Patients with type 2 diabetes are typically characterized by insulin resistance in skeletal muscle, the tissue responsible for the majority of insulin-stimulated glucose disposal1
. Therefore, it is important to understand the molecular mechanisms leading to the development of skeletal muscle insulin resistance. In the current study we tested the hypothesis that TRB3 plays a role in ER stress-induced insulin resistance in skeletal muscle. The current results support this hypothesis as indicated by: 1) expression of TRB3 and ER stress markers was increased in multiple models of insulin resistance in muscle cells, mouse skeletal muscle, and human skeletal muscle; 2) overexpression of TRB3 in C2C12 cells and skeletal muscle impaired insulin signaling and glucose uptake; 3) knockdown of TRB3 in C2C12 myotube and mouse models significantly blunted the effects of ER stressors to impair insulin signaling and glucose uptake; and 4) TRB3 knockout mice were protected from high fat diet-induced insulin resistance.
Our findings that TRB3 mediates ER stress-induced decreases in insulin signaling and glucose uptake suggest a critical role for this protein in muscle metabolism. Earlier studies have suggested that TRB3 may be important for glucose metabolism in skeletal muscle, although this hypothesis has not been tested directly. For example, overexpression of TRB3 in L6 cells resulted in impaired insulin signaling 9
, consistent with our data showing that TRB3 overexpression decreased insulin signaling in C2C12 cells. In another study an acute bout of exercise decreased TRB3 expression in ob/ob mice, which the authors proposed may have contributed to the exercise-induced improvement in glucose tolerance in these animals10
. Furthermore, muscle-specific overexpression of PGC1α in mice, which resulted in exacerbation of high fat diet-induced insulin resistance, was associated with elevated TRB3 expression34
. While all of these studies are consistent with TRB3 regulating glucose metabolism, our data directly tested this hypothesis through the use of multiple overexpression and knockdown models of TRB3. All of the current data demonstrate that TRB3 has an inhibitory role to decrease insulin-stimulated glucose uptake in skeletal muscle.
In the current study we detected TRB3 expression in mouse and human skeletal muscle, consistent with previous findings6, 10, 34, 35
. In contrast, in a study focused on mouse gastrocnemius muscle33
, TRB3 protein was only shown to be expressed when the muscle underwent denervation. This is consistent with our findings as we detected low mRNA expression of TRB3 in white gastrocnemius muscle (), but observed higher expression in muscles that express oxidative red fibers including tibialis anterior, red gastrocnemius and soleus, suggesting that TRB3 expression may be fiber-type specific. It is interesting that denervation resulted in an increase in TRB3 expression in white gastrocnemius muscle33
. Since skeletal muscle denervation results in insulin resistance in this tissue36-38
, it is possible that TRB3 plays a role in denervation-induced insulin resistance in skeletal muscle. In addition, expression of ER stress markers has been shown to be increased in the muscle from patients with myotonic dystrophy type 126
, raising the possibility that ER stress is involved in muscle wasting-associated insulin resistance in this disease. Thus, TRB3 increases under some conditions of muscle dysfunction, and could contribute to the metabolic impairment associated with these diseases.
We found that a high fat diet in mice, and both obesity and type 2 diabetes in humans increased TRB3 expression in skeletal muscle. The mechanism by which these conditions increase TRB3 expression is not known but one possibility is that the elevated circulating insulin concentrations that are present in high fat-fed mice and subjects with obesity and type 2 diabetes mediate the induction of TRB3 in the muscle. In support of this hypothesis we found that insulin incubation of C2C12 myotubes for 16hrs increased TRB3 expression along with increased expression of ER stress markers, which was abolished by inhibitors for PI3K and Akt. Studies in FAO hepatoma and 3T3-L1 adipocyte cells have also demonstrated that insulin induces the expression of TRB3 through C/EBPβ binding in the TRB3 promoter39
. Taken together, these data support the hypothesis that elevated insulin concentrations could mediate the effects of high fat feeding, obesity, and type 2 diabetes to increase TRB3 expression in mouse and human skeletal muscle.
Given the function of TRB3 in insulin signaling and glucose metabolism in liver3
and the increased insulin-stimulated glucose uptake in the skeletal muscle from TRB3KO mice (), it was somewhat surprising that whole body TRB3KO mice on a chow diet showed no alteration in glucose homeostasis33
. The lack of effect may be due to the different functions of TRB3 in various tissues, thereby offsetting any beneficial effects of ablation of TRB3. For example, overexpression of TRB3 in adipocytes promoted fatty acid oxidation and protected mice from high fat induced obesity4
, whereas overexpression of TRB3 impaired glucose metabolism in liver and skeletal muscle and inhibited insulin secretion in β-cells8
. Thus, in global TRB3 knockout mice, the beneficial effects of reduced TRB3 in liver, skeletal muscle, and pancreas on glucose homeostasis may be masked by the detrimental effects of a reduction in adipocyte TRB3 expression.
Although there was no change in whole body glucose homeostasis in TRB3 knockout mice fed a chow diet33
, we found that TRB3 knockout mice were protected from high fat diet-induced insulin resistance. The TRB3 knockout mice showed improved glucose tolerance, increased skeletal muscle insulin-stimulated glucose uptake, and less triglyceride accumulation in the liver after eight weeks on a high fat diet. These data indicate that the beneficial effects of deletion of TRB3 are more pronounced under stress conditions such as high fat feeding. It is interesting that high fat feeding resulted in 56% lower liver triglyceride concentrations in the knockout mice, which contributed to the lower liver weights in the knockout mice. This is consistent with previous studies showing that improved insulin signaling in the liver protected mice from high fat diet-induced hepatic steatosis, whereas impaired insulin signaling in the liver increased high fat diet-induced lipid accumulation40-45
. Thus, the lower lipid accumulation in liver of TRB3 knockout mice is presumably due to improved insulin signaling.
In summary, ER stress induces expression of TRB3 in skeletal muscle, which results in impairment of insulin signaling and glucose uptake. Knockdown of TRB3 significantly blunts the effects of ER stress on glucose uptake and TRB3 knockout mice are protected from high fat diet-induced insulin resistance. We conclude TRB3 plays an important role in ER stress-induced insulin resistance in skeletal muscle. Our data also suggest that inhibition of TRB3 expression in skeletal muscle may be a new therapeutic target for effectively managing insulin resistance.