AMP-activated Protein Kinase Knockout Research Articles

AMP-Activated Protein Kinase α1 but Not α2 Catalytic Subunit Potentiates Myogenin Expression and Myogenesis

The link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic α subunits, α1 and α2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the α1 but not the α2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPKα1 and AMPKα2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPKα1-specific effect on myogenesis was likely due to the dominant expression of α1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPKα1 subunit for myogenesis in vivo, we prepared both DsRed AMPKα1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wild-type myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPKα1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPKα1.

AMP‐activated protein kinase activation is associated with an inhibition of fibrotic properties of cardiac fibroblasts

PurposeWe asked whether cardiac fibroblast (CF) function might be regulated by AMP‐activated protein kinase (AMPK). We also investigated cardiac fibrosis in AMPK knockout mice (KO) after myocardial infarction.MethodsCF were treated by Transforming growth factor‐beta‐1 (TGF‐beta‐1), serum or platelet derived growth factor (PDGF‐BB) to stimulate myodifferentiation, proliferation and migration respectively. AMPK was activated prior to the different treatments with thienopyridone derivative A‐769662. For in vivo studies, KO mice and their wild‐type (WT) littermate were submitted to a ligation of the coronary artery to mimic myocardial infarction. Gene expression in the left ventricule was measured by qRT‐PCR.ResultsA‐769662 compound dose‐dependently prevented TGF‐beta‐ 1‐induced myodifferentiation. In addition, it decreased by 50% proliferation and completely prevented PDGF‐induced migration of CF, without affecting their mortality. Studies in KO mice show that RNA expression for collagen I and III was significantly increased in the infarct area of KO mice compared to WT mice, after myocardial infarction.ConclusionOur data indicate that AMPK could be a negative regulator of CF function, and by this way decrease the fibrotic component of ventricular remodelling which takes place after myocardial infarction.

Abstract 23: Loss of Astrocytic AMPK Leads to Exacerbation of Stroke in Mice

Background: AMP-activated protein kinase (AMPK) is emerging as a key sensor of brain energy balance. It acutely regulates cellular metabolism and chronically regulates gene expression, reducing energy storage and increasing energy production (glycolysis et al.). AMPK is rapidly activated in stroke. However, its role may be complex and cell dependent as the two major types of brain cells are metabolically distinct. Neurons have minimal key enzymatic activity to produce ATP via glycolysis, do not oxidize fatty acids efficiently, and have no glycogen stores. Activating AMPK in neurons may be deleterious by causing metabolic failure. In contrast, astrocytes can perform glycolysis, oxidize fatty acids to form ketones, and store glycogen providing energy supply for ischemic neurons. We hypothesize that astrocytic AMPK deletion will exacerbate the outcome following stroke. Methods: We used a Cre-lox system to generate conditional astrocytic AMPKα knockout (KO) mice. Extensive behavior screening was carried out to detect potential neurological deficits of the KO mice prior to their utilization. Stroke was induced by reversible middle cerebral artery occlusion (60 minutes) in male KO mice and WT control mice. Stroke outcome was determined with TTC staining at 72 hrs. Functional recovery following stroke was assessed using corner tests at sacrifice. Data are presented as mean±sem. Results: On baseline behavior tests, astrocytic AMPK KO mice displayed no difference from WT (n=4) in spontaneous locomotor activity by open field test (WT 4421±567 vs. KO 4850 ±879, p>0.05,), limb coordination by rotarod (WT 111.3±23 vs. KO 121.3±18, p>0.05), cognitive/memory formation using Novel Object Recognition test (WT 62.1±3.3 vs. KO 63.9±3.6, p>0.05), or integrated sensorimotor function on the corner test (WT 0.52±0.05 vs. KO 0.50±0.04, p>0.05). Following stroke, KO mice showed significantly larger infarcts than WT (n=7) (Cortex WT: 49.8±5.3% vs. KO 67.1±2.4%, p<0.05; Striatum: WT 65.2±4.8% vs. KO 77.8±2.7%, p<0.05; Total: WT 147.2±3.8% vs. 69.0±2.8%, p<0.05). Interestingly, we also observed evidence of increased hemorrhagic transformation in KO mice after stroke. We found a significant exacerbating effect on behavior as the KO mice had more sensorimotor dysfunction in the corner test (WT 0.65±0.04 vs. KO 0.90±0.04, p<0.05, n=7). There were no differences in physiological parameters between AMPK astrocytic KO and WT control groups. In addition, local cerebral blood flow measured by Laser Doppler Flow was equivalently reduced during ischemia and was restored equally in early reperfusion. Conclusion: Astrocytic AMPK is protective in experimental cerebral ischemia. Our study highlights the importance of examining AMPK cell-selectively in stroke and cast light on the complex metabolic interactions between astrocytes and neurons in stroke.

Cold tolerance, cold-induced hyperphagia, and nonshivering thermogenesis are normal in α₁-AMPK-/- mice.

Recent studies indicate that a substantial amount of metabolically active brown adipose tissue (BAT) exists in adult humans. Given the unique ability of BAT to convert calories to heat, there is intense interest in understanding the regulation of BAT metabolism in hopes that its manipulation might be an effective way of expending excess calories. Because of the established role of AMP-activated protein kinase (AMPK) as a "metabolic master switch" and its extremely high levels of activity in BAT, it was hypothesized that AMPK might play a central role in regulating BAT metabolism. To test this hypothesis, whole body α(1)-AMPK(-/-) (knockout) and wild-type mice were studied 1) under control (room temperature) conditions, 2) during chronic cold exposure (14 days at 4°C), and 3) during acute nonshivering thermogenesis (injection of a β(3)-adrenergic agonist). Under control conditions, loss of α(1)-AMPK resulted in downregulation of two important prothermogenic genes in BAT, thyrotropin-releasing hormone (-9.2-fold) and ciliary neurotrophic factor (-8.7-fold). Additionally, it caused significant upregulation of α(2)-AMPK activity in BAT, white adipose tissue, and liver, but not cardiac or skeletal muscle. During acute nonshivering thermogenesis and chronic cold exposure, body temperature was indistinguishable in the α(1)-AMPK(-/-) and wild-type mice. Similarly, the degree of cold-induced hyperphagia was identical in the two groups. We conclude that α(1)-AMPK does not play an obligatory role in these processes and that adaptations to chronic loss of α(1)-AMPK are able to compensate for its loss via several mechanisms.

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by approximately 80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.

Role of Adenosine 5′-Monophosphate-Activated Protein Kinase Subunits in Skeletal Muscle Mammalian Target of Rapamycin Signaling

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.

AMPK-Mediated AS160 Phosphorylation in Skeletal Muscle Is Dependent on AMPK Catalytic and Regulatory Subunits

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha1beta1gamma1 and alpha2beta2gamma1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha2 AMPK knockout [KO], alpha2 AMPK kinase dead [KD], and gamma3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha2 and gamma3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha2 AMPK KO and KD but not gamma3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

Role of β-Adrenoceptor Signaling and AMP-Activated Protein Kinase in Glycolysis of Postmortem Skeletal Muscle

Postmortem glycolysis is directly linked to the incidences of PSE (pale, soft, and exudative) and DFD (dark, firm, and dry) meats, which cause significant economic loss to the meat industry. However, mechanisms controlling postmortem glycolysis are unclear. The objective of this study was to determine the role of beta-adrenoceptor signaling and AMP-activated protein kinase (AMPK) in postmortem glycolysis. Eighteen 2 month old C57BL/6J female mice were randomly separated into three groups. Group I received an intraperitoneal injection of saline solution only and served as the control; group II received a saline injection and then were forced to swim for 1 min; and group III received an injection of propranolol (1 mg/kg) in saline. In addition, six C57BL/6J female AMPK knockout mice were assigned to group IV, which received a saline injection and were forced to swim for 1 min. The longissimus dorsi muscle was sampled at 0, 1, and 24 h postmortem for pH and enzyme activity measurements. The objective is to elucidate the roles of beta-adrenoceptor signaling and AMPK in the glycolysis of postmortem muscle. Results showed that AMPK activity had a major role in determining the ultimate muscle pH, with an ultimate pH for control mice of 6.16 and AMPK knockout mice of 6.48. The beta-adrenoceptor signaling is essential for initial rapid glycolysis. Blocking beta-adrenoceptor signaling prevented the initial pH decline induced by stress. Activation of beta-adrenoceptor signaling due to preslaughter stress activates glycogen phosphorylase, resulting in a rapid glycolysis shortly after slaughter. On the other hand, the activation of AMPK is important for maintaining the activity of glycogen phosphorylase and pyruvate kinase, leading to a sustained glycolysis and a low ultimate pH.