NOX2 Deficiency Protects Against Streptozotocin-Induced ��-Cell Destruction and Development of Diabetes in Mice

OBJECTIVE

The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice.

RESEARCH DESIGN AND METHODS

Five groups of mice—wild-type (WT), NOX2^−/−, WT treated with apocynin, and WT adoptively transferred with NOX2^−/− or WT splenocytes—were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2^−/− pancreatic islets were treated with cytokines for 48 h.

RESULTS

Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2^−/− mice and in WT mice adoptively transferred with NOX2^−/− splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2^−/− mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2^−/− compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2^−/− compared with WT splenocytes.

CONCLUSIONS

NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis.Type 1 diabetes is a T-cell–mediated autoimmune disease characterized by the selective destruction of insulin-secreting β-cells in the islets of Langerhans. It is a multifactorial process involving autoantigen presentation by macrophages, dentritic cells, and B-cells; activation of autoreactive CD4⁺ T-cells; and activation and recruitment of β-cell–specific CD8⁺ T-cells, leading to increased cytokine and reactive oxygen species (ROS) production and destruction of β-cells (1). The mechanisms of putative type 1 diabetes induced by multiple-low-dose streptozotocin (STZ) includes the direct β-cell destruction, which is mainly induced via DNA alkylation (2) and the indirect β-cell destruction from T-cell–dependent immune reaction (3). Furthermore, in response to cytokine stimulation including interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, β-cells also generate ROS and reactive nitrogen species, which may facilitate their destruction (4). Additionally, overexpression of antioxidant enzymes including superoxide dismutase (SOD), catalase, and glutathione peroxidase 1 (Gpx1) protects against the onset and development of diabetes and supports an important role of ROS in the pathogenesis of immune-mediated diabetes (5–12). NADPH oxidase is one of the main sources of superoxide radical formation in many cell types including phagocyte and β-cells (13). This ROS-producing enzyme consists of two membrane subunits (NOX2 and p22^phox) and at least four cytosolic components (p40^phox, p47^phox, p67^phox, and Rac1). NADPH oxidase is a highly regulated enzyme. In the resting cells, the cytosolic complex is separated from the membrane-bound catalytic core. Upon stimulation, the cytosolic component p47^phox becomes phosphorylated and the cytosolic complex migrates and binds to the membrane subunits to assemble into an active oxidase (14). It catalyzes the reduction of oxygen to superoxide anion using NADPH as a substrate and plays a major role in antimicrobial host defense as well as in tissue damage of autoimmune diseases (15,16). NOX2 is one of the critical subunits of NADPH oxidase. T-cells deficient in NOX2 exhibit an impaired ability to produce superoxide in response to anti-CD3 stimulation (17). Furthermore, glucose stimulates β-cell superoxide production, which can be inhibited by a selective NADPH oxidase inhibitor diphenylene iodonium, suggesting a functional NADPH oxidase in the islet (18). However, a definitive role of NADPH oxidase in the development of diabetes remains to be determined.In the present study, NOX2^−/− mice were used to investigate the role of NADPH oxidase in β-cell destruction induced by multiple-low-dose STZ. We demonstrated that NOX2 deficiency attenuates the severity of hyperglycemia and the loss of β-cell mass induced by STZ treatments via reduced ROS production and suppressed immune response.     

Increased phagocyte-like NADPH oxidase and ROS generation in type 2 diabetic ZDF rat and human islets: role of Rac1-JNK1/2 signaling pathway in mitochondrial dysregulation in the diabetic islet

OBJECTIVE

To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.

RESEARCH DESIGN AND METHODS

Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.

RESULTS

Levels of phosphorylated p47^phox, active Rac1, Nox activity, ROS generation, Jun NH₂-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.

CONCLUSIONS

We provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.Glucose-stimulated insulin secretion (GSIS) involves a series of metabolic and cationic events leading to translocation of insulin granules toward the plasma membrane for fusion and release of insulin into circulation (1–3). Insulin granule transport and fusion involve interplay between vesicle-associated membrane proteins on the insulin granules and docking proteins on the plasma membrane. In addition, a significant cross talk among multiple small G-proteins, including Arf6, Cdc42, and Rac1, was shown to be critical for GSIS (4–6). Several effector proteins for these G-proteins have been identified in the islet β-cell (4,7,8). We recently reported regulatory roles for Rac1 in the activation of phagocyte-like NADPH oxidase (Nox) and generation of reactive oxygen species (ROS) leading to GSIS (9).Excessive ROS generation is considered central to the development of diabetes complications. The generation of free radicals is relatively low under physiologic conditions; however, increased levels of circulating glucose promote intracellular accumulation of superoxides, leading to cellular dysfunction. Although mitochondria remain the primary source for free radicals, emerging evidence implicates Nox as a major source of extra-mitochondrial ROS. Nox is a highly regulated membrane-associated protein complex that promotes a one-electron reduction of oxygen to superoxide anion involving oxidation of cytosolic NADPH. The Nox holoenzyme consists of membrane and cytosolic components (Fig. 1). The membrane-associated catalytic core consists of gp91^phox and p22^phox, and the cytosolic regulatory core includes p47^phox, p67^phox, p40^phox, and Rac1. After stimulation, the cytosolic core translocates to the membrane for association with the catalytic core for functional activation of Nox. Immunologic localization and functional regulation of Nox have been described in clonal β-cells and in rat and human islets (10–13).Open in a separate windowFIG. 1.Schematic representation of Nox activation. Nox holoenzyme consists of cytosolic and membrane-associated components. Upon activation, Rac1, guanosine-5′-diphosphate (GDP) is converted to Rac1 guanosine-5′-triphosphate (GTP), which binds to p67^phox, and the complex translocates to the membrane. Existing evidence in other cell types suggests that phosphorylation of p47^phox also triggers its translocation to the membrane to form the Nox holoenzyme complex that culminates in the enzyme activation and associated increase in ROS.Recent findings from studies of pharmacologic and molecular biologic approaches suggest that ROS derived from Nox play regulatory “second-messenger” roles in GSIS (9–11,13,14). In addition to the positive modulatory roles for ROS in islet function, recent evidence also implicates negative modulatory roles for ROS in the induction of oxidative stress and metabolic dysregulation of the islet β-cell under the duress of glucolipotoxicity, cytokines, and ceramide (15). The generation of ROS in these experimental conditions is largely due to the activation of Nox, because inhibition of Rac1 or Nox activation markedly attenuated deleterious effects of these stimuli (15–17). Despite this compelling evidence, potential roles of Nox in islet dysfunction in animal models of type 2 diabetes remain unexplored. We therefore undertook the current study to examine the functional status of Nox in islets from the ZDF rat, which develops obesity, hyperinsulinemia, hyperglycemia, and a decline in β-cell function. We present evidence to suggest significant activation of Nox, ROS generation, and caspase-3 activation in the ZDF islets. Our findings also suggest similar metabolic defects in islets from type 2 diabetic human islets.     

Rac1 Is Required for Cardiomyocyte Apoptosis During Hyperglycemia

OBJECTIVE

Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia.

RESEARCH DESIGN AND METHODS

Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose.

RESULTS

The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91^phox or p47^phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function.

CONCLUSIONS

Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes.Diabetic cardiomyopathy has been defined as ventricular dysfunction that occurs in the absence of changes in blood pressure and coronary artery disease (1,2). Cell death by apoptosis is the predominant damage in diabetic cardiomyopathy (3,4). Diabetes increases cardiac apoptosis in animals and patients (3–7). Cardiomyocyte death causes a loss of contractile tissue, which initiates a cardiac remodeling (8). Loss of cardiomyocytes and hypertrophy of the remaining cells characterize the diabetic cardiomyopathy (9,10). Thus, suppression of cardiomyocyte apoptosis results in a significant prevention of the development of diabetic cardiomyopathy (4). However, the underlying mechanisms by which diabetes induce apoptosis remain not fully understood.All forms of diabetes are characterized by chronic hyperglycemia. Hyperglycemia induces reactive oxygen species (ROS) production in cardiomyocytes (6,11), which plays a crucial role in cardiomyocyte apoptosis in diabetes because the administration of antioxidant agents are able to rescue hyperglycemia-induced cardiomyocytes (4,6). The mechanisms activated by hyperglycemia, leading to myocardial oxidative stress and apoptosis, are not completely clarified.Although multiple sources of ROS have been demonstrated, NADPH oxidase is a critical determinant of the redox state of the myocardium (12–15). Higher myocardial NADPH oxidase activity has been detected in diabetes (16,17); more importantly, NADPH oxidase activity is markedly increased by high glucose levels (18). The NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558, which contains gp91^phox and p22^phox, the cytosolic regulatory subunits p47^phox and p67^phox, and the small guanosine triphosphate-binding protein Rac. An important step for the assembly and function of this multicomponent NADPH oxidase complex is the heterodimerization of gp91^phox with p67^phox, which is mediated by Rac (19). Three isoforms of Rac (Rac1, Rac2, and Rac3) have been identified (20), and Rac1 is the predominant isoform expressed in cardiomyocytes (21). Thus, Rac1 activation may lead to myocardial oxidative stress and apoptosis during hyperglycemia. A recent study showed that Rac1 contributes to vascular injury in diabetes (22). However, no direct evidence is available on Rac1 and NADPH oxidase activation in cardiomyocyte apoptosis in diabetes.In this study, we generated cardiomyocyte-specific Rac1 knockout (Rac1-ko) mice; analyzed the impact of Rac1 on NADPH oxidase activation, mitochondrial ROS generation, and intracellular ROS production; and investigated the role of Rac1 and NADPH oxidase activation in cardiomyocyte apoptosis during hyperglycemia.     

Nephropathy and Elevated BP in Mice with Podocyte-Specific NADPH Oxidase 5 Expression

NADPH oxidase (Nox) enzymes are a significant source of reactive oxygen species, which contribute to glomerular podocyte dysfunction. Although studies have implicated Nox1, -2, and -4 in several glomerulopathies, including diabetic nephropathy, little is known regarding the role of Nox5 in this context. We examined Nox5 expression and regulation in kidney biopsies from diabetic patients, cultured human podocytes, and a novel mouse model. Nox5 expression increased in human diabetic glomeruli compared with nondiabetic glomeruli. Stimulation with angiotensin II upregulated Nox5 expression in human podocyte cultures and increased reactive oxygen species generation. siRNA-mediated Nox5 knockdown inhibited angiotensin II–stimulated production of reactive oxygen species and altered podocyte cytoskeletal dynamics, resulting in an Rac-mediated motile phenotype. Because the Nox5 gene is absent in rodents, we generated transgenic mice expressing human Nox5 in a podocyte-specific manner (Nox5^pod+). Nox5^pod+ mice exhibited early onset albuminuria, podocyte foot process effacement, and elevated systolic BP. Subjecting Nox5^pod+ mice to streptozotocin-induced diabetes further exacerbated these changes. Our data show that renal Nox5 is upregulated in human diabetic nephropathy and may alter filtration barrier function and systolic BP through the production of reactive oxygen species. These findings provide the first evidence that podocyte Nox5 has an important role in impaired renal function and hypertension.Albuminuria is a clinical marker of kidney dysfunction that arises in most glomerulopathies and is associated with poor prognoses for ESRD, hypertension, and cardiovascular mortality. Changes to the podocyte (e.g., foot process effacement, hypertrophy, detachment, and loss) underlie the development and progression of albuminuria and thereby highlight the critical role for these cells in upholding the glomerular filtration barrier.^1,2 Therefore, identifying factors that induce podocyte injury and loss is essential to understanding the mechanisms of filtration barrier dysfunction.Of the many factors implicated in podocyte dysfunction, excessive production of reactive oxygen species (ROS; oxidative stress) may be particularly important.^3–6 Although sources of ROS are numerous, the NADPH oxidase (Nox) family of enzymes yields significant superoxide production in the kidney.^7–10 Nox-induced ROS production has been closely linked to various glomerular pathologies. In animal models of minimal change disease, membranous nephropathy, and FSGS, inhibition of Nox activity is associated with decreased podocyte effacement and amelioration of albuminuria.^11–14 In models of diabetic nephropathy, treatment with the Nox inhibitor apocynin, as well the antioxidant vitamin E, reduces oxidative stress, podocyte effacement and loss, and albuminuria.^6,15,16 Noxs are regulated by many factors, including the renin angiotensin aldosterone system.^5,9 Several studies have linked increased renin angiotensin aldosterone system activity to enhanced renal Nox activity and ROS generation.^5,17 Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers slow progression of proteinuria in models of diabetes, and these effects may be, in part, independent of their effects on systemic BP,^17–20 because direct activation of Nox enzymes through the angiotensin II (AngII)/AT1 receptor (AT1R) pathway leads to oxidative stress. In vitro studies in both human and rodent cell lines have also shown that Nox family member expression and activity are regulated by disease-associated factors, including AngII, ET-1, TGF-β, high glucose, mechanical stretch, and PDGF (factors that are upregulated in the diabetic milieu).^3,21–23The roles of Nox4, and to a lesser extent, Nox1 and -2, in the kidney have been examined, but nothing is known regarding the role of the most recently identified member of the Nox family, Nox5. The Nox5 gene is absent from the mouse and rat genomes, making the use of conventional animal models unfeasible. Unlike other Nox family members, Nox5 does not require membrane-bound or cytosolic components, such as p22phox or p47phox, for its activity, but is tightly regulated by changes in intracellular calcium levels.^24,25 Nox5 has a large amino terminal EF hand-containing domain that plays a critical role in its calcium-dependent activation along with several phosphorylation sites that alter the sensitivity of Nox5 to intracellular calcium.^26–29 Because AngII increases intracellular calcium concentrations, it seems to induce renal Nox5-dependent ROS generation, which was shown in human endothelial cells.²³ Here, we show that (1) Nox5 is upregulated in human diabetic glomeruli; (2) AngII stimulates ROS generation in human podocytes in a Nox5-dependent manner, a process associated with actin cytoskeletal reorganization and activation of Rac GTPase, which promotes podocyte motility in vitro; (3) mice that express human Nox5 in a podocyte-specific manner (Nox5β^pod+ mice) exhibit renal dysfunction, including albuminuria, podocyte effacement, glomerular basement membrane (GBM) thickening, interstitial fibrosis, and hypertension; and (4) Nox5^pod+ mice subjected to streptozotocin (STZ)-induced diabetes develop a more severe kidney phenotype than nontransgenic littermates. These novel data indicate the potential importance of podocyte Nox5 in human renal pathologies, such as diabetic nephropathy.     

Mechanisms of Podocyte Injury in Diabetes: Role of Cytochrome P450 and NADPH Oxidases

OBJECTIVE

We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo.

RESULTS

Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria.

CONCLUSIONS

Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.Diabetic nephropathy in humans is characterized by increased urinary albumin excretion (microalbuminuria), which often progresses to proteinuria, one of the most important prognostic risk factors for kidney disease progression (1). Glomerular visceral epithelial cells, or podocytes, play a critical role in maintaining the structure and function of the glomerular filtration barrier. Careful morphometric analyses of renal biopsy in subjects with type 1 and type 2 diabetes (2–4) demonstrate that the density of podocytes is reduced not only in individuals with diabetic nephropathy, but also in patients with short duration of diabetes before the onset of microalbuminuria (4,5). Studies in experimental models of type 1 and type 2 diabetes have also documented that podocyte depletion represents one of the earliest cellular lesions affecting the diabetic kidney (6,7). Among various morphologic characteristics, the decreased number of podocytes in glomeruli is the strongest predictor of progression of diabetic nephropathy, where fewer cells predict more rapid progression (3,4). Although these observations identify podocyte depletion as one of the earliest cellular features of diabetic kidney disease, the mechanisms that underlie the loss of podocytes in diabetic nephropathy remain poorly understood.High glucose induces apoptosis (8), and there is evidence that podocyte apoptosis contributes to reduced podocyte number (9). High glucose, transforming growth factor-β (TGF-β), and angiotensin II (ANGII) induce apoptosis of cultured podocytes (9–12). ANGII appears to induce apoptosis in cultured rat glomerular epithelial cells at least partially via TGF-β because its apoptotic effect is attenuated by an anti–TGF-β antibody (12). There is also evidence that reactive oxygen species (ROS) contribute to podocyte apoptosis and depletion in cells exposed to high glucose and in experimental diabetic nephropathy (7). However, the sources of ROS and the kinetics of their generation have not been well characterized. We and others (13–15) have recently identified NADPH oxidases as major sources of ROS in kidney cortex and glomeruli of rats with type 1 diabetes. Six homologs of the cytochrome subunit of the phagocyte NADPH oxidase (Nox2/gp91^phox) have been cloned (16). At least three different Nox isoforms are expressed in the kidney cortex: Nox1, Nox2, and Nox4 (16). Cytochromes P450 (CYP450s) are significant sources of ROS in many tissues (17,18). CYP450 metabolizes arachidonic acid into hydroxyeicosatetraenoic acids (20-HETEs) and EETs (epoxyeicosatrienoic acids). 20-HETE, the ω-hydroxylation product of arachidonic acid, is one of the major CYP eicosanoids produced in the kidney cortex (19–21). The predominant CYP450 in the kidney cortex that synthesizes 20-HETE is cytochrome P450 of the 4A family (CYP4A) (19–21). 20-HETE has multiple and opposing functions depending on the site of production and target cells/tissues (19,22–24).In this study, we demonstrate that high glucose induces ROS production and apoptosis in cultured mouse podocytes through the upregulation of CYP4A with increased production of 20-HETE and upregulation of NADPH oxidases. Inhibition of 20-HETE production prevented podocyte apoptosis in vitro and decreased oxidative stress, podocyte apoptosis, and proteinuria in an in vivo model of type 1 diabetes.     

NADPH-Oxidase 4 Protects against Kidney Fibrosis during Chronic Renal Injury

NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD_cl1) cell line increased TGF-β1–induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.CKD is a worldwide health problem defined as an alteration of kidney function usually estimated by GFR decline or histologic damage to the kidney.¹ CKD is associated with high mortality and morbidity as well as tremendous costs associated with renal replacement when patients reach ESRD.Interstitial fibrosis and glomerulosclerosis are hallmarks of CKD. Tubulointerstitial fibrosis is better correlated with loss of kidney function than glomerulosclerosis independently of primary injury.² Tubulointerstitial fibrosis arises from numerous, confounding events. These include tubular atrophy, via necrosis or apoptosis, and local hypoxia, due to the loss of peritubular capillaries, which are early events.^3–5 Tubular cells are both targets of tubulointerstitial fibrosis and important players in its progression via secretion of chemotactic factors and cytokines.Oxidative stress has been associated with aging and CKD progression.^6,7 Increased levels of kidney reactive oxygen species (ROS), such as angiotensin II–induced and diabetic nephropathies, are found in CKD.^8–13 However, low levels of ROS also play important roles in oxygen sensing, neoangiogenesis, and cell survival pathways, which may play protective roles against kidney fibrosis.^14,15 NADPH oxidases (NOXs) are enzymes that produce ROS as primary products.¹⁶ The NOX family is composed of seven members (NOX1–5 and DUOX 1 and 2). Among these, NOX1, NOX2, and NOX4 are expressed in both mice and human kidney, whereas NOX5 is only expressed in human kidney. NOX4 is highly expressed in the tubular cell compartment, where its physiologic role is unknown.¹⁷ This isoform is mainly regulated by its controlled abundance.¹⁸ The role of NOX4 in kidney injury is not yet elucidated. Increased renal NOX4 expression has been shown in diabetic nephropathy and its silencing has been demonstrated to be protective.^8,19 However, NOX4 may participate to oxygen sensing and exhibit anti-inflammatory properties via regulation of the NRF2 pathway in the heart.^15,20To determine the role of NOX4 and NOX2 in kidney fibrosis progression, we performed unilateral ureteral obstruction (UUO) on wild-type (WT), and NOX4 knockout (KO) mice as well as on NOX2 and NOX4/NOX2 KO mice. Indeed, UUO is a well described model of renal tubular stress leading to kidney fibrosis.^21,22 We demonstrate that deletion of NOX4 is associated with increased kidney fibrosis in obstructed kidneys. This effect was associated with enhanced tubular cell apoptosis as well as defective hypoxia-inducible factor-1α (HIF-1α) oxygen sensing and NRF2 antioxidant pathways. NOX4 is therefore crucial for kidney tubular cell survival under stressed conditions.     

Redox Modulation Protects Islets From Transplant-Related Injury

OBJECTIVE

Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant.

RESEARCH DESIGN AND METHODS

The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation.

RESULTS

Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection.

CONCLUSIONS

Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.Hypoxia is the leading cause of β-cell death during islet isolation and transplantation (1), with the highest percentage of islet graft loss and dysfunction occurring just days after transplantation (2,3). Because islets are a cellular transplant, devoid of intrinsic vasculature (1,4), they are exceptionally susceptible to ischemia-reperfusion injury. Islets are also increasingly vulnerable because they have inherently decreased antioxidant capacity (5–10), making them prone to oxidative/nitrosative/free radical damage. The antigen-independent complexities of islet transplantation increase the incidence of primary graft nonfunction and β-cell death, thus requiring protection for islets at early stages of the transplant procedure (11).In addition to antigen-independent innate-mediated inflammatory injury, islet allografts are also plagued by the antigen-dependent T-cell mediated alloimmune response, which necessitates immunosuppressive drugs for allograft survival. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response, although most are harmful to islets and do not protect well from reactive oxygen species and inflammation during islet isolation and ischemia-reperfusion injury (12–14). In their review, Balamurugan et al. (13) concluded that successful islet transplantation in type 1 diabetes necessitates islet-sparing immunosuppressive agents that combat recurrent autoimmunity with low islet toxicity. Predominantly, the field of islet transplantation is devoid of cytoprotective agents that promote islet survival and function by inhibiting nonspecific innate-mediated inflammation during islet isolation and early inflammatory events in islet transplantation (11,13,15–19).The first phase of immunity involves innate immune activation and subsequent proinflammatory signals required for optimal adaptive immune function (20–22), yet the majority of immunosuppressive drugs only target adaptive immune function (17,23), the second phase of immunity. A nontoxic, cell-permeable catalytic antioxidant (CA) redox modulator, FBC-007 [manganese(II) tetrakis (N-ethylpyridium-2-yl)porphyrin], is able to depress free radical and cytokine production by antigen-presenting cells (24) and T cells in transgenic and allospecific mouse models (20,25). Additionally, redox modulation inhibits cytotoxic lymphocyte target cell lysis by reducing the production of intracellular cytolytic molecules (perforin and granzyme B) in a mixed leukocyte reaction without toxicity (25), preserves and promotes human islet function in vitro (15,16), prevents the transfer of diabetes into young NOD.scid mice (26), and inhibits innate-immune nuclear factor (NF)-κB activation (24). Thus, islet-sparing agents, which decrease the production of free radicals and, therefore, inflammatory cytokines, may have a positive impact on islet function post-transplant.Because islet transplantation can benefit from agents that inhibit early inflammatory cascades to preserve islet function (18), we hypothesize that redox modulation holds potential as a therapy in islet transplantation to decrease the incidence of β-cell primary nonfunction. To further test the effects of redox modulation using CA we treated human islets with streptozotocin (STZ) in vitro and treated mice in vivo with STZ, both in the presence or absence of CA, to mimic antigen-independent free radical damage and inflammation of post-transplant ischemia-reperfusion injury. To examine the effects of islet-directed CA treatment on innate-mediated (antigen-independent) primary islet nonfunction in vivo, we performed syngeneic (175 islets/recipient), suboptimal syngeneic (100 islets/recipient), allogeneic (300 islets/recipient), and xenogeneic (400–500 islets/recipient) islet transplants to assess islet function. Additionally, we performed (300 islets/recipient) islet transplants in diabetic recipients to assess islet function in the presence or absence of systemic redox modulation in an allogeneic transplant setting inclusive of both innate (antigen-independent) and adaptive (antigen-dependent) immune responses. Our results demonstrate that islet-directed and systemically delivered redox modulation, administered in the absence of an additional immunosuppressive regimen, preserve islet function post-transplant.     

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.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

Human Recombinant ACE2 Reduces the Progression of Diabetic Nephropathy

OBJECTIVE

Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury, whereas renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury.

RESEARCH DESIGN AND METHODS

Male 12-week-old diabetic Akita mice (Ins2^WT/C96Y) and control C57BL/6J mice (Ins2^WT/WT) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for 4 weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity, and peptide levels were examined. The effect of hrACE2 on high glucose and angiotensin II (ANG II)–induced changes was also examined in cultured mesangial cells.

RESULTS

Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2^WT/C96Y mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased α-smooth muscle actin and collagen III expression. Human recombinant ACE2 increased ANG 1–7 levels, lowered ANG II levels, and reduced NADPH oxidase activity. mRNA levels for p47^phox and NOX2 and protein levels for protein kinase Cα (PKCα) and PKCβ1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity.

CONCLUSIONS

Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1–7 signaling.Chronic kidney disease is recognized as an increasing global public health problem due in part to the increasing prevalence of diabetes (1–3). Activation of the renin-angiotensin system (RAS) and the generation of angiotensin II (ANG II) play an important pathogenic role in diabetic nephropathy, and blockade of the RAS attenuates the development of diabetic kidney injury (4–8). The discovery of a homologue of the classical ACE, ACE2, has introduced a new enzyme in ANG peptide metabolism (9–12). Like ACE, ACE2 is membrane bound, but it is a monocarboxypeptidase that generates ANG (1–7) from the octapeptide ANG II (9,10,12,13). As such, ACE2 serves as an endogenous negative regulator of the renin-angiotensin system.In animal models of diabetes, early increases in ACE2 mRNA levels, protein expression, and ACE2 activity occurs (14,15), whereas ACE2 mRNA and protein levels have been found to decrease in older streptozotocin-induced diabetic rats (16). Loss of ACE2 is associated with age-dependent glomerulosclerosis and albuminuria (17) and exacerbation of diabetic kidney injury in Akita mice (18) and is preventable by angiotensin type 1 (AT1) receptor blockade. In patients with type 2 diabetes, glomerular and tubular ACE2 expressions are reduced in the setting of increased ACE expression (19,20). Taken together, these studies suggest that ACE2 may play an early protective role against the development of diabetic nephropathy (18,21,22). We hypothesized that treatment with human recombinant ACE2 (hrACE2) will target the diabetic glomerulus and slow progression of diabetic nephropathy in the Akita mouse (Ins2^WT/C96Y), a model of type 1 diabetes.     

Induction of Human ��-Cell Proliferation and Engraftment Using a Single G1/S Regulatory Molecule, cdk6

OBJECTIVE

Most knowledge on human β-cell cycle control derives from immunoblots of whole human islets, mixtures of β-cells and non-β-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules—cyclins D1–3 and cdk 4 and 6—in the adult human β-cell.

RESEARCH DESIGN AND METHODS

Immunocytochemistry for the five molecules and their relative abilities to drive human β-cell replication were examined. Human β-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse.

RESULTS

Human β-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human β-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control β-cells). A single molecule, cdk6, proved to be capable of driving human β-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin.

CONCLUSIONS

Human β-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent β-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal β-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human β-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human β-cell proliferation and function in vivo.While broadly similar, human pancreatic β- cells differ from their rodent counterparts in many ways. For example, rodent β-cells can be induced to replicate using many strategies, including partial pancreatectomy, induction of obesity and insulin resistance, infusion of glucose, administration of growth factors, and activation of signaling pathways downstream of these growth factors and nutrients (1–8). In contrast, while evidence suggests that human fetal and early neonatal β-cells are able to replicate (9–11), no investigator has been able to demonstrate or induce robust rates of proliferation in adult human β-cells using the same growth factors, nutrients, signaling pathways, and maneuvers that have been effective in rodents (12–19). For example, unlike events in rodents, obesity or type 2 diabetes in human adults is not associated with accelerated β-cell proliferation (19), and even partial pancreatectomy does not induce β-cell replication or regeneration in humans (17). This inability to induce substantial human β-cell replication is problematic, for it is now clear that β-cell replacement therapy for diabetes will require large numbers of human or xenogeneic β-cells (20).While much has been learned from mouse genetic models regarding the molecular control of β-cell replication (21–35), little is known regarding the molecules that regulate the G1/S transition in the human adult β-cell. We have begun to explore these molecules in adult human islets, mapping the members of the G1/S proteome that are present in human cadaveric islets (12,36). Human islets are believed to contain most of the key molecules that govern the G1/S transition in other cell types (12,36,37). These include the three members of the pocket protein family (pRb, p107, and p130); cyclins D1 and D3; cdks 1, 2, 4, and 6; the four members of the INK4 family (p15, p16, p18, and p19); the three members of the KIP/CIP family (p21, p27, and p57); several E2F family members; and p53 and its E3 ligase, HDM2 (supplementary Fig 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1776/DC1). Upstream of these G1/S molecules are other regulatory molecules such as skp2, menin, FoxM1, bmi1, and ezh2 (26–29,34). We and others have also shown that human islets differ from their rodent counterparts in that they express cdk6, a cdk that is not detectable in mouse islets (12,35). We have also shown that overexpression of cdk6 in association with a D-cyclin, cyclin D1, markedly increases the replication of adult human β-cells (12), and it does so in a way that does not lead to dedifferentiation or accelerated β-cell death but instead leads to enhanced engraftment and function of adult human cadaveric islets in vivo (12). We also have shown that cdk4 (36) and cdk6 (12) in conjunction with cyclin D1 can enhance pRb phosphorylation and replication in human β-cells. Despite these advances, it is important to underscore that, of the 30+ molecules that regulate the human β-cell G1/S transition (supplementary Fig. 1), the therapeutic potential has been explored for only three: cdk4, cdk6, and cyclin D1. For example, nothing is known about the efficacy of the other two D-cyclins, cyclins D2 and D3, in driving proliferation of human β-cells.Another difference between human and rodent islets may involve cyclin D2. In mice, excellent evidence indicates that cyclin D2 is indispensible for β-cell growth and islet development and function: animals that lack cyclin D2 develop islet hypoplasia, hypoinsulinemia, and diabetes (32,33). In contrast to mouse studies, in humans, cyclin D2 has been reported to be difficult to detect or undetectable, despite the use of multiple antisera from multiple vendors and appropriate positive controls (12,37).Here, we asked four questions. 1) Which D-cyclins are definitively present in human islets? 2) Which of the three D-cyclins might be the optimal partner for cdk4 or cdk6 for enhancing adult human β-cell replication? 3) Are cdk4, cdk6, and the three D-cyclins actually present in β-cells [in contrast to immunoblot studies of whole islet extracts described previously (12,37)], and if so, in which subcellular compartment? 4) From a therapeutic standpoint, is it essential to deliver both a D-cyclin and a cdk4/6 family member to enhance human islet engraftment, or can this be simplified by using only a single member of the cdk4/6–D-cyclin complex?We report here that 1) cyclin D1 is variably present, D2 is only marginally detectable, and D3 is readily detectable in human islets; 2) cyclin D3 appears to be surprisingly effective in inducing human β-cell proliferation and, in combination with cdk6, may be the most effective in driving human β-cell replication; 3) cdks 4 and 6, and cyclins D1 and D3, are located principally in the cytoplasm and not the nucleus; and 4) a single cdk, cdk6, is surprisingly as effective in driving human islet engraftment as the previously reported combination of cdk6 plus cyclin D1.     

Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates

OBJECTIVE

To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation.

RESEARCH DESIGN AND METHODS

Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection.

RESULTS

MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood.

CONCLUSIONS

MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.Multipotent mesenchymal stem cells (MSCs) (1,2) can deliver immunomodulatory signals (3–7) that inhibit allogeneic T-cell responses through downregulation of the proinflammatory cytokines TNF-α and IFN-γ and production of the regulatory cytokines/molecules IL-10, hepatocyte growth factor (HGF), TGF-β, vascular endothelial growth factor (VEGF), indoleamine 2,3-dioxygenase, galectin-1, prostaglandin E2, nitric oxide, and matrix metalloproteinase-2 and -9 (3,8–12). Inflammatory signals, such as IFN-γ, can activate and upregulate MSC suppressive activities (9,13). These cells are able to migrate to sites of injury after intravenous injection (14,15). Their use in clinical trials and experimental models is based on their immunomodulatory and regenerative properties (1,7,16). Clinically, MSCs have been observed to enhance donor bone marrow cell (DBMC) engraftment and chimerism (17,18). Therefore, cotransplantation of MSCs that secrete immunomodulatory cytokines and growth factors might enhance islet survival and function. In experimental mouse models, intravenously infused MSCs are capable of migrating to pancreatic islets (19,20). Systemic infusion of MSCs in murine models of diabetes was accompanied by delayed onset of diabetes, improved glycemic levels, reduced pancreatic insulitis, and pancreatic tissue regeneration (19,21–25), as well as prevention of autoimmune destruction of β-cells via induction of regulatory T-cells (Tregs) (26). Cotransplantation of syngeneic MSCs with a marginal mass of allogeneic islets under the kidney capsule of streptozotocin (STZ)-induced diabetic mice resulted in prolonged normoglycemia (11). Cotransplantation of syngeneic MSC with a marginal mass of allogeneic islets has been performed in the omentum (27) and kidney capsule (28) of STZ-induced diabetic rats, with enhanced islet graft survival as compared with animals receiving islets alone. In this study, cynomolgus monkey MSCs were characterized and donor MSCs were examined for the ability to promote intraportal islet engraftment as well as chimerism in recipients of islet/DBMC transplants. In addition, we tested the use of donor or third-party MSCs to reverse episodes of islet allograft rejection.     

Inhibition of Reactive Oxygen Species by Lovastatin Downregulates Vascular Endothelial Growth Factor Expression and Ameliorates Blood-Retinal Barrier Breakdown in db/db Mice: Role of NADPH Oxidase 4

OBJECTIVE

Oxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy.

RESEARCH DESIGN AND METHODS

Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin.

RESULTS

Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice.

CONCLUSIONS

Activation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.Diabetic retinopathy is a common complication of diabetes and one of the most frequent causes of blindness in the U.S. (1–3). Hallmark sequential pathological changes in diabetic retinopathy include increased vascular permeability, pericyte and endothelial cell death, capillary occlusion and aberrant retinal new vessel growth, or neovascularization (4). Increased vascular permeability caused by the breakdown of the blood-retinal barrier (BRB) results in diabetic macular edema, a major cause of vision loss in diabetic patients (2,5,6). Vascular endothelial growth factor (VEGF) is a potent angiogenic factor playing a crucial role in angiogenesis (7,8). VEGF is also referred as vascular permeability factor (VPF) based on its ability to induce vascular hyperpermeability (9). Significantly elevated VEGF levels in the eye have been reported in diabetic patients with diabetic macular edema and correlated with the severity of vascular leakage (10). Overexpression of VEGF is also responsible for retinal hyperpermeability in streptozotocin (STZ)-induced diabetic rats (11). These findings suggest that VEGF is a key mediator of retinal vascular leakage in diabetic retinopathy.Oxidative stress plays an important role in vascular endothelial dysfunction in diabetes (12–15). Increased level of hydrogen peroxide, a reactive oxygen species (ROS), was colocalized with VEGF expression at the inner BRB and associated with vascular leakage in the retina in diabetic BBZ/Wor rats, suggesting a role of ROS in regulation of VEGF in diabetic retinopathy (16). In addition, suppression of ROS generation by NADPH oxidase inhibitor or antioxidants significantly attenuated retinal vascular leakage in diabetic animals, suggesting that activation of NADPH oxidase contributes to retinal ROS generation and vascular damage in diabetic retinopathy (17). NADPH oxidase (Nox) 4, which is originally identified in the kidney and termed renox (renal oxidase), is a novel isoform of NADPH oxidase expressed in nonphagocytes, such as vascular endothelial cells and smooth muscle cells (18,19). In aorta isolated from the STZ-induced diabetic apolipoprotein E–deficient mice or the db/db mice, Nox4 expression is significantly upregulated, associated with increased ROS production and inflammation, indicating a potential role of Nox4 in diabetic macrovascular disease (20). Moreover, inhibition of Nox4 expression using antisense oligonucleotides attenuates ROS generation and ameliorates glomerular hypertrophy in STZ-induced diabetic mice, suggesting that Nox4 is the major source of ROS in the diabetic kidney, contributing to renal hypertrophy in diabetic nephropathy (21). However, the role of Nox4 in diabetic retinopathy has not been investigated.3-Hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis commonly used in dyslipidemia and type 2 diabetes (22). Moreover, statins have demonstrated impressive beneficial effects, such as improvement of endothelial function, neuroprotection, and anti-inflammation, which are independent of their lipid-lowering effects (23). In a previous study, we have shown that lovastatin protects retinal tight junction and ameliorates BRB breakdown in db/db mice, a type 2 diabetes model (24). However, the mechanisms remain elusive. In the present study, we have tested the hypothesis that Nox4 is a key mediator of oxidative stress and BRB breakdown in diabetic retinopathy and that inhibition of Nox4 is, at least in part, responsible for the salutary effect of lovastatin on retinal endothelial function.     

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.     

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

OBJECTIVE

The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets.

RESEARCH DESIGN AND METHODS

Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO–expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice.

RESULTS

IDO-expressing grafts survived significantly longer than controls (41.2 ± 1.64 vs. 12.9 ± 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming.

CONCLUSIONS

Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.Endocrine replacement therapy by islet transplantation represents a feasible and attractive alternative therapeutic approach for treating type 1 diabetes (1,2). Despite improvement of allogeneic islet engraftment using systemic immunosuppression, islet transplantation is still limited by high rates of rejection. Furthermore, some immunosuppressive agents are prodiabetogenic and associated with adverse side effects (3–6). Finding more efficient and less harmful strategies to protect islet graft is therefore required for improving islet transplantation outcome.Localized expression of immunoregulatory factors using gene transfer to graft is a feasible method to provide an immunoprivileged microenvironment and consequently improves graft survival. Such an on-site delivery system results in more potent local immunosuppression with less systemic side effects (7–9).IDO is a cytosolic enzyme that catalyzes essential amino acid l-tryptophan to kynurenine (10) and has profound effects on T-cell proliferation, differentiation, effector functions, and viability (11). Both the reduction in local tryptophan concentration and the production of immunomodulatory tryptophan metabolites contribute to immunosuppressive effects of IDO (12,13). Broad evidence implicates IDO and the tryptophan catabolic pathway in generation of immune tolerance to antigens in tissue microenvironments. In particular, the role of IDO in fetal tolerance in mammalian pregnancy (14,15), immunologic tolerance to tumors (16,17), and self-tolerance has been documented (18,19). The unique immunoregulatory function of IDO substantiates the application of this enzyme as a strategy to suppress alloimmune responses in transplantation.Our research group has shown that overexpression of IDO in fibroblasts suppresses immune response and improves outcome of skin grafts (20–25) and that bystander IDO-expressing fibroblasts suppress immune response to allogeneic mouse islets in vitro (26). Furthermore, in a recent study we showed that mouse islets and fibroblasts are selectively resistant to IDO-mediated activation of nutrient deficiency stress (27). Here, we engineered a three-dimensional composite islet allograft equipped with IDO-expressing fibroblasts and examined whether local expression of IDO, conferred by adenoviral-mediated gene transfer to bystander syngeniec fibroblasts, prevents the rejection of islet allograft. Our approach here is novel compared with other studies that examined the suppressive effect of IDO in islet transplantation (28,29) because 1) bystander syngeneic fibroblasts were used as the target of gene transfer instead of islets to avoid deleterious effects of adenovirus infection on islets (30–32), 2) islets were embedded within an extracellular matrix that by itself improves islet function and viability (33,34), and 3) cotransplanted fibroblasts are more than just a source of IDO and can enhance islet physiological competence (35,36).     

Additive Effects of Genetic Variation in GCK and G6PC2 on Insulin Secretion and Fasting Glucose

OBJECTIVE

Glucokinase (GCK) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) regulate the glucose-cycling step in pancreatic β-cells and may regulate insulin secretion. GCK rs1799884 and G6PC2 rs560887 have been independently associated with fasting glucose, but their interaction on glucose-insulin relationships is not well characterized.

RESEARCH DESIGN AND METHODS

We tested whether these variants are associated with diabetes-related quantitative traits in Mexican Americans from the BetaGene Study and attempted to replicate our findings in Finnish men from the METabolic Syndrome in Men (METSIM) Study.

RESULTS

rs1799884 was not associated with any quantitative trait (corrected P > 0.1), whereas rs560887 was significantly associated with the oral glucose tolerance test 30-min incremental insulin response (30′ Δinsulin, corrected P = 0.021). We found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 (P > 0.26). However, the additive effect of these single nucleotide polymorphisms was associated with fasting glucose (corrected P = 0.03) and 30′ Δinsulin (corrected P = 0.027). This additive association was replicated in METSIM (fasting glucose, P = 3.5 × 10⁻¹⁰ 30′ Δinsulin, P = 0.028). When we examined the relationship between fasting glucose and 30′ Δinsulin stratified by GCK and G6PC2, we noted divergent changes in these quantitative traits for GCK but parallel changes for G6PC2. We observed a similar pattern in METSIM.

CONCLUSIONS

Our data suggest that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion.Genome-wide association (GWA) studies have identified several loci for type 2 diabetes (1–5) and type 2 diabetes–related quantitative traits (6–20). Two of these loci, glucokinase (GCK) (21–23) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) (9,10), regulate the critical glucose-sensing mechanism within pancreatic β-cells. Mutations in GCK confer susceptibility to maturity-onset diabetes of the young (MODY)-2 (24–26), and a −30 GCK promoter variant (rs1799884) has been shown to be associated with β-cell function (21), fasting glucose, and birth weight (23). Chen et al. (9) demonstrated an association between the G6PC2 region and fasting glucose, an observation replicated by Bouatia-Naji et al. (10). Although fasting glucose levels are associated with both GCK and G6PC2, there has been no evidence that genetic variation at these two loci contribute a risk for type 2 diabetes, suggesting contribution to mild elevations in glycemia.GCK phosphorylates glucose to glucose-6-phosphate, whereas G6PC2 dephosphorylates glucose-6-phosphate back to glucose, forming a glucose cycle previously demonstrated to exist within pancreatic β-cells (27,28). The important role of GCK in glucose sensing by pancreatic islets has been demonstrated by numerous studies, and other studies suggest a role for glucose cycling in insulin secretion and diabetes (28–30), implying that the balance between GCK and G6PC2 activity is important for determining glycolytic flux, ATP production, and subsequent insulin secretion. This was validated by a demonstration that direct manipulation of glucose cycling alters insulin secretion (31,32).BetaGene is a study in which we are performing detailed phenotyping of Mexican American probands with recent gestational diabetes mellitus (GDM) and their family members to obtain quantitative estimates of body composition, insulin sensitivity (S_I), acute insulin response (AIR), and β-cell compensation (disposition index) with the goal of identifying genes influencing variations in type 2 diabetes–related quantitative traits (33–35). Based on the evidence that variation in GCK (rs1799884) and G6PC2 (rs560887) are independently associated with fasting glucose concentrations and both are crucial to glucose cycling in β-cells, we hypothesized that interaction between these loci may be associated not just with fasting glucose but also with measures of insulin secretion or β-cell function. We tested this hypothesis in the BetaGene Study and, for replication, in a separate sample of Finnish men participating in the METabolic Syndrome in Men (METSIM) Study (36).     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

Human Immune System Development and Rejection of Human Islet Allografts in Spontaneously Diabetic NOD-Rag1null IL2r��null Ins2Akita Mice

OBJECTIVE

To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell–derived β-cells in the absence or presence of a functional human immune system.

RESEARCH DESIGN AND METHODS

We backcrossed the Ins2^Akita mutation onto the NOD-Rag1^null IL2rγ^null strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts.

RESULTS

We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rγ^null, Rag1^null, and Ins2^Akita genes in NOD-Rag1^null IL2rγ^null Ins2^Akita (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rγ^null and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts.

CONCLUSIONS

NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell–derived β-cells in the absence or presence of an alloreactive human immune system.Investigators are working to develop functional human β-cells from adult or embryonic stem cells (ESCs) or from induced pluripotent stem (iPS) cells (1–4), and human ESC-derived β-cells can restore euglycemia in streptozotocin (STZ) diabetic immunodeficient mice (5). However, STZ can have detrimental effects on many organs in addition to β-cells (6,7), and there is sometimes recovery of the host β-cell function (8,9). In addition, it will be important to transplant human β-cells in the presence of an allogeneic human immune system.We have developed an immunodeficient mouse model of spontaneous hyperglycemia based on NOD-Rag1^null Prf1^null mice bearing the Ins2^Akita mutation (10). Mice heterozygous for Ins2^Akita develop spontaneous hyperglycemia at 3–6 weeks of age (11,12), and transplantation of human islets restores euglycemia (10). However, human immune system engraftment is relatively inefficient in this model (13). Immunodeficient scid and Rag1^null NOD mice bearing a mutation in the interleukin (IL)-2 receptor common γ chain (IL2rγ^null) gene are now available (14,15) and can be engrafted with functional human immune systems (14–18).We now describe the development of the NOD-Rag1^null IL2rγ^null Ins2^+/Akita (NRG-Akita) strain. These mice develop spontaneous hyperglycemia, and euglycemia can be restored following transplantation with mouse or human islets or with dissociated mouse islet cells. A human immune system develops following engraftment with human hematopoietic stem cells (HSCs), and in some mice, this immune system is capable of rejecting human islet allografts.     

Confirmation of Multiple Risk Loci and Genetic Impacts by a Genome-Wide Association Study of Type 2 Diabetes in the Japanese Population

OBJECTIVE

To identify novel type 2 diabetes gene variants and confirm previously identified ones, a three-staged genome-wide association study was performed in the Japanese population.

RESEARCH DESIGN AND METHODS

In the stage 1 scan, we genotyped 519 case and 503 control subjects with 482,625 single nucleotide polymorphism (SNP) markers; in the stage 2 panel comprising 1,110 case subjects and 1,014 control subjects, we assessed 1,456 SNPs (P < 0.0025, stage 1); additionally to direct genotyping, 964 healthy control subjects formed the in silico control panel. Along with genome-wide exploration, we aimed to replicate the disease association of 17 SNPs from 16 candidate loci previously identified in Europeans. The associated and/or replicated loci (23 SNPs; P < 7 × 10^–5 for genome-wide exploration and P < 0.05 for replication) were examined in the stage 3 panel comprising 4,000 case subjects and 12,569 population-based samples, from which 4,889 nondiabetic control subjects were preselected. The 12,569 subjects were used for overall risk assessment in the general population.

RESULTS

Four loci—1 novel with suggestive evidence (PEPD on 19q13, P = 1.4 × 10^–5) and three previously reported—were identified; the association of CDKAL1, CDKN2A/CDKN2B, and KCNQ1 were confirmed (P < 10^–19). Moreover, significant associations were replicated in five other candidate loci: TCF7L2, IGF2BP2, SLC30A8, HHEX, and KCNJ11. There was substantial overlap of type 2 diabetes susceptibility genes between the two populations, whereas effect size and explained variance tended to be higher in the Japanese population.

CONCLUSIONS

The strength of association was more prominent in the Japanese population than in Europeans for more than half of the confirmed type 2 diabetes loci.The predisposition to and the course of type 2 diabetes vary according to ethnic group (1–3). In Japan, the incidence of type 2 diabetes has increased recently and is now comparable to that of other countries; this is supposedly attributable to the gradual spread of Western habits, such as consuming a high-fat diet, and the lower insulin secretory capacity of Japanese subjects (4,5). Recent technological developments have allowed the successful identification of gene regions involved in the development of type 2 diabetes in genome-wide association (GWA) studies (6–17). Several susceptibility gene loci identified by GWA studies to date have been used to obtain reproducible evidence of disease association in different populations of European descent and Asians, but not necessarily in African Americans (18–24). A number of GWA studies on type 2 diabetes have been conducted on populations of European descent (6–12). Two GWA scans in the Japanese population simultaneously reported the discovery of type 2 diabetes susceptibility gene (KCNQ1) variants in non-European populations; this result was also obtained in Scandinavian samples (25,26). Thus far, the replicated associations for a limited number of candidate genes have broadly indicated the tendency of interethnic similarity. Even though the common (or cosmopolitan) effect of type 2 diabetes risk variants is known, the extent to which the causation of this disease differs or overlaps between populations remains unknown. Here, besides comparing the genetic associations between European-descent and Japanese populations, we aimed to identify new genetic variants using a three-staged GWA study design.     

Genetic Targeting or Pharmacologic Inhibition of NADPH Oxidase Nox4 Provides Renoprotection in Long-Term Diabetic Nephropathy

Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)–forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and Nox4). Here we show that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE^−/− mice. Deletion of Nox4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-κB in streptozotocin-induced diabetic ApoE^−/− mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify Nox4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.CKD is a major complication of diabetes. Furthermore, diabetes remains the most common cause of end stage renal failure and need for kidney transplantation.¹ The underlying mechanisms responsible for diabetic nephropathy remain to be fully defined. Therefore, effective and mechanism-based therapies are not available. It has been hypothesized that diabetes mellitus causes renal oxidative stress, that is, increased levels of reactive oxygen species (ROS), resulting in glomerular damage. Accordingly, oxidative stress is increasingly considered to be a major contributor to the development and progression of diabetic nephropathy.² Various renal sources of ROS have been suggested to be relevant in the diabetic kidney. These include auto-oxidation of glucose, advanced glycation, glycolysis, glucose-6-phosphate dehydrogenase, sorbitol/polyol pathway flux, hexosamine pathway flux, mitochondrial respiratory chain, xanthine oxidase, uncoupled nitric oxide synthase, and NADPH oxidases.^2,3Among these sources, NADPH oxidases are suggested to play a pivotal role in the development and progression of renal injury in animal models of type 1 and type 2 diabetic nephropathy^4–6 and hence represent a potentially important novel target. NADPH oxidases are the only enzymes known to be solely dedicated to ROS generation. Seven isoforms of their catalytic subunit exist (Nox1–5; Duox1 and 2). Nox isoforms depend to varying degrees on additional subunits.^7–10 Among these isoforms, Nox1, Nox2, and Nox4 are expressed in the renal cortex. In streptozotocin-induced diabetic nephropathy, expression of Nox4, Nox2, and another subunit, p22phox, are all upregulated.^11–13 With respect to Nox2, our own studies in streptozotocin-induced diabetic Nox2 knockout (KO) mice have shown increased susceptibility to infections and 100% mortality at week 20 of diabetes.¹⁴ We thus did not consider Nox2 blockade a priority in this study addressing strategies to reduce diabetic nephropathy.Nox4, originally termed Renox, is highly expressed in renal tissues.^15–18 The role of Nox4 in diabetic nephropathy remains controversial. Nox4 downregulation by systemic administration of antisense oligonucleotides, albeit for a short period of only 2 weeks, reduced renal and glomerular hypertrophy and attenuated the increased expression of fibronectin in renal cortex and glomeruli in streptozotocin-induced diabetic rats.¹⁹ However, the Nox4 antisense oligonucleotide may not be absolutely specific for Nox4. Furthermore, other authors have suggested either no effect²⁰ or a protective role of Nox4 in diabetic nephropathy or in other models of renal fibrosis.²¹ With respect to Nox1, this isoform appears to play a major role in diabetic macrovascular disease¹⁴but not much is known about the role of Nox1 in diabetic nephropathy. Thus, it remains to be determined which Nox isoform plays the most critical role in diabetic kidney disease.Here we report for the first time a direct comparison of the long-term effects of Nox1 and Nox4 deletion in the development and progression of diabetic nephropathy, by directly comparing renal injury in streptozotocin-induced diabetic Nox1^−/yApoE^−/− and Nox4^−/−ApoE^−/− double KO mice and their respective wild-type (WT) control mice. In addition, the genetic deletion studies were complemented by a pharmacologic intervention study using the currently most specific Nox inhibitor, GKT137831.²² Key findings in the in vivo studies were confirmed in vitro using human podocytes.