糖尿病中线粒体功能与内皮细胞的关系

J. Smooth Muscle Res. (2012) 48 (1): 1–26Correspondence to: Ayako Makino, Ph.D., Section of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Illinois at Chicago, 1819 West Polk Street, M/C 640, Chicago, Illinois 60612, USA Phone: +1312-355-1018 Fax: +1312-413-0437 e-mail: aymakino@https://www.wendangku.net/doc/ca14001366.html, ?2012 The Japan Society of Smooth Muscle Research

1Invited Review for the 2011 Hirosi Kuriyama Award

Mitochondrial function in vascular endothelial cell

in diabetes

Meenal P angare and Ayako M akino

University of Illinois at Chicago, USA

Received December 24, 2011; Accepted January 13, 2012

Abstract

Micro- and macrovascular complications are commonly seen in diabetic patients and en-

dothelial dysfunction contributes to the development and progression of the complications.

Abnormal functions in endothelial cells lead to the increase in vascular tension and atheroscle-

rosis, followed by systemic hypertension as well as increased incidence of ischemia and stroke

in diabetic patients. Mitochondria are organelles serving as a source of energy production

and as regulators of cell survival (e.g., apoptosis and cell development) and ion homeostasis

(e.g., H +, Ca 2+). Endothelial mitochondria are mainly responsible for generation of reactive oxy-

gen species (ROS) and maintaining the Ca 2+ concentration in the cytosol. There is increasing

evidence that mitochondrial morphological and functional changes are implicated in vascular

endothelial dysfunction. Enhanced mitochondrial fission and/or attenuated fusion lead to mi -

tochondrial fragmentation and disrupt the endothelial physiological function. Abnormal mito-

chondrial biogenesis and disturbance of mitochondrial autophagy increase the accumulation of

damaged mitochondria, such as irreversibly depolarized or leaky mitochondria, and facilitate

cell death. Augmented mitochondrial ROS production and Ca 2+ overload in mitochondria not

only cause the maladaptive effect on the endothelial function, but also are potentially detrimen-

tal to cell survival. In this article, we review the physiological and pathophysiological role of

mitochondria in endothelial function with special focus on diabetes.

Key words: fission and fusion, biogenesis, mitophagy, apoptosis, complications

Introduction

Diabetes is a metabolic disorder characterized by glucose intolerance and hyperglycemia due to deficiency of insulin and/or loss of effectiveness to insulin action. There are two main types of diabe -tes: Type-1 diabetes and Type-2 diabetes. Type-1 diabetes mellitus is mainly caused by autoimmune destruction of the beta cells in the islets of pancreas, where insulin is secreted upon glucose absorption.

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The patients with Type-1 diabetes exhibit lower or loss of plasma insulin and are characterized by total reliance on exogenous insulin for survival. On the other hand, Type-2 diabetic patients still produce and secrete insulin. However, they develop diabetes because of insufficient production/secretion of in-sulin and/or improper utilization of insulin (called insulin resistance). Type-1 diabetes usually develops in children or in young adults, whereas Type-2 diabetes mellitus is mainly seen in people over the age of 45 and accounts for nearly 90–95 percent of all diabetes cases. Not surprisingly, the prevalence of Type-2 diabetes in children and adolescents is growing worldwide, which correlates with obesity rate in the population (Rosenbloom, 1999; Broomgarden, 2004).

The common complications of diabetes are heart disease, hypertension, stroke, retinopathy, ne-phropathy, and neuropathy. Heart disease includes coronary artery disease and cardiac myopathy, which are the risk factors of the heart failure, and it is the leading cause of mortality and morbidity in patients with diabetes. Coronary artery disease is the result from narrowing the diameter of small coronary arteries by atherosclerotic lesion and increased coronary arterial tension. Hypertension is the most common complication in diabetic patients and is induced by increased vascular reactivity in the resistant arteries (e.g., mesenteric artery). Stroke and retinopathy are also caused by abnor-mal vascular reactivity. Endothelial cells serve as a key player in the development of these diseases. Vascular endothelium, which is a monolayer lining the inner surface of the blood vessels, plays an important role in a) vascular barrier function, which prevents the migration of inflammatory cells and fluid leakage into vascular media (Rao et al., 2007; Dejana et al., 2009), b) regulating vascular tone by releasing vasoconstrictors and vasodilators (Conger, 1994; Esper et al., 2006; Dora, 2010), and c) new vascular formation (Carmeliet, 2000; Madeddu, 2005). Endothelial dysfunction is implicated in many cardiovascular diseases including diabetes. It has been shown that in diabetic patients, as well as in diabetic animal models, 1) vascular tension is increased by attenuated endothelium-dependent relax-ation and increased release of vasoconstrictors from endothelium cells (Hink et al., 2001; Vinik and Flemmer, 2002; Farhangkhoee et al., 2006; Hermans, 2007), 2) vascular inflammation is augmented via increased endothelial permeability (Zhang et al., 2003; Spinetti et al., 2008) and surface adhesion molecules (Baumgartner-Parzer et al., 1995; Zou et al., 2002; Savoia and Schiffrin, 2007), and 3) endo-thelial apoptosis is increased, which is a main cause of the blood-retina barrier breakdown (Kern, 2007; Barber et al., 2011) and the decrease in capillary density in the heart (Yoon et al., 2005).

The mitochondria play a critical role in cell survival and death by regulating ATP synthesis through lipid and glucose metabolism, ROS generation, calcium homeostasis, apoptosis stimulation, and aging (McBride et al., 2006; Contreras et al., 2010). Therefore, the abnormal function of mitochon-dria leads to various cardiovascular diseases (Duchen, 2004; Ballinger, 2005; Davidson and Duchen, 2007). Endothelial cells produce the energy mainly via the anaerobic glycolytic metabolism of glucose but not through the mitochondrial ATP synthesis (Culic et al., 1997; Quintero et al., 2006), it is thus endothelial mitochondria are more like the sensor and initiator of the cell death. In this article, we will review the mitochondrial functions in the vascular endothelial cells and the pathophysiological role of mitochondria in endothelial dysfunction in diabetes mellitus.

Mitochondria in endothelium3

Mitochondrial fusion and fission

1) Mitochondrial fusion- and fission-related proteins

Mitochondria are complex organelles that move, fuse, divide, and constantly change their volume/ structure upon physiological stimulus and any stress (Frazier et al., 2006; Bereiter-Hahn et al., 2008). The definition of mitochondrial fission is the division of a mitochondrion within a cell to form two or more separate mitochondrial compartments, whereas mitochondrial fusion is merging two or more mi-tochondria within a cell to form a single compartment. Increased mitochondrial fission and decreased mitochondrial fusion result in the mitochondrial fragmentation (Detmer and Chan, 2007; Knott et al., 2008).

In mammals, mitochondrial fusion is regulated by at least three proteins: optic atrophy 1 (OPA1), mitofusin 1 (MFN1), and mitofusin 2 (MFN2) (Cipolat et al., 2004), whereas mitochondrial fission is controlled by dynamin related protein 1 (DRP1/DLP1/DNM1) and fission 1 (FIS1) (Yoon et al., 2003) (Fig. 1). A recent study identified another tail-anchored mitochondrial outer membrane protein, mito-chondrial fission factor (MFF) (Gandre-Babbe and van der Bliek, 2008), and the physiological function of MFF is for the recruitment of DRP1 to the mitochondrial membrane (Otera et al., 2010). Classically, FIS1 was the only one that recruits DRP1 from the cytosol to mitochondria upon the fission reaction (Mozdy et al., 2000; Yoon et al., 2003). OPA1 is located in the inner-mitochondrial membrane, and MFN1 and MFN2 are localized to the outer mitochondrial membrane. During mitochondrial fusion, OPA1 interacts with MFN1 and MFN2 (Cassina et al., 2000; Olichon et al., 2006). These proteins are first found to be related to neuropathy (Delettre et al., 2000; Zuchner et al., 2004; Ferre et al., 2005).

The function of fission/fusion-related proteins is regulated by various regulators, cleavage of the protein, posttranslational modifications, protein-protein interactions, and the lipid environment. GTP hydrolysis is required for fission/fusion-related proteins to be activated (Chan, 2006). DRP1 activity is negatively controlled by cyclic AMP. Phosphorylated DRP1 by cyclic AMP-dependent protein kinase increases the mitochondrial tubular formation, whereas dephosphorylation of DRP1 by calcineurin increases mitochondrial fission (Cribbs and Strack, 2007). OPA1 exhibits both long and short forms for fusion to proceed, and the balance of those forms is maintained by constitutive processing. There are two cleavage sites in OPA1, S1 and S2, and YME1L (an intermembrane space AAA protease) cleaves OPA1 at the site of S2 constitutively following mitochondrial import, whereas the loss of mitochon-drial membrane potential leads to the cleavage of the S1 site by OMA1 (zinc metalloprotease), which is followed by complete conversion of OPA1 to the short isoform and shutting off mitochondrial fusion (Griparic et al., 2007; Song et al., 2007; Head et al., 2009). Various posttranscriptional modifications of proteins regulate the activity of the fusion and fission machineries. MARCH5, a ubiquitin ligase in the outer membrane, associates with and ubiquitylates MFN1, MFN2, DRP1, and FIS1 (Yonashiro et al., 2006; Park et al., 2010). Although MARCH5 binds to both fission and fusion related proteins, knockdown of MARCH5 induces the mitochondrial elongation via notable accumulation of MFN1 protein (Park et al., 2010). DRP1 is sumoylated by mitochondrial-anchored protein ligase (MAPL, small ubiquitin-like modifier [SUMO] ligase) (Braschi et al., 2009) and desumorylated by sentrin-specific protease 5 (SENP5) (Zunino et al., 2007). Sumoylation of DRP1 stimulates mitochondrial fission (Harder et al., 2004; Zunino et al., 2007; Braschi et al., 2009). We have recently reported that high-glucose treatment leads to O-GlcNAcylation of OPA1 and mitochondrial fragmentation, while an

M. P angare et al.

4inhibition of O -GlcNAcylation by overexpression of GlcNAcase decreases the mitochondrial fragmen -tation induced by high glucose (Makino et al., 2011). S-nitrosylation of DRP1 results in mitochondrial fission (Cho et al., 2009).

An imbalance in the fusion/fission dynamics dramatically changes overall mitochondrial mor -phology (Bereiter-Hahn and Voth, 1994). Recent evidence from our laboratory, as well as others, has shown that mitochondrial dynamics play important roles in mitochondrial functions, including cell development, apoptosis, ROS generation and functional complementation of mitochondrial DNA (mtDNA) mutations by context mixing (Nakada et al., 2001; Frazier et al., 2006; Makino et al., 2010). Mitochondrial fission is essential for appropriate redistribution of mtDNA during cell division (Scott et al., 2003; Hales, 2004; Taguchi et al.

, 2007). In addition, damaged mitochondria are removed by mi-

Fig. 1. Mitochondrial fusion and fission. Mitochondrial fusion is regulated by optic atrophy 1 (OPA1), mitofusin 1 (MFN1), and mitofusin 2 (MFN2), whereas mitochondrial fission is controlled by dynamin related protein 1 (DRP1/DLP1/DNM1), fission 1 (FIS1), and mito -chondrial fission factor (MFF). OPA1 is located in the inner-mitochondrial membrane, and MFN1 and MFN2 are localized to the outer mitochondrial membrane. During mitochondrial fusion, OPA1 interacts with MFN1 and MFN2. FIS1 and MFF recruit DRP1 from the cyto-sol to mitochondria upon the fission reaction.

Mitochondria in endothelium5 tophagy through mitochondrial fission (Twig et al., 2008a; Twig et al., 2008b). Fusion also influences mitochondrial distribution in neural cells (Chen et al., 2007). Fused mitochondrial networks serve as electrically united systems that transmit the membrane potential generated by the proton pumps of the respiratory chain (Amchenkova et al., 1988; Skulachev, 2001) and also facilitate the propagation of Ca2+ wave and energy transfer in the cells (Szabadkai et al., 2004; Jou, 2008). Mitochondrial fusion is dramatically increased when mitochondrial ATP synthesis is enhanced (Tondera et al., 2009). The damaged/depolarized parts of the mitochondrial membrane are recovered by mitochondrial fusion that facilitates proper mixing of mtDNA and metabolites (Nakada et al., 2001; Twig et al., 2008b).

2) Other factors which induce mitochondrial fragmentation

The endoplasmic reticulum (ER) is an intracellular Ca2+store and releases Ca2+ via Ca2+ releasing channels upon the stimulation. The mitochondria are located close to the ER to support communication between them such as the transferring lipids, and the exchange of calcium and ATPs (Vance and Shiao, 1996; Mannella et al., 1998; Hayashi et al., 2009; Rizzuto et al., 2009). At the pathological condition, Ca2+ release from the ER causes calcium overload in the mitochondria and leads to mitochondrial frag-mentation via facilitating the DRP1 translocation to the mitochondrial outer membrane (Breckenridge et al., 2003).

Mitochondria constantly generate ROS via the electron transport chain reaction. At the physi-ological condition, majority of molecular oxygen is converted to water and less than 5% of the oxygen is incompletely reduced to O2-. Massive ROS production in the mitochondria is implicated in various cardiovascular diseases (Griendling and FitzGerald, 2003; Sugamura and Keaney, 2011), whereas ROS leads to the mitochondrial fragmentation in many cell types like rat cardiac myocytes (Yu et al., 2008; Fan et al., 2010), and mouse coronary endothelial cells (Makino et al., 2010). These data imply that mitochondrial fragmentation is enhanced by excess ROS production in pathophysiological condition.

3) Mitochondrial morphological change in endothelial cells in diabetes

There is increasing evidence showing that mitochondrial morphology is sensitive to the metabolic properties. The exposure of high glucose to endothelial cells ex vivo increases mitochondrial fragmen-tation (Makino et al., 2010; Trudeau et al., 2010; Shenouda et al., 2011). We demonstrate that mouse coronary endothelial cells isolated from Type-1 diabetic mice exhibit more fragmented mitochondrial structure and higher DRP1 protein expression levels than endothelial cells from control mice (Makino et al., 2010). The study examining the mitochondrial morphology using venous endothelial cells iso-lated from patients with Type-2 diabetes shows that FIS1 protein expression level and mitochondrial fragmentation are significantly increased in endothelial cells from diabetic patients compared with cells from control patients (Shenouda et al., 2011). Interestingly, mitochondria in retina endothelial cells are more elongated in the diabetic rat compared to the control rat, although MFN2 protein expres-sion level is significantly decreased and DRP1 expression level is increased in diabetes, implying that the mitochondrial morphological change might not be regulated by fission-fusion related proteins in this case (Zhong and Kowluru, 2011).

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4) Altered mitochondrial morphology in other cell types in diabetes

The exposure of free fatty acid (FFA) to the C2C12 muscle cells augments mitochondrial frag-mentation via increased DRP1 and FIS1 protein expression levels and mitochondrial in skeletal muscles in obesity and Type-2 diabetic mice are more fragmented (Jheng et al., 2012). In Type-2 diabetic pa-tients, MFN2 protein expression level is decreased in skeletal muscle compared with control patients (Zorzano et al., 2009). High glucose treatment increases mitochondrial fragmentation (Yu et al., 2008; Makino et al., 2011) via decreased OPA1 protein expression in the neonatal cardiac myocyte (Makino et al., 2011). High glucose or high insulin treatment increases the protein expression of DRP1 and enhances mitochondrial fragmentation in adult dorsal root ganglion neurons, suggesting that mito-chondrial morphological change might contribute to the neuropathy in Type 2 diabetes (Vincent et al., 2010). Ex vivo high glucose treatment leads to mitochondrial fragmentation in β-cell via increase in DRP1 protein expression (Men et al., 2009). Mitochondria in β-cell from Type-2 diabetic rat exhibit more fragmented structure than that in control (Dlaskova et al., 2010). Mitochondrial fission- and fusion-related proteins are cloned during the last decade and we expect to see more functional roles of these proteins in diabetic complications in the next decade.

Mitochondrial biogenesis

1) Proteins related with mitochondrial biogenesis

The cells undergo mitochondrial biogenesis process in response to various physiological stimulus and tissue- or signal-specific modification of mitochondrial gene expression and function. Mitochon-drial biogenesis is a complex process that involves the synthesis, import, and incorporation of proteins and lipids to the existing mitochondrial reticulum, as well as replication of the mtDNA (Lopez-Lluch et al., 2008). Upon the stimulation of mitochondrial biogenesis, mitochondrial genes in the nucleus and in mitochondria will be transcribed. Majority of genes required for mitochondrial biogenesis and function are in the nucleus, and the few genes crucial for oxidative phosphorylation, are on the mito-chondrial gene. The peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is the nucleus genome-encoded protein and a transcriptional coactivator of nuclear respiratory factor (NRF)-1, GA-binding protein (GABP) (also known as NRF-2), and peroxisome proliferator-activated receptors (PPARs) (Puigserver et al., 1998; Wu et al., 1999; Duncan et al., 2007). The activation of NRF-1 and 2 by PGC-1α leads to the expression of mitochondrial transporters, mitochondrial ribosomal proteins, oxidative phosphorylation components, and mitochondrial transcription factor (TFAM) (Feige and Au-werx, 2007; Scarpulla, 2008). PPARs regulate a broad set of genes which are required for lipid homeo-stasis and glucose and lipid oxidation (Djouadi et al., 1998; Burkart et al., 2007; Yang and Li, 2007).

PGC-1α activity is regulated by various kinds of posttranslational modifications. Phosphoryla-tion is required for the activation of PGC-1α and its phosphorylation is regulated by AMP-activated protein kinase (Jager et al., 2007), mitogen-activated protein kinase p38 (Knutti et al., 2001; Akimoto et al., 2005), and glycogen synthase kinase-3 (GSK-3) (Anderson et al., 2008), acetylation by Sirtuin 1 (SIRT1) (Rodgers et al., 2005; Gerhart-Hines et al., 2007), and arginine methylation by protein argi-nine N-methyltransferase 1 (PRMT1) (Teyssier et al., 2005). In the muscle cells, the increase in cyto-solic Ca2+ leads to PGC-1α activation through Ca2+/calmodulin-dependent kinases and p38 activation

Mitochondria in endothelium7 (Ojuka et al., 2003; Wright et al., 2007). Nitric oxide (NO) increases PGC-1α expression via cyclic GMP dependent pathway (Nisoli et al., 2003). On the other hand, PGC-1α is negatively regulated via deacetylation by GCN5 (Lerin et al., 2006) and ubiquitination by SCF cdc4 (Olson et al., 2008).

2) Mitochondrial biogenesis in endothelial cells in diabetes

Since the energy in endothelial cells is mainly generated by glycolytic metabolism instead of via mitochondrial ATP synthesis, mitochondrial biogenesis is not as critical as in muscle cells or in adipo-cytes. It is, however, important to maintain good quality and quantity of mitochondria in endothelial cells for cell survival. mtDNA copy number, PGC-1α and NRF-1 protein expression in nuclear extract, PGC-1α activity, and TFAM protein expression level in mitochondria are commonly used to determine the mitochondrial biogenesis. Hyperglycemia significantly decreases PGC-1α protein expression in retinal endothelial cells (Zheng et al., 2010). Santos et al. (2011) demonstrate that retinal mtDNA copy number is decreased in Type-1 diabetic mice and the mitochondrial number is lowered in retina in dia-betic patients, and in retinal endothelial cells treated with high glucose.

PPARγ activator, thiazolidinedione (TZD), is approved for use in Type-2 diabetic patients to im-prove insulin sensitivity by several mechanisms, including increased uptake and metabolism of free fatty acids in adipose tissue (Saltiel and Olefsky, 1996; Spiegelman, 1998; Kalaitzidis et al., 2009). Recent reports demonstrate that TZD induces mitochondrial biogenesis via the activation of PGC-1α in human umbilical vein endothelial cells (Fujisawa et al., 2009) and other cell types (see next paragraph). PGC-1α is the coactivator of PPARγ and the activation of PPARγ by TZD induces PGC-1α expression. Further studies are required to identify the role of TZD on mitochondrial biogenesis and the regulatory mechanisms of the positive feedback by PPARγ activation in endothelial cells.

3) Mitochondrial biogenesis in other cell types in diabetes

mtDNA copy number and mRNA expression of PGC-1α and TFAM are significantly decreased in aorta from Type-2 diabetic mice compared with the control (Csiszar et al., 2009). Lowered mtDNA copy number is observed in skeletal muscle from the patient with Type-2 diabetes (Hsieh et al., 2011) and muscles from Type-2 diabetic rats compared with the control (Shen et al., 2008). On the other hand, it has been reported that there is increased mitochondrial area, mitochondrial number and mtDNA in the heart of Type-1 diabetic mice, but the function of mitochondria is attenuated (Shen et al., 2004).

PGC-1α is induced by TZD in white and brown adipocyte cells (Wilson-Fritch et al., 2004; Hond-ares et al., 2006), and neuronal cells (Miglio et al., 2009). The adipose tissue obtained from the patient with Type-2 diabetes exhibits lower mitochondrial number and TFAM mRNA expression level com-pared with the control; TZD treatment restores the mitochondrial abnormality (Bogacka et al., 2005; Hakansson et al., 2011). Mitochondrial biogenesis is significantly attenuated in adipose tissue from Type-2 diabetic mice compared with the control, whereas TZD increases PGC-1α mRNA expression and restores the mitochondrial biogenesis in diabetic mice (Rong et al., 2007). PGC-1β also serves as a key regulator in energy metabolism by promoting mitochondrial biogenesis. There is increasing evidence showing that TZD enhances mitochondrial biogenesis by increase in PGC-1β expression, but not PGC-1α, in adipocyte cells (Deng et al., 2011; Pardo et al., 2011) and osteocytes (Wei et al., 2010).

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Mitochondrial autophagy/mitophagy

1) Molecular mechanisms of mitophagy

Autophagy is a cellular degradation system through the encapsulation by a double membrane structure called an autophagosome (Kelekar, 2005). There are two types of autophagy, non-selective autophagy and cargo-specific autophagy. At the low level of energy demand or at the starvation condi-tion, cells undergo non-selective autophagy to supply/re-use the metabolic component and energy by degradation of their organelles, whereas the cargo-specific autophagy could be initiated independent from the nutrient level (Kundu and Thompson, 2005; Komatsu and Ichimura, 2010; Rabinowitz and White, 2010). The selective elimination of mitochondria is called mitophagy (Lemasters, 2005). The purpose of mitophagy is primarily 1) to maintain the mitochondrial integrity in the cells and 2) to eliminate the damaged mitochondria (Narendra et al., 2008; Twig et al., 2008a). The excess amount of mitochondria at the low energy demand is the source of excessive ROS. The damaged mitochondria re-lease various apoptosis-promoting factors and lead to further damage of neighboring mitochondria and entire cell (Crompton et al., 1999). Therefore, mitophagy is the well-designed cytoprotective pathway.

Depolarized mitochondrial membrane is the hallmark of damaged mitochondria and sustains the mitochondrial fission status (Song et al., 2007; Twig et al., 2008a). Mitochondrial fusion is the physi-ological function to fuse the damaged membrane with intact membrane and minimize the damage. Damaged mitochondria that failed to be fused will be the target of the mitophagy. The PTEN-induced putative kinase protein 1 (PINK1) is a voltage-sensitive kinase, and it will be accumulated on the outer membrane in the mitochondria upon the membrane depolarization (Narendra et al., 2008; Jin et al., 2010; Matsuda et al., 2010). The accumulation of PINK1 facilitates the recruitment of Parkin, an E3 ubiquitin ligase, to the mitochondrial surface (Sha et al., 2010; Vives-Bauza et al., 2010). Ubiquitinated mitochondrial proteins by Parkin interact with the autophagy adaptor p62, and subsequently lead to the autophagosomal degradation of the mitochondria (Geisler et al., 2010; Okatsu et al., 2010) (Fig. 2). An-other protein which regulates mitophagy in mammalian cells is NIX (Kanki, 2010), although detailed mechanism is not clear.

Extensive damage by sustained membrane depolarization facilitates the opening of the mitochon-drial permeability transition pore (mPTP), increases mitochondrial membrane permeability and re-leases of pro-apoptotic molecules, and results in cell apoptosis. This will be described in the following section (Mitochondria-induced cell apoptosis).

The autophagy induced by starvation (non-selective autophagy) is mediated by mammalian target of rapamycin (mTOR)/AMP-activated protein kinase (AMPK) pathway. At the high nutrient, mTOR phosphorylates UNC-51-like kinase (ULK) that has inhibitory effects on the kinase activity of ULK. Starvation increases AMPK activation, which promotes mTOR inhibition and activates ULK, and sub-sequently leads to autophagy (Lee et al., 2010; Egan et al., 2011; Kim et al., 2011). It has to be noted that AMPK increases SIRT1 activity, which deacetylates PGC-1, and results in mitochondrial biogenesis as described above (2. Mitochondrial biogenesis). Therefore, mitochondrial autophagy and biogenesis are coordinately regulated.

There are other factors which possibly regulate the autophagy, including ROS and Bcl-2. During autophagic process, autophagy-regulating protein (ATG) 8 conjugates to the autophagosomal mem-brane through an ubiquitin-like conjugation system. ATG4 negatively regulates ATG8 function by

Mitochondria in endothelium 9

cleavage of ATG8, which releases ATG8 from the autophagosomal membrane and inhibits autophagy (Kaminskyy and Zhivotovsky, 2012). ATG4 is redox sensitive and oxidation of ATG4 inhibits the cleav -age activity of ATG4 and stabilizes the ATG8-mediated autophagosomal expansion (Scherz-Shouval et al., 2007). Beclin1, the mammalian ortholog of yeast ATG6, was identified as a Bcl-2-interacting protein (Kabeya et al., 2000) and it induces the formation of autophagosomes and promotes autophagy (Sinha and Levine, 2008). Anti-apoptotic protein Bcl-2 binds to Beclin1 and inhibits autophagy (Pat-tingre et al., 2005; Kang et al., 2011).

2) Mitophagy in diabetes

Interestingly, there is no report that demonstrates the change in mitochondrial autophagy/mi-tophagy in endothelial cells in diabetes. The exposure of oxidized LDL (ox-LDL) leads to autophagic pathway in HUVECs and HMECs (Zhang et al., 2010; Muller et al., 2011). High-glucose treatment augments autophagy in H9c2 cardiomyoblasts via increase in Beclin1 and LC3 protein expression level (Younce et al., 2010). Cardiac myocytes in Type-1 diabetic mice exhibit decreased autophagy deter-mined by the number of autophagic vacuoles in the cells (Xie et al.

, 2011), whereas skeletal muscles Fig. 2. Mitophagy. At the healthy condition, the Pten-induced novel kinase 1 (PINK1) is degraded by presenilins-associated rhomboid-like protein (PARL). Upon mitochondrial damage or loss of mitochondrial membrane potential, PINK1 accumulates on the outer mitochondrial membrane (OMM) without degradation and recruits Perkin from the cytosol to the OMM. Perkin ubiquitylates OM proteins and these ubiquitylated proteins are recognized by the adaptor protein p62, targeted by autophagosomes, and eventually degraded in lysosomes.

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from Type-2 diabetic rats show increased autophagy assessed by the protein expression level of LC3 and Beclin1 (Yan et al., 2011). Mitochondrial autophagy is increased in pancreatic cell of Type-2 dia-betic mice (Lo et al., 2010) and in adipose tissue in Type-2 diabetic patients (Ost et al., 2010). The reason for these inconsistent results might be due to the differences of the diabetic model, tissues used for the experiments, and methods to determine the mitochondrial autophagy.

Mitochondria-induced cell apoptosis

Mitochondria serve as the key organelles which maintain the cell function via generating ATP, regulating cellular Ca2+ homeostasis and heme biosynthesis, whereas mitochondria also determine the cell fate, as well as terminate the cell life, through several mitochondria-induced cell apoptosis path-ways. In this section, we discuss the cell apoptosis pathway induced by mitochondria (e.g., regulation of Bcl-2 protein family, mitochondrial ROS generation and mitochondrial Ca2+ overload).

1) Mitochondria-mediated apoptosis

There are two main apoptotic pathways: the extrinsic pathway and intrinsic pathway (Fig. 3). The extrinsic pathway is initiated by the binding of the ligands to the cell surface-specific receptors, called “death receptors”, whereas the intrinsic pathway is initiated by mitochondria. Both pathways are over-lapped at the point of mitochondrial outer membrane permeabilization (MOMP). MOMP is triggered by the formation of pores in the outer mitochondrial membrane (OMM), and the Bcl-2 protein family regulates the pore formation. Bcl-2 protein families can be classified into four groups based on their functions: 1) effectors whose oligomerization creates pores [Bax and Bak], 2) inhibitors of Bax and Bak [Bcl-2, Mcl1 and BclxL], 3) activators of Bax and Bak [Bid and Bim], and 4) sensitizers which antagonize antiapoptotic Bcl-2 like proteins [Bad, Bik, Noxa and Bmf]. The extrinsic pathway involves formation of a death-inducing signal complex (DISC) in the plasma membrane. DISCs contain multiple adaptor proteins that recruit and promote the activation of initiator procaspases, including procaspase 8. Activated caspase 8 induces cell apoptosis through two pathways; 1) direct cleavage of procaspase 3 followed by the cleavage of a variety of substrates and the cell apoptosis, 2) truncation of Bid to tBid, which leads to Bax/Bak oligomers, creates pores in OMM, and releases proapoptotic peptides such as cytochrome c (Liu et al., 1996), apoptosis inducing factor (AIF) (Susin et al., 1999), endonuclease G (EndoG) (Li et al., 2001), Smac/Diablo (Du et al., 2000), and Omi/HtrA2 (Hedge et al., 2002). These molecules activate both caspase dependent and independent cell death pathways (Donovan and Cotter, 2004).

The intrinsic pathway involves both MOMP and the opening of the mPTP. The mPTP is a trans-membrane channel formed between the inner and outer mitochondrial membranes and composed of voltage-dependent anion channel (VDAC) in OMM, adenine nucleotide translocator (ANT) in the in-ner mitochondrial membrane, and cyclophilin D in the mitochondrial matrix (Crompton et al., 1999; Halestrap and Pasdois, 2009). VDAC is a bidirectional transporter and permeable to solutes of up to 5 kDa. Under physiological conditions, VDAC serves as a shuttle of ATP and other small morecules. On the other hand, ANT is impermeable under normal conditions. During apoptosis, excess Ca2+ influx triggers the increase of ANT conductivity, followed by an inward flux of protons and ions through

Mitochondria in endothelium 11

ANT. The increase in matrix osmolality leads to water influx, mitochondrial swelling, and apoptogenic protein release from the mitochondrial storage to the cytosol though the mPTP opening, BAX/BAK-VDAC channel, and/or ruptured OMM (Ott et al., 2002; Tsujimoto and Shimizu, 2002; Baines, 2011). It has been reported that the opening of mPTP is regulated by Bcl-2 and pH change in inner mitochon-drial membrane (Matsuyama and Reed, 2000).

2) Mitochondrial O 2-generation and apoptosis

Mitochondria continuously generate superoxide anion (O 2-) through reduction of molecular oxy-gen by the electron transport chain (ETC) to water. The ETC is composed of four multiple subunit complexes; complex I (NADH-ubiquinone oxidoreductase), II (succinate-dehydrogenease), III (ubiqui-nol-cytochrome c oxidoreductase), and IV (cytochrome c oxidase), and the main function is to oxidize NADH and FADH 2 to NAD + and FAD +, that will be used in the tricarboxylic acid cycle (TCA cycle) to generate ATPs. The protons transported across the membrane in the ETC will serve as a motive force Fig. 3. Apoptotic Pathways . Extrinsic Pathway . The binding of the death ligands to the death re-ceptors forms a death-inducing signal complex (DISC). Procaspase 8 is autoactivated at the DISC and converted to the active form, caspase 8. Caspase 8 leads to cell apoptosis via caspase 3 activation and by truncation of Bid to tBid. Intrinsic Pathway . Intrinsic pathway is activated by intracellular stimuli (e.g., ROS) and involves the formation of the pore in the OMM (Bax/Bak oligomer) and the opening of the mitochondrial permeability transition

pore (mPTP). These pores release proapoptotic peptides and induce cell apoptosis.

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in complex V to synthesize ATPs. O2- is primarily generated at complexes I and III; complex I releases O2- predominately into the matrix, while complex III releases O2- to both sides of the mitochondrial in-ner membrane (Han et al., 2001; Muller et al., 2004; Lenaz et al., 2006). O2- is dismutated to hydrogen peroxide by CuZn-superoxide dismutase (SOD, in the intermembrane space and cytosol) and Mn-SOD (in the matrix) (Faraci and Didon, 2004), and subsequently reduced to water by catalase (in the cytosol) and glutathione peroxidase (in mitochondria and cytosol) (Chance et al., 1979; Phung et al., 1994). Ma-jority of O2- is reduced to water and very few O2- is leaked out from the ETC in normal cells, whereas excess O2- production in mitochondria is implicated in the pathogenesis of cardiovascular diseases (Li and Shah, 2004; Ballinger, 2005).

mtDNA is more sensitive than genomic DNA to ROS-induced damage, as it is not protected by histones and its repair capabilities are limited (Wei and Lee, 2002). Damaged mtDNA promotes outer membrane permeabilization and the release of cytochrome c, AIF, or Smac/Diablo from mitochondria to the cytosol and leads to cell apoptosis (Ryter et al., 2007). ROS also stimulates the extrinsic or in-trinsic apoptotic signaling via activation of JNK (Dhanasekaran and Reddy, 2008). The translocation of activated JNK to nucleus initiates activator protein 1-mediated expression of proapoptotic factors, such as TNFα, FasL, and Bak (Fan et al., 2001), while the translocation to mitochondria promotes to release cytochrome c (Kharbanda et al., 2000). Furthermore, the interaction of ROS with NO regulates the cell apoptotic pathway (Cassina et al., 2000; Jang and Han, 2006; Nakagawa et al., 2007; Wang et al., 2008).

3) Mitochondrial Ca2+ homeostasis and apoptosis

There are several Ca2+-sensitive intramitochondrial enzymes that regulate physiological cell func-tions (e.g., pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase) (McCor-mack and Denton, 1984). It is thus important to maintain appropriate level of Ca2+ in the mitochondria for their routine work, while Ca2+ overload in mitochondria causes the maladaptive effect on mitochon-drial function, as well as cell function, and it is implicated in variety of different disease processing (Esper et al., 2006; Halestrap and Pasdois, 2009). Mitochondrial Ca2+ overload leads to an opening of mPTP and it is followed by cell apoptosis and necrosis (see detailed in section 4.1.). Excess mitochon-drial [Ca2+] causes an increase in mitochondrial O2- via several mechanisms including by stimulation of the ETC to increase electron leak, by facilitating cytochrome c dislocation and by enhancing NO gen-eration which blocks complex IV and causes electron leak from complex III (Peng and Jou, 2010), and subsequently leads to mitochondria-mediated cell apoptosis (see detailed in section 4.2.). Ca2+ overload in mitochondria decreases mitochondrial membrane potential, which leads to mitochondrial fission. The fragmented/damaged mitochondria will be the target of the mitophagy, but too much fission will lead to more caspase release and cause cell apoptosis (Jeong and Seol, 2008; Suen et al., 2008; Liesa et al., 2009; Jahani-Asl et al., 2010; Westermann, 2011).

Where is the source of Ca2+ which accumulates in mitochondria? Mitochondria increase their [Ca2+] in response to elevated cytosolic [Ca2+] (Szabadkai et al., 2001; Pitter et al., 2002), and this Ca2+ transfer might be required for the physiological mitochondrial function such as ATP synthesis. There is increasing evidence showing that Ca2+ released from the ER is the main source of mitochondrial Ca2+ overload under pathophysiological condition (reviewed in Contreras et al., 2010; de Brito and Scorrano, 2010; Patergnani et al., 2011). Mitochondrial Ca2+ uptake is achieved by VDAC in the OMM and mitochondrial Ca2+ uniporter (MCU) in the IMM. VDAC is a channel permeable to both anions

Mitochondria in endothelium13 and cations, and the selectivity of the channel depends on the mitochondrial membrane potential; low potential is more preferable to anion transfer and high potential to cation. It has been demonstrated that VDAC is more permeable to Ca2+ in the closed states of the channel, and thus VDAC closure is a proapoptotic signal (Rostovtseva et al., 2005; Tan and Colombini, 2007). MCU is the highly selective ion channel and Ca2+ uptake by MCU is also driven by the membrane potential (Gunter and Gunter, 1994). It has been shown that Ca2+ has a biphasic effect on the MCU activity. Before reaching a certain level, cytosolic Ca2+ inactivates the uniporter and prevents further Ca2+ uptake. This mechanism al-lows the mitochondrial Ca2+ oscillation, but it prevents an excessive mitochondrial Ca2+ accumulation. Above the certain range of [Ca2+]cyt, Ca2+ activates MCU by the Ca2+-dependent calmodulin activation (Moreau et al., 2006).

4) Mitochondria-induced endothelial cell apoptosis in diabetes

Pathophysiological changes of metabolic parameters in diabetes are related with, or lead to, the increase in endothelial apoptosis (Nakagami et al., 2005; Piconi et al., 2006; Leduc et al., 2010; van den Oever et al., 2010; Barber et al., 2011). As described above, cell apoptosis could be induced in a mitochondria-dependent or mitochondria-independent manner, and the mitochondria-dependent cell apoptosis is modulated by mitochondrial functional and morphological changes including the increase in mitochondrial ROS formation, mitochondrial fission, mitochondrial Ca2+ overload, and the opening of mPTP. In addition, these mitochondrial pathophysiological changes interact and regulate each other. Although the initiation of mitochondria-mediated apoptosis could be varied and complex, it seems to be one common downstream, which is the opening of mPTP and the release of the proapoptotic fac-tors from mitochondrial to the cytosol. In diabetes, increased mitochondrial O2- is well documented in endothelial cells (Nishikawa and Araki, 2007; Di Lisa et al., 2009; Giacco and Brownlee, 2010; Cheng et al., 2011). Hyperglycemia leads to increased BAX expression (Meng et al., 2008; Yang et al., 2008; Guan et al., 2011), mitochondrial Ca2+ overload (Paltauf-Doburzynska et al., 2004), opening of mPTP in endothelial cells (Detaille et al., 2005; Huang et al., 2010) and releasing the proapoptotic proteins from the mitochondria (Kowluru and Abbas, 2003; Detaille et al., 2005; Kowluru, 2005; Leal et al., 2009; Li et al., 2009; Trudeau et al., 2010; Chong et al., 2011; Li et al., 2011), and subsequently in-creases endothelial apoptosis. Type-2 diabetes mellitus is usually accompanied by hyperlipidemia and ox-LDL accumulation (Shimada et al., 2004). Increased ox-LDL also induces mitochondria-mediated apoptosis in endothelial cells (Zhang et al., 2003; Chen et al., 2004; Vindis et al., 2005; Takabe et al., 2010; Chang et al., 2011). These data suggest that the changes of metabolic parameter in diabetes lead to endothelial cell apoptosis via mitochondrial dysfunction that may regulate the vascular permeability and capillary density as well as the vascular tone in diabetes.

5) Mitochondria-mediated apoptosis in other cell types in diabetes

Although there are many reports describing the mitochondria-mediated cell apoptosis in diabetes (reviewed in Duchen, 2004; Allen et al., 2005; Joza et al., 2009 Szabadkai and Duchen, 2009), there is a limited number of reports in which the actual assessment of the mPTP activity is carried out. Most prominent cell types which were examined for mPTP opening in diabetes are the cardiac myocyte. Cardiac myocytes isolated from diabetic patients (Anderson et al., 2010) and diabetic animal models (Oliveira, 2005; Bhamra et al., 2008; Williamson et al., 2010; Lumini-Oliveira et al., 2011) exhibit

M. P angare et al.

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augmented mPTP activity compared with the control. On the other hand, the increase in proapoptotic protein in the cytosol has been demonstrated in many cell types in diabetes, which is the downstream cascade of the mPTP opening (reviewed in (Adeghate, 2004; Duchen, 2004).

Apoptosis plays an important role in biological processes and various pathophysiological events. An alteration of mitochondrial function is heavily involved in the death pathway, which results in the cardiovascular dysfunction in many diseases. The molecular mechanisms of mitochondria-mediated cell apoptosis has been extensively studied during the past 10 years and it greatly helps understanding the cell fate determined by mitochondria in diabetes.

Conclusion

Mitochondria are small organelles in the cytosol and have been known as an ATP-producing or-ganelle. During past decades, their role has been expanded not only in the physiological cell function, but also in the development and progression of many diseases including diabetes. The main function of mitochondrial morphological changes is to ensure proper inheritance and distribution of mitochondria and to maintain them in a healthy state. Mitochondrial autophagy takes care of damaged mitochondria to minimize the maladaptive effect on cell functions, and mitochondrial biogenesis keeps energy sup-ply to the cell demands. Any abnormal alteration in these steps affects cell fate and tissue functions.

Endothelial dysfunction is the key risk factor of complications seen in diabetes, and here we demonstrate that mitochondrial dysfunction in endothelial cells represent a crucial step in the develop-ment of endothelial dysfunction. There are still many things to be examined to define mitochondrial pathophysiological role in endothelial function in diabetes, such as the relation between mitochondrial autophagy and endothelial dysfunction in diabetes and the contribution of mitochondrial abnormality to decreased quantity and quality of circulating endothelial progenitor cells in diabetes. Mitochondrial morphological and functional alteration in endothelial cells will remain an exciting field of diabetic research in another decade.

Acknowledgments

This work was supported by grant DK083506 (A. Makino) from the National Institutes of Health.

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线粒体与细胞凋亡

万方数据

万方数据

万方数据

线粒体与细胞凋亡 作者：周艺群， 谷志远， ZHOU Yi-qun， GU Zhi-yuan 作者单位：浙江大学医学院附属口腔医院口腔颌面外科,浙江,杭州,310006 刊名： 解剖科学进展 英文刊名：PROGRESS OF ANATOMICAL SCIENCES 年，卷(期)：2006,12(1) 被引用次数：14次 参考文献(17条) 1.樊廷俊;夏兰;韩贻仁线粒体与细胞凋亡[期刊论文]-生物化学与生物物理学报 2001(01) 2.赵云罡;徐建兴线粒体,活性氧和细胞凋亡[期刊论文]-生物化学与生物物理进展 2001(02) 3.蔡循;陈国强;陈竺线粒体跨膜电位与细胞凋亡[期刊论文]-生物化学与生物物理进展 2001(01) 4.Hortelano S;Dallaporte B;Zamzami N Nitric oxide induces apoptosis via triggering mitochondrial permeability transition[外文期刊] 1997(2-3) 5.Marchetti P;Hirsch T;Zamzami N Mitochondrial permeability transition triggers lymphocyte apoptosis 1996(11) 6.Marchetti P;Castodo M;Susin SA Mitochondrial permeability transition is a central coordinating event of apoptosis[外文期刊] 1996(03) 7.Susin SA;Zamzami N;Castedo M Bcl-2 inhibits the mitochondrial release of an apoptogenic protease [外文期刊] 1996(04) 8.Chou JJ;Li H;Salvesen GS Solution structure of BID,an intracellular amplifier of apoptotic signaling[外文期刊] 1999 9.Ji HB;Zhai QW;Liu XY Transcription regulation of bcl-2gene 2000(02) 10.Tsujimoto Y;Shimizu S Bcl-2 family:Life or death switch 2000(01) 11.Zamzami N;Susin SA;Marchetti P Mitochondrial control of nuclear apoptosis 1996(04) 12.Ruth MK;Ella BW;Douglas RG The release of cytochrome c from apoptosis[外文期刊] 1997(5303) 13.Narita M;Shimizu S;ItoT Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondrial 1998(25) 14.Cosulich SC;Savory PJ;Clarke PR Bcl-2 regulates amplification of caspase activation by cytochrome C[外文期刊] 1999(03) 15.Bossy-Wetzel E;Green DR Caspases induce cytochrome C release from mitochondria by activating cytosolic factors[外文期刊] 1999(25) 16.Sutton VR;Davis JE;Cancilla M Initiation of apoptosis by granzyme B requires direct cleavage of bid,but not direct granzyme B-mediated caspase activation[外文期刊] 2000(10) 17.Stoka V;Turk B;Schendel SL Lysosomal protease pathways to apoptosis.Cleavage of bid,not pro-caspases,is the most likely route[外文期刊] 2001(05) 本文读者也读过(10条) 1.杨胜细胞凋亡机制简述[期刊论文]-科技信息（学术版）2007(26) 2.冯俊奇.李秀兰.白人骁.FENG Jun-qi.LI Xiu-lan.BAI Ren-xiao细胞凋亡机制研究进展[期刊论文]-国际生物医学工程杂志2006,29(1)

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《中国2型糖尿病防治指南（2020版）》要点 2020年11月25-27日，中华医学会糖尿病学分会第二十四次全国学术会议（CDS）在苏州市以线上线下相结合的形式火热召开。《中国2型糖尿病防治指南（2020版）》的发布无疑是本次大会最为引人注目的焦点之一，更新版指南结合了最新的国际糖尿病管理指南和临床证据，为我国2型糖尿病的临床诊疗提供指导。 更新要点一：糖尿病患病率 2020版指南：根据最新的流调数据，依WHO诊断标准，我国糖尿病患病率上升至11.2%。

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线粒体与细胞凋亡

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[整理]中国2型糖尿病防治指南科普版部分.

糖尿病 什么是糖尿病？ 糖尿病是一种遗传因素和环境因素长期共同作用所导致的慢性、全身性、代谢性疾病，以血浆葡萄糖水平增高为特征，主要是因体内胰岛素分泌不足或作用障碍引起的糖、脂肪、蛋白质代谢紊乱而影响正常生理活动的一种疾病。 糖尿病有哪些特点？ 1、常见病 2、终身疾病 3、可控制疾病 4、需配合部分管理的疾病 5、病情不断变化的疾病 哪些人容易患上糖尿病呢？ 1、糖尿病家族史 2、超重、肥胖 3、多食少动 4、年龄>45岁 5、出生时低体重<5斤 6、有异常分娩史。如有原因不明的多次流产史、死胎、死产、早产、畸形儿或巨大儿等。

糖尿病有哪临床表现？ 一、糖尿病的典型症状：“三多一少”，即多饮、多尿、多食和消瘦（体重下降）。 有典型症状的糖尿病病友通常会主动就诊，而绝大多数的糖尿病病友，特别是2型糖尿病病友都没有任何症状，或者只有一些不引人注意的不舒服，若不加以注意，则慢慢地随着糖尿病的发展，才会出现一些其他并发症症状。 二、糖尿病的不典型症状： 1、反复生痔长痈、皮肤损伤或手术后伤口不愈合； 2、皮肤瘙痒，尤其是女性外阴瘙痒或泌尿系感染； 3、不明原因的双眼视力减退、视物模糊； 4、男性不明原因性功能减退、勃起功能障碍（阳痿）者； 5、过早发生高血压、冠心病或脑卒中； 6、下肢麻木、烧灼感； 7、尿中有蛋白（微量或明显蛋白尿）。

2型糖尿病会出现在孩子身上吗？ 在临床中发现，前来就诊的2型糖尿病患儿多数是“小胖墩”，他们的生活方式有很大的共性，如偏食、嗜食肉类、薯片等油炸类食品，排斥蔬菜、水果，喜欢吃肯德基、麦当劳等“洋快餐”，贪睡，不爱运动，一有时间不是玩电子游戏就是看电视。正是这些不良的生活习惯使“小胖墩”越来越多，也使2型糖尿病离孩子们越来越近。 夫妻，母子、祖孙之间会“传染”糖尿病吗? 有血缘关系的人可能具有相同的遗传基因，即父母与孩子之问、爷爷奶奶与孙子孙女之间无论哪一个人得了糖尿病，同家族的人得糖尿病的可能性也比较大。遗传因素另一方面的含义是，没有血缘关系的人不会传染糖尿病，如夫妻之间、朋友之间或同事之间，即使有紧密接触，也没有传染糖尿病的可能。 当然，妻子得了糖尿病，不会因为做家务、做饭就把疾病传染给丈夫和孩子，奶奶也不会因为带孙子就把糖尿病传染给孙子。但是，与糖尿病病友有血缘关系的人到底会不会得糖尿病，还取决于环境因素。大量的流行病学资料显示，环境因素是发生糖尿病的重要因素，其中生活方式、饮食习惯、运动习惯．性格等都与糖尿病的发生有关。

第八版-内科学-糖尿病-诊断与鉴别诊断(糖尿病)

诊断与鉴别诊断 在临床工作中要善于发现糖尿病，尽可能早期诊断和治疗。糖尿病诊断以血糖异常升高作为依据，血糖的正常值和糖代谢异常的诊断切点是依据血糖值与糖尿病和糖尿病特异性并发症（如视网膜病变）发生风险的关系来确定。应注意如单纯检查空腹血糖，糖尿病漏诊率高，应加验餐后血糖，必要时进行OGTT。诊断时应注意是否符合糖尿病诊断标准、分型、有无并发症（及严重程度）和伴发病或加重糖尿病的因素存在。 （一)诊断线索 ①三多一少症状。②以糖尿病各种急、慢性并发症或伴发病首诊的患者。③高危人群:有IGR史;年龄≥45岁;超重或肥胖;T2DM的一级亲属;有巨大儿生产史或GDM史;多囊卵巢综合征；长期接受抗抑郁症药物治疗等。 此外，30-40岁以上健康体检或因各种疾病、手术住院时应常规排除糖尿病。 （二）诊断标准 我国目前采用国际上通用的WHO糖尿病专家委员会（1999）提出的诊断和分类标准，要点如下： 1.糖尿病诊断是基于空腹（FPG）、任意时间或OGTT中2小时血糖值（2h PG）。空腹指至少8小时内无任何热量摄入；任意时间指一日内任何时间，无论上一次进餐时间及食物摄入量。糖尿病症状指多尿、烦渴多饮和难于解释的体重减轻。 2.糖尿病的临床诊断推荐采用葡萄糖氧化酶法测定静脉血浆葡萄糖。 3.严重疾病或应激情况下，可发生应激性高血糖；但这种代谢紊乱常为暂时性和自限性，因此在应激时，不能据此时血糖诊断糖尿病，必须在应激消除后复查才能明确其糖代谢状况。 4.儿童糖尿病诊断标准与成人相同。 5.妊娠糖尿病:强调对具有高危因素的孕妇(GDM个人史、肥胖、尿糖阳性或有糖尿病家族史者)，孕期首次产前检查时，使用普通糖尿病诊断标准筛查孕前未诊断的T2DM，如达到糖尿病诊断标准即可判断孕前就患有搪尿病。如初次检查结果正常，则在孕24一28周行75g OGTT,筛查有无GDM, GDM的诊断定义为达到或超过下列至少一项指标:FPG ≥ 5. 1 mmol/L,1 h PG≥10.Ommo1/L和(或)2h PG≥8.5mmolL。 6.关于应用HbA1c诊断糖尿病 HbAlc能稳定和可靠地反映患者的预后。ADA已经把HbA1c≥6.5%作为糖尿病的诊断标准，WHO也建议在条件成熟的地方采用HbA1c作为糖尿

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10.以动物细胞摄入LDL为例，概述受体介导胞吞的组成结构、运行过程及生理意义。 组成结构：衔接蛋白、网格蛋白、发动蛋白、受体、膜 过程：低密度脂蛋白LDL，先与细胞表面的互补性受体相结合，形成受体-配体复合物并引起细胞膜的局部内化作用，先是质膜在网格蛋白的参与作用下内陷形成有被小窝，然后是深陷的小窝脱离质膜形成有被小泡。即完成胞吞过程（后又脱包被，胞内体作用等）。生理意义：作为一种选择性浓缩机制，既保证了细胞大量的摄入特定的大分子，同时又避免了吸入胞外大量的液体。 11.比较两种胞吐途径的特点及功能。 类型特点功能 组成型合成就外排补充膜成分；信号介导完成其他生命活动；可形成外周 蛋白、基质等 调节型合成先储存，等信号刺激 短时间内大量释放，维持机体平衡 12. 甾类激素是如何通过胞内受体介导的信号通路去调节基因表达？ 甾类激素与受体结合时，导致抑制性蛋白脱离，暴露出受体上DNA结合位点而被激活。受体结合的DNA序列是转录增强子，可增加某些相邻基因的转录水平。甾类激素诱导的基因活化分两个阶段: 1）初级反应阶段：直接活化少数特殊基因，发生迅速 2）延迟的次级反应：由初级反应的基因产物，再活化其他基因，对初级反应起放大作用。NO是自由基性质的气体，具脂溶性，可快速扩散透过细胞膜，对邻近靶细胞起作用。血管内皮细胞和神经细胞中有一氧化氮合酶（NOS），能催化合成NO，当血管神经末释放乙酰胆碱作用于血管内皮，使其合成释放NO，所以才快速缓解心绞痛。 13. 以突触处神经递质作用为例，说明离子通道偶联受体介导的信号通路特点。离子通道偶联受体本身具信号结合点，又是离子通道，其跨膜信号转导无需中间步骤。神经递质（胞外化学信号）与受体结合而引起通道蛋白变构，导致离子通道开启，使突触后细胞膜出现过膜离子流（如Na＋和Ca2＋），从而将胞外化学信号转换成胞内电信号，导致突触出后细胞的兴奋。当胆碱脂酶将神经递质水解后，离子通道关闭，信号传递中断。 14. 概述G蛋白偶联受体介导的信号通路的组成、特点及主要功能。组成：细胞外配体、细胞表面受体、G蛋白（分子开关）、第二信使、靶蛋白 G蛋白偶联受体介导的信号通路整体的传递过程：细胞外配体—→细胞表面受体—→G蛋白（分子开关）—→第二信使—→靶蛋白（酶或离子通道）—→细胞应答根据第二信使的不同，信号通路可以分为两类： （1）cAMP信号通路信号通路信号通路信号通路cAMP的产生有腺苷酸环化酶催化完成，而该酶的活性由激活性激素（肾上腺素、胰高血糖素）或抑制性激素（前列腺素、腺苷）调控。激素－→G蛋白偶联受体－→G蛋白－→腺苷酸环化酶－（激素作用）→cAMP－→cAMP依赖的蛋白激酶A（PKA）产生PKA后，他可以激活下游的靶酶以及开启基因表达：（前者是快速反应，后者是慢速反应）a. 活化的PKA—>靶酶蛋白磷酸化—>细胞代谢核细胞行为（如肾上腺素刺激骨骼肌细胞导致糖原分解） b. 活化的PKA—>基因调控蛋白—>基因转录 （2）磷脂酰肌醇信号通路磷脂酰肌醇信号通路磷脂酰肌醇信号通路磷脂酰肌

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无论从原始的生物线虫到高级的哺乳动物乃至人类，还是从生物体外周器官到中枢神经系统，细胞凋亡都广泛存在。它作为生命的基本现象之一，可以发生在生理或病理条件下[1]。最初人们仅从形态学表现的特征上加以认识，如胞膜对称性丧失、染色质凝集、细胞皱缩、DNA破碎、线粒体肿胀和凋亡小体形成。随着科学的发展，人们开始发现线粒体在细胞凋亡中的起着重要的作用。随着对细胞凋亡研究的深入，人们对线粒体与体细胞凋亡的关系有了新的认识。

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中国2型糖尿病防治指南（2017年版）(四) 高血糖的药物治疗要点提示●生活方式干预是糖尿病治疗的基础，如血糖控制不达标（HbA1c≥7.0%）则进入药物治疗（A）●二甲双胍、α-糖苷酶抑制剂或胰岛素促泌剂可作为单药治疗的选择，其中，二甲双胍是单药治疗的首选（A）●在单药治疗疗效欠佳时，可开始二联治疗、三联治疗或胰岛素多次注射（B） 一口服降糖药物高血糖的药物治疗多基于纠正导致人类血糖升高的两个主要病理生理改变——胰岛素抵抗和胰岛素分泌受损。根据作用效果的不同，口服降糖药可分为主要以促进胰岛素分泌为主要作用的药物（磺脲类、格列奈类、DPP-4抑制剂）和通过其他机制降低血糖的药物（双胍类、TZDs、α-糖苷酶抑制剂、SGLT2抑制剂）。磺脲类和格列奈类直接刺激胰岛b细胞分泌胰岛素；DPP-4抑制剂通过减少体内GLP-1的分解、增加GLP-1浓度从而促进胰岛b细胞分泌胰岛素；双胍类的主要药理作用是减少肝脏葡萄糖的输出；TZDs的主要药理作用为改善胰岛素抵抗；α-糖苷酶抑制剂的主要药理作用为延缓碳水化合物在肠道内的消化吸收。SGLT2抑制剂的主要药理作用为通过减少肾小管对葡萄糖的重吸收来增加肾脏葡萄糖的排出。 糖尿病的医学营养治疗和运动治疗是控制2型糖尿病高血糖

的基本措施。在饮食和运动不能使血糖控制达标时应及时采用药物治疗。

2型糖尿病是一种进展性的疾病。在2型糖尿病的自然病程中，对外源性的血糖控制手段的依赖会逐渐增大。临床上常需要口服药物间及口服药与注射降糖药间（胰岛素、GLP-1受体激动剂）的联合治疗。 （一）二甲双胍目前临床上使用的双胍类药物主要是盐酸二甲双胍。双胍类药物的主要药理作用是通过减少肝脏葡萄糖的输出和改善外周胰岛素抵抗而降低血糖。许多国家和国际组织制定的糖尿病诊治指南中均推荐二甲双胍作为2型糖尿病患者控制高血糖的一线用药和药物联合中的基本用药。对临床试验的系统评价显示，二甲双胍的降糖疗效（去除安慰剂效应后）为HbA1c下降1.0%～1.5%，并可减轻体重[101-103]。在我国2型糖尿病人群中开展的临床研究显示，二甲双胍可使HbA1c下降0.7%～1.0%[104-105]。在500～2 000 mg/d剂量范围之间，二甲双胍疗效呈现剂量依赖效应[104,106]，在低剂量二甲双胍治疗的基础上联合DPP-4抑制剂的疗效与将二甲双胍的剂量继续增加所获得的血糖改善程度和不良事件发生的比例相似[107-108]。UKPDS结果证明，二甲双胍还可减少肥胖的2型糖尿病患者心血管事件和死亡[78]。在我国伴冠心病的2型糖尿病患者中开展的针对二甲双胍与磺脲类药物对再发心血管事件影响的临床随机分组对照试验结果显示，二甲双胍的治疗与主要心血管事件的显著下降相关[109]。单独使用二甲双胍不导致低血糖，但二

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