W W Winder’s research while affiliated with Brigham Young University and other places

To determine effects of iron deficiency on LKB1/AMPK signaling, rats were fed a control (C=0.29 mg/g Fe) or iron deficient (ID=0 mg/g Fe) diet for 8 wks resulting in hematocrits of 47.5% ± 1.0% and 16.5% ± 0.6% respectively. Iron deficiency resulted in 28.3% greater muscle fatigue (p<0.01) in response to 10 min of stimulation (1 twitch/sec) and was associated with a greater reduction in phosphocreatine (C: 45.6%; ID: 85.9%) and ATP levels (C: 0%; ID: 24.0%). AMPK activation increased with stimulation in muscles of C and ID animals. A reduction in Cytochrome c and other heme‐containing mitochondrial proteins was observed in ID animals (p<0.01). The two isoforms of the AMPK catalytic subunit (α) are known to sense and respond differently to energy challenges. In ID animals, the AMPKα2 subunit protein content was reduced by 28.4% (p<0.05), however this did not result in a significant difference in resting AMPKα2 activity. AMPKα1 protein was unchanged, however an overall increase in AMPKα1 activity was observed (C: 1375.8 pmol/mg/min; ID: 2229.8 pmol/mg/min, p<0.01). Resting phospho Acetyl CoA Carboxylase (pACC) was also increased by 17‐fold (p<0.05), indicating greater AMPK activity that was not determined by the AMPK activity assay. This study indicates that chronic iron deficiency causes a shift in the expression of AMPKα subunit composition and potentially altered sensitivity to cellular energy challenges.

In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25α/β), and Ste-20-related adaptor protein (STRADα/β); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1(-/-)) mouse muscle. LKB1 detection in untreated control and LKB1(-/-) muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1(-/-) samples [55 ± 2.5 and 13 ± 1.5 arbitrary units (AU) in control and LKB1(-/-), respectively, P < 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 ± 5 and 8.4 ± 4 AU in control and LKB1(-/-), respectively, P < 0.01) and ion-exchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25α, and STRADβ mRNA in LKB1(-/-) muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.

The liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signalling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm. Two methods of AMPK activation were used to characterize LKB1/AMPK signalling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation of the diaphragm. Diaphragms were excised 60 min post-AICAR injection and immediately after electrical stimulation. AMPK phosphorylation increased with AICAR and electrical stimulation in control but not KO mice. Acetyl CoA carboxylase phosphorylation increased with AICAR in control but not KO mice, but increased in both genotypes with electrical stimulation. While the majority of mitochondrial protein levels were lower in KO diaphragms, uncoupling protein 3, complex I and cytochrome oxidase IV protein levels were not different between genotypes. KO diaphragms have a lower percentage of IIx fibres and an elevated percentage of IIb fibres when compared with control diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover. LKB1 regulates AMPK phosphorylation, mitochondrial protein expression, fibre type distribution, as well as recovery of the diaphragm from fatigue.

Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMP-activated protein kinase (AMPK), calcium, and circulating free fatty acids (FFAs). Chronic treatment with either 5-aminoimidazole-4-carboxamide riboside (AICAR), a chemical activator of AMPK, or increasing circulating FFAs with a high-fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high-fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high-fat diet (HF), AICAR injections (AICAR), or a high-fat diet and AICAR injections (HF + AICAR) for 6 wk. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on long-chain acyl-CoA dehydrogenase, cytochrome c, and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein, as well as on citrate synthase and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) activity. In contrast, no additive effect was noted in the soleus, and in the red quadriceps only beta-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high-fat diet-induced posttranscriptional increase in PGC-1alpha protein and AMPK-mediated increase in PGC-1alpha protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high-fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

Liver kinase B1 (LKB1) is a tumor-suppressing protein that is involved in the regulation of muscle metabolism and growth by phosphorylating and activating AMP-activated protein kinase (AMPK) family members. Here we report the development of a myopathic phenotype in skeletal and cardiac muscle-specific LKB1 knockout (mLKB1-KO) mice. The myopathic phenotype becomes overtly apparent at 30-50 wk of age and is characterized by decreased body weight and a proportional reduction in fast-twitch skeletal muscle weight. The ability to ambulate is compromised with an often complete loss of hindlimb function. Skeletal muscle atrophy is associated with a 50-75% reduction in mammalian target of rapamycin pathway phosphorylation, as well as lower peroxisome proliferator-activated receptor-alpha coactivator-1 content and cAMP response element binding protein phosphorylation (43 and 40% lower in mLKB1-KO mice, respectively). Maximum in situ specific force production is not affected, but fatigue is exaggerated, and relaxation kinetics are slowed in the myopathic mice. The increased fatigue is associated with a 30-78% decrease in mitochondrial protein content, a shift away from type IIA/D toward type IIB muscle fibers, and a tendency (P=0.07) for decreased capillarity in mLKB1-KO muscles. Hearts from myopathic mLKB1-KO mice exhibit grossly dilated atria, suggesting cardiac insufficiency and heart failure, which likely contributes to the phenotype. These findings indicate that LKB1 plays a critical role in the maintenance of both skeletal and cardiac function.

Cushing's syndrome is characterized by marked central obesity and insulin insensitivity, effects opposite those seen with chronic AMP-activated protein kinase (AMPK) activation. This study was designed to determine whether chronic exposure to excess glucocorticoids influences LKB1/AMPK signaling in skeletal muscle. Corticosterone pellets were implanted subcutaneously in rats (hypercorticosteronemia, Hypercort) for 2 wk. Controls were sham operated and fed ad libitum or were sham operated and food restricted (pair-weighted group, Pair) to produce body weights similar to Hypercort rats. At the end of the 2-wk treatment period, rats were anesthetized, and the right gastrocnemius-plantaris (gastroc) and soleus muscles were removed. Left muscles were removed after electrical stimulation for 5 min. No significant differences were noted between treatment groups in ATP, creatine phosphate, or LKB1 activity. The alpha- and beta-subunit isoforms were not significantly influenced in gastroc by corticosterone treatment. Expression of the gamma3-subunit decreased, and gamma1- and gamma2-subunit expression increased. Both alpha2-AMPK and alpha1-AMPK activities were increased in the gastroc in response to electrical stimulation, but the magnitude of the increase was less for alpha2 in the Hypercort rats. Despite elevated plasma insulin and elevated plasma leptin in the Hypercort rats, phosphorylation of TBC1D1 was lower in both resting and stimulated muscle compared with controls. Malonyl-CoA content was elevated in gastroc muscles of resting Hypercort rats. These changes in response to excess glucocorticoids could be responsible, in part, for the decrease in insulin sensitivity and adiposity seen in Cushing's syndrome.

AMP-activated protein kinase (AMPK) has emerged as a key regulator of skeletal muscle fat metabolism. Because abnormalities in skeletal muscle metabolism contribute to a variety of clinical diseases and disorders, understanding AMPK’s role in the muscle is important. It was originally shown to stimulate fatty acid (FA) oxidation decades ago, and since then much research has been accomplished describing this role. In this brief review, we summarize much of these data, particularly in relation to changes in FA oxidation that occur during skeletal muscle exercise. Potential roles for AMPK exist in regulating FA transport into the mitochondria via interactions with acetyl-CoA carboxylase, malonyl-CoA decarboxylase, and perhaps FA transporter/CD36 (FAT/CD36). Likewise, AMPK may regulate transport of FAs into the cell through FAT/CD36. AMPK may also regulate capacity for FA oxidation by phosphorylation of transcription factors such as CREB or coactivators such as PGC-1α.

The LKB1/AMP‐Activated Protein Kinase (AMPK) signaling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1‐mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been characterized in the diaphragm. Two methods of AMPK activation were used to characterize LKB1/AMPK signaling in diaphragms from muscle‐specific LKB1 knockout (KO) and littermate control (C) mice: (1) acute injection of 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) and (2) 5‐min direct electrical stimulation (ES) of the diaphragm. Diaphragms were excised 60 minutes post‐AICAR injection and immediately after ES. AMPK phosphorylation increased with AICAR and ES in C but not KO mice. Acetyl CoA Carboxylase (ACC) phosphorylation increased with AICAR in C but not KO mice, but increased in both genotypes with ES. No differences in Hexokinase 2 (HK2) levels were evident. CREB phosphorylation was lower in KO diaphragms. While Cytochrome C (Cyto C) levels were lower in KO diaphragms, Uncoupling Protein 3 (UCP‐3) levels were not different between genotypes. In conclusion, AMPK is activated in an LKB1‐dependent manner in the diaphragm by AICAR injection and direct ES. This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR‐051928.

LKB1 and its target, AMPK, are regulators of muscle metabolism and play an important role in the control of mitochondrial biogenesis. Mitochondrial dysfunction leads to myopathic conditions in rodents and humans. We report here that the muscle‐specific knockout of LKB1 (KO) in female mice leads to a dysfunctional muscle phenotype and signs of congestive heart failure similar to other mitochondrial myopathies. At 9‐11 mo. of age, body weight (BW) was lower (p = 0.05) for KO vs control littermate (C) mice (21.6 ± 0.7 vs 26.2 ± 0.6 g). Absolute gastrocnemius (GAST) weight was lower for KO vs C mice (77.3 ± 2.9 vs 101.1 ± 3.7 mg), but not different as expressed relative to BW. Heart (HRT) weight relative to BW was greater in KO vs C mice (5.6 ± 0.3 vs 4.4 ± 0.1 mg). Cytochrome C (CytoC) and UCP‐2 levels were 40.1% and 59.3% lower, respectively, in KO vs C GAST muscles. CytoC, UCP‐2, UCP‐3, and cytoC oxidase‐1 (COX‐1) were all significantly lower in KO vs C HRT (33.8%, 50.8%, 49.5%, and 61.1% lower, respectively). Phosphorylation of CREB, one transcription factor controlling the expression of mitochondrial enzymes, was also decreased in HRT (40.1%) and GAST (35.6%) of KO vs C mice. Our findings suggest that impaired mitochondrial function may underlie the myopathic phenotype of LKB1‐deficient mice. This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR‐051928.