AMP-Activated Protein Kinase�CDeficient Mice Are Resistant to the Metabolic Effects of Resveratrol

OBJECTIVE

Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5′ AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKα1 or -α2 to determine whether the metabolic effects of resveratrol are mediated by AMPK.

RESEARCH DESIGN AND METHODS

Mice deficient in the catalytic subunit of AMPK (α1 or α2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro.

RESULTS

Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKα1^−/− mice. In the absence of either AMPKα1 or -α2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly.

CONCLUSIONS

We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.Resveratrol is a natural polyphenolic compound found in grapes and red wine and has been shown to extend lifespan in many organisms, including yeast (1), flies (2), and worms (2–4). Resveratrol extended lifespan in mice on a high-fat diet (5) but not a regular diet (6). In mice with diet-induced obesity, resveratrol reduced fat accumulation and improved glucose tolerance and insulin sensitivity (5,7). In addition, resveratrol increases mitochondrial biogenesis and physical endurance. A resveratrol derivative with higher bioavailability is being tested in clinical trials for treating type 2 diabetes.Given its potential as a lead molecule for the development of drugs that treat metabolic disorders, it is critical to understand how resveratrol modulates metabolism. It is widely accepted that Sirt1, the founding member of the Sirtuin family (8) of NAD-dependent deacetylase, is the target of resveratrol (1,5,7). However, whether the putative Sirt1 activators such as resveratrol actually target Sirt1 in vivo is controversial because resveratrol increases Sirt1 activity in vitro only if the substrate is modified with a fluorescent tag (9,10). Resveratrol appears to increase the deacetylation rate by enhancing the affinity of Sirt1 for fluorescent-tagged peptides.Resveratrol also has a number of indirect effects (11), including stimulation of 5′ AMP-activated protein kinase (AMPK) (5,12,13). AMPK is a heterotrimeric protein consisting of an α-catalytic subunit and two regulatory subunits, β and γ (14). AMPK is a fuel-sensing kinase, which is activated by ATP-depleting conditions such as physical exercise, ischemia, and glucose deprivation. The catalytic subunit of AMPK has two isoforms, α1 and α2, which have different tissue expression patterns. Muscle expresses predominantly the α2-isoform (15), whereas fat and brain express predominantly the α1 isoform (16,17), and liver expresses both α1 and α2 isoforms (18). AMPKα1 and AMPKα2 knockout mice are viable, but AMPKα1/α2 double knockout causes embryonic lethality. Like resveratrol, activation of AMPK has been shown to reduce fat accumulation and increase glucose tolerance, insulin sensitivity, mitochondrial biogenesis, and physical endurance (19–23). Therefore, it is possible that the metabolic effects of resveratrol are mediated by AMPK. Supporting this possibility, resveratrol-mediated extension of lifespan in worms requires AMPK (24).Resveratrol may activate AMPK in several different ways. Resveratrol, as well as other polyphenols, can reduce ATP levels by inhibiting ATP synthase (25). Resveratrol can also activate AMPK without altering the AMP-to-ATP ratio. Dasgupta et al. (12) showed that, at lower doses, resveratrol can activate AMPK through a Sirt1-independent manner. Interestingly, Hou et al. (26) and Lan et al. (27) reported that the activity of liver kinase B (LKB)-1, one of the AMPK kinases that is important for AMPK activity, is activated by resveratrol in a Sirt1-dependent manner.     

Direct Effects of TNF-α on Local Fuel Metabolism and Cytokine Levels in the Placebo-Controlled,Bilaterally Infused Human Leg: Increased Insulin Sensitivity,Increased Net Protein Breakdown,and Increased IL-6 Release

Tumor necrosis factor-α (TNF-α) has widespread metabolic actions. Systemic TNF-α administration, however, generates a complex hormonal and metabolic response. Our study was designed to test whether regional, placebo-controlled TNF-α infusion directly affects insulin resistance and protein breakdown. We studied eight healthy volunteers once with bilateral femoral vein and artery catheters during a 3-h basal period and a 3-h hyperinsulinemic-euglycemic clamp. One artery was perfused with saline and one with TNF-α. During the clamp, TNF-α perfusion increased glucose arteriovenous differences (0.91 ± 0.17 vs. 0.74 ± 0.15 mmol/L, P = 0.012) and leg glucose uptake rates. Net phenylalanine release was increased by TNF-α perfusion with concomitant increases in appearance and disappearance rates. Free fatty acid kinetics was not affected by TNF-α, whereas interleukin-6 (IL-6) release increased. Insulin and protein signaling in muscle biopsies was not affected by TNF-α. TNF-α directly increased net muscle protein loss, which may contribute to cachexia and general protein loss during severe illness. The finding of increased insulin sensitivity, which could relate to IL-6, is of major clinical interest and may concurrently act to provide adequate tissue fuel supply and contribute to the occurrence of systemic hypoglycemia. This distinct metabolic feature places TNF-α among the rare insulin mimetics of human origin.Originally, tumor necrosis factor-α (TNF-α) was identified as an endogenous pyrogen or “cachectin” (1) because of its biological properties of inducing fever, cachexia, and muscle protein loss in various states of disease (2–4). TNF-α is a key component of an inflammatory response and one of the most potent proinflammatory cytokines released by innate immune cells that induces release of other cytokines, including interleukin-6 (IL-6) (5,6). TNF-α plays an important role in the pathophysiology of sepsis, and there seems to be a relation between the TNF-α level and the severity of disease (7–9). Finally, TNF-α has been associated with states of constant low-grade inflammation, eventually leading to insulin resistance and overt diabetes (10,11). In line with this, it has been shown that plasma levels of TNF-α are correlated with BMI; weight loss leads to a decrease in plasma levels of TNF-α (12,13).Systemic infusion of TNF-α induces insulin resistance and increased lipolysis in humans (6,14,15), whereas the effects on protein metabolism are less clear (16). A number of studies have shown that anti–TNF-α treatment increases insulin sensitivity in patients with inflammatory chronic diseases (17–19), whereas other reports have failed to confirm this relationship (20–23). Furthermore, studies investigating TNF-α neutralization in type 2 diabetic patients and in patients with metabolic syndrome show no effect of anti–TNF-α treatment on insulin sensitivity (24,25). TNF-α activates the hypothalamopituitary axis and stimulates the release of stress hormones, such as epinephrine, glucagon, cortisol, and growth hormone into the blood (26,27); all of these counter-regulatory stress hormones generate insulin resistance (27–29), and glucocorticoids generate muscle loss (30). Thus, TNF-α invariably generates release of both other cytokines and stress hormones, and it is uncertain to which extent the metabolic actions of TNF-α are intrinsic or caused by other cytokines or stress hormones in humans.The current study was therefore designed to define the direct metabolic effects of TNF-α in human muscle. Since all previous human studies assessing the metabolic actions of TNF-α have used systemic administration, making discrimination between direct and indirect effects impossible, we infused TNF-α directly into the femoral artery and compared the effects to the saline-infused contralateral leg.     

C-Peptide Activates AMPKα and Prevents ROS-Mediated Mitochondrial Fission and Endothelial Apoptosis in Diabetes

Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.C-peptide and insulin are cosecreted in equimolar amounts into the circulation from the pancreatic β-cells of Langerhans (1). C-peptide deficiency is a prominent attribute of type 1 diabetes (1). Deficiencies of C-peptide and insulin may also occur in the late stages of type 2 diabetes as a result of progressive loss of β-cells (2–4). Recent evidence demonstrates a beneficial role for C-peptide in diabetic neuropathy (1,5,6), nephropathy (1,6,7), and vascular dysfunction (1,5) and inflammation (1). C-peptide protects against diabetic vascular damage by promoting nitric oxide (NO) release (8) and suppressing nuclear factor-κB (9), which suppresses leukocyte-endothelium interactions (8,9). C-peptide may prevent atherosclerosis by inhibiting vascular smooth muscle proliferation and migration (10) and reducing venous neointima formation (11). However, the molecular mechanism by which C-peptide prevents diabetes complications is not understood well enough to permit its clinical implementation.Generation of reactive oxygen species (ROS) in response to high glucose is the leading cause of endothelial damage and diabetic vasculopathy (12). Protein kinase C (PKC)-dependent NADPH oxidase is considered a major cytosolic mediator of ROS generation in endothelial cells (13,14) that play a central role in hyperglycemia-induced endothelial cell apoptosis and vascular complications (15–17). Overproduction of intracellular ROS by mitochondria also occurs during the development of hyperglycemia-induced vascular complications (12,18,19). Altered mitochondrial dynamics due to mitochondrial fission were recently linked with endothelial dysfunction in diabetes (20,21). However, the mechanisms regulating production of cytosolic and mitochondrial ROS and their individual functions in regulating mitochondrial dynamics and apoptosis remain to be elucidated.AMP-activated protein kinase (AMPK) is an intracellular energy and stress sensor (22) and is an emerging target for preventing diabetes complications (23), as exhibited by the most common antihyperglycemic drugs, rosiglitazone (24) and metformin (25). AMPK prevents apoptosis of endothelial cells (26–28) by inhibiting ROS generation by NADPH oxidase (24,29) and mitochondria (30). Additionally, AMPK dephosphorylation is associated with diabetes (22,31,32). It has been reported that C-peptide inhibits high glucose–induced mitochondrial superoxide generation in renal microvascular endothelial cells (7). We recently demonstrated a key role for C-peptide in preventing high glucose–induced ROS generation and apoptosis of endothelial cells through inhibition of transglutaminase (17). However, the mechanism underlying C-peptide–mediated inhibition of intracellular ROS production and subsequent apoptosis remains unclear. Thus, we hypothesized that the potential protective role of C-peptide could be attributed to activation of AMPK, which results in reduced hyperglycemia-induced production of intracellular ROS and altered mitochondrial dynamics that suppress apoptosis of endothelial cells.In this study, we sought to elucidate the mechanism by which C-peptide protects against hyperglycemia-induced ROS production and subsequent endothelial cell damage. We examined the beneficial effect of C-peptide through AMPKα activation and subsequent protection against hyperglycemia-induced production of intracellular ROS, dysregulation of mitochondrial dynamics, mitochondrial membrane potential (∆Ψ_m) collapse, and apoptosis of endothelial cells. These studies were confirmed in vivo in mice with streptozotocin-induced diabetes using C-peptide supplement therapy delivered through osmotic pumps. Thus, our study implicates C-peptide replacement therapy as a potentially significant approach for preventing diabetes complications.     

Exercise Alleviates Lipid-Induced Insulin Resistance in Human Skeletal Muscle–Signaling Interaction at the Level of TBC1 Domain Family Member 4

Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation.Studies in human and rodent models have revealed deleterious effects of excess lipid availability on peripheral insulin sensitivity (1,2). Intracellular increases in fatty acid metabolites, such as diacylglycerol (DAG) and ceramide, may play critical roles in mediating lipid-induced insulin resistance by inducing serine phosphorylation of insulin-receptor substrate 1 (IRS-1) (3–6) and consequently inhibiting downstream signaling to GLUT4 translocation. However, recent reports challenge such causality. These studies revealed unaltered signal transduction at the level of IRS-1, IRS-1–associated phosphatidylinositol-3-kinase (PI3K) activity, Akt, and TBC1 domain family member 4 (TBC1D4) phosphorylation (phospho-Akt-substrate [PAS] an unspecific antibody recognizing phosphorylated Akt substrate motifs), after 2–7 h of lipid infusion (7–11). When DAG and/or ceramide levels were reported, no changes in skeletal muscle DAG or ceramide levels were found after lipid infusion (7,11).We recently showed that lactate release in human skeletal muscle is augmented along with reduced respiratory exchange ratio (RER) values during lipid infusion (11). This could indicate suppressed activity of the pyruvate dehydrogenase (PDH) complex, which in turn could lead to a reduction in glucose uptake according to the Randle cycle (12). Here, we wished to investigate whether this increase in leg lactate release and reduced RER values were accompanied by altered regulation of PDH, measured by site-specific phosphorylation.Exercise increases peripheral insulin sensitivity (13–15). After an acute bout of exercise, the ability for insulin to stimulate glucose uptake in skeletal muscle is increased several hours into recovery (14,16). This effect can be ascribed to adaptations in the exercised muscle rather than changes in systemic factors (13,17,18) and is observed in both healthy and insulin-resistant states (e.g., obesity) (19) and type 2 diabetes (20). A recent study has shown that a single bout of exercise can prevent subsequent lipid-induced impairments in whole-body glucose tolerance assessed by an intravenous glucose tolerance test (IVGTT) (2). It was hypothesized that repartitioning fatty acids toward intramuscular triacylglycerol (IMTG) synthesis and storage rather than DAG or ceramide might be a primary mediator of the beneficial effects of exercise on lipid-induced impairments in glucose tolerance (2). Enhanced insulin sensitivity after a bout of exercise is associated with increased GLUT4 recruitment to the plasma membrane (21) and not with altered protein synthesis (e.g., GLUT4 protein) (22), but has not been associated with altered signal transduction through the insulin receptor, IRS-1, PI3K, or Akt (13,22,23). Recently, the hypothesis was put forward (24) that the guanosine triphosphatase (GTPase) activating proteins TBC1 domain family member 1 (TBC1D1) and 4 (TBC1D4) might serve as points of convergence for insulin dependent and independent signaling pathways to GLUT4 translocation. In agreement with this hypothesis, PAS phosphorylation of TBC1D4 is elevated along with insulin-stimulated glucose uptake for up to 27 h after exercise in skeletal muscle of rats (25), and we recently showed that phosphorylation of TBC1D4 on specific residues was elevated 4 h after a single bout of exercise in human skeletal muscle (26).TBC1D4/D1 are multikinase substrates proposed to be involved in contraction- and insulin-stimulated glucose uptake in mice (27,28), and exercise and insulin both substantially increase TBC1D4/D1 phosphorylation in human skeletal muscle (29,30). TBC1D4/D1 contain several phosphorylation sites distinctly phosphorylated by various kinases, including Akt and 5′AMP-activated protein kinase (AMPK) (28,31–33). Phosphorylation of TBC1D4/D1 and subsequent 14-3-3 binding is proposed to lead to inactivation of the GTPase-activating proteins, decreasing their inhibitory function on the GLUT4 translocation process and thus, potentially, increasing the GLUT4 capacity of the surface membrane.In the current study we tested the hypothesis that prior exercise prevents subsequent lipid-induced insulin resistance in human skeletal muscle through regulation of the signaling molecules TBC1D4/TBC1D1.     

NAD(P)H Oxidase Mediates TGF-β1–Induced Activation of Kidney Myofibroblasts

TGF-β1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase–derived ROS in TGF-β1–induced activation of rat kidney fibroblasts and expression of α-smooth muscle actin (α-SMA) and Fn-ED-A. In vitro, TGF-β1 stimulated formation of abundant stress fibers and increased expression of both α-SMA and Fn-ED-A. In addition, TGF-β1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-β1–induced stimulation of NADPH oxidase activity and reduced α-SMA and Fn-ED-A expression. Inhibition of TGF-β1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-β1–enhanced NADPH oxidase activity; and decreased expression of Nox4, α-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-β1–induced Smad3 but reduced both α-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked α-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-β1 stimulated phosphorylation of extracellular signal–regulated kinase (ERK1/2), and this was inhibited by blocking TGF-β1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased α-SMA and Fn-ED-A. Taken together, these results suggest that TGF-β1–induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.Progression of renal fibrosis involves expansion of interstitial myofibroblasts and extracellular matrix accumulation, resulting in the loss of function and ultimately renal failure.^1,2 The origin of myofibroblasts is under extensive investigation, and evidence indicates the cells may be derived from several sources, including an expansion of activated resident fibroblasts, perivascular adventitial cells, blood-borne stem cells that migrate into the glomerular mesangial or interstitial compartment, or tubular epithelial-to-mesenchymal transition and migration into the peritubular interstitial space. Regardless of their origin, there is common agreement that the myofibroblast is the cell most responsible for interstitial expansion and matrix accumulation during the course of renal fibrosis. TGF-β1 is the predominant growth factor responsible for matrix synthesis by mesenchymal cells such as fibroblasts in vitro and during renal fibrosis.^3,4 Indeed, there is a close correlation in the cellular expression of TGF-β1, a fibroblast transition to an activated, α-smooth muscle actin (α-SMA)-positive myofibroblast phenotype, and synthesis of an alternatively spliced isoform of fibronectin, Fn-ED-A.⁵ TGF-β1 differentially regulates the expression of Fn-ED-A in fibroblasts^6–8 and induces expression of α-SMA in a variety of mesenchymal cells in culture.^9,10 Indeed, a functional ED-A domain is mandatory for α-SMA induction by TGF-β1.^7,8,10 Moreover, TGF-β1 is frequently associated with a myofibroblast phenotype in liver, lung, and kidney disease,^1,11–13 and all three proteins frequently co-localize in these disease settings. In addition, a co-localization of α-SMA and Fn-ED-A is frequently observed in fibrotic disease as well as in glomerular and interstitial lesions in kidney diseases previously investigated in our laboratory.^14–17Accumulating evidence also indicates that reactive oxygen species (ROS), mainly in the form of superoxide, play a significant role in the initiation and progression of cardiovascular^18,19 and renal^20–25 disease. ROS are involved in distinct cell functions, including hypertrophy, migration, proliferation, apoptosis, and regulation of extracellular matrix.^25–28 More specific, the NAD(P)H oxidases of the Nox family have gained heightened attention as mediators of injury associated with vascular diseases, including hypertension, atherosclerosis, heart disease, and diabetes.^18,19,29,30 NAD(P)H oxidase generation of superoxide is recognized as an important mediator of cell proliferation in glomerulonephritis²² and matrix accumulation in diabetic nephropathy^25,31–33 and fibrosis.^21,24 Adventitial fibroblasts are also a major source of superoxide in the aorta,^19,34–36 therefore being highly relevant to renal disease. This is because the renal perivascular space is noticeably reactive and is the site where myofibroblasts may first appear during the course of renal disease and fibrosis.^17,37–39The observations that both TGF-β1 and ROS induce fibroblasts to α-SMA–positive myofibroblast phenotype^40–42 suggest that these two pathways are interrelated and may share signaling pathways in kidney disease. TGF-β signaling occurs through a well-established process involving two downstream pathways: Smad and extracellular signal–regulated kinase (ERK).^43–45 TGF-β/Smad signaling (Smad2 and Smad3) is tightly controlled by mitogen-activated protein kinase (MAPK; ras/MEK/ERK) signaling cascades.⁴⁶ A regulatory role for ROS in PDGF and angiotensin II–induced signal transduction has gained recognition^47,48; however, a role for ROS in TGF-β signaling is less well understood. It is also unknown whether kidney myofibroblasts express NAD(P)H oxidase homologs or generate ROS in response to TGF-β1. Given TGF-β1–induced myofibroblast activation and matrix synthesis during renal disease may be linked to ROS, we examined a role for NAD(P)H oxidase in TGF-β1–induced Smad3 and ERK signaling as well as kidney myofibroblast activation, as assessed by a switch to an α-SMA–positive phenotype and expression of Fn-ED-A expression in vitro.     

Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo

Recent studies suggest that interleukin 6 (IL-6) is released from contracting skeletal muscles; however, the cellular origin, secretion kinetics, and signaling mechanisms regulating IL-6 secretion are unknown. To address these questions, we developed imaging methodology to study IL-6 in fixed mouse muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP–containing vesicles and protein by 62% (P < 0.05), occurring rapidly and progressively over 25 min of contraction. However, contraction-mediated IL-6-EGFP reduction was normal in muscle-specific AMP-activated protein kinase (AMPK) α2-inactive transgenic mice. In contrast, the AMPK activator AICAR decreased IL-6-EGFP vesicles, an effect that was inhibited in the transgenic mice. In conclusion, resting skeletal muscles contain IL-6–positive vesicles that are expressed throughout myofibers. Contractions stimulate the rapid reduction of IL-6 in myofibers, occurring through an AMPKα2-independent mechanism. This novel imaging methodology clearly establishes IL-6 as a contraction-stimulated myokine and can be used to characterize the secretion kinetics of other putative myokines.Skeletal muscle is a critical tissue for whole-body glucose metabolism during both normal and pathological conditions. There is increasing evidence that skeletal muscles express myokines, hormone-like factors that are released into the serum to function in an autocrine, paracrine, or endocrine manner (1–5). In recent years, numerous myokines have been proposed to be secreted from muscle, including interleukin-6 (IL-6) (1), fibroblast growth factor 21 (3), follistatin-like 1 (2), insulin-like 6 factor (4), and most recently irisin (5). Thus, skeletal muscle is potentially the largest endocrine organ in the body, and myokine release may provide a significant mechanism for crosstalk with other tissues.Of these putative myokines, IL-6 has been the most extensively studied (1,6). IL-6 has been proposed to be secreted from skeletal muscle and to function in an autocrine manner to activate signaling proteins mediating glucose uptake (7), glycogen metabolism (8), fat metabolism (9), and muscle hypertrophy (10). Despite considerable investigation of IL-6, the exact cellular origin of IL-6 within the muscle tissue is not well understood. In fact, previous studies have not clearly detected IL-6 protein within the muscle fibers from human biopsies (11) or mouse muscle sections (10) unless a state of inflammation (12,13) or injury (10) was present. It is possible that the biopsy procedure itself causes IL-6 release and contamination from invading macrophages (14), interfering with the ability to determine the exact level and localization of IL-6 within the muscle fibers. Thus, whether IL-6 is present in skeletal muscle fibers under normal, resting conditions is not fully understood.There is considerable evidence that exercise increases circulating concentrations of IL-6 based on studies demonstrating an increased arterial/venous IL-6 difference across contracting skeletal muscles (1,6,15–17). However, studies analyzing the cellular localization of IL-6 within muscle fibers during exercise are limited. In one study, bicycle ergometer exercise for 2 h resulted in increased detection of IL-6 protein near the sarcolemma region of vastus lateralis muscle (11). Since light microscopy cannot distinguish the sarcolemma from the interstitial space, one interpretation of this finding is that the detected IL-6 did not originate from muscle fibers but instead arose from biopsy- and/or exercise-induced macrophage infiltration (14). If muscle fibers are the source of increased circulating IL-6 during exercise, then the number of secretory vesicles containing IL-6 in the muscle fibers might be expected to decrease with contractions, not increase. Given the ambiguities of previous data, one aim of the current study was to determine the kinetics and time course of a putative IL-6 release from contracting skeletal muscle fibers.Exercise increases AMP-activated protein kinase (AMPK) activity in skeletal muscle, and AMPK signaling pathways have been proposed to mediate multiple metabolic effects (18). Exercise-stimulated AMPK activity in muscle has been associated with an increase in circulating IL-6 during exercise (19), although a direct link between AMPK activation and IL-6 protein release from muscle fibers has not been reported (19,20). AMPK stimulation has also been reported to alter IL-6 expression, albeit with conflicting results (21–23). In one report, 24 h of incubation of C2C12 muscle cells with the AMPK activator AICAR increased IL-6 mRNA (21). In another report, 2–4 h of AICAR incubation of soleus and extensor digitorum longus muscles decreased IL-6 mRNA (22,23) and IL-6 secretion into the incubation media (23). Thus, the role of AMPK in the regulation of IL-6 in skeletal muscle has not been established.In the current study, we determined if intact muscle fibers express IL-6 under basal, resting conditions. In addition, we determined if muscle contraction and AICAR regulate IL-6 secretion in vivo. Finally, we investigated the potential role of AMPKα2 in contraction-stimulated IL-6 release. To address these questions, we developed novel imaging techniques that allow for kinetic analysis of IL-6–containing vesicles within intact muscle fibers in vivo. These studies establish that IL-6 is a contraction-induced myokine in intact muscle fibers, but that AMPKα2 activity does not mediate contraction-stimulated IL-6 secretion.     

Prevention of Diabetes With Pioglitazone in ACT NOW: Physiologic Correlates

We examined the metabolic characteristics that attend the development of type 2 diabetes (T2DM) in 441 impaired glucose tolerance (IGT) subjects who participated in the ACT NOW Study and had complete end-of-study metabolic measurements. Subjects were randomized to receive pioglitazone (PGZ; 45 mg/day) or placebo and were observed for a median of 2.4 years. Indices of insulin sensitivity (Matsuda index [MI]), insulin secretion (IS)/insulin resistance (IR; ΔI_0–120/ΔG_0–120, ΔIS rate [ISR]_0–120/ΔG_0–120), and β-cell function (ΔI/ΔG × MI and ΔISR/ΔG × MI) were calculated from plasma glucose, insulin, and C-peptide concentrations during oral glucose tolerance tests at baseline and study end. Diabetes developed in 45 placebo-treated vs. 15 PGZ-treated subjects (odds ratio [OR] 0.28 [95% CI 0.15–0.49]; P < 0.0001); 48% of PGZ-treated subjects reverted to normal glucose tolerance (NGT) versus 28% of placebo-treated subjects (P < 0.005). Higher final glucose tolerance status (NGT > IGT > T2DM) was associated with improvements in insulin sensitivity (OR 0.61 [95% CI 0.54–0.80]), IS (OR 0.61 [95% CI 0.50–0.75]), and β-cell function (ln IS/IR index and ln ISR/IR index) (OR 0.26 [95% CI 0.19–0.37]; all P < 0.0001). Of the factors measured, improved β-cell function was most closely associated with final glucose tolerance status.The prevalence of type 2 diabetes mellitus (T2DM) has risen to epidemic proportions in the United States and worldwide (1), and is being driven by the epidemic of obesity (2). In high-risk individuals, it is reasonable to consider interventions that reduce the incidence of T2DM. Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) are “high-risk” states with annual diabetes conversion rates ranging from 3 to 11% per year (3). Individuals with IGT have moderate-to-severe insulin resistance (IR) in muscle and impaired second-phase insulin secretion (IS), while those with IFG are characterized by hepatic IR and impaired first-phase IS with intact second-phase IS and normal/near-normal muscle insulin sensitivity (4–7). IGT conversion to T2DM is associated with a further and progressive decline in β-cell function with little worsening of IR, which is near maximally established in IGT (4,5,8–10). Treatment with thiazolidinediones improves IS (11) and IR (12–15), ameliorates lipotoxicity (12,14), and redistributes fat from muscle/liver/β-cells to subcutaneous fat depots (12,16). Therefore, they represent a logical choice for the treatment of IGT and IFG.In the ACT NOW Study (17,18), over a 2.4-year period, the annual conversion rate of IGT to T2DM was 7.6% in placebo-treated vs. 2.1% in pioglitazone (PGZ)-treated subjects (hazard ratio 0.28, P < 0.0001) (18). PGZ is a potent insulin-sensitizing agent in muscle, liver, and adipocytes (reviewed in 12–14); augments IS; and preserves β-cell function (11,19). These effects are mediated, in part, via peroxisome proliferator–activated receptor-γ (PPAR-γ) receptor (13,20) and via reversal of lipotoxicity (12,14) and changes in adipocytokines (20). PGZ reduces plasma free fatty acid (FFA) levels, mobilizes fat out of muscle (21) and liver (22), and redistributes fat from visceral to subcutaneous depots (12,16).In the current study, we examined which physiologic/metabolic/anthropometric changes (end-of-study versus baseline) in the ACT NOW Study (18) were associated with IGT progression to diabetes and reversion to normal glucose tolerance (NGT) in PGZ- and placebo-treated subjects.     

Kinetics of Contraction-Induced GLUT4 Translocation in Skeletal Muscle Fibers From Living Mice

OBJECTIVE

Exercise is an important strategy for the treatment of type 2 diabetes. This is due in part to an increase in glucose transport that occurs in the working skeletal muscles. Glucose transport is regulated by GLUT4 translocation in muscle, but the molecular machinery mediating this process is poorly understood. The purpose of this study was to 1) use a novel imaging system to elucidate the kinetics of contraction-induced GLUT4 translocation in skeletal muscle and 2) determine the function of AMP-activated protein kinase α2 (AMPKα2) in this process.

RESEARCH DESIGN AND METHODS

Confocal imaging was used to visualize GLUT4-enhanced green fluorescent protein (EGFP) in transfected quadriceps muscle fibers in living mice subjected to contractions or the AMPK-activator AICAR.

RESULTS

Contraction increased GLUT4-EGFP translocation from intracellular vesicle depots to both the sarcolemma and t-tubules with similar kinetics, although translocation was greater with contractions elicited by higher voltage. Re-internalization of GLUT4 did not begin until 10 min after contractions ceased and was not complete until 130 min after contractions. AICAR increased GLUT4-EGFP translocation to both sarcolemma and t-tubules with similar kinetics. Ablation of AMPKα2 activity in AMPKα2 inactive transgenic mice did not change GLUT4-EGFP′s basal localization, contraction-stimulated intracellular GLUT4-EGFP vesicle depletion, translocation, or re-internalization, but diminished AICAR-induced translocation.

CONCLUSIONS

We have developed a novel imaging system to study contraction-stimulated GLUT4 translocation in living mice. Contractions increase GLUT4 translocation to the sarcolemma and t-tubules with similar kinetics and do not require AMPKα2 activity.Skeletal muscle is critical in the regulation of glucose homeostasis, being the major site of whole-body glucose disposal (1). In skeletal muscle fibers, the GLUT4 protein mediates increases in glucose uptake. Upon stimulation with insulin or muscle contraction, GLUT4 is translocated from intracellular vesicle compartments to the two main muscle membrane surfaces, the sarcolemma and t-tubules (2–5). The kinetics of GLUT4 intracellular trafficking in skeletal muscle and how signaling molecules regulate GLUT4 translocation are poorly understood, especially for contraction-mediated GLUT4 translocation. The majority of studies analyzing GLUT4 translocation dynamics have been carried out in adipocytes (6–10) or in muscle cell cultures (11–13), cell types that do not resemble fully differentiated muscle (14).Recently, intravital imaging techniques have allowed detailed analysis of the spatial-temporal dynamics of GLUT4 translocation in response to insulin stimulation in skeletal muscle fibers of living animals (5,15,16). Direct imaging of insulin-stimulated GLUT4-EGFP translocation has shown that GLUT4 is translocated to both the sarcolemma and t-tubules. Both GLUT4 translocation and re-internalization were delayed in the t-tubules compared with the sarcolemma due to a lag in insulin diffusion (5,16). These imaging studies have also shown that in states of insulin resistance, the t-tubules, and not the sarcolemma, are the primary site of impaired insulin signaling and GLUT4 translocation (16). Collectively, these findings illustrate that insulin-mediated signaling and GLUT4 translocation are compartmentalized in mature skeletal muscle fibers.It is unknown whether a similar type of compartmentalization exists for contraction-mediated GLUT4 translocation. It is well established that rapid spreading of membrane depolarization throughout the t-tubule network results in simultaneous activation of contraction throughout the muscle fiber (17).Biochemical studies have suggested that in response to muscle contraction, GLUT4 can translocate to both the sarcolemma and t-tubules (4,18). However, imaging studies have never been done in intact contracting skeletal muscle to analyze the kinetics of GLUT4 translocation in high resolution.The signaling mechanisms mediating contraction-induced GLUT4 translocation are not fully understood, but differ from those triggered by insulin (19). Muscle contractions increase AMPK activity (20), and pharmacological activation of AMPK results in increased glucose transport in skeletal muscle (20–22), although one report suggested that AICAR only caused GLUT4 translocation to the sarcolemma (23). Surprisingly, studies directly assessing the role of AMPK in contraction-mediated glucose transport using animal models with ablated AMPK activity have been ambiguous. Contraction-mediated glucose transport was not impaired in whole-body knockouts of the AMPKα1 or AMPKα2 catalytic subunits (24). In muscle-specific AMPKα2 inactive transgenic mice, contraction- or exercise-mediated glucose transport was either partially reduced (25,26) or unaffected (27,28), but the effects of AMPK activity on GLUT4 translocation kinetics are not known.In the current study, we used intravital imaging to determine GLUT4 translocation kinetics in contracting skeletal muscle. To understand the intracellular signals that regulate this effect, we used muscle-specific AMPKα2 inactive transgenic mice (27). We found that contraction-stimulated GLUT4 translocation occurs at both the sarcolemma and t-tubules with similar kinetics. Our data demonstrate that contractions elicited by a higher voltage resulted in a higher degree of translocation, and that GLUT4 remains at the cell surface for up to 2 h after the cessation of contraction. Finally, our data show that contraction-stimulated GLUT4 translocation is AMPKα2-independent.     

Therapeutic Effects of PPARα Agonists on Diabetic Retinopathy in Type 1 Diabetes Models

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator–activated receptor α (PPARα) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARα dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARα agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-κB in the retinas of OIR and diabetic models. Fenofibrate’s beneficial effects were blocked by a specific PPARα antagonist. Furthermore, Pparα knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARα-dependent mechanism.With the rising incidence of diabetes, the prevalence of the vascular complications of diabetes are increasing, in spite of recent advances in therapies targeting hyperglycemia, hypertension, and dyslipidemia (1,2). Diabetic retinopathy (DR) is a feared and common microvascular complication of diabetes and one of the most common sight-threatening conditions in developed countries (3). DR is a chronic, progressive, and multifactorial disorder, primarily affecting retinal capillaries (4,5). Diabetes induces retinal inflammation, blood-retinal barrier breakdown, and increased retinal vascular permeability, leading to diabetic macular edema (DME) (6). In proliferative DR, overproliferation of capillary endothelial cells results in retinal neovascularization (NV), which can cause severe vitreous cavity bleeding, retinal detachment, and vision loss (7,8).Unlike type 2 diabetes, in type 1 diabetes, obesity, the metabolic syndrome, and dyslipidemia are less common, although when present in people with type 1 diabetes, they are risk factors for micro- and macrovascular complications (9,10). Retinopathy in both type 1 and type 2 diabetes develops retinal vascular leakage, inflammation, NV, and fibrosis (11). Even though it is well established that vascular endothelial growth factor (VEGF) mediates the pathologic processes of vascular leakage and angiogenesis in DR, anti-VEGF compounds are not always effective in all patients with DR (12). This may be ascribed to the fact that DR is mediated by multiple angiogenic, inflammatory, and fibrogenic factors such as VEGF, tumor necrosis factor-α (13), intercellular adhesion molecule-1 (ICAM-1) (14), and connective tissue growth factor (15), and thus, blockade of VEGF alone is not sufficient to ameliorate all of the perturbed signaling.Fenofibrate, a peroxisome proliferator–activated receptor α (PPARα) agonist, available clinically for >30 years for the treatment of dyslipidemia (16,17), is particularly effective in improving the lipid profile in hypertriglyceridemia and low HDL syndromes (18), and for reducing some cardiovascular events (19). Recent studies reported that activation of PPARα suppresses transforming growth factor-α–induced matrix metalloproteinase-9 expression in human keratinocytes (20), blocks tumor angiogenesis via vascular NADPH oxidase (21), modulates endothelial production of inflammatory factors (22), and improves wound healing in pediatric burn patients (23). In the retinal pigment epithelium, fenofibrate modulates cell survival signaling (24) and reduces diabetic stress–induced fibronectin and type IV collagen overexpression (25). Moreover, fenofibrate also prevents interleukin-1β–induced retinal pigment epithelium disruption through inhibition of the activation of AMP-activated protein kinase (26).Recent studies suggest that PPARα is an emerging therapeutic target in diabetic microvascular complications (27–29). Two recent, large, prospective, placebo-controlled clinical trials have demonstrated protective effects of fenofibrate against DR in type 2 diabetic patients. The Fenofibrate Intervention in Event Lowering in Diabetes (FIELD) Study reported that fenofibrate monotherapy significantly reduced the cumulative need for laser therapy for DR by 37% (30), nephropathy progression by 14% (31), and amputations (23), including microvascular amputations, by 37% (32) in type 2 diabetic patients. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) Lipid Study of combination simvastatin and fenofibrate demonstrated a 40% reduction in progression of proliferative DR in type 2 diabetic patients over simvastatin only (33). Despite these exciting clinical findings, several unanswered questions remain. Is fenofibrate effective against DR in type 1 diabetes? Is the fenofibrate effect on DR a direct action on retinal vasculature or through the systemic lipid-lowering effect? Are the ocular fenofibrate effects PPARα dependent? This study was designed to address these important questions.In the current study, we explore whether fenofibrate has therapeutic effects on DR in type 1 diabetes animal models, and on ischemia-induced retinal NV, and whether such effects are dependent on PPARα.     

Cyclic GMP kinase I modulates glucagon release from pancreatic α-cells

OBJECTIVE

The physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis.

RESEARCH DESIGN AND METHODS

Gene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKIα and Iβ rescue mice [α/βRM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²⁺ ([Ca²⁺]_i) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo.

RESULTS

Immunohistochemistry showed that cGKI is present in α- but not in β-cells in islets of Langerhans. Mice that lack α-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²⁺]_i levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²⁺]_i or hormone release in cGKI-deficient islets.

CONCLUSIONS

We propose that cGKI modulates glucagon release by suppression of [Ca²⁺]_i in α-cells.The complex and tightly controlled process of glucagon secretion from pancreatic α-cells is important for the maintenance of blood glucose homeostasis (1). Glucagon release is physiologically regulated by multiple signaling pathways that include neuronal control of α-cell function, paracrine factors such as insulin (2,3), and/or γ-aminobutyric acid (GABA) (4) released from β-cells, somatostatin (SST) secreted from adjacent δ-cells (5), and the inhibitory role of high blood glucose itself that directly acts on α-cells to suppresses glucagon release (3,6).Controversial data have been reported for the physiologic significance of the nitric oxide (NO) pathway for islet functions. Both types of constitutive NOS (eNOS, nNOS) isozymes have been identified in islets (7–11). It was suggested that NO stimulates glucose-induced insulin release (7,10), was a negative modulator of insulin release (8,12,13) or had no effect (14). These discrepant results are probably caused by the analysis of various β-cell–derived cell lines compared with intact islets, the use of different types and concentrations of NO-donors and NOS-inhibitors. Additional data suggested that iNOS-derived NO is involved in autoimmune reactions that cause β-cell–dysfunction leading to insulin-dependent diabetes (15,16).It has been difficult to discriminate between a direct action of NO on α-cells and an indirect effect of NO via β-cells since β-cell factors are potent inhibitors of α-cell activity (12,17,18). An important target of NO is the soluble guanylyl cyclase (sGC) that generates the second messenger cyclic guanosine-3′-5′-monophosphate (cGMP) (19). Some studies detected increased islet cGMP levels upon treatment with cytokines and l-arginine (12,15,20). cGMP analogues were reported to potentiate insulin release directly (21), suggesting that cGMP-dependent effectors are involved in the control of islet activity. The cGMP-dependent protein kinase type I (cGKI) is an important intracellular mediator of NO/cGMP signaling in many cells (22). The analysis of cGKI-deficient mice revealed that cGKI mediates the inhibitory effects of NO on platelet aggregation, the negative inotropic effect of NO/cGMP in the murine heart, and the NO-induced relaxation of blood vessels (22). However, cGKI knockout mice could not be analyzed reliably for a distorted islet function because they display abnormalities of various organ systems and die within the first 6 weeks (23). Recently, we generated an improved mouse model to study the specific roles of cGKI in vivo (24,25). These mice express either the cGKIα or cGKIβ isozyme selectively in smooth muscle cells (SMCs) on a cGKI-deficient genetic background. Since the animals show a prolonged life expectancy and normal SMC functions they were termed cGKIα and cGKIβ rescue mice (αRM and βRM, respectively).We examined the role of cGKI for the regulation of glucose homeostasis using cGKI-KO mice (23) and rescue mice (RM) (24,25) models. We show that cGKI is expressed in the α-cells of the endocrine pancreas, whereas in different gene-targeted animals the cGKI protein was not detectable. Furthermore, we demonstrate that islet cGKI regulates glucagon release by modulation of the glucose-dependent changes of [Ca²⁺]_i that trigger exocytosis. These ex-vivo findings were supported by elevated serum levels of basal glucose and glucagon in intact RM animals.     

TSG-6 Produced by hMSCs Delays the Onset of Autoimmune Diabetes by Suppressing Th1 Development and Enhancing Tolerogenicity

Genetic and immunological screening for type 1 diabetes has led to the possibility of preventing disease in susceptible individuals. Here, we show that human mesenchymal stem/stromal cells (hMSCs) and tumor necrosis factor-α–stimulated gene 6 (TSG-6), a protein produced by hMSCs in response to signals from injured tissues, delayed the onset of spontaneous autoimmune diabetes in NOD mice by inhibiting insulitis and augmenting regulatory T cells (Tregs) within the pancreas. Importantly, hMSCs with a knockdown of tsg-6 were ineffective at delaying insulitis and the onset of diabetes in mice. TSG-6 inhibited the activation of both T cells and antigen-presenting cells (APCs) in a CD44-dependent manner. Moreover, multiple treatments of TSG-6 rendered APCs more tolerogenic, capable of enhancing Treg generation and delaying diabetes in an adoptive transfer model. Therefore, these results could provide the basis for a novel therapy for the prevention of type 1 diabetes.Recent advances in the use of genetic and immunological screening for identification of prediabetic patients (1–3) have opened up the opportunity to prevent, delay, or halt disease progression before the diagnosis of diabetes. Based on the success in animal models (4–6), clinical trials of oral or nasal insulin (7,8) and nicotinamide (9,10) have been conducted in humans to prevent type 1 diabetes. However, despite all efforts, these clinical trials have failed to show any improvement in the prevention of type 1 diabetes.Recently, we found that intravenously administered human mesenchymal stem/stromal cells (hMSCs) were activated to express the anti-inflammatory protein tumor necrosis factor (TNF)-α–stimulated gene 6 (TSG-6), which reduced excessive inflammatory response in the myocardial-infarcted heart in mice (11), chemically and mechanically injured cornea in rodent models (12,13), and zymosan-induced peritonitis in mice (14). Specifically, our recent observation revealed that TSG-6 attenuated zymosan-induced mouse peritonitis by decreasing TLR2-mediated NF-κB signaling in resident macrophages (14). This suppressive effect of TSG-6 on NF-κB signaling could provide the rationale for TSG-6 as a potential therapy for the prevention of type 1 diabetes, since several studies have already shown that inflammation and the innate immune system contribute to induction, amplification, and maintenance of the immune cell infiltrate as well as β-cell destruction during this preclinical period (15–17). Particularly, antigen-presenting cells (APCs) from NOD mice, mainly dendritic cells (DCs) and macrophages, have been shown to secrete substantially elevated levels of interleukin-12 (IL-12) and TNF-α (18,19) due to NF-κB hyperactivity (18,20), which leads to T-helper 1 (Th1) development and overt diabetes (21).Here, we tested whether a new treatment for the prevention of type 1 diabetes could be developed using TSG-6, which hMSCs produce in response to signals from injured tissues. Our data showed that systemic administration of hMSCs to prediabetic mice delayed the onset of type 1 diabetes in NOD mice in part by secreting TSG-6.