mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue

Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon β-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation

The relationship between insulin binding, insulin activation of insulin-receptor tyrosine kinase, and insulin stimulation of glucose uptake in isolated rat adipocytes

We have studied the relationship between insulin activation of insulin-receptor kinase and insulin stimulation of glucose uptake in isolated rat adipocytes. Glucose uptake was half-maximally or maximally stimulated, respectively, when only 4% or 14% of the maximal kinase activity had been reached. To investigate this relationship also under conditions where the insulin effect on activation of receptor kinase was decreased, the adipocytes were exposed to 10 microM-isoprenaline alone or with 5 micrograms of adenosine deaminase/ml. An approx. 30% (isoprenaline) or approx. 50% (isoprenaline + adenosine deaminase) decrease in the insulin effect on receptor kinase activity was found at insulin concentrations between 0.4 and 20 ng/ml, and this could not be explained by decreased insulin binding. The decreased insulin-effect on kinase activity was closely correlated with a loss of insulin-sensitivity of glucose uptake. Moreover, our data indicate that the relation between receptor kinase activity and glucose uptake (expressed as percentage of maximal uptake) remained unchanged. The following conclusions were drawn. (1) If activation of receptor kinase stimulates glucose uptake, only 14% of the maximal kinase activity is sufficient for maximal stimulation. (2) Isoprenaline decreases the coupling efficiency between insulin binding and receptor-kinase activation, this being accompanied by a corresponding decrease in sensitivity of glucose uptake. (3) Our data indicate that the signalling for glucose uptake is closely related to receptor-kinase activity, even when the coupling efficiency between insulin binding and kinase activation is altered. They thus support the hypothesis that receptor-kinase activity reflects the signal which originates from the receptor and which is transduced to the glucose-transport system

Cardiosphere-derived cells demonstrate metabolic flexibility that Is influenced by adhesion status

Adult stem cells demonstrate metabolic flexibility that is regulated by cell adhesion status. The authors demonstrate that adherent cells primarily utilize glycolysis, whereas suspended cells rely on oxidative phosphorylation for their ATP needs. Akt phosphorylation transduces adhesion-mediated regulation of energy metabolism, by regulating translocation of glucose transporters (GLUT1) to the cell membrane and thus, cellular glucose uptake and glycolysis. Cell dissociation, a pre-requisite for cell transplantation, leads to energetic stress, which is mediated by Akt dephosphorylation, downregulation of glucose uptake, and glycolysis. They designed hydrogels that promote rapid cell adhesion of encapsulated cells, Akt phosphorylation, restore glycolysis, and cellular ATP levels

Effects of [beta]-amyloid on glucose uptake by cultured hippocampal neurons

Alzheimer\u27s disease (AD) is the most common type of dementia in the elderly. The deposit of extracellular amyloid [beta]-peptide (A[beta]) is a distinct feature of the disease. Recent studies have shown that A[beta] interferes with glucose uptake in cultured neurons; however, how A[beta] inhibits glucose uptake is not known. This study proposed a pathway in which A[beta]25-35, a neurotoxic portion of the A[beta] peptide identical to the 25-35 amino acid-sequence in A[beta], decreases neuronal glucose uptake. The inhibitory effect of A[beta]25-35 on neuronal glucose uptake was reduced by a G protein antagonist (GPAnt-2). In the first part of the study, we demonstrated a signaling pathway in which A[beta]25-35, a neurotoxic portion of the A[beta] peptide corresponding to amino acids 25-35, inhibits neuronal glucose uptake by hippocampal neurons. The GPAnt-2, which blocks Gs, prevented the inhibitory effect of A[beta] on the glucose uptake. Cholera toxin, which stimulates adenylyl cyclase by activating Gs protein, also inhibited neuronal glucose uptake. Furthermore, the inhibitory effect of cholera toxin on glucose uptake was potentiated by A[beta]. Exposure of cells to A[beta] resulted in a transitory increase in intracellular levels of cAMP. Addition of dibutyryl cAMP (Bt2cAMP) or an adenylyl cyclase activator, forskolin, to the culture medium inhibited neuronal glucose uptake, and a protein kinase A inhibitor (KT-5720) blocked the A[beta]-mediated inhibition of glucose uptake. Thus, our findings suggest that A[beta] inhibits glucose uptake by activating the Gs-coupled receptors and involves the cAMP-PKA system. The second part of this study examined the effects of insulin and insulin-like growth factor 1 (IGF-I) on the inhibitory effect of A[beta] on neuronal glucose uptake. Insulin and IGF-I elevated neuronal glucose uptake, but the effect of IGF-I was more potent than insulin. Neurons exposed to A[beta]25-35 showed 38% less glucose uptake than the control. However, IGF-I and insulin prevented this inhibitory effect of A[beta]. To study the signaling pathway of IGF-I that stimulates glucose uptake, hippocampal neurons were studied for their glucose uptake following exposure to a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) or a mitogen-activated protein kinase (MAPK) inhibitor (PD-98059). The LY294002 decreases the stimulatory effect of IGF-I on neuronal glucose uptake while PD-98059 has no effect on the IGF-I increase neuronal glucose uptake. The results demonstrates that (1) both IGF-I and insulin are effective in elevating neuronal glucose uptake and preventing the inhibitory effect of A[beta] and (2) PI3K plays a regulatory role in neuronal glucose uptake. The present study shows that A[beta] may inhibit neuronal glucose uptake via G protein and cAMP-dependent protein kinase pathway and insulin and IGF-I reverse the AP inhibition effect on neuronal glucose uptake. However, how the two hormones interact to the A[beta] inhibition effect need to be further investigated

Characterization of the role of myosin II during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of the nonmuscle motor protein myosin II. Our previous studies using pharmacological inhibitors suggest that insulin signaling results in the phosphorylation of myosin IIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Since pharmacological inhibitors are not specific, we wanted to use a siRNA approach to complement our previous studies. In this report we demonstrate that knockdown of myosin IIA using a myosin IIA-specific siRNA resulted in impaired insulin-stimulated glucose uptake. To delineate the signaling pathway involved we asked if siRNA specific to myosin light chain kinase (MLCK), an upstream regulator of myosin IIA, would have the same effect. While we did not observe a reduction in MLCK or impairment of insulin-stimulated glucose uptake, we were able to observe that MLCK is phosphorylated upon insulin stimulation suggesting a role for MLCK in insulin-stimulated glucose uptake. Next, we used siRNA specific to extracellular-signal regulated kinase 2 (ERK2) to establish a role for this kinase in insulin-stimulated glucose uptake. Our results revealed that knockdown of ERK2 resulted in reduced phosphorylation of MLCK. Knockdown of ERK2 also impaired insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Lastly, we used siRNA specific to the calcium/calmodulin kinase II delta isoform to explore its role as a potential upstream activator of ERK2. Only a slight decrease in CaMKIIδ expression was achieved, but this did not result in a significant change in insulin-stimulated glucose uptake. Taken together, our results suggest that myosin IIA is involved in insulin-stimulated glucose uptake and that it is regulated via MLCK phosphorylation by ERK2 resulting in regulation of glucose uptake upon insulin stimulation in 3T3-L1 adipocytes

Effects of oil palm (elais guineensis) fruit extracts on glucose uptake activity of muscle, adipose and liver cells

The effect of oil palm (Elaeis guineensis) fruit aqueous extract (OPF) on glucose uptake activity of three different cell lines was investigated. The cell lines were incubated with different concentrations of OPF to evaluate the stimulatory effect of OPF towards glucose uptake activity of L6 myotubes, 3T3F442A adipocytes and Chang liver cell line. The glucose uptake activities of all tested cells were enhanced in the presence of OPF extract (basal condition). Nevertheless in combination of OPF extract and 100 nM insulin, the glucose uptake activity was only significantly enhanced in L6 myotubes and 3T3F442A adipocytes cell lines. The extracts enhanced the glucose uptake into cells through either insulin-mimetic or insulin-sensitizing property or combination of these two properties. It can be suggested that the OPF extract exerts its antihyperglycemic action partly by mediated glucose uptake into the glucose-responsive disposal cells, muscle, adipose and liver

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for (14)C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in (14)C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of (14)C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate (14)C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate (14)C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.These results suggest that AMPK-mediated PLD1 activation is required for (14)C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake

The Role of Myosin II in GLUT4 Activity and Membrane Fusion during Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes.

Insulin-stimulated glucose uptake is a vital physiological process, which requires the translocation and fusion of insulin sensitive glucose transporter (GLUT4) vesicles from intracellular pools to the plasma membrane. Previous studies have implicated cortical actin reorganization in proper GLUT4-mediated glucose uptake. However, not much is known about how cortical actin is reorganized to allow GLUT4 vesicle fusion to the plasma membrane. A recent study found that myosin II is necessary in insulin-stimulated glucose uptake and implicated myosin II as a possible mechanism for cortical actin reorganization. Our study further examined the role of myosin II in insulin-stimulated glucose uptake. We found that myosin II associates with GLUT4 vesicles upon insulin stimulation. This study also found that myosin II is necessary for proper GLUT4 vesicle fusion and activation. The findings in this study are the first to demonstrate the dual role myosin II plays in GLUT4 vesicle fusion and activation during insulin-stimulated glucose uptake. Our results provide further understanding into cellular and molecular mechanisms necessary for proper glucose uptake

Ciliary Neurotrophic Factor Stimulates Muscle Glucose Uptake by a PI3-Kinase–Dependent Pathway That Is Impaired With Obesity

OBJECTIVE: Ciliary neurotrophic factor (CNTF) reverses muscle insulin resistance by increasing fatty acid oxidation through gp130-LIF receptor signaling to the AMP-activated protein kinase (AMPK). CNTF also increases Akt signaling in neurons and adipocytes. Because both Akt and AMPK regulate glucose uptake, we investigated muscle glucose uptake in response to CNTF signaling in lean and obese mice. RESEARCH DESIGN AND METHODS: Mice were injected intraperitoneally with saline or CNTF, and blood glucose was monitored. The effects of CNTF on skeletal muscle glucose uptake and AMPK/Akt signaling were investigated in incubated soleus and extensor digitorum longus (EDL) muscles from muscle-specific AMPKalpha2 kinase-dead, gp130(DeltaSTAT), and lean and obese ob/ob and high-fat-fed mice. The effect of C2-ceramide on glucose uptake and gp130 signaling was also examined. RESULTS: CNTF reduced blood glucose and increased glucose uptake in isolated muscles in a time- and dose-dependent manner with maximal effects after 30 min with 100 ng/ml. CNTF increased Akt-S473 phosphorylation in soleus and EDL; however, AMPK-T172 phosphorylation was only increased in soleus. Incubation of muscles from AMPK kinase dead (KD) and wild-type littermates with the PI3-kinase inhibitor LY-294002 demonstrated that PI3-kinase, but not AMPK, was essential for CNTF-stimulated glucose uptake. CNTF-stimulated glucose uptake and Akt phosphorylation were substantially reduced in obesity (high-fat diet and ob/ob) despite normal induction of gp130/AMPK signaling--effects also observed when treating myotubes with C2-ceramide. CONCLUSIONS: CNTF acutely increases muscle glucose uptake by a mechanism involving the PI3-kinase/Akt pathway that does not require AMPK. CNTF-stimulated glucose uptake is impaired in obesity-induced insulin resistance and by ceramide

Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes

BACKGROUND: Phytanic acid (PA) is a chlorophyll metabolite with potentials in regulating glucose metabolism, as it is a natural ligand of the peroxisome proliferator-activated receptor (PPAR) that is known to regulate hepatic glucose homeostasis. This study aimed to establish primary porcine myotubes as a model for measuring glucose uptake and glycogen synthesis, and to examine the impact of physiological amounts of PA on glucose uptake and glycogen synthesis either alone or in combination with insulin. METHODS: Porcine satellite cells were cultured into differentiated myotubes and tritiated 2-deoxyglucose (2-DOG) was used to measure glucose uptake, in relation to PA and 2-DOG exposure times and also in relation to PA and insulin concentrations. The MIXED procedure model of SAS was used for statistical analysis of data. RESULTS: PA increased glucose uptake by approximately 35%, and the presence of insulin further increased the uptake, but this further increase in uptake was non- additive and less pronounced at high insulin concentrations. There was no effect of PA alone on glycogen synthesis, while the insulin stimulation of glycogen was increased by 20% in the presence of PA. PA neither stimulated glucose uptake nor glycogen synthesis in insulin-resistant myotubes generated by excess glucose exposure. CONCLUSIONS: Primary porcine myotubes were established as a model of skeletal muscles for measuring glucose uptake and glycogen synthesis, and we showed that PA can play a role in stimulating glucose uptake at no or inadequate insulin concentrations