Deficiency of Rac1 Blocks NADPH Oxidase Activation, Inhibits Endoplasmic Reticulum Stress, and Reduces Myocardial Remodeling in a Mouse Model of Type 1 Diabetes

OBJECTIVE

Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes.

RESEARCH DESIGN AND METHODS

Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose.

RESULTS

In diabetic hearts, NADPH oxidase activation, its subunits'' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers'' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose–induced ER stress was inhibited by blocking Rac1 or NADPH oxidase.

CONCLUSIONS

Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy.Diabetic cardiomyopathy has been defined as ventricular dysfunction that occurs in the absence of changes in blood pressure and coronary artery disease (1). Cardiac structural phenotypes of diabetic cardiomyopathy include cardiomyocyte apoptosis, cardiac hypertrophy, myocardial fibrosis, and interstitial inflammation (2,3), all of which significantly contribute to myocardial dysfunction. Three evident characteristic metabolic disturbances in diabetes, including hyperglycemia, hyperlipidemia, and hyperinsulinemia, are attributable to altered myocardial structure and function in diabetic cardiomyopathy (4). However, the signaling pathways associated with these metabolic triggers remain not fully understood in diabetic hearts.Several mechanisms involved in diabetic myocardial dysfunction have been suggested, which include increased oxidative stress, impaired calcium homeostasis, upregulation of the renin-angiotensin system, altered substrate metabolism, and mitochondrial dysfunction (3). These changes are closely related to reactive oxygen species (ROS) production. ROS is mainly produced by mitochondria and NADPH oxidase in cardiomyocytes. A cross-talk between mitochondria and NADPH oxidase has been suggested to sustain cellular ROS production under stresses (5–9). Selective inhibition of mitochondrial ROS has been shown to prevent diabetic cardiac changes in type 1 diabetic mice, confirming an important role of mitochondrial ROS (10). Our recent study has revealed that Rac1 via NADPH oxidase activation induces mitochondrial ROS production and plays an essential role in cardiomyocyte apoptosis and myocardial dysfunction in streptozotocin (STZ)-induced diabetes (8). Cell death by apoptosis is the predominant damage in diabetic cardiomyopathy (2). Cardiomyocyte death causes a loss of contractile tissue, which initiates a cardiac remodeling (11). Furthermore, Rac1/NADPH oxidase signaling has also been demonstrated to directly induce cardiac hypertrophy (12,13) and skin fibrosis (14,15). However, direct evidence is lacking as for the contribution of Rac1/NADPH oxidase to myocardial remodeling in the development of diabetic cardiomyopathy.In this study, we took advantage of the availability of mice with cardiomyocyte-specific Rac1 knockout to analyze the impact of Rac1 on NADPH oxidase activation, endoplasmic reticulum (ER) stress, hypertrophy, fibrosis, and inflammatory response in diabetic hearts. We further investigated the therapeutic effect of the NADPH oxidase inhibitor apocynin on diabetic cardiomyopathy in STZ-induced type 1 diabetic mice.     

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 NOX2 Defines a New Antagonistic Role for Reactive Oxygen Species and cAMP/PKA in the Regulation of Insulin Secretion

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform–specific knockout mice. Both human and mouse β-cells express NOX, in particular NOX2. With use of Nox isoform–specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic β-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.NOX enzymes generate superoxide by transferring one electron from NADPH to oxygen (1). The best known NOX isoform is the phagocyte NADPH oxidase (NOX), a multicomponent complex comprising a membrane catalytic heterodimer, the flavocytochrome b_558, formed by gp91^phox (also referred to as NOX2) and p22^phox (where phox is phagocyte oxidase). The cytosolic regulatory subunits are composed of p40^phox, p47^phox, p67^phox, and GTPases Rac1 or Rac2 (1). Assembly of cytosolic elements to membrane catalytic core initiates the activation of NOX. To date, seven isoforms of NOX (NOX1–5 and dual oxidases DUOX1–2) have been identified with different activation mechanisms and heterogeneous tissue distribution (1). In addition to microbial attack by professional phagocytes, physiopathological roles of NOX have recently attracted attention in nonphagocytic cells, including pancreatic β-cells (2–7). Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, might participate in β-cell dysfunction (8). The redox imbalance favored by high metabolic rate and a relatively low detoxifying system has contributed to the general concept that β-cells are sensitive to ROS, although they can handle rather high concentrations of H₂O₂ (9).NOX family represents one of the potential sources of ROS in insulin-secreting cells (4). Both rat islets and insulinoma express membrane-associated catalytic components Nox1, Nox2, Nox4, and p22^phox, as well as cytosolic regulators p40^phox, p47^phox, and p67^phox and their homologs Noxo1 and Noxa1 (3,5,6). Regarding their putative function in the β-cell, NOXs have been implicated in glucose-induced ROS production in MIN-6 cells (10). Knockdown of p47^phox results in total inhibition of glucose-stimulated insulin secretion and lowers ROS (11). In animal models of type 2 diabetes, islets exhibit increased NOX components Nox2 and p22^phox, correlating with increased oxidative stress (12). Activation of Nox and accompanying ROS generation were demonstrated in Zucker diabetic fatty (ZDF) rat and diabetic human islets (13). However, inhibition of islet NOX using diphenyleneiodonium (DPI) impairs glucose-stimulated insulin secretion (6) along with blunted glucose-induced superoxide production (5,10). These conflicting findings regarding NOX activity and β-cell function might be attributed to poor specificity of old-generation NOX inhibitors, such as apocynin and DPI (14). The former has been shown to function as a general ROS scavenger, and the latter is a nonspecific inhibitor of electron transporters (1,15).In the current study, we first investigated relative expression levels of the different catalytic subunits of NOXs in both human and mouse pancreatic islets. Then, subcellular distribution of the identified predominant NOX isoform NOX2 was assessed in human islet cells. For avoidance of poor specificity of NOX inhibitors, islets isolated from Nox isoform–specific–deficient mice were used to investigate the contribution of NOXs in insulin secretory function.     

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.     

Rac1 Is a Novel Regulator of Contraction-Stimulated Glucose Uptake in Skeletal Muscle

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.Muscle contraction, like insulin, increases glucose uptake into skeletal muscle (1,2). Insulin and muscle contraction both stimulate the translocation of vesicles containing the glucose transporter GLUT4 from intracellular compartments to the sarcolemma and T tubules, allowing glucose to enter the cell via facilitated diffusion (3–5). However, the proximal signaling pathways of contraction and insulin are distinct. Muscle contraction has no effect on the insulin-signaling pathway (6,7), and muscle-specific knockout of the insulin receptor (8), or inhibition of phosphatidyl inositol 3-kinase with wortmannin (9) does not impair contraction-stimulated glucose uptake. Exercise has an insulin-sensitizing effect, suggesting that these two pathways may regulate similar unidentified distal signaling steps (10,11).Activation of AMP-activated protein kinase (AMPK) and calcium-dependent signaling, such as protein kinase Cs (PKCs) and calcium-calmodulin–dependent kinases, has traditionally been believed to induce glucose uptake during muscle contraction (3,12). However, the functional significance of these pathways is not fully understood. It is likely that so far unrecognized mechanisms regulated by AMPK, PKCs, calcium, liver kinase B1, stretch, reactive oxygen, and nitrogen species, or other yet unidentified pathways, participate in the regulation of glucose uptake during muscle contraction (3,13).One such candidate is Rac1 (Ras-related C3 botulinum toxin substrate 1), a small Rho family GTPase that regulates various cellular processes, including dynamic assembly and disassembly of the actin cytoskeleton (14,15). Rac1 is activated by insulin and induces actin cytoskeleton remodeling at the plasma membrane (15,16). Rac1-dependent rearrangement of the actin cytoskeleton is necessary for insulin-stimulated GLUT4 translocation in L6 myotubes (16–18).Even though Rac1 has traditionally only been implicated in insulin signaling, the contraction-related protein, AMPK, has been proposed to activate Rac1 in cultured muscle cells (19), endothelial cells (20), and macrophages (21). The primary aim of the present investigation was to explore whether insulin-independent stimuli, such as exercise in vivo and muscle contractions in vitro, activate Rac1 in skeletal muscle. Because Rac1 activation is necessary for insulin-stimulated GLUT4 translocation (22), we further aimed to investigate the involvement of Rac1 in AICAR- and contraction-stimulated glucose uptake. We hypothesized that Rac1 is activated by muscle contraction and that this activation plays a role in contraction-induced glucose uptake.     

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.     

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

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

Hypothalamic Reactive Oxygen Species Are Required for Insulin-Induced Food Intake Inhibition: An NADPH Oxidase�CDependent Mechanism

OBJECTIVE

Insulin plays an important role in the hypothalamic control of energy balance, especially by reducing food intake. Emerging data point to a pivotal role of reactive oxygen species (ROS) in energy homeostasis regulation, but their involvement in the anorexigenic effect of insulin is unknown. Furthermore, ROS signal derived from NADPH oxidase activation is required for physiological insulin effects in peripheral cells. In this study, we investigated the involvement of hypothalamic ROS and NADPH oxidase in the feeding behavior regulation by insulin.

RESEARCH DESIGN AND METHODS

We first measured hypothalamic ROS levels and food intake after acute intracerebroventricular injection of insulin. Second, effect of pretreatment with a ROS scavenger or an NADPH oxidase inhibitor was evaluated. Third, we examined the consequences of two nutritional conditions of central insulin unresponsiveness (fasting or short-term high-fat diet) on the ability of insulin to modify ROS level and food intake.

RESULTS

In normal chow-fed mice, insulin inhibited food intake. At the same dose, insulin rapidly and transiently increased hypothalamic ROS levels by 36%. The pharmacological suppression of this insulin-stimulated ROS elevation, either by antioxidant or by an NADPH oxidase inhibitor, abolished the anorexigenic effect of insulin. Finally, in fasted and short-term high-fat diet–fed mice, insulin did not promote elevation of ROS level and food intake inhibition, likely because of an increase in hypothalamic diet-induced antioxidant defense systems.

CONCLUSIONS

A hypothalamic ROS increase through NADPH oxidase is required for the anorexigenic effect of insulin.The hypothalamus is a cerebral area involved in the regulation of energy homeostasis. In this context, insulin plays a pivotal role. By acting in the hypothalamus, insulin reduces food intake and body weight (1,2), activates sympathetic nerve outflow to the brown adipose tissue (3), and suppresses hepatic endogenous glucose production (4,5). In a situation of excess nutrient intake, the hypothalamus rapidly becomes resistant to insulin before obesity and diabetes onset (6,7). Moreover, inactivation of the neuronal insulin receptor leads to the development of diet-induced obesity with an increase in body fat, mild insulin resistance, and elevated plasma insulin levels (8). Therefore, it is of prime importance to elucidate cellular mechanisms by which insulin acts on hypothalamic cells to understand central early-onset diet-induced obesity and diabetes.In recent years, emerging data point to a pivotal role of reactive oxygen species (ROS) in the energy homeostasis regulation by the hypothalamus. In response to an acute overload of nutrients, a subtle rise of the ROS concentration within this area is sufficient to reduce food intake (9) or stimulate the parasympathetic nervous system as well as pancreatic insulin secretion (10). Furthermore, the gut-derived hormone ghrelin exerts its central effect on feeding behavior by controlling hypothalamic ROS levels (11). However, the role of ROS in the anorexigenic effect of insulin and more generally in the brain insulin signaling remains unknown. In peripheral insulin-sensitive cells, ROS have been shown to control the crucial early steps in insulin signaling (12–14). Moreover, they contribute to propagation of the insulin cascade in these cells (15–17). Thus, transient bursts of small amounts of ROS triggered in response to insulin facilitate both the early and distal insulin signaling pathway. Reinforcing this concept, it has been recently shown that this transient insulin-induced ROS production is also required in neuronal cells for the enhancement of insulin receptor autophosphorylation, the first step in the insulin signaling pathway (18). These data lead us to hypothesize that insulin might trigger ROS elevation within the hypothalamus, which, in turn, would inhibit food intake.In peripheral cells, it is well established that both the insulin-induced ROS rise and insulin cascade activation are dependent on NADPH oxidase activity (12–17,19). Blocking insulin-induced ROS production with an inhibitor of NADPH oxidase activity (diphenyleneidonium) dramatically reduces insulin signaling pathway activation and thus its physiological effects (15).Despite the demonstration of the crucial role of ROS signaling and NADPH oxidase in peripheral insulin effects, their involvement in brain insulin signaling has not yet been investigated. Therefore, in this study we tested whether the anorexigenic insulin effect required a ROS-dependent signaling pathway within the hypothalamus. For this purpose, we measured hypothalamic ROS levels and food intake after intracerebroventricular insulin injection. The involvement of ROS and NADPH oxidase in insulin-induced food intake inhibition was evaluated by pretreatment with a ROS scavenger or an NADPH oxidase inhibitor. Because central insulin responsiveness is known to be altered by a short-term deficit (20) as well as an excess (7) of nutrient availability, we examined the consequence of 18-h fasting or 3 days'' high-fat diet on insulin''s ability to modify hypothalamic ROS levels and food intake.     

Unacylated Ghrelin Rescues Endothelial Progenitor Cell Function in Individuals With Type 2 Diabetes

OBJECTIVE

Acylated ghrelin (AG) is a diabetogenic and orexigenic gastric polypeptide. These properties are not shared by the most abundant circulating form, which is unacylated (UAG). An altered UAG/AG profile together with an impairment of circulating endothelial progenitor cell (EPC) bioavailability were found in diabetes. Based on previous evidence for the beneficial cardiovascular effects of AG and UAG, we investigated their potential to revert diabetes-associated defects.

RESEARCH DESIGN AND METHODS

Healthy human subjects, individuals with type 2 diabetes, and ob/ob mice were AG or UAG infused. EPC mobilization in patients and mice was evaluated, and the underlying molecular mechanisms were investigated in bone marrow stromal cells. Recovered EPCs were also evaluated for the activity of senescence regulatory pathways and for NADPH oxidase activation by knocking down p47^phox and Rac1. Finally, UAG modulation of human EPC vasculogenic potential was investigated in an in vivo mouse model.

RESULTS

Neither AG nor UAG had any effect in healthy subjects. However, systemic administration of UAG, but not AG, prevented diabetes-induced EPC damage by modulating the NADPH oxidase regulatory protein Rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob/ob mice. In addition, unlike AG, UAG facilitated the recovery of bone marrow EPC mobilization. Crucial to EPC mobilization by UAG was the rescue of endothelial NO synthase (eNOS) phosphorylation by Akt, as UAG treatment was ineffective in eNOS knockout mice. Consistently, EPCs expressed specific UAG-binding sites, not recognized by AG.

CONCLUSIONS

These data provide the rationale for clinical applications of UAG in pathologic settings where AG fails.Ghrelin is a 28–amino acid peptide that circulates in both acylated (AG) and, more abundantly, unacylated forms (UAG) (1). Historically, AG, usually referred to as ghrelin, has been considered the only active form of the peptide. It recognizes the Gq-coupled growth hormone secretagogue receptor type 1a, denoted as GHS-R1a (2), mediating its growth hormone–releasing properties as well as other significant neuroendocrine actions (3). In contrast, UAG does not bind the GHS-R1a and is devoid of growth hormone secretagogue activity (3). Nevertheless, both AG and UAG share affinity for common binding sites that mediate vascular activities in terms of vasodilation and inhibition of cardiomyocyte and endothelial cell apoptosis (3). Furthermore, UAG effects, different from those elicited by AG, have also been demonstrated (4), suggesting the existence of an additional unidentified receptor for UAG.Besides expression in several tissues (5), including the cardiovascular system (6,7), ghrelin is produced mostly by the stomach. In particular, in humans, AG reduces insulin sensitivity and exerts orexigenic activity, whereas UAG has opposite effects (8). Circulating total ghrelin levels are negatively associated with BMI (9), and ghrelin secretion is reduced in obese (10) and type 2 diabetic (11) individuals, possibly as a compensatory mechanism protecting against hyperglycemia. Interestingly, a relative excess of AG compared with UAG has been reported in clinical conditions marked by insulin resistance (12), raising the possibility that the altered UAG/AG ratio could play a role in the altered glucose metabolism and its ongoing complications.Among such complications, accelerated vascular disease is widely recognized as the major cause of disability and death in individuals with type 2 diabetes. Endothelial injury is thought to represent a crucial step in the initiation and progression of atherosclerotic vascular disease in this setting (13). Previous data support the central role of advanced glycosylation end products (AGEs) (14) and of NADPH oxidase–mediated reactive oxygen species (ROS) production in impaired vascular remodeling associated with diabetes (15). NADPH oxidase (Nox) consists of a membrane-bound catalytic subunit and several cytosolic regulatory subunits (p47^phox and p67^phox). Moreover, the GTPase-bound Rac1 is required for the functional assembly of the holoenzyme (15).Vascular remodeling relies on resident endothelial cells and on circulating endothelial progenitor cells (EPCs): early circulating angiogenic cells (CACs) and late EPCs (16,17). Although they share several common features, they have distinct features with respect to morphology, proliferative potential, and functional characteristics (18). Compelling evidence indicates that changes in their number and functional activities are closely associated with cardiovascular risk factor profiles (19,20), impacting on their delivery to sites of ischemia where angiogenesis might be required. Indeed, treatment with certain cytokines to induce bone marrow mobilization of EPCs has been shown to be cardioprotective (21). EPC mobilization strictly depends on local secretion and activation of the matrix metalloproteinase 9 (MMP9) in the hematopoietic and stromal compartments of the bone marrow (22). In turn, activated MMP9 converts the membrane-bound form of the Kit ligand (mbKitL) into a soluble form (sKitL) that promotes hematopoietic and endothelial progenitor cell proliferation and facilitates their mobilization into the circulation (22). Because an impairment of progenitor cell mobilization has also been reported in mice lacking endothelial NO synthase (eNOS) (23), eNOS may also be involved in the control of progenitor cell delivery to sites of neovascularization. Consistently, pathologic settings, characterized by reduced systemic NO bioavailability, also show defective EPC mobilization and compromised vascular regenerative processes (24,25).Based on previous evidence for cardiovascular protective effects of both AG and UAG, the aim of the present study was to investigate the therapeutic potential of ghrelin isoforms in diabetes-associated vascular disease and related mechanisms. Herein, we demonstrate that UAG, but not AG, systemic administration protects diabetic EPCs from senescence and restores their vasculogenic potential by regulating the small GTPase Rac1 activity. Finally, we show that UAG, unlike AG, rescues defective EPC mobilization in individuals with type 2 diabetes, but has no effect in healthy subjects.     

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.     

C-Peptide Prevents Hyperglycemia-Induced Endothelial Apoptosis Through Inhibition of Reactive Oxygen Species–Mediated Transglutaminase 2 Activation

C-peptide is a bioactive peptide with a potentially protective role in diabetes complications; however, its molecular mechanism of protection against cardiovascular damage caused by hyperglycemia-induced apoptosis remains unclear. We investigated the protective mechanism of C-peptide against hyperglycemia-induced apoptosis using human umbilical vein endothelial cells and streptozotocin diabetic mice. High glucose (33 mmol/L) induced apoptotic cell death in endothelial cells via sequential elevation of intracellular Ca²⁺ and reactive oxygen species (ROS) as well as subsequent activation of transglutaminase 2 (TG2). C-peptide (1 nmol/L) prevented endothelial cell death by inhibiting protein kinase C– and NADPH oxidase–dependent intracellular ROS generation and by abolishing high glucose–induced TG2 activation, without affecting intracellular Ca²⁺ levels. Consistently, in the aorta of streptozotocin diabetic mice, hyperglycemia stimulated transamidating activity and endothelial cell apoptosis that was inhibited by C-peptide replacement therapy (35 pmol/min/kg) using osmotic pumps (control and diabetes, n = 8; diabetes + C-peptide, n = 7). In addition, C-peptide prevented hyperglycemia-induced activation of transamidation activity and apoptosis in the heart and renal cortex of streptozotocin diabetic mice. Thus, C-peptide protects endothelial cells from hyperglycemia-induced apoptotic cell death by inhibiting intracellular ROS-mediated activation of TG2. Furthermore, TG2 may be a promising avenue of therapeutic investigation to treat diabetic vasculopathies.Lack of C-peptide, along with insulin, is the main feature of type 1 diabetes mellitus (DM) and is also observed in progressive β-cell loss in later stage of type 2 DM (1,2). The subsequent hyperglycemia in diabetes is the foremost risk factor for vascular complications due to enhanced rates of cellular apoptosis as observed in retinal pericytes, renal podocytes, and vascular endothelial cells (3,4). Apoptosis in the vasculature has also been associated with pathogenesis and progression of atherosclerosis (5) that causes cardiovascular disease, the leading cause of death worldwide (6).Exposure of endothelial cells to high glucose in diabetes triggers apoptosis, leading to vascular dysfunction (7–10). In hyperglycemia, reactive oxygen species (ROS) generation plays a critical role in mediating endothelial cell apoptosis (11,12). Although activation of NADPH oxidase downstream of protein kinase C (PKC) appears to be a major cytosolic source of ROS generation in diabetic vasculature and kidney (9,11,13), mitochondrial ROS production is also involved in hyperglycemia (14,15). Elevation of intracellular Ca²⁺ is another attribute of apoptotic cell death upon high glucose exposure (16,17). Indeed, intracellular Ca²⁺ and/or ROS can enhance the activation of transglutaminase 2 (TG2), which plays a diverse role in a variety of cellular processes, including cell death, proliferation, differentiation, and migration (18,19). TG2 transamidating activity has been paradoxically reported to either facilitate or attenuate apoptosis in various cell types (20–23). Nevertheless, the role of intracellular TG2 in high glucose–induced endothelial cell apoptosis is not clear.Human C-peptide is a 31-amino acid peptide that is released into the peripheral circulation in an equimolar concentration with insulin (24). C-peptide is considered a bioactive peptide with diverse tissue- and cell-specific protective roles in various physiologic states and diseases, including diabetic neuropathy, nephropathy, vascular dysfunctions, and inflammation in type 1 DM (2,24–27). C-peptide is potentially beneficial in type 1 DM, as well as in type 2 DM, by preventing smooth muscle cell proliferation, macroangiopathy, and neointima formation (28–30). In addition, C-peptide is believed to exhibit antiapoptotic effects in diabetic rat hippocampus and in SH-SY5Y cells (31,32). C-peptide was recently reported to decrease NADPH oxidase generation of intracellular ROS in human aortic endothelial cells (33). However, the molecular mechanism(s) underlying the protective role of C-peptide in endothelial cells in diabetes and subsequent vascular complications is still unclear.In this study, we sought to determine the molecular mechanism by which C-peptide could protect endothelial cells against high glucose–induced apoptosis. We hypothesized that high glucose–induced elevation of intracellular Ca²⁺ and ROS could enhance TG2 activation to mediate endothelial cell apoptosis and that C-peptide might protect endothelial cells from high glucose–induced apoptosis by inhibiting intracellular ROS-mediated activation of TG2. To validate our in vitro findings, we generated streptozotocin diabetic mice and investigated the effects of C-peptide by continuous subcutaneous delivery of human C-peptide as a supplement therapy. We then investigated the role of C-peptide in hyperglycemia-induced activation of transamidating activity and apoptosis in aorta, heart, and renal cortex of diabetic mice.     

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 Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

The actin cytoskeleton–regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAK^Thr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.Insulin increases glucose uptake in skeletal muscle by stimulating translocation of GLUT4 from intracellular compartments to the plasma membrane and transverse tubuli (1–4). Skeletal muscle accounts for up to 75% of postprandial glucose disposal in humans (5), and normal insulin action in skeletal muscle is therefore crucial for maintaining glucose homeostasis.The Rho family GTPase Rac1 has been shown to regulate insulin-stimulated GLUT4 translocation and glucose transport in cultured muscle cells (6–8). Insulin activates Rac1, which leads to reorganization of the cortical actin cytoskeleton. Downregulation of Rac1 by small interfering RNA prevents this process (7,9) and also abolishes insulin-stimulated glucose uptake and GLUT4 translocation in L6 myoblasts (6,7). In addition, expression of a constitutively active Rac1 increases GLUT4 translocation to the same level seen after maximal insulin stimulation in this cell line (6).Even though cultured muscle cell lines are powerful tools to understand intracellular mechanisms, they differ from mature skeletal muscle in the expression and reliance of various proteins in the regulation of insulin-stimulated glucose uptake (10). Cultured muscle myoblasts, although able to fuse into myotubes, do not reach the same end-stage differentiation (e.g., do not have cross striations and do not develop transverse tubules) as muscles in vivo and therefore do not fully mature into a system that mimics fully developed skeletal muscles (11,12). Furthermore, the location, expression, and insulin-stimulated GLUT4 translocation are very different in cultured cells compared with mature muscle and may not require the same trafficking steps (2,3,13,14). As a consequence, it is imperative to investigate the role of Rac1 in insulin-stimulated glucose uptake in fully matured skeletal muscle in order to understand its role in glucose metabolism. Furthermore, the importance of skeletal muscle Rac1 on whole-body glucose homeostasis has not been determined.Rac1 activates p21-activated kinase (PAK) by facilitating autophosphorylation of PAK on threonine 423 (p-PAK^Thr423), and this pathway induces actin remodeling of the actin cytoskeleton (15). Accordingly, disruption of the actin cytoskeleton by actin-depolymerizing agents, such as latrunculin B, inhibits insulin-stimulated GLUT4 translocation in L6 myotubes (16,17). Dynamic rearrangement of the actin cytoskeleton is thus necessary for insulin to induce GLUT4 translocation in these cells (18).These findings also apply to mature skeletal muscle, since latrunculin B inhibits insulin-stimulated glucose uptake in rat epitrochlearis muscle (19). Furthermore, Ueda et al. (20) recently showed that Rac1 is activated by insulin in mouse skeletal muscle and that insulin-stimulated GLUT4 translocation is decreased in muscle-specific Rac1 knockout (KO) mice. PAK1 was also recently shown to be implicated in the regulation of insulin-stimulated GLUT4 translocation in mouse skeletal muscle (21). However, GLUT4 translocation does not always mimic glucose uptake, and numerous studies have reported experimental conditions where GLUT4 translocation and transport can be clearly dissociated (22–27), suggesting that GLUT4 translocation is not always an adequate measure of the functional end point, glucose uptake. Thus, the involvement of Rac1 and its downstream signaling in insulin-stimulated glucose uptake in mature skeletal muscle has not yet been investigated, and Rac1-dependent signaling has not been characterized in animal or human models of insulin resistance.A decreased ability to rearrange the cortical actin cytoskeleton in response to insulin has been proposed as a central defect in insulin-resistant muscle cells (28–30). Although exposure to insulin resistance–inducing agents decreased Rac1 activation and GLUT4 translocation (7), only small reductions in Akt signaling were observed in L6 myotubes (8). It is therefore possible that Rac1 is a major regulator of glucose uptake in mature skeletal muscle, and its dysregulation might contribute to the phenotype of muscular insulin resistance and type 2 diabetes (T2D). In the current study, we hypothesized that activation of Rac1 and its downstream target, PAK, is crucial for insulin-induced glucose uptake in mature skeletal muscle and for maintaining whole-body glucose homeostasis. We further hypothesized that Rac1-dependent signaling is downregulated in insulin-resistant states.     

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.     

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.     

Activation of p53 Promotes Renal Injury in Acute Aristolochic Acid Nephropathy

Ingestion of aristolochic acid (AA) can cause AA nephropathy (AAN), in which excessive death of tubular epithelial cells (TECs) characterize the acute phase. AA forms adducts with DNA, which may lead to TEC apoptosis via p53-mediated signaling. We tested this hypothesis both by studying p53-deficient mice and by blocking p53 in TECs with its inhibitor pifithrin-α. AA induced acute AAN in wild-type mice, resulting in massive apoptotic and necrotic TEC death and acute renal failure; p53 deficiency or pharmacologic inhibition attenuated this injury. In vitro, AA induced apoptotic and necrotic death of TEC in a time- and dosage-dependent manner, with apoptosis marked by a 10-fold increase in cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling–positive/Annexin V-positive propidium iodide–negative TECs (all P < 0.001). AA induced dephosphorylation of STAT3 and the subsequent activation of p53 and TEC apoptosis. In contrast, overexpression of STAT3, p53 inhibition, or p53 knockdown with small interfering RNA all attenuated AA-induced TEC apoptosis. Taken together, these results suggest that AA induces TEC death via apoptosis by dephosphorylation of STAT3 and posttranslational activation of p53, supporting the hypothesis that p53 promotes renal injury in acute AAN.Chinese herb nephropathy was first reported in Belgium in patients with prolonged intake of Chinese herbs during a slimming regimen and is recognized as one of the most severe complications caused by traditional Chinese medicine.^1–3 It is now clear that the major substance that causes Chinese herb nephropathy is the plant nephrotoxin aristolochic acid and its metabolism products.^4–6 Thus, the term aristolochic acid nephropathy (AAN), instead of Chinese herbal nephropathy, is used today.^7,8 AAN has emerged as an important cause of drug-associated renal failure worldwide.⁹Patients with AAN exhibit a rapidly progressive renal deterioration, resulting in acute renal failure that could lead to ESRD.^1–3,10,11 A similar clinical course was observed in experimental animals treated with AA.^12,13 Pathologically, chronic AAN is characterized by extensive interstitial fibrosis with atrophy and loss of renal tubules.^{1–3,10–13} The lesions of chronic AAN are mainly in the cortex involving proximal tubular epithelial cells (TECs)^10–13; glomeruli are relatively spared with minimal inflammation.^9–12 In contrast, progressive TEC death occurs early in the clinical course with an absence of renal fibrosis and inflammation in experimental models and patients with acute AAN.^10,14,15 Although apoptosis is an important pathologic feature in in vivo and in vitro studies of acute AAN,^16–18 the underlying mechanisms remain unclear.In considering the genotoxic effect of AA with the formation of AA-DNA adducts and the importance of the p53 signaling pathway in DNA damage and cell apoptosis,^19–21 we hypothesized that TEC apoptosis in acute AAN is dependent on p53 signaling. We investigated this by inducing acute AAN in p53 knockout (KO) and p53 wild-type (WT) mice and by blocking the p53 activities with a pharmacologic inhibitor. We further studied the toxicity of AA on TEC apoptosis by examining a panel of apoptotic biomarkers. The mechanism that AA induced TEC apoptosis by activating p53 via a STAT3-dependent posttranslational modification was identified.     

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.     

Kallikrein 5-Mediated Inflammation in Rosacea: Clinically Relevant Correlations with Acute and Chronic Manifestations in Rosacea and How Individual Treatments May Provide Therapeutic Benefit

Rosacea is a chronic inflammatory condition of facial skin estimated to affect more than 16 million Americans. Although the pathogenesis of rosacea is not fully understood, recent evidence in vitro as well as in vivo has supported the role of increased levels of the trypsin-like serine protease, kallikrein 5, in initiating an augmented inflammatory response in rosacea. The increase in the quantity and magnitude of biological activity of kallikrein 5 leads to production of greater quantities of cathelicidin (LL-37), an antimicrobial peptide associated with increases in innate cutaneous inflammation, vasodilation, and vascular proliferation, all of which are characteristic features of rosacea. In this article, the authors review the literature supporting the role of kallikrein 5 in the pathophysiology of rosacea, including how therapeutic interventions modulate the effects of kallikrein 5, thus providing further support for this pathophysiological model that at least partially explains many of the clinical features of cutaneous rosacea.Cutaneous rosacea (rosacea) is a chronic inflammatory facial skin disorder noted most commonly in individuals of northern European descent, although people of any ethnicity or skin color may be affected.1-4 The visible manifestations with central facial predominance are characteristic of rosacea, including erythema, papules, pustules, telangiectasias, and phymatous changes.1-4 However, persistent (nontransient) erythema involving the central face that intensifies during flares and the presence of telangiectasias, which are also accentuated mostly on the central face, are the core clinical features that support a diagnosis of rosacea.1-9 Papules and pustules are not consistently present in rosacea, characterizing only those individuals with rosacea who exhibit the papulopustular subtype of the disease.3-6 In fact, papulopustular lesions never emerge in many individuals affected by rosacesa, and phymatous changes affect only a relatively small number of the rosacea-affected population; however, central facial erythema is present to some extent in essentially all people with rosacea.1-8Why do some people get rosacea and others do not? Although the entire explanation that would fully answer this question remains elusive, current evidence suggests that individuals affected by rosacea exhibit rosacea-prone skin, which inherently displays dysregulation of two main systems present within skin—the neurovascular/neuroimmune system and the immune detection/response system (innate immunity).3,5-8,1–9 Both of these systems normally serve physiological functions related to how skin responds to exogenous changes or insults (i.e., changes in temperature, exposure to microbial pathogens). However, in rosacea, both the cutaneous neurovascular/neuroimmune system and the immune detection/response system are dysregulated, with both demonstrating augmented responses that correlate with clinical manifestations commonly seen in patients with cutaneous rosacea.Neurovascular/neuroimmune dysregulation, which includes both anatomic and physiochemical differences present in rosacea-prone skin as compared to healthy facial skin, appears to be a major contributor that exacerbates the vasodilation of facial skin vasculature with increased facial blood flow that occurs during a rosacea flare.3,5-7,17,18,20 This increased vasodilation in rosacea-affected skin, which can be acute or subacute in onset, is commonly referred to as flushing.1,3,4,6,7,17,20 Neurosensory symptoms (i.e., stinging, burning) are often associated with or exacerbated during a rosacea flare.1,3-8 Exogenous factors that are commonly recognized by patients as triggers, which seem to induce a flare, include increased ambient heat/warmth and certain spices (i.e., capsaicin), all of which can induce signaling of neurogenic inflammation via specific receptor channels (transient receptor potential vanilloid [TRPV] subfamily) shown to be increased in rosacea-prone skin.3,6,17,18 The immune detection/response dysregulation of rosacea is evidenced by the upregulation of the pattern recognition receptor, toll-like receptor 2 (TLR2) and the cathelicidin innate immunity pathway.3,5-17,19,21,22 Ultraviolet light (UV) exposure, another recognized trigger factor associated with flares of rosacea, produces changes that induce ligand-binding of TLR2, which signals innate inflammation.3,5-16,19,21 Lastly, upregulated production of several matrix metalloproteases (MMPs) has been demonstrated in rosacea, further contributing to cascades of inflammation and degradation of the dermal matrix.1,3,5-7,19 Accentuated immune detection/response as a major component of the pathophysiology of rosacea has been discussed extensively in the literature and is addressed in more detail as a major subject of this article.5-7,10-16,19,21,22Although the pathophysiology of rosacea is not completely understood, dysregulation of the innate immune detection/response system plays a significant role in the inflammatory and vascular responses seen in this condition.5-7,10-16,19,21,22 As a known inducer of innate and cellular inflammation, increased vascularity, and angiogenesis, cathelicidin (LL-37), an antimicrobial peptide that physiologically provides near-immediate innate defense against several microbial organisms, has been investigated to determine its potential role in the pathophysiology of rosacea.10,11,23,24 Results have shown that patients with rosacea express elevated levels of LL-37 in facial skin, with this increased expression attributed to abnormally high levels of the trypsin-like serine protease enzyme, kallikrein 5 (KLK5), which selectively cleaves an inactive precursor protein (hCAP18) to form the biologically active antimicrobial peptide (LL-37).10,22 Investigations of the mechanism of action of two agents proven to be effective in reducing papulopustular lesions and perilesional erythema in rosacea, topical azelaic acid (AzA) and oral doxycycline, demonstrated direct and indirect inhibition of KLK5, respectively.25-29 In one study with AzA 15% gel, the reduction in KLK5 activity correlated with clinical improvement of rosacea.29 In this review, the authors further describe the role of KLK5 in the pathophysiology of rosacea, including the inflammatory cascades that result from increased KLK5 expression, as well as a more detailed discussion of different therapies shown to inhibit the progression of this cascade.