Autophagy regulates inflammation following oxidative injury in diabetes

Abstract

T1D (type 1 diabetes) is an autoimmune disease characterized by lymphocytic infiltration, or inflammation in pancreatic islets called ‘insulitis.’ Comparatively speaking, T2D (type 2 diabetes) is traditionally characterized by insulin resistance and islet β cell dysfunction; however, a number of studies have clearly demonstrated that chronic tissue inflammation is a key contributing factor to T2D. The NLR (Nod-like receptor) family of innate immune cell sensors such as the NLRP3 inflammasome are implicated in leading to CASP1 activation and subsequent IL1B (interleukin 1, β) and IL18 secretion in T2D. Recent developments reveal a crucial role for the autophagy pathway under conditions of oxidative stress and inflammation. Increasingly, research on autophagy has begun to focus on its role in interacting with inflammatory processes, and thereby how it potentially affects the outcome of disease progression. In this review, we explore the pathophysiological pathways associated with oxidative stress and inflammation in T2D. We also explore how autophagy influences glucose homeostasis by modulating the inflammatory response. We will provide here a perspective on the current research between autophagy, inflammation and T2D.

Keywords: :

• autophagy
• diabetes
• inflammation
• NLRP3 inflammasome
• oxidative stress

Introduction

T1D is a well-known autoimmune disease in which pancreatic β cells are killed by the immune system. Inflammation is the first response of the immune system to infection or tissue injury. Low-grade systemic inflammation may contribute to pancreatic β cell apoptosis and the building up of ‘insulitis’ in T1D.¹ Recent years have seen remarkable growth in our understanding of the cellular and molecular mechanisms that control the inflammatory response, and this is due in part to the information we have gained about the NLR family of innate immune cell sensors. Among the NLR family members, the NLRP3 inflammasome, which regulates the release of pro-inflammatory cytokines such as IL1B and IFNG (interferon, gamma), is the best-characterized receptor that activates the inflammasome. The NLRP3 inflammasome can be triggered by a variety of situations of host ‘danger,’ including infection and metabolic dysregulation.² In addition, T2D is another disease that is mediated by a sterile inflammatory mechanism (i.e., an inflammatory response that occurs in the absence of microorganisms), which, until recently, was not well understood.³ Mounting evidence indicates that the NLRP3 inflammasome plays a role in T2D. A reduction in the functioning of islet β cells in the pancreas is characteristic of T2D, and pro-inflammatory cytokines, such as IL1B andIFNG, activate signaling pathways resulting in pancreatic β-cell death and dysfunction. IL1B is involved in the autoimmune processes leading to T1D.¹ Recent reports have shown that expression of IL1B is upregulated in the islets of patients with T2D.⁴ NLRP3-dependent IL1B production during metabolic stress, in this case chronic hyperglycemia, may contribute to the progression of T2D.⁵

T2D is a common multifactorial disease associated with oxidative stress, which through the formation of ROS (reactive oxygen species) results in cell damage and insulin resistance induced by hyperglycemia and hyperlipidemia. Chronic islet exposure to elevated glucose triggers ROS generation, through mechanisms that are currently unclear. The increased level of ROS induced by oxidative stress was demonstrated to trigger NLRP3 activation and promote CASP1-dependent IL1B secretion.⁶ Chronic low-grade inflammation resulting from oxidative stress and imbalances in the innate immune system has been associated with obesity, metabolic syndrome and insulin resistance,⁷ whereas pro-inflammatory signaling pathways can inhibit insulin signaling,⁸ thus providing a link between inflammation, oxidative stress and insulin resistance.

In conjunction with this increased knowledge on inflammasomes, the role of autophagy in the regulation of inflammatory responses has started to emerge. Autophagy is a significant regulator of innate immunity responses in host defense. There are now convincing experimental data demonstrating that efficient autophagic activity can prevent the activation of inflammasomes and induction of inflammatory responses.⁹ Normally, the autophagic uptake of dysfunctional mitochondria prevents excessive ROS production and inflammasome activation; however, excessive autophagy may lead to autophagic cell death.¹⁰ It is now well recognized that autophagy can exert a critical influence on systemic immune and inflammatory responses. It might be beneficial to investigate the potential for therapeutic targeting of autophagic processes to mitigate inflammatory diseases including T2D.

Connecting Type 1 and Type 2 Diabetes through Inflammatory Responses

Diabetes is an increasing health problem worldwide. T1D is a multifactorial disease where a chronic autoimmune assault results in a progressive β-cell loss. It is a T cell-driven disease and β cells are destroyed by the immune system, causing insulin dependence for life. In the course of T1D, the pro-inflammatory cytokines IL1B, IFNG and TNF (tumor necrosis factor) produced by infiltrating immune cells play a critical role in the progression of β-cells, leading to local inflammation and β-cell apoptosis.¹¹ This cytokine-induced response is a low-grade inflammation that occurs through activation of the innate immune system. A study has previously shown that pancreatic β-cells respond to the pro-inflammatory cytokines by modifying the expression of complex gene networks under the regulation of at least two master transcription factors, namely NFKB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) and STAT1 (signal transducer and activator of transcription 1, 91 kDa).¹² Cytokine-induced STAT1 activation in β-cells is associated with the induction of apoptosis and diabetes progression in murine models of T1D.¹³ These data indicate that STAT1 is a key player in immune-mediated early β-cell dysfunction and death. Thus, a better understanding of the signaling pathways involved in cytokine-induced β-cell apoptosis during ‘insulitis’ may help to define potential therapeutic targets to interfere with T1D development. Research presently combines RNA interference and array analysis through three key pathways to clarify the gene networks regulated by the TNF-STAT1-IRF1 (interferon regulatory factor 1) pathway in β-cells.

T1D results from cellular-mediated autoimmune destruction of pancreatic β-cells, which usually leads to total loss of insulin secretion; in contrast, type 2 diabetes is caused by resistance to insulin combined with a failure to produce enough additional insulin to compensate for the resistance.¹⁴ Islet inflammation promotes β-cell destruction in T1D as well as in T2D, and systemic inflammation is involved in the development of insulin resistance in T2D. In addition, cytokines which are crucially involved in the etiopathology of T1D also play a role in islet dysfunction in T2D. A recent study brings evidence for the development of a clear inflammatory process in pancreatic islets from Zucker fa/fa rats, a model of obesity-associated insulin resistance.¹⁵

NLRP3 Inflammasome Contributes to Insulin Resistance of Pancreatic Islets in T2D

Pancreatic β-cells play a key role in glucose homeostasis in mammals. The NLR family of innate immune cell sensors like NLRP3 is implicated in insulin resistance of pancreatic islets in T2D. Reduction in adipose tissue expression of NLRP3 is coupled with decreased inflammation and improved insulin sensitivity in obese T2D patients.¹⁶ Chronic low-grade inflammation, which is present in both TID and T2D, contributes to the pathogenesis of insulin resistance.¹⁷ The NLRP3 inflammasome is an intracellular, multiprotein complex composed of an NLR protein consisting of NRLP3, PYCARD/ASC (PYD and CARD domain containing) with a caspase activation and recruitment domain, and CASP1. Activation induces oligomerization of the NLRP3 inflammasome and leads to the recruitment of PYCARD-pro-CASP1, which results in CASP1 activation and processing of cytoplasmic targets, including the pro-inflammatory cytokines IL1B and IL18. IL1B is an important inflammatory mediator of T2D. IAPP (islet amyloid polypeptide), a protein that forms amyloid deposits in the pancreas during T2D, triggers the NLRP3 inflammasome and generates mature IL1B, which has been found to have profound effects on the function of pancreatic β cells, inducing them to undergo apoptosis.¹⁸ An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host ‘danger,’ including infection and metabolic dysregulation.¹⁹ Experiments in mouse models indicate that mice deficient in NLRP3, PYCARD and CASP1 are resistant to the obesity-induced insulin resistance.¹⁶

Obesity, which itself induces the assembly of the NLRP3 inflammasome in adipose tissue macrophages (ATMs), is frequently the basis for insulin resistance in early T2D,¹⁶ and is associated with increased concentrations of pro-inflammatory cytokines like IL1B, which has been found to have profound effects on the function of pancreatic β-cells.²⁰ IL1B is induced by pro-inflammatory signaling through TLRs (toll-like receptors) or by cytokines, such as TNFAor IL1B itself. Inflammatory cytokines converge on inhibitors of NFKB, IKBKB (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase β), and MAPK8/JNK1 (mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1) to directly inhibit insulin action via serine phosphorylation of IRS1 and IRS2 (insulin receptor substrate 1 and 2).¹⁷ In confirmation of published data,²¹ inhibition of IL1B has been shown to ameliorate T2D, which suggests an important role of this pro-inflammatory cytokine in disease progression. Elevated IL1B is also a risk factor for the development of T2D, and contributes to insulin resistance by antagonizing insulin signaling. Thus, targeting individual inflammasome components may be an alternative approach for treating T2D.

Oxidative Stress and Inflammation Interactions in Human Obesity

Obesity is characterized by the activation of an inflammatory process in metabolically active sites such as adipose tissue, liver and immune cells.²² The FTO (fat mass and obesity associated) genes, which are key obesity susceptibility genes, predict persistent central obesity. Inflammatory markers [e.g.,CRP (C-reactive protein, pentraxin-related)]²³ and inflammatory cytokines (e.g., IL6)²⁴ are increased in patients with diabetes. AGTR2 (angiotensin II receptor, type 2) is to a large degree responsible for triggering inflammation by inducing oxidative stress in obese states, which supports the relationship between inflammation and obesity in humans.²⁵ AGTR2 also downregulates pro-inflammatory transcription factors such as NFKB, resulting in the generation and secretion of ROS. When moderately produced, ROS are involved in important physiological processes that lead to desired cellular responses. However, high ROS production is negatively associated with different biological signaling pathways.²⁶

The link between obesity and inflammation has been derived from the finding that pro-inflammatory cytokines are overexpressed in obesity.²⁷ One of the major contributions in the understanding of the inflammatory nature of obesity was the identification of the cytokine TNF. An increase in TNF promotes the secretion of other pro-inflammatory agents, and evidence suggests that TNF promotes insulin resistance by the inhibition of the IRS1 signaling pathway.²² IL6 is also implicated in the development of obesity-related systemic inflammation and insulin resistance.

Chronic hyperglycemia-associated oxidative stress and low-grade inflammation are considered to play critical roles in disease initiation and progression of diabetes. The oxidative stress mechanism and inflammatory signal are interrelated, and their impairment leads to an inhibition of insulin responses as well as a higher risk of diabetes. Inflammation is a physiological response of the organism to harmful stimuli. TXNIP (thioredox ininteracting protein), encoding an endogenous inhibitor of the antioxidant thioredoxin, is an early response gene highly induced by diabetes and hyperglycemia.²⁸ Interactions between TXNIP and NLRP3-NLRP3 inflammasome in human embryonic kidney T cells have been confirmed, and suggested that the TXNIP interaction is a specific feature of NLRP3-NLRP3 inflammasome. In addition, the absence of NLRP3 or TXNIP might affect the secretion of cytokines other than IL1B.⁶ Recent findings further demonstrate a potential role for TXNIP in innate immunity via activation of the NLR-NLRP3-CASP1 inflammasome and release of IL1B in diabetes and oxidative stress.^28,29 The involvement of TXNIP in inflammasome activation is also consistent with the reported requirement for ROS in activation of the NLRP3 inflammasome. In β cells, TXNIP is downregulated by insulin, and TXNIP expression is consistently higher in humans with T2D.³⁰

Autophagy

There are three types of cell death; apoptosis, necrosis and autophagy. In the past decades, studies of autophagy have been vastly expanded. Autophagy is a cellular degradation pathway which maintains cellular homeostasis through the degradation and recycling of cytoplasm constitutively, and in response to environmental conditions, such as starvation, growth factor deprivation, ER (endoplasmic reticulum) stress and pathogen infection.³¹ Autophagy is initiated by the formation of a double-membrane bound vesicle known as the autophagosome. The autophagosome then fuses with the lysosome to degrade the sequestered cargoes and to recycle nutrients and membranes. Autophagy is basically a cell-protection mechanism, since it maintains the cell’s energy level under nutrient-depleted conditions, regulates the turnover of aged or abnormal proteins, and eliminates damaged organelles. However, it may also promote cell death through excessive degradation of cellular constituents, depending on the cellular and environmental context.¹⁰ Several molecules are involved in the regulation and execution of autophagy. In the presence of adequate nutrients, growth factors such as insulin can activate class II PI3K (phosphoinositide 3-kinase) proteins, which in turn activate MTOR (mechanistic target of rapamycin),³² which inhibits the activation of autophagy. The suppression of MTOR by specific inhibitors such as rapamycin also induces autophagy.^33-35 In general, the process of autophagy is beneficial for cells. However, an increase in autophagy can cause autophagic cell death distinct from apoptosis. Fraenkel et al.³⁶ show that chronic inhibition of MTOR by rapamycin dramatically worsens the metabolic syndrome in nutrition-dependent T2D. Rapamycin, an immunosuppressant used in human transplantation, impairs β-cell function,³⁷ but the mechanism is unclear.

The main targets of MTOR with regard to autophagy are the ATG (autophagy-related) genes, which were first discovered in yeast. These genes constitute the core molecular machinery of autophagy, which regulate the formation of autophagosomes and subsequent fusion of these vesicles with lysosomal or vacuolar compartments for cargo degradation.³⁸ Atg7 is an E1-like gene that is essential for the formation and completion of autophagosomes. Fujitani et al.³⁹ reported that compared with wild-type mice, Atg7^f/f:RIP-Cre mice (β cells specific Atg7-deficient mice) show progressive degeneration of pancreatic β cells, which currently is considered the primary determinant of progression of insulin resistance in diabetes. The formation of autophagosomes is reliant upon two novel conjugation systems, LC3 (microtubule-associated protein 1 light chain 3) is a ubiquitin-like protein that is part of one of the systems, and it undergoes post-translational modifications involving the conversion of a soluble form (LC3-I) to a membrane-associated form (LC3-II) that is conjugated to phosphatidylethanolamine.

The Dual Nature of Autophagy in Pancreatic β Cells

Pancreatic β cells, which play a key role in glucose homeostasis, have the main functions of insulin synthesis and secretion. β cell failure is currently considered the primary determinant of progression of insulin resistance to diabetes. Recent in vivo studies, including our recent unpublished study showing that LC3-I and LC3-II are obviously increased when INS-1 cells are treated with insulin at 100 nmol/L for 24 h compared with the control, indicate that high levels of insulin upregulate the expression of autophagy. When treated with 3-MA (3-methyladenine), an autophagy inhibitor, apoptosis was significantly increased when compared tocells treated with high levels of insulin only. These results indicate that autophagy, a protein degradation system, plays an important role in the maintenance of normal β cell function and survival. However, a different group reported no such protective role of autophagy in Pdx1 KD MIN6 cells (knockdown expression of pancreatic and duodenal homeobox 1 in mouse insulinoma cell line). The possibility that activation of autophagy may increase β cell death is addressed in a recent in vitro and in vivo study. Fujimoto et al.⁴⁰ reported that autophagy is increased in Pdx1 KD MIN6 cells; autophagy is activated in Pdx1-reduced MIN6 cells and in Pdx1^+/− β cells in vivo. The autophagy inhibitor 3-MA prevents Pdx1 KD-induced MIN6 cell death, but 3-MA-treated Pdx1 KD MIN6 cells finally die by CASP3-dependent cell death, which indicate that an increase in autophagy appears to occur early, and this is followed by an increase in apoptosis. Thus, it is still unclear as to whether autophagy plays a protective or harmful role in diabetes.

Autophagy is a highly dynamic, multistep process. A previous study revealed a low level of constitutive autophagy in pancreatic β cells under steady-state conditions and its enhancement when mice are fed a high-fat diet.⁴¹ The study also reported that autophagy was reduced but not completely inhibited in Pdx1^+/−Becn1^{+/ −} (autophagy-related) mice islets, which suggested a persistence of basal autophagy in Pdx1^+/−Becn1^+/− β cells.⁴¹ Basal autophagy can be viewed as a defense mechanism against cellular damage in β cells. Interestingly, lipotoxicity induces impaired autophagy in human β cells with reduction of lysosome-associated membrane protein 2 (LAMP2, a protein involved in later steps of autophagy) and lysosomal hydrolases leading to vacuole accumulation and cell death.⁴² The study indicated that enhanced autophagy can result in deterioration of β cell function by allowing intracellular accumulation of damaged organelles, particularly dysfunctional mitochondria, the study does not necessarily mean that enhanced autophagy is harmful in patients with T2D. Enhanced autophagy may contribute to reduced β cell mass under certain conditions by accelerating β cell death.

The above study suggests that both basal levels of autophagy and stress-induced increases in autophagy are likely to be important in promoting mammalian health. The activation of autophagy, however, is not without potential risks. The induction of autophagy by rapamycin may be abnormally magnified to exceed the compatibility of the cells.⁴³ Accumulation of autophagic vacuoles and autophagosomes in human T2D β cells may contribute to β cell dysfunction.⁴⁴ Autophagic structure overload might be due to their increased production and/or reduced removal. Thus, the dual nature of autophagy can either promote cell death or protect cells from diverse types of injuries depending on the cellular and environmental context.

The Emerging Role of Autophagy in Inflammation-Associated T2D

The autophagy machinery interfaces with most cellular stress-response pathways such as programmed cell death, inflammation and adaptive immune mechanisms, and thereby potentially influences disease pathogenesis.⁴⁵ Recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. However, there is little information on whether the regulation of autophagy in T2D is associated with pro-inflammatory cytokines through an anti-inflammatory function. Crisan et al.⁴⁶ show that autophagy has strong inhibitory effects on IL1B production. These results show that basal autophagy in physiological conditions has important modulatory effects on inflammation. However, the effect of autophagy on IL1B production was demonstrated only in murine cells, and no information was available on whether similar effects may be exerted in human cells.

There is a complex reciprocal relationship between the autophagy pathway/proteins and immunity and inflammation. The signaling pathways that regulate inflammatory processes now apparently have a role in the regulation of autophagy and vice versa. Thus, the autophagy pathway and autophagy proteins may function in a central role that balances the beneficial and harmful effects of the host response in immunity and inflammation. Defects in autophagy may contribute to inflammation-associated metabolic diseases such as diabetes and obesity, which are both linked to insulin resistance.⁴⁷ A recent study demonstrates that depletion of the autophagic proteins LC3B and BECN1 enhances CASP1 activation and secretion of IL1B and IL18.⁴⁸ It is also demonstrated that autophagy stimulates pro-inflammatory cytokine secretion in response to LPS (lipopolysaccharide) and ATP in Atg16 deleted mice, and requires the NLRP3 inflammasome pathway.^2,48 Recent observations have revealed a relationship between autophagic proteins and inflammasome-associated pro-inflammatory cytokine maturation in macrophages. The mechanism through which autophagy deficiency enhances NLRP3 inflammasome pathway activation includes direct interactions between autophagy proteins and inflammasome components, and indirect activation of inflammasome activity through enhanced production of mitochondrial ROS.⁴⁹ The study shows that depletion of autophagy proteins enhances CASP1 activation and secretion of IL1B and IL18 in macrophages in response to LPS and ATP.⁵⁰ The pathway to CASP1-dependent IL18 secretion in macrophages was further shown to be blocked by mitochondrial targeting antioxidants.⁵¹ The amplification of CASP1-dependent IL18 secretion responses to LPS and ATP were attributed to loss of mitochondrial quality control and enhanced mitochondrial ROS production in response to impaired autophagic processing. These experiments, taken together, suggest that autophagic proteins inhibit inflammasome pathway activation by stabilizing mitochondria and/or maintaining mitochondrial quality control through autophagy. Current studies indicate that autophagic processes can exert a significant impact on the regulation of inflammation. It might be useful to investigate the potential of autophagic processes to mitigate inflammation-associated T2D as a therapeutic target.

Conclusion

Diabetes is a health problem that is increasing worldwide. Over-nutrition promotes insulin resistance. To cope with this increased demand of insulin secretion, the pancreatic islet needs to enhance its secretory activity, and adapt to the enhanced demand. When this adaptation fails, diabetes occurs. A reduction in functional pancreatic β cell mass leads to both major forms of diabetes (T1D and T2D). There is consistent evidence supporting the hypothesis that inflammation status is related to the development of T1D and T2D. Chronic inflammation can enhance the appearance of insulin resistance, which plays a pivotal role in T2D. The NLRP3 inflammasome is an intracellular multiprotein sensor that can recognize a large set of danger signals, and once activated, it subsequently stimulates inflammatory responses. Obesity has been considered a state of chronic low-grade systemic inflammation and chronic oxidative stress, which is caused by an imbalance between increased production of ROS and/or reduced antioxidant activity, leading to oxidative damage to cells or tissue.

Current studies indicate that autophagic processes can exert a significant impact on the regulation of inflammation. The mechanism of autophagy initiation is complex, and recent studies have shown a double effect in pancreatic β cells. Although most components of the metabolic inflammasome promote autophagy, the induction of autophagy by this signaling complex would be expected to serve as a negative-feedback mechanism that limits ER stress and disease progression. Autophagy has principal roles in the cellular adaptation to stress, in innate immune responses, and as a quality control mechanism.⁴⁵ Thus, autophagy represents an essential cytoprotective pathway. However, altered autophagy is often associated with pathologies with an altered inflammatory response. Defective autophagy-induced inflammation subsequently leads to obesity and insulin resistance.⁵²

Autophagy is suggested to be involved in the regulation of inflammation. Several studies have clearly indicated that autophagy can suppress inflammatory reactions,^53,54 and loss of autophagy proteins potentiates pro-inflammatory cytokine production.⁴⁸ This may not be the case, however, when excessive stimulation of autophagy is associated with cell death. Defects in autophagy may contribute to inflammation-associated metabolic diseases such as diabetes and obesity. Studies have shown that cells dying through autophagy, unlike apoptotic cells, can induce a pro-inflammatory response and can induce IL1B production and release which initiates a pro-inflammatory cytokine response in human macrophages⁵⁵ and in human β cells.⁵⁶

Clearly, our understanding of the molecular mechanisms of the plethora of functions of autophagy in inflammation processes is still quite primitive. Further research will advance our understanding of the molecular mechanisms of activation, and roles, of inflammation in diabetes. The clinical observational discussion of this study has several limitations. It is an open question as to what role—and under what conditions—an inflammatory response to autophagic dying cells may play in various physiological environments and settings. Nonetheless, further research is needed to define whether autophagy is beneficial or pathological in more specific disease contexts. Future studies specifically designed to investigate the role of autophagy in T2D using inflammation as the main outcome are urgently needed in order to provide a link between autophagy, inflammation and T2D.

Abbreviations:
ATG	=	autophagy-related
Atg7f/f:RIP-Cre	=	β-cell-specific Atg7-deficient
AGTR2	=	angiotensin II receptor, type 2
ATMs	=	adipose tissue macrophages
BECN1	=	Beclin 1, autophagy-related
CRP	=	C-reactive protein
ER	=	endoplasmic reticulum
IL18	=	interleukin 18
IL6	=	interleukin 6
IRF1	=	interferon regulatory factor 1
IAPP	=	islet amyloid polypeptide
IFNG	=	interferon, gamma
IRS1	=	insulin receptor substrate 1
IRS2	=	insulin receptor substrate 2
LAMP2	=	lysosome-associated membrane protein 2
LAMP2	=	lysosome-associated membrane protein 2
LC3	=	microtubule-associated protein 1 light chain 3
MAPK8	=	mitogen-activated protein kinase 8
MTOR	=	mechanistic target of rapamycin
NFKB	=	nuclear factor of kappa light polypeptide gene enhancer in B-cells
PI3K	=	phosphoinositide 3-kinase
PDX1	=	pancreatic and duodenal homeobox 1
TXNIP	=	thioredox ininteracting protein
3-MA	=	3-methyladenine

Acknowledgements

This work was supported by Scientific and Technological Project of Heilongjiang Province (No.D201340), and a grant from the Health Department of Heilongjiang Province (No.2012-540).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.