consequences of intermuscular adipogenesis.

© 2011 by the American Diabetes Association.

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Figures

FIG. 1.

FIG. 1.

Adipocyte-myocyte coculture model. Computed tomography images of the thigh comparing (A) a young healthy female subject with (B) an older, obese female subject. a: Thigh muscle; b: subcutaneous adipose tissue; c: intermuscular adipose tissue. C: Free fatty acid concentration of the culture medium after 24-h exposure to basal (unstimulated) or stimulated (+100 μM IBMX) human adipocytes compared with no adipocytes. D: Individual free fatty acids in culture medium exposed to stimulated adipocytes for 24 h compared with no adipocytes. E: Adipokine concentrations in the culture medium after 24-h exposure to basal (unstimulated) or stimulated (+100 μM IBMX) human adipocytes. Results are expressed as mean ± SEM and representative of at least two experiments performed in triplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test. FA, fatty acid. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

FIG. 2.

Adipocyte exposure alters glucose metabolism in primary human myotubes. Primary human skeletal myotubes from lean (A–D) and severely obese (E–H) women were maintained 24 h in the absence or presence of human adipocytes under basal or stimulated (100 μmol/L IBMX) conditions. After 24 h of coculture, adipocytes were removed, and myotubes were washed and incubated 2 h in α-MEM containing 2 μCi/mL

d

-[U-14C]glucose. Rates of glycogen synthesis and glucose oxidation were determined by measuring label incorporation into glycogen (A, B, E, and F) and CO2 (C, D, G, and H). Results are expressed as mean ± SEM and representative of at least three experiments performed in triplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test.

FIG. 3.

FIG. 3.

Effects of adipocyte exposure on fatty acid metabolism in primary human myotubes. Primary human skeletal myotubes from lean (A, B, and E) and severely obese (C, D, and F) women were maintained 24 h in the absence or presence of human adipocytes under basal or stimulated (100 μmol/L IBMX) conditions. After 24 h of coculture, adipocytes were removed, and myotubes were washed and incubated 2 h with α-MEM containing 100 μmol/L [14C]oleate (A–D) or 5 mmol/L [14C]glucose (E and F). Label incorporation into CO2 was measured to assess oxidation rates of fatty acid and glucose, respectively. The addition of 100 μmol/L etomoxir during the glucose oxidation assay (E and F) was used to block oxidation of intracellular lipids. Results are expressed as mean ± SEM and representative of at least three experiments performed in triplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test.

FIG. 4.

FIG. 4.

Adipocyte exposure alters acylcarnitine accumulation in primary human myotubes. Primary human skeletal myotubes from lean (A) and severely obese (B) women were maintained 24 h in the absence or presence of human adipocytes under basal conditions. After 24 h of coculture, adipocytes were removed, myotubes were washed, and cell lysates were prepared for acylcarnitine profiling by mass spectrometry. C3DC and C5OH are isobaric species. The ratio of total medium-chain relative to total long-chain acylcarnitines was calculated to assess potential flux limitations at the medium-chain acyl-CoA dehydrogenase step of β-oxidation. Results are expressed as mean ± SEM and representative of at least three experiments performed in triplicate. Abbreviations reflect carbon chain length (e.g., C2, acetylcarnitine). DC, dicarboxylic acid; OH, hydroxylated species. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test.

FIG. 5.

FIG. 5.

Effect of stimulated adipocyte exposure on acylcarnitine accumulation in primary human myotubes. Primary human skeletal myotubes from lean (A) and severely obese (B) women were maintained 24 h in the absence or presence of human adipocytes under stimulated (100 μmol/L IBMX) conditions. After 24 h of coculture, adipocytes were removed, myotubes were washed, and cell lysates were prepared for acylcarnitine profiling by mass spectrometry. C3DC and C5OH are isobaric species. The ratio of total medium-chain relative to total long-chain acylcarnitines was calculated to assess potential flux limitations at the medium-chain acyl-CoA dehydrogenase step of β-oxidation. Results are expressed as mean ± SEM and are representative of at least three experiments performed in triplicate. Abbreviations reflect carbon chain length (e.g., C2, acetylcarnitine). DC, dicarboxylic acid; OH, hydroxylated species. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test.

FIG. 6.

FIG. 6.

Adipocyte exposure alters myotube mRNA expression. Primary human skeletal myotubes from lean (A and C) and severely obese (B and D) women were maintained 24 h in the absence or presence of human adipocytes under basal or stimulated (100 μmol/L IBMX) conditions. After 24 h of coculture, adipocytes were removed and myotubes were washed and harvested for RNA extraction. Myotube expression of candidate mRNAs involved in fatty acid oxidation (A and B) or glucose metabolism (C and D) was measured by real-time quantitative PCR and normalized to mRNA levels of 18S. Data are expressed as mean ± SEM of the fold change in mRNA abundance relative to the control cells that were not exposed to adipocytes (dashed line). Results are representative of at least three experiments performed in triplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test. CS, citrate synthase; Glut1, glucose transporter 1; Glut4, glucose transporter 4; HK1, hexokinase 1; HK2, hexokinase 2; LDHa, lactate dehydrogenase α; LDHb, lactate dehydrogenase β; MCD, malonyl-CoA decarboxylase; VLAD, very long-chain acyl-CoA dehydrogenase; TXNIP, thioredoxin-interacting protein.

FIG. 7.

FIG. 7.

Coculture exposure for 24 h enhances myotube insulin response. Primary human skeletal myotubes from lean (A–D) and severely obese (E–H) women were maintained 24 h in the absence or presence of human adipocytes under basal or stimulated (100 μmol/L IBMX) conditions. Adipocytes were removed, and myotubes were washed and incubated with α-MEM ± 100 nmol/L insulin for 10 min. Protein extracts were used for immunoblot analysis of Akt Ser473 phosphorylation, corrected for total protein loading using Memcode staining. Measurement of total Akt protein showed no treatment effects. Rates of glycogen synthesis were measured after a 2-h incubation with α-MEM containing 5 mmol/L [14C]glucose ± 100 nmol/L insulin. Results are expressed as mean ± SEM and representative of at least three experiments performed in triplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test.

FIG. 8.

FIG. 8.

Chronic coculture exposure dampens myotube insulin response. Primary human skeletal myotubes from lean women were maintained 96 h in the absence or presence of human adipocytes under stimulated (100 μmol/L IBMX) conditions. Adipocytes were removed, and myotubes were washed and incubated with α-MEM ± 100 nmol/L insulin for 10 min. A: Protein extracts were used for immunoblot analysis of Akt Ser473 phosphorylation, corrected for total protein loading using Memcode staining. Measurement of total Akt protein showed no treatment effects. B: GSK-3β Ser9 phosphorylation, corrected for total protein using tubulin staining. C–F: Myotube extracts were used for measurement of total triacylglycerol (C), total diacylglycerol (D), total ceramide (E), and acylcarnitine profiling (F). C3DC and C5OH are isobaric species. The ratio of total medium-chain relative to total long-chain acylcarnitines was calculated to assess potential flux limitations at the medium-chain acyl-CoA dehydrogenase step of β-oxidation. Results are expressed as mean ± SEM and representative of at least three experiments performed in triplicate, except for the TAG, DAG, and ceramides, which represent two independent experiments performed in quadruplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test. Abbreviations reflect carbon chain length (e.g., C2, acetylcarnitine). DC, dicarboxylic acid; OH, hydroxylated species.

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