Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells

Both type 2 diabetes (T2D) and obesity are characterized by excessive hyperlipidaemia and subsequent lipid droplet (LD) accumulation in adipose tissue. To investigate whether LDs also accumulate in β‐cells of T2D patients, we assessed the expression of PLIN2, a LD‐associated protein, in non‐diabetic (ND) and T2D pancreata. We observed an up‐regulation of PLIN2 mRNA and protein in β‐cells of T2D patients, along with significant changes in the expression of lipid metabolism, apoptosis and oxidative stress genes. The increased LD buildup in T2D β‐cells was accompanied by inhibition of nuclear translocation of TFEB, a master regulator of autophagy and by down‐regulation of lysosomal biomarker LAMP2. To investigate whether LD accumulation and autophagy were influenced by diabetic conditions, we used rat INS‐1 cells to model the effects of hyperglycaemia and hyperlipidaemia on autophagy and metabolic gene expression. Consistent with human tissue, both LD formation and PLIN2 expression were enhanced in INS‐1 cells under hyperglycaemia, whereas TFEB activation and autophagy gene expression were significantly reduced. Collectively, these results suggest that lipid clearance and overall homeostasis is markedly disrupted in β‐cells under hyperglycaemic conditions and interventions ameliorating lipid clearance could be beneficial in reducing functional impairments in islets caused by glucolipotoxicity.     

Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.     

14‐3‐3ζ binds to and stabilizes phospho‐beclin 1S295 and induces autophagy in hepatocellular carcinoma cells

Data from The Cancer Genome Atlas (TCGA) indicate that the expression levels of 14‐3‐3ζ and beclin 1 (a key molecule involved in cellular autophagy) are up‐regulated and positively correlated with each other (R = .5, P < .05) in HCC tissues. Chemoresistance developed in hepatoma cancer cells is associated with autophagy initiation. This study aimed to explore 14‐3‐3ζ’s role in regulating autophagy in HCC cells, with a focus on beclin 1. The co‐localization of 14‐3‐3ζ and beclin 1 was detectable in primary HCC tissues. To simulate in vivo tumour microenvironment (hypoxia), CSQT‐2 and HCC‐LM3 cells were exposed to 2% oxygen for 24 hours. The protein levels of 14‐3‐3ζ and phospho‐beclin 1^S295 peaked at 12 hours following hypoxia. Meanwhile, the strongest autophagy flux occurred: LC3II was increased, and p62 was decreased significantly. By sequencing the coding area of BECN 1 gene of CSQT‐2 and HCC‐LM3 cells, we found that the predicted translational products of BECN 1 gene contained RLPS²⁹⁵VP (R, arginine; L, leucine; P, proline; S, serine; V, valine), a classic 14‐3‐3ζ binding motif. CO‐IP results confirmed that 14‐3‐3ζ bound to beclin 1, and this connection was markedly weakened when S²⁹⁵ was mutated into A²⁹⁵ (alanine). Further, 14‐3‐3ζ overexpression prevented phospho‐beclin 1^S295 from degradation and enhanced its binding to VPS34, whilst its knockdown accelerated the degradation. Additionally, 14‐3‐3ζ enhanced the chemoresistance of HCC cells to cis‐diammined dichloridoplatium by activating autophagy. Our work reveals that 14‐3‐3ζ binds to and stabilizes phospho‐beclin 1^S295 and induces autophagy in HCC cells to resist chemotherapy.     

Interplay between FGFR2b‐induced autophagy and phagocytosis: role of PLCγ‐mediated signalling

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b‐induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor‐triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation.     

HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Calpain‐2 plays a pivotal role in the inhibitory effects of propofol against TNF‐α‐induced autophagy in mouse hippocampal neurons

Calpains are calcium‐dependent proteases and play critical roles in neuronal autophagy induced by inflammation. Propofol has been reported to exert anti‐inflammatory effects in neurons. We aimed to identify whether and how propofol‐modulated calpain activity and neuron autophagy in response to tumour necrosis factor‐α (TNF‐α). Mouse hippocampal neurons were pre‐treated with propofol and exposed to TNF‐α. Autophagy was evaluated by fluorescent autophagy assay and by measuring LC3I and LC3II expression. Intracellular calcium concentration was measured by fluorescent assay. Calpain activation was measured by calpain activity assay. The protein expression of intracellular signalling molecules was detected by Western blot analysis. Compared with untreated control neurons, 40 ng/mL TNF‐α treatment for 2 hours induced neuron autophagy, which was attenuated by 25 μmol/L propofol. TNF‐α induced intracellular calcium accumulation, the phosphorylation of calcium/calmodulin‐dependent protein kinase II (CAMK II) and calpain‐2, calpain activation and lysosomal cathepsin B release as well as tyrosine kinase receptor B (TrkB) truncation. These effects were alleviated by propofol, calcium chelator, CAMK II inhibitor, calpain‐2 inhibitor, calpain‐2 siRNA transfection and N‐Methyl‐d ‐aspartic acid (NMDA) receptor antagonist. Propofol, via NMDA receptor, inhibited TNF‐α‐mediated hippocampal neuron autophagy. The mechanism may involve calcium and calcium‐dependent signalling pathway, especially CAMK II and calpain‐2.     

Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol,which induces autophagy in cancer cells

Resveratrol has many proposed health benefits, including the prevention of cancers, but its low bioavailability is considered a limiting factor in translating these effects to humans. Based on in vivo and clinical studies we have shown that resveratrol is indeed rapidly metabolized by phase II enzymes, and that resveratrol sulfates are deconjugated by steroid sulfatases to afford free resveratrol in vitro and in vivo and hence act as an intracellular reservoir for resveratrol. Further, we have demonstrated that at clinically achievable concentrations of resveratrol sulfate, parent resveratrol is regenerated within human colorectal cancer, but not normal epithelial cells, and is responsible for inducing autophagy with senescence selectively in cancer cells.     

Resveratrol-sulfates provide an intracellular reservoir for generation of parent resveratrol,which induces autophagy in cancer cells

Resveratrol has many proposed health benefits, including the prevention of cancers, but its low bioavailability is considered a limiting factor in translating these effects to humans. Based on in vivo and clinical studies we have shown that resveratrol is indeed rapidly metabolized by phase II enzymes, and that resveratrol sulfates are deconjugated by steroid sulfatases to afford free resveratrol in vitro and in vivo and hence act as an intracellular reservoir for resveratrol. Further, we have demonstrated that at clinically achievable concentrations of resveratrol sulfate, parent resveratrol is regenerated within human colorectal cancer, but not normal epithelial cells, and is responsible for inducing autophagy with senescence selectively in cancer cells.     

Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy

The majority of studies on autophagy, a cytoplasmic homeostatis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3‐kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3‐phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI‐1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.     

Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β‐amyloid peptide

Alzheimer's disease (AD) is an aging‐related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP‐ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up‐regulated the lysoso‐mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3‐methylade‐nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ‐induced apoptosis.     

SB‐216763, a GSK‐3β inhibitor,protects against aldosterone‐induced cardiac,and renal injury by activating autophagy

Cardiovascular and renal inflammation induced by Aldosterone (Aldo) plays a pivotal role in the pathogenesis of hypertension and renal fibrosis. GSK‐3β contributes to inflammatory cardiovascular and renal diseases, but its role in Aldo‐induced hypertension, and renal damage is not clear. In the present study, rats were treated with Aldo combined with SB‐216763 (a GSK‐3β inhibitor) for 4 weeks. Hemodynamic, cardiac, and renal parameters were assayed at the indicated time. Here we found that rats treated with Aldo presented cardiac and renal hypertrophy and dysfunction. Cardiac and renal expression levels of molecular markers attesting inflammation and fibrosis were increased by Aldo infusion, whereas the treatment of SB‐216763 reversed these alterations. SB‐216763 suppressed cardiac and renal inflammatory cytokines levels (TNF‐a, IL‐1β, and MCP‐1). Meanwhile, SB‐216763 increased the protein levels of LC3‐II in the cardiorenal tissues as well as p62 degradation, indicating that SB‐216763 induced autophagy activation in cardiac, and renal tissues. Importantly, inhibition of autophagy by 3‐MA attenuated the role of SB‐216763 in inhibiting perivascular fibrosis, and tubulointerstitial injury. These data suggest that SB‐216763 protected against Aldo‐induced cardiac and renal injury by activating autophagy, and might be a therapeutic option for salt‐sensitive hypertension and renal fibrosis.     

XIAP inhibits autophagy via XIAP‐Mdm2‐p53 signalling

The primary role of autophagy is adaption to starvation. However, increasing evidence suggests that autophagy inhibition also plays an important role in tumorigenesis. Upregulation of X‐linked inhibitor of apoptosis (XIAP) has been associated to a variety of human cancers, yet the underlying mechanisms remain obscure. Here, we report that XIAP suppresses autophagy by exerting a previously unidentified ubiquitin E3 ligase activity towards Mdm2, which is a negative regulator of p53. XIAP controls serum starvation‐induced autophagy downstream of the PI3K/Akt pathway. In mouse models, inhibition of autophagy by XIAP promotes tumorigenecity of HCT116 cells. XIAP‐mediated autophagy inhibition is also largely validated in clinical tumour samples. These findings reveal a novel XIAP‐Mdm2‐p53 pathway that mediates the inhibition of autophagy, by which XIAP may contribute to tumorigenesis.