Zhiyong Cheng

Insulin signaling meets mitochondria in metabolism

Insulin controls nutrient and metabolic homeostasis via the IRS-PI3K-AKT signaling cascade that targets FOXO1 and mTOR. Mitochondria, as the prime metabolic platform, malfunction during insulin resistance in metabolic diseases. However, the molecular link between insulin resistance and mitochondrial dysfunction remains undefined. Here we review recent studies on insulin action and the mechanistic association with mitochondrial metabolism. These studies suggest that insulin signaling underpins mitochondrial electron transport chain integrity and activity by suppressing FOXO1/HMOX1 and maintaining the NAD(+)/NADH ratio, the mediator of the SIRT1/PGC1α pathway for mitochondrial biogenesis and function. Mitochondria generate moderately reactive oxygen species (ROS) and enhance insulin sensitivity upon redox regulation of protein tyrosine phosphatase and insulin receptor. However, chronic exposure to high ROS levels could alter mitochondrial function and thereby cause insulin resistance.

Foxo1 integrates insulin signaling with mitochondrial function in the liver

Type 2 diabetes is a complex disease that is marked by the dysfunction of glucose and lipid metabolism. Hepatic insulin resistance is especially pathogenic in type 2 diabetes, as it dysregulates fasting and postprandial glucose tolerance and promotes systemic dyslipidemia and nonalcoholic fatty liver disease. Mitochondrial dysfunction is closely associated with insulin resistance and might contribute to the progression of diabetes. Here we used previously generated mice with hepatic insulin resistance owing to the deletion of the genes encoding insulin receptor substrate-1 (Irs-1) and Irs-2 (referred to here as double-knockout (DKO) mice) to establish the molecular link between dysregulated insulin action and mitochondrial function. The expression of several forkhead box O1 (Foxo1) target genes increased in the DKO liver, including heme oxygenase-1 (Hmox1), which disrupts complex III and IV of the respiratory chain and lowers the NAD+/NADH ratio and ATP production. Although peroxisome proliferator–activated receptor-γ coactivator-1α (Ppargc-1α) was also upregulated in DKO liver, it was acetylated and failed to promote compensatory mitochondrial biogenesis or function. Deletion of hepatic Foxo1 in DKO liver normalized the expression of Hmox1 and the NAD+/NADH ratio, reduced Ppargc-1α acetylation and restored mitochondrial oxidative metabolism and biogenesis. Thus, Foxo1 integrates insulin signaling with mitochondrial function, and inhibition of Foxo1 can improve hepatic metabolism during insulin resistance and the metabolic syndrome.

Targeting Forkhead Box O1 from the Concept to Metabolic Diseases: Lessons from Mouse Models

Forkhead box O (FOXO) transcription factors have been implicated in regulating the metabolism, cellular proliferation, stress resistance, apoptosis, and longevity. Through the insulin receptor substrate → phosphoinositide 3-kinase → Akt signal cascade, FOXO integrates insulin action with the systemic nutrient and energy homeostasis. Activation of FOXO1 in liver induces gluconeogenesis via phosphoenolpyruvate carboxykinase (PEPCK)/glucose 6-phosphate pathway, and disrupts mitochondrial metabolism and lipid metabolism via heme oxygenase 1/sirtuin 1/Ppargc1α pathway. In skeletal muscle, FOXO1 activation underpins the carbohydrate/lipid switch during fasting state. Inhibition of FOXO1 under physiological conditions accounts for maintenance of skeletal muscle mass/function and adipose differentiation. In pancreatic β-cells, nuclear translocation of FOXO1 antagonizes pancreatic and duodenal homeobox 1 and attenuates β-cells proliferation and insulin secretion. Regardless, FOXO1 promotes the proliferation of β-cells through induction of Cyclin D1 in low nutrition, and elicits antioxidant mechanism to protect against β-cell failure during oxidative insults. In the brain, FOXO1 controls food intake through transcriptional regulation of the orexigenic neuropeptide Y, agouti-related protein, and carboxypeptidase E. In this article, we review the role of FOXO1 in the regulation of metabolism and energy expenditure based on recent findings from mouse models, and discuss the therapeutic value of targeting FOXO1 in metabolic diseases.

The Irs1 branch of the insulin signaling cascade plays a dominant role in hepatic nutrient homeostasis.

ABSTRACT We used a Cre-loxP approach to generate mice with varied expression of hepatic Irs1 and Irs2 to establish the contribution of each protein to hepatic nutrient homeostasis. While nutrient-sensitive transcripts were expressed nearly normally in liver lacking Irs2 (LKO2 mice), these transcripts were significantly dysregulated in liver lacking Irs1 (LKO1 mice) or Irs1 and Irs2 together (DKO mice). Similarly, a set of key gluconeogenic and lipogenic genes was regulated nearly normally by feeding in liver retaining a single Irs1 allele without Irs2 (DKO/1 mice) but was poorly regulated in liver retaining one Irs2 allele without Irs1 (DKO/2 mice). DKO/2 mice, but not DKO/1 mice, also showed impaired glucose tolerance and insulin sensitivity—though both Irs1 and Irs2 were required to suppress hepatic glucose production during hyperinsulinemic-euglycemic clamp. In contrast, either hepatic Irs1 or Irs2 mediated suppression of HGP by intracerebroventricular insulin infusion. After 12 weeks on a high-fat diet, postprandial tyrosine phosphorylation of Irs1 increased in livers of control and LKO2 mice, whereas tyrosine phosphorylation of Irs2 decreased in control and LKO1 mice. Moreover, LKO1 mice—but not LKO2 mice—that were fed a high-fat diet developed postprandial hyperglycemia. We conclude that Irs1 is the principal mediator of hepatic insulin action that maintains glucose homeostasis.

Insulin Receptor Substrates Irs1 and Irs2 Coordinate Skeletal Muscle Growth and Metabolism via the Akt and AMPK Pathways

ABSTRACT Coordination of skeletal muscle growth and metabolism with nutrient availability is critical for metabolic homeostasis. To establish the role of insulin-like signaling in this process, we used muscle creatine kinase (MCK)-Cre to disrupt expression of insulin receptor substrates Irs1 and Irs2 in mouse skeletal/cardiac muscle. In 2-week-old mice, skeletal muscle masses and insulin responses were slightly affected by Irs1, but not Irs2, deficiency. In contrast, the combined deficiency of Irs1 and Irs2 (MDKO mice) severely reduced skeletal muscle growth and Akt→mTOR signaling and caused death by 3 weeks of age. Autopsy of MDKO mice revealed dilated cardiomyopathy, reflecting the known requirement of insulin-like signaling for cardiac function (P. G. Laustsen et al., Mol. Cell. Biol. 27:1649-1664, 2007). Impaired growth and function of MDKO skeletal muscle were accompanied by increased Foxo-dependent atrogene expression and amino acid release. MDKO mice were resistant to injected insulin, and their isolated skeletal muscles showed decreased insulin-stimulated glucose uptake. Glucose utilization in MDKO mice and isolated skeletal muscles was shifted from oxidation to lactate production, accompanied by an elevated AMP/ATP ratio that increased AMP-activated protein kinase (AMPK)→acetyl coenzyme A carboxylase (ACC) phosphorylation and fatty acid oxidation. Thus, insulin-like signaling via Irs1/2 is essential to terminate skeletal muscle catabolic/fasting pathways in the presence of adequate nutrition.

Mitochondria and Metabolic Homeostasis

Mitochondrial function is fundamental to metabolic homeostasis. In addition to converting the nutrient flux into the energy molecule ATP, the mitochondria generate intermediates for biosynthesis and reactive oxygen species (ROS) that serve as a secondary messenger to mediate signal transduction and metabolism. Alterations of mitochondrial function, dynamics, and biogenesis have been observed in various metabolic disorders, including aging, cancer, diabetes, and obesity. However, the mechanisms responsible for mitochondrial changes and the pathways leading to metabolic disorders remain to be defined. In the last few years, tremendous efforts have been devoted to addressing these complex questions and led to a significant progress. In a timely manner, the Forum on Mitochondria and Metabolic Homeostasis intends to document the latest findings in both the original research article and review articles, with the focus on addressing three major complex issues: (1) mitochondria and mitochondrial oxidants in aging-the oxidant theory (including mitochondrial ROS) being revisited by a hyperfunction hypothesis and a novel role of SMRT in mitochondrion-mediated aging process being discussed; (2) impaired mitochondrial capacity (e.g., fatty acid oxidation and oxidative phosphorylation [OXPHOS] for ATP synthesis) and plasticity (e.g., the response to endocrine and metabolic challenges, and to calorie restriction) in diabetes and obesity; (3) mitochondrial energy adaption in cancer progression-a new view being provided for H(+)-ATP synthase in regulating cell cycle and proliferation by mediating mitochondrial OXPHOS, oxidant production, and cell death signaling. It is anticipated that this timely Forum will advance our understanding of mitochondrial dysfunction in metabolic disorders.

IRS2 increases mitochondrial dysfunction and oxidative stress in a mouse model of Huntington disease

Aging is a major risk factor for the progression of neurodegenerative diseases, including Huntington disease (HD). Reduced neuronal IGF1 or Irs2 signaling have been shown to extend life span in mice. To determine whether Irs2 signaling modulates neurodegeneration in HD, we genetically modulated Irs2 concentrations in the R6/2 mouse model of HD. Increasing Irs2 levels in the brains of R6/2 mice significantly reduced life span and increased neuronal oxidative stress and mitochondrial dysfunction. In contrast, reducing Irs2 levels throughout the body (except in β cells, where Irs2 expression is needed to prevent diabetes onset; R6/2•Irs2+/-•Irs2βtg mice) improved motor performance and extended life span. The slower progression of HD-like symptoms was associated with increased nuclear localization of the transcription factor FoxO1 and increased expression of FoxO1-dependent genes that promote autophagy, mitochondrial function, and resistance to oxidative stress. Mitochondrial function improved and the number of autophagosomes increased in R6/2•Irs2+/-•Irs2βtg mice, whereas aggregate formation and oxidative stress decreased. Thus, our study suggests that Irs2 signaling can modulate HD progression. Since we found the expression of Irs2 to be normal in grade II HD patients, our results suggest that decreasing IRS2 signaling could be part of a therapeutic approach to slow the progression of HD.

Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase.

Thiol-disulfide reactions are crucial for redox homeostasis in the cell.1-2 As a disulfide oxidoreductase, thioredoxin (Trx, ∼12 kDa) regulates a wide variety of biological molecules in both eukaryotic and prokaryotic species, including ribonucleotide reductase, peroxiredoxin, methionine sulfoxide reductase, phosphatase and tensin homolog (PTEN), transcription factors such as nuclear factor-κB (NF-κB), redox factor-1 (Ref-1), and activator protein-1 (AP-1).3 Thus, Trx has been implicated in such diverse processes as antioxidant defense, DNA repair and synthesis, redox regulation, and apoptosis.4-9 In the evolution of Trx catalysis important physical factors (e.g., a hydrophobic binding groove) have appeared to make Trx bind the disulfide substrate in a specific fashion and generate stabilizing interactions.10-11 The interactions and substrate binding were shown to regulate the geometry and orientation of the target disulfide in the catalytic site of the enzyme, and account for Michaelis-Menten–type kinetics of disulfide reduction by Trx.10-11 Trx molecules contain two cysteines in the active site in a CXXC motif. The chemistry of Trx-catalyzed protein disulfide reduction following substrate-binding includes a nucleophilic attack on the disulfide of target proteins by the N-terminal cysteine thiolate of Trx (which is Cys32 in the E. coli numbering) to form an intermolecular mixed disulfide, and a subsequent attack on the disulfide intermediate by the thiolate of the C-terminal cysteine of Trx (Cys35), producing the reduced target protein and oxidized Trx (Figure 1).12-13 To date a number of factors, including the identity of the amino acid residues spanning the CXXC motif, the pKa of the redox active thiols, the redox potential of the disulfide/dithiol couple, the acid/base catalysts, the molecular interactions and the local conformational changes after substrate binding, have been implicated in the regulation of Trx activity as a protein disulfide oxidoreductase, albeit with controversies and debates. In this work, we review the emerging evidence that support or dispute the regulating roles of these factors, and propose the likely prime determinants of Trx activity that deserve more attention in future research, with the hope to promote a better understanding of the mechanistic factors of Trx reactivity. Figure 1 A schematic cartoon view of a Trx-catalyzed disulfide reduction. A number of factors are involved in the regulation of Trx activity, including the amino acid residues spanning the redox active CXXC motif, molecular interaction (e.g., electrostatic force), ... The 3-D structures of Trx proteins are highly conserved, with the five central β-strands surrounded by four α-helices (Figure 2, A and B). Part of the redox active center (-CGPC- motif) protrudes at the surface of the molecule at the very N-terminus of helix α2, with Cys35 largely covered by the N-terminal portion of the helix α2. Only one side of the Cys32-SH is accessible to solvent for the transfer of reducing equivalents in Trxox but the side chain of Cys32 turns into the solvent upon reduction.14-16 The Trx fold is also found in several other classes of enzymes that interact with substrates containing either disulfides or dithiols. Protein families that have a Trx-fold include the thioredoxin, Dsb (disulfide bond formation protein) proteins, glutaredoxin (Grx), glutathione S-transferase, and protein disulfide isomerase (PDI) families.2,16-17 We performed a structural bioinformatics study on 515 sequences of the Trx family, 495 sequences of the DsbA family, and 382 sequences of the PDI family (from the Conserved Domain Database (CDD),18 http://www.ncbi.nlm.nih.gov/sites/entrez?db=cdd). This search confirmed a recent observation that the two components that are most conserved across these 3 families of proteins include an active site CXXC motif, and a conserved proline that is distant in sequence from this active site but immediately adjacent in 3 dimensional space.19 In the Trx family, the CXXC motif has the sequence CGPC and is present at position 32–35, while the proline residue is at position 76 in its best-studied member, E. coli thioredoxin 1 (EcTrx; PDB: 2TRX_A) (Figure 2C). The highly conserved proline is at position 151 in E. coli DsbA (PDB: 1DSB_A) and at position 83 in human PDI (PDB: 1MEK) (Figure 2, D and E). In EcTrx, the distances between the N-atom of Pro76 and the two sulfur atoms of the cysteine pair are 4.07 A (Cys32) and 3.62A (Cys35) (Figure 2F). The next closest residue is Asp26, and the shortest distance between an O-atom of Asp26 and the sulfur atom of Cys35 is 5.59 A (Figure 2G). Pro76 and Asp26 have been shown to play important roles in the redox reactions due to their close contact with the Cys pair.20-21 Figure 2 The structure of EcTrx and conserved amino acid residues in the Trx family. (A) and (B) are the ribbon diagrams of oxidized and reduced EcTrx, respectively. They show the spatial distances between Trp28 and Cys32, and between Trp28 and Asp26. The color ... Conserved structural features such as the Trx-fold and secondary structure in Trx family members, never-the-less, allow diverse reactivities in catalyzing protein disulfide interchange reactions. Quantum mechanical calculations suggest that the relative stability of thiolates in the CXXC motif determines whether these enzymes catalyze oxidation, reduction, or isomerization.2,22-23 Because the cysteine thiolates in Trx are both poorly stabilized, Trx is a good reducing agent. In contrast, DsbA stabilizes both cysteine thiolates and thus is a good oxidizing agent; isomerases such as PDI have one thiolate relatively solvent exposed and poorly stabilized while the other (relatively buried) thiolate is highly stabilized.22-23 Static calculations, such as the quantum mechanical calculations, provide insights into the thermodynamics of thiol-disulfide reactions, but as previously noted and as recently further investigated,1,24 the reactivity in an actual reaction is more complex due to dynamics and conformational changes that occur during substrate binding. For instance, studies with site-specific mutagenesis indicate that the relative stability of the oxidized versus the reduced form determines the difference in the redox potentials of Trx from Staphylococcus aureus (SaTrx).25-27 Replacement of the conserved proline in the CXXC motif by threonine or serine greatly reduced the relative stability value and made SaTrx less reducing.25 Moreover, the activation energy barrier for forming a transition state complex must be overcome by dynamics or other properties to accomplish a thermodynamically favorable thiol-disulfide reaction.1 However, DsbA from Staphylococcus aureus shows identical stabilities in oxidized and reduced forms, suggesting that alternative mechanisms beyond thermodynamic stability underlie the activity of SaDsbA in thiol-disulfide reactions.28-29 In this work, we review recent studies on the reactivity of thioredoxins as a protein disulfide oxidoreductase. Our goal is to provide an updated view and a better understanding of the determinants of Trx reactivity and their critical roles in redox homeostasis.

Mitochondrial alteration in type 2 diabetes and obesity: An epigenetic link

The growing epidemic of type 2 diabetes mellitus (T2DM) and obesity is largely attributed to the current lifestyle of over-consumption and physical inactivity. As the primary platform controlling metabolic and energy homeostasis, mitochondria show aberrant changes in T2DM and obese subjects. While the underlying mechanism is under extensive investigation, epigenetic regulation is now emerging to play an important role in mitochondrial biogenesis, function, and dynamics. In line with lifestyle modifications preventing mitochondrial alterations and metabolic disorders, exercise has been shown to change DNA methylation of the promoter of PGC1α to favor gene expression responsible for mitochondrial biogenesis and function. In this article we discuss the epigenetic mechanism of mitochondrial alteration in T2DM and obesity, and the effects of lifestyle on epigenetic regulation. Future studies designed to further explore and integrate the epigenetic mechanisms with lifestyle modification may lead to interdisciplinary interventions and novel preventive options for mitochondrial alteration and metabolic disorders.

Inhibition of TNF-α Improves the Bladder Dysfunction That Is Associated With Type 2 Diabetes

Diabetic bladder dysfunction (DBD) is common and affects 80% of diabetic patients. However, the molecular mechanisms underlying DBD remain elusive because of a lack of appropriate animal models. We demonstrate DBD in a mouse model that harbors hepatic-specific insulin receptor substrate 1 and 2 deletions (double knockout [DKO]), which develops type 2 diabetes. Bladders of DKO animals exhibited detrusor overactivity at an early stage: increased frequency of nonvoiding contractions during bladder filling, decreased voided volume, and dispersed urine spot patterns. In contrast, older animals with diabetes exhibited detrusor hypoactivity, findings consistent with clinical features of diabetes in humans. The tumor necrosis factor (TNF) superfamily genes were upregulated in DKO bladders. In particular, TNF-α was upregulated in serum and in bladder smooth muscle tissue. TNF-α augmented the contraction of primary cultured bladder smooth muscle cells through upregulating Rho kinase activity and phosphorylating myosin light chain. Systemic treatment of DKO animals with soluble TNF receptor 1 (TNFRI) prevented upregulation of Rho A signaling and reversed the bladder dysfunction, without affecting hyperglycemia. TNFRI combined with the antidiabetic agent, metformin, improved DBD beyond that achieved with metformin alone, suggesting that therapies targeting TNF-α may have utility in reversing the secondary urologic complications of type 2 diabetes.