The major findings of our study are that a novel, synthetic small molecule, DHPO alleviates impaired glucose intolerance in genetic (leptin deficient ob/ob) and dietary (high-fat fed) mouse models of insulin resistance. DHPO-treatment markedly improved the impaired glucose-uptake in the gastrocnemius muscles in obese mice, suggesting that DPHO-treatment augments peripheral glucose utilization. Interestingly, DHPO treatment was also associated with a reduction in the gain in body mass in the high-fat-fed mice. In addition to improving carbohydrate metabolism, DHPO-treatment corrected the lipid abnormalities associated with obesity and high-fat diet. Skeletal muscles from obese mice showed significantly attenuated glucose uptake compared to lean mice which was reversed in mice that were treated with DHPO. Of great interest are the experiments that demonstrate the lack of effect of DHPO on glucose-uptake in AMPK-KD mice. These results strongly suggest that DHPO may be mediating its effects by modulating the AMPK-signaling pathway.
To further ascertain the role of AMPK and insulin signaling pathways in DHPO–mediated augmentation of glucose uptake we investigated the effects of DHPO on glucose transport in cultured myotubes in the presence of compound C and Wortmanin. As expected, in cultured myotubes DHPO augmented glucose-uptake in a concentration-dependent manner. There was however a discrepancy in the concentrations mediating the in-vivo and in-vitro activity. Whereas in-vivo serum concentrations of DHPO ranged in the nanomolar levels, micromolar concentrations were necessary for in-vitro activity. Although we are unable to reconcile this difference a potential explanation may be the accumulation of the drug on chronic treatment and/or the presence of active metabolites of DHPO that may be responsible for the activity. In addition to augmenting glucose uptake, DHPO caused an increase in phosphorylation of AMPK and its downstream substrate ACC. However, contrary to our expectations both compound C and Wortmanin only modestly inhibited DHPO's effect, while these compounds completely blocked the effects of insulin and AICAR. In addition, DHPO failed to alter the activity of AMPK in an in-vitro kinase assay. Taken together, these results seem to suggest that DHPO may not be mediating its activity by directly activating AMPK, although it is likely that AMPK is necessary for its actions. It is also plausible that other unknown mechanisms in addition to augmenting the AMPK signaling may contribute to the observed biological effects of DHPO.
Insulin mediates it’s action by binding to the e insulin receptor (IR), a disulfide-bonded heterotetrameric membrane protein [12
]. Binding of insulin to the alpha-subunit of IR causes conformational changes that lead to autophosphorylation of the membrane-spanning beta-subunit and activation of the receptor’s intrinsic tyrosine kinase activity [13
]. Thus activated insulin receptor transphosphorylates several downstream substrates (on Tyr residues) including insulin receptor substrate (IRS) proteins, causing the activation of Akt and subsequent translocation of Glut-4 glucose transporter vesicles to the cell-surface resulting in glucose uptake. In our studies DHPO failed to augment any of these pathways suggesting that DHPO may not affect the insulin signaling pathway. Interestingly however, we did observe an augmentation of Glut-4 translocation in response to DHPO treatment.
Recent studies have shown that in skeletal muscle, glucose uptake can be induced in an insulin-independent manner via the activation of AMPK [14
], which plays a pivotal role in the regulation of carbohydrate and fat metabolism [15
]. Consequently, AMPK has emerged as a novel and attractive therapeutic target to treat or prevent diseases and symptoms associated with impaired carbohydrate and fat metabolism. Muscle-specific expression of a kinase-dead form of the catalytic α2-subunit of AMPK in mice results in an insulin resistant phenotype whereas, overexpression of this subunit results in lowering of blood glucose and increased fatty acid oxidation [17
]. At the cellular level, AMPK is thought to mediated cellular-glucose uptake by increasing glut-4 translocation to the cell surface [18
]. Interestingly, insulin sensitizing activity of the antidiabetic drugs such as metformin and thiazolidinediones has been attributed, at least in part, to their stimulation of AMPK activity. The ability of DHPO to cause Glut-4 translocation independent of insulin and apparent lack of glucose-uptake in skeletal muscle of AMPK-KO mice suggest that DHPO may be mediating its effects, at least in part, by activating the AMPK-pathway.
AMPK is activated by known antidiabetic drugs such as metformin and rosiglitazone [19
] and by a variety of structurally unrelated molecules that include, AICAR [20
], A-769662 [21
], resveratrol [22
] and caffeic acid phenylethyl ester [24
]. Cool and coworkers have characterized novel thienopyridones as selective activators of AMPK, and have demonstrated that these molecules alleviate metabolic symptoms and dyslipidemia in mice [21
]. More recently, Pang and coworkers have characterized a novel small-molecule activator of AMPK called PT1 capable of docking with AMPK near the auto-inhibitory domain and directly relieve the auto-inhibition [26
]. Activation of AMPK requires phosphorylation of Thr172 in the catalytic α-subunit activation T-loop which is regulated by several upstream kinases and downstream phosphatases [27
]. The diversity in the chemical structure of compounds that have been reported thus far to cause specific activation of AMPK suggests that these molecules may be working by different mechanisms or at multiple targets in the AMP-signaling cascade. Both compound C (to a greater extent) and Wortmannin (to a lesser extent) suppressed glucose up-take induced by DHPO in myotubes although both compounds failed to completely negate the effects of DHPO suggesting that DHPO may be mediating its effect by targeting multiple pathways. On the other hand, the increase in phosphorylation of Thr-172 and lack of phosphorylation of Akt, in cells treated with DHPO suggest that AMPK-pathway may be playing a predominant role in mediating the effects of DHPO, although contributions from other unknown pathways cannot be ruled out.
In the present study we also found that treatment with DHPO results in a robust attenuation of serum LDL and triglyceride levels which are elevated in obese mice. The role AMPK-ACC pathway in lipid metabolisms has been well documented [28
]. ACC catalyzes the biosynthesis of malonyl-CoA, the primary substrate for fatty acid biosynthesis as well as a potent inhibitor of carnitine palmitoyltransferase I, the rate-limiting step for mitochondrial fatty acid oxidation. Previous studies have demonstrated that the lipid lowering properties of leptin and metformin may be mediated via the activation of AMPK [30
]. However, hepatic p-ACC levels did not differ significantly (although a trend towards reduction was observed) between obese mice treated with vehicle and DHPO suggesting that the reduction in LDL-levels caused by DHPO may be mediated by other factors beyond ACC phosphorylation that affect fatty acid synthesis or oxidation, such as sterol regulatory element-binding proteins. Interestingly, there were no changes in serum HDL-levels following treatment with DHPO in both the ob/ob and high-fat fed mice. Ob/ob
mice used in this study may not be an ideal model to study HDL owing to the fact that the basal HDL-cholesterol levels are significantly higher in these animals compared to the lean controls [32
]. It is however interesting that high-fat feeding did not show a significant reduction of HDL in our experiments. It is likely that the short duration of feeding may be the reason for this lack of change in serum HDL levels. Further extensive studies, with long duration feeding of high-fat diet will be needed to understand the effects of DHPO on HDL levels and to reveal the critical mediators of the lipid-lowering effect of DHPO via AMPK.
Although DHPO altered AMPK pathway in the peripheral tissues, treatment with DHPO failed to change insulin signaling pathway or AMPK signaling pathway in the liver -the phosphorylation status of AMPK, ACC and IRβ remained unchanged following DHPO treatment in the hepatic tissues of obese and lean mice. This suggests that DHPO may not be affecting insulin/AMPK signaling in the liver and may be mediating its effect on glucose uptake by acting predominantly in the periphery. A discrepancy however is the IRS-1 phosphorylation in the liver tissue which was significantly lowered by DHPO in lean mice compared to vehicle treatment. This attenuation of IRS-1 phosphorylation did not alter the phosphorylation status of its downstream effector Akt and therefore the significance of the reduced IRS-1 phosphorylation in response to DHPO-treatment is difficult to explain.
In summary, these results suggest that DHPO may be used as a template to develop newer, more potent and selective pharmacological agents to treat or control insulin resistance observed with obesity and type 2 diabetes. It also supports the view that activation of AMPK may represent a potential therapeutic option in treatment of diabetes and its complications.