Strong evidence suggests that DAG may be an important link between tissue lipids and insulin resistance. Skeletal muscle DAG concentration is elevated in individuals with obesity and type 2 diabetes [22
], and has been shown to be negatively related to insulin sensitivity [6
]. Most studies report whole cell DAG content, yet there are a large number of potential molecular species of DAG, as well as variability in intracellular localisation that could influence biological action. We hypothesised that both membrane DAG localisation and saturated molecular species would be independently related to skeletal muscle insulin action. Indeed, major findings from the present study confirm our hypotheses. Of the 16 measurable molecular species of DAG in human skeletal muscle, the majority are localised to membranes, and only membrane DAG were positively related to PKC activation and insulin resistance. Three particular species of DAG were significantly higher in individuals with type 2 diabetes than the other groups, but only di-C18:0 was significantly related to insulin resistance. Decreasing DAG localised to skeletal muscle membranes, or decreasing stearate-containing DAG may be a novel therapeutic target for the treatment and prevention of insulin resistance in humans.
Currently, all DAG in human skeletal muscle is thought to promote insulin resistance. Consistent with previous investigations, total intramuscular DAG concentration correlated inversely with insulin sensitivity in this study. Nevertheless, because DAG induces insulin resistance by activating PKC in membranes, we hypothesised that only membrane DAG would be related to PKC activation and insulin resistance in skeletal muscle. Supportive data were reported in a recent study demonstrating increased skeletal muscle membrane DAG content during aging, which was associated with increased PKC activation and insulin resistance in rodents [17
]. Similarly, liver samples from morbidly obese participants also revealed compartmentalisation of DAG, with cytosolic, but not membrane, DAG correlated with PKCε activation [23
]. Our data show the importance of DAG compartmentalisation in human skeletal muscle, as only membrane DAG was related to PKC activation and decreased insulin sensitivity. In contrast, cytosolic DAG was inversely related to insulin resistance and PKCε activation. Together, these data are the first to reveal that membrane localisation of DAG in skeletal muscle, rather than total intramuscular concentration, drives insulin resistance in humans.
Alterations in DAG production induced by obesity and/or type 2 diabetes may explain differences in DAG localisation. For example, hyperglycaemia increases phospholipase C activity [24
], which degrades membrane phospholipids and would produce membrane DAG. Increased C18:0/C20:4 DAG in type 2 diabetes is consistent with phospholipase DAG generation. Hyperinsulinaemia and hyperglycaemia also increase de novo DAG synthesis [25
], which occurs at the endoplasmic reticulum [26
], and could be an important mechanism promoting membrane DAG accumulation in obesity and diabetes. In contrast, DAG formed during IMTG degradation [27
] would promote cytosolic accumulation and may be less prominent in type 2 diabetes. The combination of enhanced de novo DAG synthesis driven by elevated plasma concentrations of glucose and insulin, high saturated fat intake [28
], and less muscle lipid desaturation form a plausible explanation for how saturated membrane DAG accumulate in obesity and diabetes. Exploiting pathways dictating intracellular DAG localisation may prove a novel target for insulin sensitisation.
Whether decreasing membrane DAG increases insulin sensitivity is not known, but would be supported by these data. Altered abundance of DGKδ, an enzyme responsible for converting DAG into phosphatidic acid to terminate DAG signalling [29
], may be one manner of doing so. DGKδ exists in many locations of the cell, including endoplasmic reticulum [30
], neuromuscular junction [31
], cytoskeletal compartments [32
] and the nucleus, which confirms intracellular DAG compartmentalisation [29
]. In a previous study, hyperglycaemia downregulated DGKδ and explained increased DAG concentration in individuals with type 2 diabetes [33
]. We found no differences in whole cell DGKδ between groups, suggesting that changes in DAG content and/or localisation was not due to downregulation of this enzyme. Of note, hyperglycaemia in our participants with type 2 diabetes was ~2.2 mmol/l lower than in the previous report, and may contribute to the lack of differences in this study. Membrane DAG content may also be influenced by muscle oxidative capacity. Preventing mitochondrial oxidative damage preserved oxidative capacity and precluded membrane DAG accumulation and insulin resistance in aging mice [17
]. Similarly, muscle oxidative capacity is increased in endurance-trained athletes, who also had decreased membrane DAG content in the present study. These studies suggest a possible link between oxidative capacity, membrane DAG localisation and insulin sensitivity. Nevertheless, further studies are needed to determine if membrane DAG content can be decreased, and if this change results in increased insulin sensitivity.
In addition to DAG localisation, DAG composition appears to discriminate DAG function, and therefore plays an important role linking muscle lipids to insulin resistance. Of the 16 measurable membrane DAG species, only di-C18:0 was significantly related to insulin sensitivity. Interestingly, these data corroborate previous data from our laboratory and others suggesting that saturated DAG has a particularly negative impact on insulin sensitivity [4
]. This is exemplified by the observation that disaturated membrane DAGs were negatively related to insulin sensitivity in the cohort as a whole, and were also significantly lower in our insulin-sensitive endurance-trained athletes. Similar to other reports [5
], SCD1 content was increased in athletes, which may be one mechanism explaining less saturated skeletal muscle DAG in this group. Less saturated membrane DAG in endurance-trained athletes may help explain how they maintain insulin sensitivity, despite a high IMTG concentration, the so-called ‘athletes paradox’ [34
]. Further, these data highlight that DAG species are not homogeneous and probably have dissimilar impacts on insulin sensitivity. Similar data showing that unique DAG species correlated with PKC activation and insulin sensitivity were recently reported in human liver [23
]. However, not all data agree, as two recent studies [10
] did not observe the relationship between skeletal muscle DAG molecular species and insulin sensitivity observed in the present study. This apparent discrepancy can be reconciled when considering the relative importance of DAG species differs by their subcellular location. Similar to the data of Coen et al [10
] and Dube et al [35
], we also did not find a significant relationship between insulin sensitivity and individual DAG species when DAG species from the whole cell were analysed. However, when only membrane species were examined, the relationship between di-C18:0 DAG and insulin sensitivity was revealed. Therefore our data agree with previous studies in this area, but, importantly, extend what is known, highlighting the importance of DAG based on location and species.
DAG is thought to decrease insulin sensitivity in skeletal muscle by promoting activity of conventional and novel PKC isoforms [11
]. Localisation of DAG species was important for PKC activation, as C16:1/C18:1 was positively related to PKCε activation in the membrane, with no relationship in the cytosol. The prevailing paradigm would contend that polyunsaturated fatty acid-containing DAG activate PKC [36
]; however, the literature contains reports suggesting that saturated DAGs are related to PKC activation as well [16
]. Nevertheless, we found no significant relationships between polyunsaturated fatty acid containing-DAG and PKCθ and PKCε activation, but rather an effect of monounsaturated fatty acid containing-DAG in the membrane on PKCε. Divergence between membrane DAG molecular species related to insulin sensitivity and PKC activation may due to involvement of PKC isoforms PKCßII and PKCδ, which we did not measure [14
], and/or implicate non-PKC-mediated mechanisms for the effect of DAG on insulin resistance in humans [40
]. Alternatively, while there are data suggesting that PKC isoforms are involved in acute insulin resistance [14
], other data suggest that they may not be related to chronic insulin resistance [44
]. Our data can also be interpreted as placing less importance on DAG-induced PKC activation in chronic insulin resistance in humans. These data are also consistent with the interpretation that saturated DAG are only a marker of insulin resistance, and may reflect increased saturated lipid content of other species, such as ceramides [9
] and/or long-chain acyl-CoA [8
], which may play a more direct role.
There are several limitations to this study. It is well known that dietary fat influences muscle lipid composition [47
], and a recent study showed that a 1-week dietary intervention changed the composition of DAG in healthy lean men and women [28
]. Therefore our 3-day dietary control, designed to ensure energy balance, may have minimised differences between groups. Although others have reported PKC activation using membrane/cytosol ratios [15
], variability between participants and a small sample size in this study may have precluded finding relationships that exist between PKC isoforms and DAG species. We did not have a lean control group, which does not allow us to discern the influence of BMI from exercise training when comparing athletes with the other two groups. In addition, skeletal muscle was only separated into two fractions. Therefore alterations in DAG localised to endoplasmic reticulum from de novo synthesis cannot be delineated from plasma membrane DAG with our methods. Owing to the hydrophobic nature of DAG, the cytosolic fraction may also not truly represent ‘cytosolic’ distribution, but rather DAG localised to small membrane structures or in cytosolic lipid droplets pelleting during the first centrifugation step. In addition, we measured insulin sensitivity using an IVGTT, which does not isolate the influence of muscle on insulin sensitivity as well as a hyperinsulinaemic/euglycaemic clamp. Further, we are assuming that DAG promotes insulin resistance by binding to C1-containing domains of PKC [40
]. However, other molecules with C1-containing domains may be stimulated by DAG, including chimaerins, protein kinase D, ras guanyl nucleotide-releasing proteins (RasGRPs), mammalian homologues of C. elegans Unc-13 (Munc13s) and DAG kinase γ [40
In summary, these data are the first report of cellular localisation of molecular species of DAG in human skeletal muscle. Together, they challenge the existing paradigm that all DAG species negatively impact insulin action in skeletal muscle. Only membrane, not cytosolic, DAG were found to be related to insulin resistance in the present study. Further, this relationship was largely driven by the amount of di-C18:0 DAG. Therefore therapeutic strategies to alter DAG composition and/or decrease membrane DAG localisation may be a novel target to promote insulin sensitivity in humans.