Pancreatic ß-cell mitophagy as an adaptive response to metabolic stress and the underlying mechanism that involves lysosomal Ca2+ release.

Mitophagy is an excellent example of selective autophagy that eliminates damaged or dysfunctional mitochondria, and it is crucial for the maintenance of mitochondrial integrity and function. The critical roles of autophagy in pancreatic ß-cell structure and function have been clearly shown. Furthermore, morphological abnormalities and decreased function of mitochondria have been observed in autophagy-deficient ß-cells, suggesting the importance of ß-cell mitophagy. However, the role of authentic mitophagy in ß-cell function has not been clearly demonstrated, as mice with pancreatic ß-cell-specific disruption of Parkin, one of the most important players in mitophagy, did not exhibit apparent abnormalities in ß-cell function or glucose homeostasis. Instead, the role of mitophagy in pancreatic ß-cells has been investigated using ß-cell-specific Tfeb-knockout mice (TfebΔß-cell mice); Tfeb is a master regulator of lysosomal biogenesis or autophagy gene expression and participates in mitophagy. TfebΔß-cell mice were unable to adaptively increase mitophagy or mitochondrial complex activity in response to high-fat diet (HFD)-induced metabolic stress. Consequently, TfebΔß-cell mice exhibited impaired ß-cell responses and further exacerbated metabolic deterioration after HFD feeding. TFEB was activated by mitochondrial or metabolic stress-induced lysosomal Ca2+ release, which led to calcineurin activation and mitophagy. After lysosomal Ca2+ release, depleted lysosomal Ca2+ stores were replenished by ER Ca2+ through ER→lysosomal Ca2+ refilling, which supplemented the low lysosomal Ca2+ capacity. The importance of mitophagy in ß-cell function was also demonstrated in mice that developed ß-cell dysfunction and glucose intolerance after treatment with a calcineurin inhibitor that hampered TFEB activation and mitophagy.

Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic ß-cell dysfunction.

Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic ß-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master regulator of lysosomal biogenesis and autophagy gene expression, through dephosphorylation. We studied whether PPP3/calcineurin inhibitors can affect the mitophagy of pancreatic ß-cells and pancreatic ß-cell function employing FK506, an immunosuppressive drug against graft rejection. FK506 suppressed rotenone- or oligomycin+antimycin-A-induced mitophagy measured by Mito-Keima localization in acidic lysosomes or RFP-LC3 puncta colocalized with TOMM20 in INS-1 insulinoma cells. FK506 diminished nuclear translocation of TFEB after treatment with rotenone or oligomycin+antimycin A. Forced TFEB nuclear translocation by a constitutively active TFEB mutant transfection restored impaired mitophagy by FK506, suggesting the role of decreased TFEB nuclear translocation in FK506-mediated mitophagy impairment. Probably due to reduced mitophagy, recovery of mitochondrial potential or quenching of mitochondrial ROS after removal of rotenone or oligomycin+antimycin A was delayed by FK506. Mitochondrial oxygen consumption was reduced by FK506, indicating reduced mitochondrial function by FK506. Likely due to mitochondrial dysfunction, insulin release from INS-1 cells was reduced by FK506 in vitro. FK506 treatment also reduced insulin release and impaired glucose tolerance in vivo, which was associated with decreased mitophagy and mitochondrial COX activity in pancreatic islets. FK506-induced mitochondrial dysfunction and glucose intolerance were ameliorated by an autophagy enhancer activating TFEB. These results suggest that diminished mitophagy and consequent mitochondrial dysfunction of pancreatic ß-cells contribute to FK506-induced ß-cell dysfunction or glucose intolerance, and autophagy enhancement could be a therapeutic modality against post-transplantation diabetes mellitus caused by PPP3/calcineurin inhibitors.

A novel NLRP3 inhibitor as a therapeutic agent against monosodium urate-induced gout.

Background: Since NEK7 is critical for NLRP3 inflammasome activation, NEK7 inhibitors could be employed as therapeutic agents against gout, a representative disease caused by NLRP3 inflammasome. Methods: We designed NEK7 inhibitors based on biochemical kinome profiling of 2,7-substituted thieno[3,2-d]pyrimidine derivatives (SLC3031~3035 and SLC3037). Inflammasome activation was assessed by ELISA of IL-1b and immunoblotting of IL-1b maturation after treatment of bone marrow-derived macrophages with LPS+monosodium urate (MSU). NLPR3 binding to NEK7 and oligomerization were examined using immunoprecipitation and Blue Native gel electrophoresis, respectively. In vivo effect was investigated by studying gross and histopathological changes of food pad tissue of MSU-injected mice, together with assays of maturation of IL-1b and ASC speck in the tissue. Results: SLC3037 inhibited inflammasome by MSU and other inflammasome activators through blockade of NLRP3 binding to NEK7 or oligomerization, and subsequent ASC oligomerization/phosphorylation. SLC3037 significantly reduced foot pad thickness and inflammation by MSU, which was superior to the effects of colchicine. SLC3037 significantly reduced content or maturation of IL-1b and ASC speck in the food pad. The number and height of intestinal villi were decreased by colchicine but not by SLC3037. Conclusion: SLC3037, a NLRP3 inhibitor blocking NEK7 binding to NLRP3, could be a novel agent against diseases associated with NLRP3 inflammasome activation such as gout, cardiovascular diseases, metabolic syndrome or neurodegenerative diseases.

Essential role of lysosomal Ca2+-mediated TFEB activation in mitophagy and functional adaptation of pancreatic ß-cells to metabolic stress.

Although the role of pancreatic ß-cell macroautophagy/autophagy is well known, that of ß-cell mitophagy is unclear. We investigated the changes of lysosomal Ca2+ by mitochondrial or metabolic stress that can modulate TFEB activation and, additionally, the role of TFEB-induced mitophagy in ß-cell function. Mitochondrial or metabolic stress induces mitophagy, which is mediated by lysosomal Ca2+ release, increased cytosolic [Ca2+] and subsequent TFEB activation. Lysosomal Ca2+ release is replenished by ER→lysosome Ca2+ refilling through ER Ca2+ exit channels, which is important for the increase of cytosolic [Ca2+] and mitophagy by mitochondria stressors. High-fat diet (HFD) feeding augments pancreatic ß-cell mitophagy, probably as an adaptation to metabolic stress. HFD-induced increase ofß-cell mitophagy is reduced by tfeb KO, leading to increased ROS and decreased mitochondrial complex activity or oxygen consumption in tfeb-KO islets. In tfeb Δß-cell mice, HFD-induced glucose intolerance and ß-cell dysfunction are aggravated. Expression of mitophagy receptor genes including Optn or Calcoco2 is increased by mitochondrial or metabolic stressors in a TFEB-dependent manner, likely contributing to increased mitophagy. These results suggest that lysosomal Ca2+ release in conjunction with ER→lysosome Ca2+ refilling is important for TFEB activation and mitophagy induction, which contributes to pancreatic ß-cell adaptation to metabolic stress.

Current Status of Autophagy Enhancers in Metabolic Disorders and Other Diseases.

Autophagy is pivotal in the maintenance of organelle function and intracellular nutrient balance. Besides the role of autophagy in the homeostasis and physiology of the individual tissues and whole organism in vivo, dysregulated autophagy has been incriminated in the pathogenesis of a variety of diseases including metabolic diseases, neurodegenerative diseases, cardiovascular diseases, inflammatory or immunological disorders, cancer and aging. Search for autophagy modulators has been widely conducted to amend dysregulation of autophagy or pharmacologically modulate autophagy in those diseases. Current data support the view that autophagy modulation could be a new modality for treatment of metabolic syndrome associated with lipid overload, human-type diabetes characterized by deposition of islet amyloid or other diseases including neurodegenerative diseases, infection and cardiovascular diseases. While clinically available bona fide autophagy modulators have not been developed yet, it is expected that on-going investigation will lead to the development of authentic autophagy modulators that can be safely administered to patients in the near future and will open a new horizon for treatment of incurable or difficult diseases.

Lysosomal Ca2+-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic ß-cells to metabolic stress.

Although autophagy is critical for pancreatic ß-cell function, the role and mechanism of mitophagy in ß-cells are unclear. We studied the role of lysosomal Ca2+ in TFEB activation by mitochondrial or metabolic stress and that of TFEB-mediated mitophagy in ß-cell function. Mitochondrial or metabolic stress induced mitophagy through lysosomal Ca2+ release, increased cytosolic Ca2+ and TFEB activation. Lysosomal Ca2+ replenishment by ER- > lysosome Ca2+ refilling was essential for mitophagy. ß-cell-specific Tfeb knockout (TfebΔß-cell) abrogated high-fat diet (HFD)-induced mitophagy, accompanied by increased ROS and reduced mitochondrial cytochrome c oxidase activity or O2 consumption. TfebΔß-cell mice showed aggravation of HFD-induced glucose intolerance and impaired insulin release. Metabolic or mitochondrial stress induced TFEB-dependent expression of mitophagy receptors including Ndp52 and Optn, contributing to the increased mitophagy. These results suggest crucial roles of lysosomal Ca2+ release coupled with ER- > lysosome Ca2+ refilling and TFEB activation in mitophagy and maintenance of pancreatic ß-cell function during metabolic stress.

An autophagy enhancer ameliorates diabetes of human IAPP-transgenic mice through clearance of amyloidogenic oligomer.

We have reported that autophagy is crucial for clearance of amyloidogenic human IAPP (hIAPP) oligomer, suggesting that an autophagy enhancer could be a therapeutic modality against human diabetes with amyloid accumulation. Here, we show that a recently identified autophagy enhancer (MSL-7) reduces hIAPP oligomer accumulation in human induced pluripotent stem cell-derived ß-cells (hiPSC-ß-cells) and diminishes oligomer-mediated apoptosis of ß-cells. Protective effects of MSL-7 against hIAPP oligomer accumulation and hIAPP oligomer-mediated ß-cell death are significantly reduced in cells with knockout of MiTF/TFE family members such as Tfeb or Tfe3. MSL-7 improves glucose tolerance and ß-cell function of hIAPP+ mice on high-fat diet, accompanied by reduced hIAPP oligomer/amyloid accumulation and ß-cell apoptosis. Protective effects of MSL-7 against hIAPP oligomer-mediated ß-cell death and the development of diabetes are also significantly reduced by ß-cell-specific knockout of Tfeb. These results suggest that an autophagy enhancer could have therapeutic potential against human diabetes characterized by islet amyloid accumulation.

ß-cell autophagy: Mechanism and role in ß-cell dysfunction.

BACKGROUND: Elucidation of the basic molecular mechanism of autophagy was a breakthrough in understanding various physiological events and pathogenesis of diverse diseases. In the fields of diabetes and metabolism, many cellular events associated with the development of disease or its treatment cannot be explained well without taking autophagy into account. While a grand picture of autophagy has been established, detailed aspects of autophagy, particularly that of selective autophagy responsible for homeostasis of specific organelles or metabolic intermediates, are still ambiguous and currently under intensive research. SCOPE OF REVIEW: Here, results from previous and current studies on the role of autophagy and its dysregulation in the physiology of metabolism and pathogenesis of diabetes are summarized, with an emphasis on the pancreatic ß-cell autophagy. In addition to nonselective (bulk) autophagy, machinery and significance of selective autophagy such as mitophagy of pancreatic ß-cells is discussed. Novel findings regarding autophagy types other than macroautophagy are also covered, since several types of autophagy or lysosomal degradation pathways other than macroautophagy coexist in pancreatic ß-cells. MAJOR CONCLUSION: Autophagy plays a critical role in cellular metabolism, homeostasis of the intracellular environment and function of organelles such as mitochondria and endoplasmic reticulum. Impaired autophagic activity due to aging, obesity or genetic predisposition could be a factor in the development of ß-cell dysfunction and diabetes associated with lipid overload or human-type diabetes characterized by islet amyloid deposition. Modulation of autophagy of pancreatic ß-cells is likely to be possible in the near future, which would be valuable in the treatment of diabetes associated with lipid overload or accumulation of islet amyloid.

A novel autophagy enhancer as a therapeutic agent against metabolic syndrome and diabetes.

Autophagy is a critical regulator of cellular homeostasis, dysregulation of which is associated with diverse diseases. Here we show therapeutic effects of a novel autophagy enhancer identified by high-throughput screening of a chemical library against metabolic syndrome. An autophagy enhancer increases LC3-I to LC3-II conversion without mTOR inhibition. MSL, an autophagy enhancer, activates calcineurin, and induces dephosphorylation/nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy gene expression. MSL accelerates intracellular lipid clearance, which is reversed by lalistat 2 or Tfeb knockout. Its administration improves the metabolic profile of ob/ob mice and ameliorates inflammasome activation. A chemically modified MSL with increased microsomal stability improves the glucose profile not only of ob/ob mice but also of mice with diet-induced obesity. Our data indicate that our novel autophagy enhancer could be a new drug candidate for diabetes or metabolic syndrome with lipid overload.

Obesogenic diet-induced gut barrier dysfunction and pathobiont expansion aggravate experimental colitis.

Consumption of a typical Western diet is a risk factor for several disorders. Metabolic syndrome is the most common disease associated with intake of excess fat. However, the incidence of inflammatory bowel disease is also greater in subjects consuming a Western diet, although the mechanism of this phenomenon is not clearly understood. We examined the morphological and functional changes of the intestine, the first site contacting dietary fat, in mice fed a high-fat diet (HFD) inducing obesity. Paneth cell area and production of antimicrobial peptides by Paneth cells were decreased in HFD-fed mice. Goblet cell number and secretion of mucin by goblet cells were also decreased, while intestinal permeability was increased in HFD-fed mice. HFD-fed mice were more susceptible to experimental colitis, and exhibited severe colonic inflammation, accompanied by the expansion of selected pathobionts such as Atopobium sp. and Proteobacteria. Fecal microbiota transplantation transferred the susceptibility to DSS-colitis, and antibiotic treatment abrogated colitis progression. These data suggest that an experimental HFD-induced Paneth cell dysfunction and subsequent intestinal dysbiosis characterized by pathobiont expansion can be predisposing factors to the development of inflammatory bowel disease.