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      Opposing Activity Changes in AMP Deaminase and AMP-Activated Protein Kinase in the Hibernating Ground Squirrel

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          Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.

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          Most cited references 41

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          Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator.

          Hibernating arctic ground squirrels, Spermophilus parryii, were able to adopt and spontaneously arouse from core body temperatures as low as -2.9 degrees C without freezing. Abdominal body temperatures of ground squirrels hibernating in outdoor burrows were recorded with temperature-sensitive radiotransmitter implants. Body temperatures and soil temperatures at hibernaculum depth reached average minima during February of -1.9 degrees and -6 degrees C, respectively. Laboratory-housed ground squirrels hibernating in ambient temperatures of -4.3 degrees C maintained above 0 degree C thoracic temperatures but decreased colonic temperatures to as low as -1.3 degrees C. Plasma sampled from animals with below 0 degree C body temperatures had normal solute concentrations and showed no evidence of containing antifreeze molecules.
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            Metformin activates AMP kinase through inhibition of AMP deaminase.

            The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action.
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              Counteracting Roles of AMP Deaminase and AMP Kinase in the Development of Fatty Liver

              Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation –either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.

                Author and article information

                Role: Academic Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                9 April 2015
                : 10
                : 4
                [1 ]Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO, 80045, United States of America
                [2 ]Department of Cell and Developmental Biology, Aurora, CO, 80045, United States of America
                [3 ]Department of Biochemistry and Molecular Genetics, Aurora, CO, 80045, United States of America
                [4 ]Laboratory of Renal Physiopathology and Nephrology Dept, INC Ignacio Chavez, Mexico City, Mexico
                [5 ]Division of Renal Medicine, Department of Clinical Intervention and Technology, Karolinska Institutet, Stockholm, Sweden
                [6 ]Division of Nephrology, Eastern Colorado Health System, Department of Veteran Affairs, Denver, CO, United States of America
                University of Santiago de Compostela School of Medicine - CIMUS, SPAIN
                Author notes

                Competing Interests: Drs MAL, CJR, GEG and RJJ are listed as inventors on a patent for the use of AMPD2 inhibitors to treat obesity and insulin resistance (US 8,697, 628). MAL, CJR, LGSL, CARJ and RJJ are also members of Colorado Research Partners LLC that is interested in developing novel treatments for obesity. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

                Conceived and designed the experiments: MAL LEE GEG MS AJ SLM RJJ. Performed the experiments: MAL LEE NL CC GEG CARJ SJ JT CTM. Analyzed the data: MAL CJR TI LGSL TN PS SLM RJJ JT. Contributed reagents/materials/analysis tools: MAL GEG CR SLM RJJ. Wrote the paper: MAL LEE NL CC SLM RJJ.


                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                Page count
                Figures: 7, Tables: 1, Pages: 19
                This work is supported by NIH grants HL68607, RC4 DK90859 and startup funds from the University of Colorado to RJJ and grant HL089049 to SLM for support of the animal samples. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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