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. 2012 Apr;55(4):1140-50.
doi: 10.1007/s00125-011-2419-7. Epub 2012 Jan 15.

Localisation and composition of skeletal muscle diacylglycerol predicts insulin resistance in humans

B C Bergman  1 , D M Hunerdosse, A Kerege, M C Playdon, L Perreault
Affiliations

Localisation and composition of skeletal muscle diacylglycerol predicts insulin resistance in humans

B C Bergman et al. Diabetologia. 2012 Apr.

Abstract

Aims/hypothesis: We sought to evaluate if the cellular localisation and molecular species of diacylglycerol (DAG) were related to insulin sensitivity in human skeletal muscle.

Methods: Healthy sedentary obese controls (Ob; n = 6; mean±SEM age 39.5 ± 2.3 years; mean ± SEM BMI 33.3 ± 1.4 kg/m(2)), individuals with type 2 diabetes (T2D; n = 6; age 44 ± 1.8 years; BMI 30.1 ± 2.3 kg/m(2)), and lean endurance-trained athletes (Ath; n = 10; age 35.4 ± 3.1 years; BMI 23.3 ± 0.8 kg/m(2)) were studied. Insulin sensitivity was determined using an IVGTT. Muscle biopsy specimens were taken after an overnight fast, fractionated using ultracentrifugation, and DAG species measured using liquid chromatography/MS/MS.

Results: Total muscle DAG concentration was higher in the Ob (mean ± SEM 13.3 ± 1.0 pmol/μg protein) and T2D (15.2 ± 1.0 pmol/μg protein) groups than the Ath group (10.0 ± 0.78 pmol/μg protein, p = 0.002). The majority (76-86%) DAG was localised in the membrane fraction for all groups, but was lowest in the Ath group (Ob, 86.2 ± 0.98%; T2D, 84.2 ± 1.2%; Ath, 75.9 ± 2.7%; p = 0.008). There were no differences in cytoplasmic DAG species (p > 0.12). Membrane DAG species C18:0/C20:4, Di-C16:0 and Di-C18:0 were significantly more abundant in the T2D group. Cytosolic DAG species were negatively related to activation of protein kinase C (PKC)ε but not PKCθ, whereas membrane DAG species were positively related to activation of PKCε, but not PKCθ. Only total membrane DAG (r = -0.624, p = 0.003) and Di-C18:0 (r = -0.595, p = 0.004) correlated with insulin sensitivity. Disaturated DAG species were significantly lower in the Ath group (p = 0.001), and significantly related to insulin sensitivity (r = -0.642, p = 0.002).

Conclusions/interpretation: These data indicate that both cellular localisation and composition of DAG influence the relationship to insulin sensitivity. Our results suggest that only saturated DAG in skeletal muscle membranes are related to insulin resistance in humans.

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Figures

Fig. 1
Fig. 1
Western blot showing enrichment of membrane and cytosolic proteins in human skeletal muscle biopsy specimens separated by ultracentrifugation. The membrane marker antibody is Na/K ATPase α1, and the cytosolic marker is glyceraldehyde-3-phosphate dehydrogenase. Cyto, cytosol; Mem, membrane; T2D, type 2 diabetes
Fig. 2
Fig. 2
DAG concentration by group in a total muscle homogenate, b cytosolic fraction and c membrane fraction and d percentage DAG in membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05; significantly different from Ath, ‡p<0.05. T2D, type 2 diabetes
Fig. 2
Fig. 2
DAG concentration by group in a total muscle homogenate, b cytosolic fraction and c membrane fraction and d percentage DAG in membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05; significantly different from Ath, ‡p<0.05. T2D, type 2 diabetes
Fig. 2
Fig. 2
DAG concentration by group in a total muscle homogenate, b cytosolic fraction and c membrane fraction and d percentage DAG in membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05; significantly different from Ath, ‡p<0.05. T2D, type 2 diabetes
Fig. 2
Fig. 2
DAG concentration by group in a total muscle homogenate, b cytosolic fraction and c membrane fraction and d percentage DAG in membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05; significantly different from Ath, ‡p<0.05. T2D, type 2 diabetes
Fig. 3
Fig. 3
Relationship between insulin sensitivity (Si) and a total cytosolic DAG (r=0.217, p=0.34) and b total membrane DAG, r=−0.624, p=0.003 in T2D, Ath and Ob groups. To convert Si values to SI units multiply by 0.167
Fig. 3
Fig. 3
Relationship between insulin sensitivity (Si) and a total cytosolic DAG (r=0.217, p=0.34) and b total membrane DAG, r=−0.624, p=0.003 in T2D, Ath and Ob groups. To convert Si values to SI units multiply by 0.167
Fig. 4
Fig. 4
DAG molecular species in T2D (light grey), Ath (dark grey) and Ob (speckled) groups localised in the a cytosolic fraction and b membrane fraction, and low-abundance DAG species in the c cytosolic fraction and d membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from Ath, †p<0.05
Fig. 4
Fig. 4
DAG molecular species in T2D (light grey), Ath (dark grey) and Ob (speckled) groups localised in the a cytosolic fraction and b membrane fraction, and low-abundance DAG species in the c cytosolic fraction and d membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from Ath, †p<0.05
Fig. 4
Fig. 4
DAG molecular species in T2D (light grey), Ath (dark grey) and Ob (speckled) groups localised in the a cytosolic fraction and b membrane fraction, and low-abundance DAG species in the c cytosolic fraction and d membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from Ath, †p<0.05
Fig. 4
Fig. 4
DAG molecular species in T2D (light grey), Ath (dark grey) and Ob (speckled) groups localised in the a cytosolic fraction and b membrane fraction, and low-abundance DAG species in the c cytosolic fraction and d membrane fraction. Values are means±SEM. Significantly different from Ob, *p<0.05; significantly different from Ath, †p<0.05
Fig. 5
Fig. 5
Ratio of membrane (Mem) to cytosolic (Cyto) content of a PKCε and b PKCθ in T2D (light grey), Ath (dark grey), and Ob (speckled) groups. Values are means±SEM
Fig. 5
Fig. 5
Ratio of membrane (Mem) to cytosolic (Cyto) content of a PKCε and b PKCθ in T2D (light grey), Ath (dark grey), and Ob (speckled) groups. Values are means±SEM
Fig. 6
Fig. 6
Relationship between PKCε activation and a cytosolic DAG (r=−0.552, p=0.009) and b membrane DAG (r=0.507, p=0.019) in T2D, Ath and Ob groups
Fig. 6
Fig. 6
Relationship between PKCε activation and a cytosolic DAG (r=−0.552, p=0.009) and b membrane DAG (r=0.507, p=0.019) in T2D, Ath and Ob groups
Fig. 7
Fig. 7
a Content of disaturated DAG in membrane fraction in T2D, Ath and Ob groups. b Relationship between disaturated membrane DAG and insulin sensitivity (Si) (r=−0.642, p=0.002). c Percentage of disaturated DAG relative to total membrane DAG. d Relationship between percentage disaturated DAG relative to total membrane DAG and Si (r=−0.633, p=0.002). To convert Si values to SI units multiply by 0.167. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05. T2D, type 2 diabetes
Fig. 7
Fig. 7
a Content of disaturated DAG in membrane fraction in T2D, Ath and Ob groups. b Relationship between disaturated membrane DAG and insulin sensitivity (Si) (r=−0.642, p=0.002). c Percentage of disaturated DAG relative to total membrane DAG. d Relationship between percentage disaturated DAG relative to total membrane DAG and Si (r=−0.633, p=0.002). To convert Si values to SI units multiply by 0.167. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05. T2D, type 2 diabetes
Fig. 7
Fig. 7
a Content of disaturated DAG in membrane fraction in T2D, Ath and Ob groups. b Relationship between disaturated membrane DAG and insulin sensitivity (Si) (r=−0.642, p=0.002). c Percentage of disaturated DAG relative to total membrane DAG. d Relationship between percentage disaturated DAG relative to total membrane DAG and Si (r=−0.633, p=0.002). To convert Si values to SI units multiply by 0.167. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05. T2D, type 2 diabetes
Fig. 7
Fig. 7
a Content of disaturated DAG in membrane fraction in T2D, Ath and Ob groups. b Relationship between disaturated membrane DAG and insulin sensitivity (Si) (r=−0.642, p=0.002). c Percentage of disaturated DAG relative to total membrane DAG. d Relationship between percentage disaturated DAG relative to total membrane DAG and Si (r=−0.633, p=0.002). To convert Si values to SI units multiply by 0.167. Significantly different from Ob, *p<0.05; significantly different from T2D, †p<0.05. T2D, type 2 diabetes
Fig. 8
Fig. 8
Skeletal muscle protein levels of a DGKδ, b DGAT1, c lipin 1 and d SCD1 in T2D (light grey), Ath (dark grey) and Ob (speckled) groups. Values are means±SEM. Significantly different from Ob, *p<0.05. T2D, type 2 diabetes
Fig. 8
Fig. 8
Skeletal muscle protein levels of a DGKδ, b DGAT1, c lipin 1 and d SCD1 in T2D (light grey), Ath (dark grey) and Ob (speckled) groups. Values are means±SEM. Significantly different from Ob, *p<0.05. T2D, type 2 diabetes
Fig. 8
Fig. 8
Skeletal muscle protein levels of a DGKδ, b DGAT1, c lipin 1 and d SCD1 in T2D (light grey), Ath (dark grey) and Ob (speckled) groups. Values are means±SEM. Significantly different from Ob, *p<0.05. T2D, type 2 diabetes
Fig. 8
Fig. 8
Skeletal muscle protein levels of a DGKδ, b DGAT1, c lipin 1 and d SCD1 in T2D (light grey), Ath (dark grey) and Ob (speckled) groups. Values are means±SEM. Significantly different from Ob, *p<0.05. T2D, type 2 diabetes
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