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Despite its position as the first-line drug for treatment of type 2 diabetes mellitus, the mechanisms underlying the plasma glucose level-lowering effects of metformin (1,1-dimethylbiguanide) still remain incompletely understood. Metformin is thought to exert its primary antidiabetic action through the suppression of hepatic glucose production. In addition, the discovery that metformin inhibits the mitochondrial respiratory chain complex 1 has placed energy metabolism and activation of AMP-activated protein kinase (AMPK) at the centre of its proposed mechanism of action. However, the role of AMPK has been challenged and might only account for indirect changes in hepatic insulin sensitivity. Various mechanisms involving alterations in cellular energy charge, AMP-mediated inhibition of adenylate cyclase or fructose-1,6-bisphosphatase 1 and modulation of the cellular redox state through direct inhibition of mitochondrial glycerol-3-phosphate dehydrogenase have been proposed for the acute inhibition of gluconeogenesis by metformin. Emerging evidence suggests that metformin could improve obesity-induced meta-inflammation via direct and indirect effects on tissue-resident immune cells in metabolic organs (that is, adipose tissue, the gastrointestinal tract and the liver). Furthermore, the gastrointestinal tract also has a major role in metformin action through modulation of glucose-lowering hormone glucagon-like peptide 1 and the intestinal bile acid pool and alterations in gut microbiota composition.

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

Metformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMP-activated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK- and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARgamma coactivator 1alpha (PGC-1alpha) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state.

During the early stages of Alzheimer’s disease (AD) in both mouse models and human patients, soluble forms of Amyloid-β 1–42 oligomers (Aβ42o) trigger loss of excitatory synapses (synaptotoxicity) in cortical and hippocampal pyramidal neurons (PNs) prior to the formation of insoluble amyloid plaques. In a transgenic AD mouse model, we observed a spatially restricted structural remodeling of mitochondria in the apical tufts of CA1 PNs dendrites corresponding to the dendritic domain where the earliest synaptic loss is detected in vivo. We also observed AMPK over-activation as well as increased fragmentation and loss of mitochondrial biomass in Ngn2-induced neurons derived from a new APP Swe/Swe knockin human ES cell line. We demonstrate that Aβ42o-dependent over-activation of the CAMKK2-AMPK kinase dyad mediates synaptic loss through coordinated phosphorylation of MFF-dependent mitochondrial fission and ULK2-dependent mitophagy. Our results uncover a unifying stress-response pathway causally linking Aβ42o-dependent structural remodeling of dendritic mitochondria to synaptic loss. Loss of excitatory synapses occur prior to the formation of amyloid plaques in Alzheimer’s disease. Here the authors show in an animal model that the loss of synapses induced by amyloid-beta oligomers requires over-activation of a stress-response pathway inducing structural remodelling of mitochondria in dendrites of cortical and hippocampal neurons.

Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5' AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKalpha1 or -alpha2 to determine whether the metabolic effects of resveratrol are mediated by AMPK. Mice deficient in the catalytic subunit of AMPK (alpha1 or alpha2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro. Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKalpha1(-/-) mice. In the absence of either AMPKalpha1 or -alpha2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly. We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.

Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD. Urinary flow is sensed by renal cells but its intensity is dysregulated in renal diseases. Here, the authors report that physiological flow inhibits YAP to promote autophagy, while pathological flow leads to YAP activation and autophagy inhibition.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH. Parathyroid hormone (PTH) is an endogenous hormone and osteoporosis therapeutic that suppresses sclerostin activity. Here the authors develop SIK inhibitors as potential therapeutic tools and use them to show that PTH-cAMP signalling in osteocytes inhibits SIK2 from driving Hdac4/5 nuclear shuttling to suppress sclerostin.

Nicotine acts on adipocytes to induce activation of AMPK, promoting excess lipolysis and weight loss but also the development of whole-body insulin resistance. Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen–activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.

AMPK has been reported to facilitate hypoxic pulmonary vasoconstriction but, paradoxically, its deficiency precipitates pulmonary hypertension. Here we show that AMPK-α1/α2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the new-born. Accordingly, dual AMPK-α1/α2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularisation and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no oedema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. K V 1.5 potassium currents of pulmonary arterial myocytes were markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation was also evident. Importantly, there was no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction was attenuated by AMPK-α1 or AMPK-α2 deletion without triggering pulmonary hypertension. AMPK modulates idiopathic and hypoxic pulmonary hypertension in adults, largely via unknown mechanisms. Here, the authors show that AMPK deficiency in smooth muscles causes persistent pulmonary hypertension of the new born and premature death in mice, due to increased muscularisation and remodeling of pulmonary arteries, reduced alveolar numbers and alveolar membrane thickening