Ectopic fat deposition in non-adipose tissues such as skeletal muscle is a common feature of insulin resistance in obesity and type 2 diabetes (3
). The mechanisms underlying IMTG accumulation and elevated lipotoxicity in insulin-resistant states are not yet fully understood. We show for the first time a novel mechanism by which an altered lipolytic balance in skeletal muscle might contribute to lipotoxicity and insulin resistance in humans. An imbalance of ATGL relative to HSL promotes DAG accumulation and induces insulin resistance at least in part through a DAG/PKC pathway.
We first showed that ATGL protein expression in vastus lateralis samples obtained from a wide range of subjects was negatively associated with whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamp. It is interesting to note that the glucose disposal rate during this clamp is mostly accounted by skeletal muscle and thus mostly reflects skeletal muscle insulin sensitivity in lean subjects. Higher doses of insulin are required to fully suppress hepatic glucose production in obese and type 2 diabetic subjects as previously shown (34
). Thus, the relationship between muscle ATGL and insulin sensitivity is stronger in lean subjects than in obese and type 2 diabetic subjects. When the data were examined by group, only obese type 2 diabetic subjects displayed increased muscle ATGL protein content. This finding is slightly in contrast with recent data showing an increased skeletal muscle ATGL protein expression in nondiabetic obese versus age-matched lean individuals (35
). The discrepancy could be explained by an effect of aging because the type 2 diabetic subjects were older than the lean and obese subjects in our study. Thus, age-related changes in skeletal muscle lipases with respect to insulin sensitivity should be further explored. Elevated muscle ATGL protein in obese type 2 diabetic subjects is in agreement with a higher muscle DAG content previously reported in these subjects (36
). We next evaluated the causal relationship between elevated ATGL expression and insulin resistance in primary culture of skeletal muscle cells.
We overexpressed ATGL in human primary myotubes using an adenovirus and assessed the consequences on lipids and insulin action. Elevated expression of ATGL reduced TAG content and simultaneously increased DAG and ceramide content. Ceramides are potentially produced de novo through the action of serine palmitoyl-transferase I as previously shown (8
). ATGL-mediated lipotoxicity was paralleled by impairment in insulin-stimulated glycogen synthesis and insulin signaling possibly due to PKC-mediated Ser1101-IRS-1 phosphorylation and downstream inhibition of Akt Ser473 phosphorylation. Of note, increased ATGL expression induced baseline Akt Ser473 phosphorylation independently of insulin. This suggests that ATGL may activate potential regulators of Ser473 Akt, such as mTORC2, by yet unknown mechanisms (37
). Future studies will be required to dissect the precise mechanism by which ATGL elicits baseline Akt activation. IRS-1 phosphorylation at Ser1101 is primarily mediated by PKCθ and induces general inhibition of IRS-1 function (13
). Together, our data show that ATGL mediates insulin resistance at least in part through DAG and PKC activation. Itani et al. (38
) have previously shown that membrane-associated PKCβ and θ protein content and activity increased the skeletal muscle of obese versus lean and obese diabetic versus nondiabetic matched control subjects, respectively. Even if the exact nature of DAG stereoisomers produced by the action of ATGL is currently unknown, the data support an important role for DAG in mediating skeletal muscle insulin resistance in this model. This observation is consistent with other studies showing a critical role of DAG in mediating insulin resistance in liver (40
) and skeletal muscle (10
) in response to high-fat diets and lipid infusions. Our results are also consistent with a study by Bell et al. (41
) suggesting that AML12 liver cells lacking the lipid coat protein adipophilin and tail-interacting protein of 47 kDa develop insulin resistance with increased recruitment of ATGL to lipid droplets.
Of interest, ATGL-mediated insulin resistance was fully rescued by HSL after restoring a proper cellular lipolytic balance. Overexpression of HSL by itself did not cause lipotoxicity and insulin resistance contrary to ATGL. HSL displays a high DAG substrate specificity and is considered the major DAG hydrolase in several tissues (42
). This supports the concept that the functional balance between ATGL and HSL may influence intracellular DAG concentrations and insulin action in skeletal muscle. Altogether, these data highlight a potential protective role of DAG hydrolases against intramyocellular lipotoxicity by their ability to rapidly hydrolyze DAG. The importance of DAG turnover in insulin resistance is also illustrated by the protective role of DGAT1 against intramyocellular lipotoxicity and fat-induced insulin resistance by increasing lipid partitioning into IMTG (43
). Along these lines, reduced DAG kinase-δ activity, which converts DAG into phosphatidic acid, might also contribute to skeletal muscle insulin resistance by increasing total DAG level (45
Consistent with recent studies that reported a robust reduction in skeletal muscle HSL protein expression in insulin-resistant obese subjects (35
), our study confirmed that both HSL Ser660 phosphorylation and HSL protein content were reduced in skeletal muscle of obese compared with lean subjects. The trend for increased HSL protein content in skeletal muscle of patients with type 2 diabetes could be explained by the hyperglycemic milieu because glucose was shown to induce HSL transcription in adipocytes (47
). To study the impact of reduced HSL expression/activity in skeletal muscle, we next evaluated the consequences of inhibiting HSL activity on lipid pools and insulin sensitivity in myotubes. HSL activity is regulated by phosphorylation on serine residues in response to muscle contraction and catecholamines in vivo (48
). Jocken et al. (46
) found that reduced HSL phosphorylation at Ser563, Ser565, and Ser659 was entirely due to lower HSL protein content and associated with reduced resting glycerol release from the forearm muscle of obese subjects. We report that selective inhibition of HSL increases specifically total DAG levels and consequently disrupts insulin receptor signaling and action. This observation is somehow consistent with data on HSL knockout mice that are insulin-resistant at the level of the skeletal and cardiac muscles when fed a chow diet (49
) and accumulate DAG in their muscles (16
). Further studies will be required to unravel the precise mechanism by which reduced HSL expression/activity induces insulin resistance in skeletal muscle.
In conclusion, the current study highlights a new mechanism by which an altered lipolytic balance between ATGL and HSL induces DAG and insulin resistance in skeletal muscle. The molecular mechanism involves at least in part DAG-mediated PKC activation. Future studies should explore the cause-effect relationship between altered lipase expression and insulin resistance in vivo in animal models with targeted modulations of lipase expression in skeletal muscle. Targeting skeletal muscle lipases might be of potential therapeutic interest for improving insulin resistance in obesity and type 2 diabetes.