AMPD1: a novel therapeutic target for reversing insulin resistance
© Cheng et al.; licensee BioMed Central Ltd. 2014
Received: 7 October 2014
Accepted: 9 December 2014
Published: 15 December 2014
Insulin resistance is one of the hallmark manifestations of obesity and Type II diabetes and reversal of this pathogenic abnormality is an attractive target for new therapies for Type II diabetes. A recent report that metformin, a drug known to reverse insulin resistance, demonstrated in vitro the metformin can inhibit AMP deaminase (AMPD) activity. Skeletal muscle is one of the primary organs contributing to insulin resistance and that the AMPD1 gene is selectively expressed at high levels in skeletal muscle.
Recognizing the background above, we asked if genetic disruption of the AMPD1 gene might ameliorate the manifestations of insulin resistance. AMPD1 deficient homozygous mice and control mice fed normal chow diet or a high-fat diet, and blood analysis, glucose tolerance test and insulin tolerance test were performed. Also, skeletal muscle metabolism and gene expression including nucleotide levels and activation of AMP activated protein kinase (AMP kinase) were evaluated in both conditions.
Disruption of the AMPD1 gene leads to a less severe state of insulin resistance, improved glucose tolerance and enhanced insulin clearance in mice fed a high fat diet. Given the central role of AMP kinase in insulin action, and its response to changes in AMP concentrations in the cell, we examined the skeletal muscle of the AMPD1 deficient mice and found that they have greater AMP kinase activity as evidenced by higher levels of phosphorylated AMP kinase.
Taken together these data suggest that AMPD may be a new drug target for the reversal of insulin resistance and the treatment of Type II diabetes.
KeywordsAMP deaminase Adenine nucleotide Diabetes Insulin resistance AMP kinase Glucose metabolism
Type II diabetes and the associated disorder referred to as the metabolic syndrome affect more than 380 million people in the world and the prevalence of these disorders is predicted to increase in the future . The fundamental abnormality in glucose metabolism underlying these disorders is insulin resistance . While insulin resistance can be caused by a number of factors, there is a large body of evidence that a high fat diet with resultant fat accumulation in various organs is a major predisposing factor to the development of insulin resistance . One of the predominant organs in which fat accumulates in the setting of a high fat diet is skeletal muscle, and numerous studies have documented that skeletal muscle which is a major site of insulin action contributes to the state of insulin resistance observed in animals and humans consuming a high fat diet .
Given that skeletal muscle is one of the major organs that contributes to insulin resistance it stands to reason that therapies directed to targets expressed in this tissue may offer novel approaches to treating these disorders. A recent report demonstrated that AMP deaminase (AMPD) isolated from skeletal muscle is inhibited by metformin, one of the best known drugs for reversing the pathologic state of insulin resistance . While the results of this study are intriguing it is difficult to assess if inhibition of AMPD is the target responsible for the in vivo effects of this drug on insulin action since many studies have reported that metformin affects the activity of numerous enzymatic activities .
To address the question of whether AMPD represents a valid new target for drug development to reverse or ameliorate insulin resistance we have taken the approach of gene disruption to test this hypothesis more definitively. In particular we elected to reduce the level of AMPD enzyme activity in skeletal muscle through disruption of the AMPD1 gene, the predominant AMPD gene expressed in skeletal muscle [6, 7]. The results described in this report demonstrate that mice rendered deficient in AMPD1 enzyme activity have a milder state of insulin resistance, improved glucose tolerance, and enhanced insulin clearance when fed a high fat diet when compared to controls with normal skeletal muscle AMPD enzyme activity.
We previously described the generation of AMPD1 deficient mice . Heterozygous AMPD1 deficient mice (A1(+/−) mice) were backcrossed with C57BL/6 control mice more than 10 times. Genotyping was performed as described before . Animals had free access to food (CE-2, CLEAR) and water, and were housed in a controlled SPF environment with a 12-h light–dark cycle and constant temperature (25°C).
AMPD1 deficient homozygous mice (A1(−/−) mice) and control mice (Wt mice) at 5 weeks old were fed normal chow diet (CD: CE-2) or a high-fat diet (HFD: HFD32) (CLEAR, Tokyo, Japan) with 60% fat calories for 12 weeks.
All animal experiments were approved by the Committee on Animal Research of National Cerebral and Cardiovascular Center and were performed according to the guidelines for the protection of experimental animals of National Cerebral and Cardiovascular Center.
The adiposity of mice was examined radiographically using CT (LaTheta LCT-100, ALOKA) according to the manufacturer’s protocol. Two-mm intervals were scanned from the diaphragm to bottom of the abdominal cavity.
Blood analysis: insulin, FFA, triglyceride, cholesterol, adiponectin and leptin
Samples were obtained from the mice fasted for 17 h. Plasma insulin levels were measured using an ELISA kit (Morinaga, Yokohama, Japan). Serum FFA, triglyceride, cholesterol were detected by ACS-ACOD methods, GPO-HDAOS methods and HDAOS methods. The levels of leptin, adiponectin were measured using a mouse leptin ELISA kit (Morinaga, Yokohama, Japan), a mouse adiponectin ELISA kit (Ootuka, Tokyo, Japan), respectively.
Glucose and insulin tolerance
Groups of male mice before and after HFD were used for intraperitoneal glucose injection. After a 17-hr fast, plasma insulin and glucose levels were measured and d-glucose (2 g/kg body weight) was administered. Blood samples were taken from the tail vein at indicated times. Blood glucose levels were detected at 15, 30, 60, 90, 120 min and blood glucose concentration was determined using a glucometer.
Groups of male mice before and after HFD were used for insulin tolerance tests. Mice were fasted for 5 h. Blood glucose levels were detected at 0, 15, 30, 60, 90 min after intraperitoneal injection 0.75 units insulin/kg body weight.
Western blot analysis
Tissues were sonicated and homogenized in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulforyl fluoride, 45 μg/ml aprotinin, 100 mM sodium orthovanadate). The supernatant of the homogenate was used for protein determination with a BCA Protein Assay Kit (Pierce, IL, USA) and electrophoresis. Samples were loaded on SDS-PAGE gel with the same amount of total protein. Electrophoretic transfer to polyvinylidene difluoride membranes was followed by immunoblotting with different primary antibodies. Signals were developed with an ECL kit (Amersham, Piscataway, USA).
Glucose and insulin dynamics
Body weight and adiposity
The parameters of lipid metabolism in blood
41 ± 8
42 ± 3
512 ± 95
473 ± 63
271 ± 11
240 ± 22
50 ± 4
43 ± 3
15 ± 4
15 ± 5
Skeletal muscle metabolism and gene expression
Nucleotides, adenosine and cAMP in skeletal muscles
10.00 ± 0.25
9.90 ± 0.28
2.79 ± 0.13
2.78 ± 0.13
0.36 ± 0.01
0.40 ± 0.03
0.26 ± 0.04
9.54 ± 0.71
9.86 ± 0.44
1.00 ± 0.10
0.99 ± 0.09
The results of this study demonstrate that disruption of the AMPD1 gene with reduction of AMPD activity in skeletal muscle protects mice from a number of the adverse metabolic consequences of a high fat diet. The following indices of glucose metabolism are significantly improved in A1(−/−) mice on a high fat diet: fasting blood glucose is lower, fasting and post-prandial insulin levels are lower, glucose and insulin tolerance tests are improved, and insulin resistance is less pronounced in the A1(−/−) animals. In addition to these measures of insulin action A1(−/−) animals exhibited a higher rate of insulin clearance on a high fat diet. While insulin clearance is determined by many factors including renal excretion it is also influenced by insulin uptake by peripheral organs such as skeletal muscle .
These studies do not indicate which organ(s) are responsible for these various measures of enhanced insulin action but given the central importance of skeletal muscle in contributing to the state of insulin resistance in animals and humans on a high fat diet [3, 11] and recognizing that AMPD1 is only highly expressed in skeletal muscle of humans and rodents , it points to changes in skeletal muscle metabolism as the most likely tissue to produce the observed changes in insulin and glucose metabolism in the whole animal.
As expected, fat content of skeletal muscle increased in mice on a high fat diet, as did fat content in other tissues. But there were no demonstrable differences in fat content of skeletal muscle, or fat content of other tissues, in the A1(−/−) animals, suggesting that the mechanism for enhanced insulin sensitivity is not the result of reduced levels of fat in skeletal muscle or other organs. Consistent with these findings A1(−/−) mice exhibited a comparable increment in weight gain resultant from the high fat diet.
Many interventions and drugs have been reported to improve insulin sensitivity through affecting the activity of AMP kinase . Reduction of AMPD activity with resultant alternations in adenine nucleotide metabolism might be expected to lead to activation AMP kinase which is known to be regulated by adenine nucleotides . Consistent with this hypothesis we observed an increase in the levels of phosphorylated AMP kinase in skeletal muscle of A1(−/−) mice. In sedentary animals we did not detect any changes in the total concentration of AMP or any other adenine nucleotides but we were not able to quantitate the levels of free AMP, the most critical effector of AMP kinase activation, and we postulate that the small fraction of total AMP that is in the pool of free AMP may have been increased leading to the activation of AMP kinase. This conclusion is supported by the failure to detect any IMP, a product of the AMPD reaction, in the skeletal muscle of the A1(−/−) animals.
An unexpected and potentially interesting finding with regard to the phenotypic changes observed in AMPD deficient mice is the increase in expression of the leptin receptor in the skeletal muscle of these animals. Leptin is known to increase the amount phosphorylated AMP kinase in skeletal muscle  and it is possible that enhanced activity of the leptin pathway may also have contributed to the observed increase in phosphorylated AMP kinase in the skeletal muscle of the A1(−/−) mice. How alterations in AMPD activity affect expression of the leptin receptor remain to be determined but this observation suggests that alterations in AMPD activity may lead to changes in other pathways that affect insulin and glucose metabolism in myocytes. In this regards, the crossing A1(−/−) mice with db/db mice will provide the further evidence for the interaction between AMPD activity and the leptin receptor.
Decreased levels of IMP in skeletal muscles in A1(−/−) mice indicated that other AMPD genes were not upregulated in muscles. In fact, total AMPD activity was quite low in muscles of A1(−/−) mice as reported before . Therefore, skeletal muscles in A1(−/−) mice should have striking difference in metabolic conditions if there were any change of endogenous AMPD1 at baseline and after 12-week HFD in Wt mice. Also, besides the skeletal muscle, liver, adipose tissue, and others have an important role in regulating glucose metabolism at baseline and after HFD challenge. However, we found that no difference of AMPD2 mRNA expression was observed in liver between Wt and A1(−/−) mice at base line or after HFD challenge (Additional file 2: Figure S2), while AMPD2 mRNA expression was slightly increased in liver of both mice after HFD. Therefore, we thought that the AMPK activity at baseline or after HFD challenge in other organs was not appreciably affected by AMPD1 deficiency.
Regarding AMPK activation in A1(−/−) mice, it could be possible that other metabolites including IMP, hypoxanthine, or uric acid in the pathway downstream to AMPD might play a role for it. Further investigation will be awaited for delineation of precise mechanisms for AMPK activation in AMPD1 deficiency.
During the revision of this report, other reports indicated that AMPD1 deficient mice showed only moderate increase in AMPK activation despite increase in AMP levels and AMP/ATP ratio and that there were no favorable metabolic phenotype in AMPD1 deficient mice after HFD challenge [13, 14]. We think that different protocol for HFD challenge created different results and that further study is awaited for delineating the importance of AMPD1 on insulin metabolism.
In conclusion, the results of this study validate AMP deaminase as potential new drug target for the amelioration of insulin resistance, which is one of the underlying causes of the metabolic syndrome and Type II diabetes. The results indicate that inhibition of AMPD may lead to improved insulin dynamics in the resting state and without the requirement of concomitant weight loss or reduction in fat content of skeletal muscle. While the mechanism(s) by which reductions in AMPD activity lead to this beneficial metabolic profile need more study the data are consistent with activation of AMP kinase, a known pathway for decreasing insulin resistance, and potentially a novel mechanism of action that may involve the leptin receptor. The effects of reduced AMPD activity in altering insulin action may not be unique to the mouse as two studies have reported that AMPD1 deficient human subjects have a lower incidence of type II diabetes [15, 16], an intriguing result that needs to be confirmed in a larger group of individuals, and the demonstration that AMPD1 haplotype is associated with changes in insulin clearance in man . AMPD1 deficiency is one of the most common inherited defects in the Caucasian population, with approximately 2% of individuals in this ethnic group being homozygous for this gene defect, and few if any of these individuals experience any adverse effects from this enzyme deficiency  suggesting that not only would inhibition of AMPD have beneficial effects in patients with the metabolic syndrome and diabetes but inhibition of this enzyme may lead to few deleterious effects.
- AMP kinase:
AMPK: AMP activated protein kinase
Control wild type
High fat diet
We express our thanks to the members of the Department of Bioscience and Genetics for their technical support. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from the Japan Science and Technology Corporation, Ministry of Health, Labour and Welfare of Japan, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and the National Natural Science Foundation of China.
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