CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

TBC1D4-S711 Controls Skeletal Muscle Insulin Sensitization After Exercise and Contraction.

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.

Timing and Causes of Death in Acute Myocardial Infarction Complicated by Cardiogenic Shock (from the RETROSHOCK Cohort).

Acute myocardial infarction complicated by cardiogenic shock (AMICS) comprises a heterogeneous population with high mortality. Insight in timing and cause of death may improve understanding of the condition and aid individualization of treatment. This was assessed in a retrospective, multicenter observational cohort study based on 1,716 patients with AMICS treated during the period of 2010 to 2017, of whom 904 died before hospital discharge. Patients with AMICS were identified through national registries and review of individual patients charts. In 904 patients with AMICS who died before hospital discharge (median age 72 years [interquartile range (IQR) 63 to 79], 70% men), 342 (38%) had suffered out-of-hospital cardiac arrest. The most frequent cause of death was primary cardiac (54%), whereas 24% died of neurologic injury, and 20% of multiorgan failure (MOF). Time to death was 13 hours (IQR 5 to 43) for heart failure; 140 hours (IQR 95 to 209) in neurologic injury; and 137 hours (IQR 59 to 321) in MOF, p <0.001. The causes of death in patients presenting with out-of-hospital cardiac arrest (OHCA) were: neurologic injury in 57%, as opposed to 4% in patients not presenting with OHCA, p <0.001. In conclusion, in patients with AMICS, cause of death was mainly primary heart failure followed by neurologic injury and MOF. Median time from first medical contact to death was only 13 hours in patients dying from cardiac causes. The risk of dying of neurologic injury was low in patients without OHCA.

Illumination of the Endogenous Insulin-Regulated TBC1D4 Interactome in Human Skeletal Muscle.

Insulin-stimulated muscle glucose uptake is a key process in glycemic control. This process depends on the redistribution of glucose transporters to the surface membrane, a process that involves regulatory proteins such as TBC1D1 and TBC1D4. Accordingly, a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with an increased risk of type 2 diabetes, and observations from carriers of a TBC1D1 variant associate this protein to a severe obesity phenotype. Here, we identified interactors of the endogenous TBC1D4 protein in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors. The binding of 12 of these interactors was regulated by insulin, including proteins known to be involved in glucose metabolism (e.g., 14-3-3 proteins and α-actinin-4 [ACTN4]). TBC1D1 also coprecipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin or exercise in young, healthy, lean individuals. Similarly, the exercise- and insulin-regulated phosphorylation of the TBC1D1-TBC1D4 complex was intact. In contrast, we observed an altered interaction as well as compromised insulin-stimulated phosphoregulation of the TBC1D1-TBC1D4 complex in muscle of obese individuals with type 2 diabetes. Altogether, we provide a repository of TBC1D4 interactors in human and mouse skeletal muscle that serve as potential regulators of TBC1D4 function and, thus, insulin-stimulated glucose uptake in human skeletal muscle.

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake. Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method applied possesses some caveats under certain conditions when studying muscle energy metabolism.

TBC1D4 Is Necessary for Enhancing Muscle Insulin Sensitivity in Response to AICAR and Contraction.

Muscle insulin sensitivity for stimulating glucose uptake is enhanced in the period after a single bout of exercise. We recently demonstrated that AMPK is necessary for AICAR, contraction, and exercise to enhance muscle and whole-body insulin sensitivity in mice. Correlative observations from both human and rodent skeletal muscle suggest that regulation of the phosphorylation status of TBC1D4 may relay this insulin sensitization. However, the necessity of TBC1D4 for this phenomenon has not been proven. Thus, the purpose of this study was to determine whether TBC1D4 is necessary for enhancing muscle insulin sensitivity in response to AICAR and contraction. We found that immediately after contraction and AICAR stimulation, phosphorylation of AMPKα-Thr172 and downstream targets were increased similarly in glycolytic skeletal muscle from wild-type and TBC1D4-deficient mice. In contrast, 3 h after contraction or 6 h after AICAR stimulation, enhanced insulin-stimulated glucose uptake was evident in muscle from wild-type mice only. The enhanced insulin sensitivity in muscle from wild-type mice was associated with improved insulin-stimulated phosphorylation of TBC1D4 (Thr649 and Ser711) but not of TBC1D1. These results provide genetic evidence linking signaling through TBC1D4 to enhanced muscle insulin sensitivity after activation of the cellular energy sensor AMPK.