Can Patients with Type 2 Diabetes Be Treated with AMPK-Activators?

Abstract

AMP-activated protein kinase (AMPK) has been identified as a target for development of pharmaceuticals for prevention and treatment of type 2 diabetes. Chemical activation of AMPK in experimental animals using 5-aminoimidazole-4-carboxamide (AICAR) has previously been reported to activate AMPK, stimulate fat oxidation, stimulate glucose uptake into muscle, inhibit lipogenesis and cholesterogenesis, increase hexokinase and GLUT-4 in muscle, and increase insulin sensitivity. Animals with metabolic conditions similar to type 2 diabetes show improvement in glucoregulation with chronic AICAR injections. A more recent study of normal human subjects demonstrated increased uptake of a glucose analogue into skeletal muscle in response to AICAR infusion. The current study by Boon, et al in this issue of Diabetologia demonstrates reduction of glucose production and improvement in glucoregulation in patients with type 2 diabetes infused with AICAR at much lower doses than were previously used in rats and mice. This study provides additional evidence that AMPK-activating pharmaceuticals may be useful in prevention and treatment of type 2 diabetes.

Why is AMP-activated protein kinase a reasonable therapeutic target?

AMP-activated protein kinase (AMPK) has emerged as a key regulatory protein expressed in most cells of the body. It is an energy sensing protein that is activated in response to a decrease in the AMP/ATP ratio, a condition resulting form hypoxia or muscle contraction[1]. It can also be activated by hormones such as adiponectin, leptin, and IL-6. AMPK must be phosphorylated at Thr172 of the alpha subunit by an upstream kinase in order to be active in phosphorylating downstream targets. Three upstream kinases have been identified: LKB1-STRAD-MO25, calmodulin kinase kinase beta (CaMKKβ), and transforming growth factor β activated kinase (TAK1). Once phosphorylated AMPK is also subject to allosteric activation by 5′-AMP. In general, activated AMPK phosphorylates proteins that increase ATP production and decrease ATP utilization. Such processes as cholesterol and fatty acid synthesis, protein synthesis, and gluconeogenesis in liver are decreased, whereas glucose uptake and fatty acid oxidation in muscle and fatty acid oxidation in liver are enhanced. Expression of proteins involved in ATP production also increase, including GLUT-4, hexokinase, and mitochondrial oxidative enzymes. Specific transcription factors or coactivators such a PGC-1alpha are phosphorylated by the active AMPK. Insulin sensitivity is enhanced. Because of its capacity to inhibit hepatic gluconeogenesis, fatty acid synthesis, and cholesterogenesis, to stimulate glucose uptake, and fatty acid oxidation, to increase expression of hexokinase and GLUT4 and to improve insulin sensitivity in muscle, AMP-activated protein kinase (AMPK) has been targeted for development of a new class of pharmaceuticals for prevention and treatment of type 2 diabetes.

Evidence from animal studies that AMPK activation could help in treatment of type 2 diabetes?

The first evidence suggesting AMPK activators might be useful in treatment of type 2 diabetes came from animal studies [2, 3]. AMPK was found to be activated in skeletal muscle during exercise and in response to electrical stimulation of contraction. Accompanying the AMPK activation was an increase in phosphorylation/inactivation of acetyl-CoA carboxylase (ACC), the first downstream target identified in skeletal muscle. Chemical activation of AMPK with 5-aminoimidazole-4-carboxamide (AICAR) in perfused rat hindlimbs resulted in not only a stimulation of fatty acid oxidation, but a stimulation of glucose uptake[4]. AICAR, an analogue of adenosine that is taken into the muscle and phosphorylated to form ZMP, can activate AMPK similarly to AMP. Subsequent studies showed that AICAR stimulated uptake of non-metabolizable glucose analogues into isolated epitrochlearis and that the AICAR stimulated glucose transport was due to translocation of GLUT4 into sarcolemmal membranes from a microsome fraction of perfused muscles[2]. Subcutaneous injection of AICAR into rats was shown to activate skeletal muscle AMPK, to induce an increase in muscle GLUT4 and hexokinase, and to cause an increase in glycogen content of the muscle [5].

Studies in animal models of type 2 diabetes provide hope that AMPK activators may be useful for treatment of type 2 diabetes [6–8]. Obese ZDF rats, a model of type 2 diabetes, show marked decreases in plasma glucose and plasma insulin concentrations in response to administration of AICAR for 7 wk. Glucose tolerance was normalized 24 hr following the last AICAR injection. These changes were accompanied by increases in GLUT4 expression in the muscles and improvement in 3-methylglucose uptake in incubated epitrochlearis and extensor digitorum longus. Both exercise training and AICAR treatment prevented the rise in blood glucose at 9 wk of age in the ZDF rat. Deterioration of pancreatic islet cells was also attenuated in these rats by the AICAR treatment. Two mouse models (KKAy-CETP, and ob/ob) of type 2 diabetes also show improvement in glucoregulation upon treatment with AICAR [9, 10]. In all of these animal studies, relatively high doses of AICAR were required (0.5 – 1.0 mg/g body weight). The 1 mg/g dose caused liver hypertrophy and lactic acidosis in rats[4].

Human studies on AMPK activation

After the beneficial effects of chemical AMPK activation were demonstrated in animal models, the next question became, can these effects be seen in humans with tolerable doses of AICAR? In the first study of this nature, healthy human subjects were infused with AICAR at a rate of 10 mg/Kg body weight/hr[11]. This produced a muscle ZMP concentration of 68 μmole/Kg dry weight. Values achieved in rat hindlimbs perfused with 0.5–2 mM AICAR and found to be effective in activation of AMPK were 200 – 500 μmole/Kg wet weight [4]. In rats injected subcutaneously with AICAR the concentration of ZMP achieved in muscle was in the 1 mM range[5]. The 10 mg/Kg body wt/hr infusion rate stimulated a significant increase in infused 2-deoxyglucose uptake by muscle without a detectable increase in AMPK. It is also conceivable that the phosphorylated fraction of AMPK was allosterically activated by ZMP. This would not be detected either in the pAMPK western blot or in the AMPK activity assays which are conducted in the presence of saturating amounts of AMP. Phosphorylation of ERK1/2 was increased in response to AICAR infusion. No cause-effect relationship could be established however between 2-deoxyglucose uptake and ERK1/2 activation.

Major findings in the Boon, et al study

In the current study by Boon, et al in this issue of Diabetologia, patients with type 2 diabetes were infused with AICAR or saline at a rate of 45 mg/kg/hr, a rate 450% of the infusion rate used by Cuthbertson, et al. This infusion rate produced a maximal plasma AICAR concentration of 0.16 mM. Plasma glucose rate of appearance (Ra) and plasma glucose rate of disappearance (Rd) were quantitated using stable isotope methodology. At this infusion rate, no effects were noted on Rd, but Ra was reduced during AICAR infusion, resulting in a greater rate of decline in blood glucose. The content of pAMPK in muscle biopsies was not changed, but the pACC was increased possibly indicating the prevailing pAMPK fraction was allosterically activated by ZMP. The AICAR-induced increase in blood lactate concentration could have resulted from increased glucose uptake by muscle and increased glycolytic flux or from muscle glycogenolysis, but there was not strong evidence for stimulation of glucose uptake by muscle. In rat studies, muscle glycogen was increased, not decreased after AICAR injection [5]. It is possible that glucose uptake was increased in muscle, but decreased in other tissues, resulting in a stable whole body Rd during the AICAR infusion. It appears that the major effect of AICAR infusion in this study was on the liver, decreasing the rate of glucose production. This effect on the liver could conceivably have resulted from AMPK-independent inhibition of fructose-1,6-bisphosphatase (gluconeogenic enzyme) by ZMP [12]. Non-esterified fatty acid (NEFA) Ra and Rd were both reduced in response to AICAR infusion but Rd remained slightly higher than Ra with the consequence that plasma NEFA concentration decreased considerably. Although it not possible to predict the mechanism with available data, authors suggested increased uptake and oxidation of fatty acids by the liver and inhibition of lipolysis in adipose tissue could be responsible.

Where do we go from here?

This work by Boon, et al represents a very important step in development of AMPK activators for treating type 2 diabetes. Glucose production was reduced even with these relatively low doses of AICAR. The fact that this infusion rate is well-tolerated by the patients with no untoward side effects is also encouraging. It would certainly be of interest to see if doses effective in producing a detectable increase in muscle AMPK would be more effective in normalizing glucoregulation in patients with type 2 diabetes. Some of the pharmaceuticals (metformin, and thiazolidinediones) currently used for treatment of type 2 diabetes have been shown to be AMPK activators, but there is some question whether concentrations required for AMPK activation in tissue incubation studies are actually achieved in muscle and liver of patients at pharmacological doses. One new AMPK activator (A-769662, a thienopyridone) has been developed recently which is effective in activating AMPK in the low micromolar range (EC50 = 0.8 μM). Treatment of ob/ob mice with A-769662 (30 mg/Kg) decreased plasma glucose by 40% and decreased plasma and liver triglyceride levels [13].

The development of an ideal AMPK activator will include several attributes. The drug will activate AMPK at a much lower concentration than is required for AICAR. The drug will activate AMPK in specific target tissues, such as liver, adipose tissue, and skeletal muscle. The drug will be effective when administered by the oral route. It will have minimal and tolerable side effects even with long term administration. It is probably worthwhile noting that exercise specifically activates AMPK in muscle and heart and perhaps in liver at high work rates. For those who are able to exercise, it is not necessary to wait for development of more potent AMPK activators to launch this stategy. The beneficial effects of exercise in prevention and treatment of type 2 diabetes, postulated to be due in part to AMPK activation in working muscle, are well-documented [14].

Figure 1.

Effects of AMPK activation in liver, adipose tissue, and skeletal muscle. Phosphorylation of target proteins results in activation (represented by solid line arrow) or inhibition (represented by solid line with bar) of key metabolic processes in the cells. AMPK must be phosphorylated on Thr 172 of the alpha subunit to be active. AMP increases in response to muscle contraction or hypoxia and can make AMPK a poorer substrate for the phosphatase thus increasing the fraction that is phosphorylated and which also activates pAMPK allosterically. AICAR can be taken up by cells and phosphorylated to form ZMP which can then mimic the effects of AMP in AMPK activation. ZMP can also inhibit gluconeogenesis independently of AMPK activation.

Abbreviations

ACC

acetyl-CoA carboxylase

AICAR

5-aminoimidazole-4-carboxamide

AMP

5′-adenosine-monophosphate

AMPK

AMP-activated protein kinase

ATP

adenosine triphosphate

ERK1/2

extracellular signal-regulated mitogen-activated protein kinases1/2

GLUT4

glucose transporter 4

IL-6

interleukin 6

LKB1

serine-threonine kinase 11

MO25

mouse protein 25

NEFA

non-esterified fatty acids

pAMPK

AMPK phosphorylated on Thr 172 of the alpha subunit

PGC-1alpha

peroxisome proliferator-activated receptor-gamma coactivator

STRAD

Ste20-related adaptor

ZMP

monophosphorylated AICAR