Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes.

Background: Excessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia. Methods: The ROS levels were analyzed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia. The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes. Results: Exposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD)-dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1). The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes. Conclusions: We conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use. Funding: This work was supported by grants from the Swedish Research Council, Stockholm County Research Council, Stockholm Regional Research Foundation, Bert von Kantzows Foundation, Swedish Society of Medicine, Kung Gustaf V:s och Drottning Victorias Frimurarestifelse, Karolinska Institute's Research Foundations, Strategic Research Programme in Diabetes, and Erling-Persson Family Foundation for S-B.C.; grants from the Swedish Research Council and Swedish Heart and Lung Foundation for T.A.S.; and ERC consolidator grant for M.M.

L-Carnosine Stimulation of Coenzyme Q10 Biosynthesis Promotes Improved Mitochondrial Function and Decreases Hepatic Steatosis in Diabetic Conditions.

Mitochondrial dysfunction in type 2 diabetes leads to oxidative stress, which drives disease progression and diabetes complications. L-carnosine, an endogenous dipeptide, improves metabolic control, wound healing and kidney function in animal models of type 2 diabetes. Coenzyme Q (CoQ), a component of the mitochondrial electron transport chain, possesses similar protective effects on diabetes complications. We aimed to study the effect of carnosine on CoQ, and assess any synergistic effects of carnosine and CoQ on improved mitochondrial function in a mouse model of type 2 diabetes. Carnosine enhanced CoQ gene expression and increased hepatic CoQ biosynthesis in db/db mice, a type 2 diabetes model. Co-administration of Carnosine and CoQ improved mitochondrial function, lowered ROS formation and reduced signs of oxidative stress. Our work suggests that carnosine exerts beneficial effects on hepatic CoQ synthesis and when combined with CoQ, improves mitochondrial function and cellular redox balance in the liver of diabetic mice. (4) Conclusions: L-carnosine has beneficial effects on oxidative stress both alone and in combination with CoQ on hepatic mitochondrial function in an obese type 2 diabetes mouse model.

Treatment with a ß-2-adrenoceptor agonist stimulates glucose uptake in skeletal muscle and improves glucose homeostasis, insulin resistance and hepatic steatosis in mice with diet-induced obesity.

AIMS/HYPOTHESIS: Chronic stimulation of ß2-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of ß2-adrenoceptors. In the current study we further explored the potential therapeutic relevance of ß2-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect. METHODS: C57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen. RESULTS: Consistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment, p < 0.01) and these effects persisted up to 42 days of treatment. These favourable metabolic effects could be achieved with doses as low as 0.025 mg kg-1 day-1 (40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%, p < 0.001) as well as during chronic treatment in diet-induced obese mice (by 74%, p < 0.001). Notably, prolonged treatment with low-dose clenbuterol improved whole-body insulin sensitivity (glucose disposal rate after insulin injection increased up to 1.38 ± 0.31%/min in comparison with 0.15 ± 0.36%/min in control mice, p < 0.05) and drastically reduced hepatic steatosis (by 40%, p < 0.01) and glycogen (by 23%, p < 0.05). CONCLUSIONS/INTERPRETATION: Clenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective ß2-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans. Graphical abstract.

Protective Actions of Anserine Under Diabetic Conditions.

BACKGROUND/AIMS: In rodents, carnosine treatment improves diabetic nephropathy, whereas little is known about the role and function of anserine, the methylated form of carnosine. METHODS: Antioxidant activity was measured by oxygen radical absorbance capacity and oxygen stress response in human renal tubular cells (HK-2) by RT-PCR and Western-Immunoblotting. In wildtype (WT) and diabetic mice (db/db), the effect of short-term anserine treatment on blood glucose, proteinuria and vascular permeability was measured. RESULTS: Anserine has a higher antioxidant capacity compared to carnosine (p < 0.001). In tubular cells (HK-2) stressed with 25 mM glucose or 20⁻100 µM hydrogen peroxide, anserine but not carnosine, increased intracellular heat shock protein (Hsp70) mRNA and protein levels. In HK-2 cells stressed with glucose, co-incubation with anserine also increased hemeoxygenase (HO-1) protein and reduced total protein carbonylation, but had no effect on cellular sirtuin-1 and thioredoxin protein concentrations. Three intravenous anserine injections every 48 h in 12-week-old db/db mice, improved blood glucose by one fifth, vascular permeability by one third, and halved proteinuria (all p < 0.05). CONCLUSION: Anserine is a potent antioxidant and activates the intracellular Hsp70/HO-1 defense system under oxidative and glycative stress. Short-term anserine treatment in diabetic mice improves glucose homeostasis and nephropathy.

Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift.

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.

Coenzyme Q10 and oxidative stress, the association with peripheral sensory neuropathy and cardiovascular disease in type 2 diabetes mellitus.

OBJECTIVE: Our study aimed to explore associations between metabolic control, oxidative stress and coenzyme Q10 (CoQ10) in relation to diabetes complications in a representative population of type 2 diabetes. RESEARCH DESIGN AND METHODS: A geographic cohort of 156 subjects was recruited. Serum concentrations of CoQ10 and vitamin E were measured by HPLC. ROS was determined by free oxygen radicals testing (FORT). Glutaredoxin (Grx) activity, oxidized LDL cholesterol (oxLDLc), high sensitive CRP (hsCRP), HbA1c, urine albumin, serum creatinine, serum cystatin C, and plasma lipids were assayed with routine laboratory protocols. RESULTS: Serum CoQ10 was higher than in nondiabetics. HbA1c, fP-glucose, hyperlipidemia, inflammation (hsCRP), and increased BMI were associated with signs of oxidative stress as increased levels of FORT, Grx activity and/or increased levels of oxLDLc Oxidative stress was found to be strongly correlated with prevalence of cardiovascular disease (CVD) and peripheral sensory neuropathy (PSN). In both gender groups there were positive correlations between CoQ10 and oxLDLc, and between BMI and the ratio CoQ10/chol. Grx activity was inversely correlated to oxLDLc and CoQ10. Women with CVD and PSN had higher waist index, oxLDLc, and FORT levels compared to men but lower CoQ10 levels. Men had worse kidney function and lower vitamin E. Multiple regression analysis showed increased levels of CoQ10 to be significantly correlated with increased levels of cholesterol, triglycerides, vitamin E, fB-glucose and BMI. CONCLUSIONS: Hyperlipidemia, hyperglycemia and inflammation were associated with oxidative stress, which was correlated to the prevalence of diabetes complications. CoQ10 was increased in response to oxidative stress. There were gender differences in the risk factors associated with diabetes complications.

Carnosine metabolism in diabetes is altered by reactive metabolites.

Carnosinase 1 (CN1) contributes to diabetic nephropathy by cleaving histidine-dipeptides which scavenge reactive oxygen and carbonyl species and increase nitric oxide (NO) production. In diabetic mice renal CN1 activity is increased, the regulatory mechanisms are unknown. We therefore analysed the in vitro and in vivo regulation of CN1 activity using recombinant and human CN1, and the db/db mouse model of diabetes. Glucose, leptin and insulin did not modify recombinant and human CN1 activity in vitro, glucose did not alter renal CN1 activity of WT or db/db mice ex vivo. Reactive metabolite methylglyoxal and Fenton reagent carbonylated recombinant CN1 and doubled CN1 efficiency. NO S-nitrosylated CN1 and decreased CN1 efficiency for carnosine by 70 % (p < 0.01), but not for anserine. Both CN1 cysteine residues were nitrosylated, the cysteine at position 102 but not at position 229 regulated CN1 activities. In db/db mice, renal CN1 mRNA and protein levels were similar as in non-diabetic controls, CN1 efficiency 1.9 and 1.6 fold higher for carnosine and anserine. Renal carbonyl stress was strongly increased and NO production halved, CN1 highly carbonylated and less S-nitrosylated compared to WT mice. GSH and NO2/3 concentrations were reduced and inversely related with carnosine degradation rate (r = -0.82/-0.85). Thus, reactive metabolites of diabetes upregulate CN1 activity by post-translational modifications, and thus decrease the availability of reactive metabolite-scavenging histidine dipeptides in the kidney in a positive feedback loop. Interference with this vicious circle may represent a new therapeutic target for mitigation of DN.

Carnosine decreases IGFBP1 production in db/db mice through suppression of HIF-1.

IGF binding protein 1 (IGFBP1) is a member of the binding proteins for the IGF with an important role in glucose homeostasis. Circulating IGFBP1 is derived essentially from the liver where it is mainly regulated negatively by insulin. Carnosine, a natural antioxidant, has been shown to improve metabolic control in different animal models of diabetes but its mechanisms of action are still not completely unraveled. We therefore investigate the effect of carnosine treatment on the IGFBP1 regulation in db/db mice. Db/db mice and heterozygous non-diabetic mice received for 4 weeks regular water or water supplemented with carnosine. Igfbp1 mRNA expression in the liver was evaluated using qPCR and the protein levels in plasma by western blot. Plasma IGF1 and insulin were analyzed using immunoassays. HepG2 cells were used to study the in vitro effect of carnosine on IGFBP1. The modulation of hypoxia inducible factor-1 alpha (HIF-1α) which is the central mediator of hypoxia-induction of IGFBP1 was analyzed using: WB, reporter gene assay and qPCR. Carnosine decreased the circulating IGFBP1 levels and the liver expression Igfbp1, through a complex mechanism acting both directly by suppressing the HIF-1α-mediated IGFBP1 induction and indirectly through increasing circulating insulin level followed by a decrease in the blood glucose levels and increased the plasma levels or IGF1. Reduction of IGFBP1 in diabetes through insulin-dependent and insulin-independent pathways is a novel mechanism by which carnosine contributes to the improvement of the metabolic control in diabetes.

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes.

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100 mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.

Carnosine treatment largely prevents alterations of renal carnosine metabolism in diabetic mice.

Recently, we identified an allelic variant of human carnosinase 1 (CN1) that results in increased enzyme activity and is associated with susceptibility for diabetic nephropathy in humans. Investigations in diabetic (db/db) mice showed that carnosine ameliorates glucose metabolism effectively. We now investigated the renal carnosinase metabolism in db/db mice. Kidney CN1 activity increased with age and was significantly higher in diabetic mice compared to controls. Increased CN1 activity did not affect renal carnosine levels, but anserine concentrations were tenfold lower in db/db mice compared to controls (0.24±0.2 vs. 2.28±0.3 nmol/mg protein in controls; p<0.001). Homocarnosine concentrations in kidney tissue were low in both control and db/db mice (below 0.1 nmol/mg protein, p=n.s.). Carnosine treatment for 4 weeks substantially decreased renal CN1 activity in diabetic mice (0.32±0.3 in non-treated db/db vs. 0.05±0.05 µmol/mg/h in treated db/db mice; p<0.01) close to normal activities. Renal anserine concentrations increased significantly (0.24±0.2 in non-treated db/db vs. 5.7±1.2 µmol/mg/h in treated db/db mice; p<0.01), while carnosine concentrations remained unaltered (53±6.4 in non-treated vs. 61±15 nmol/mg protein in treated db/db mice; p=n.s.). Further, carnosine treatment halved proteinuria and reduced vascular permeability to one-fifth in db/db mice. In renal tissue of diabetic mice carnosinase activity is significantly increased and anserine concentrations are significantly reduced compared to controls. Carnosine treatment largely prevents the alterations of renal carnosine metabolism.

Genetic and functional effects of membrane metalloendopeptidase on diabetic nephropathy development.

BACKGROUND/AIMS: Vasopeptidase as an agent inhibits membrane metalloendopeptidase (MME, also known as neutral endopeptidase). MME is widely distributed in the body and particularly abundant in the kidney. The MME gene is located on chromosome 3q25.1 within a linkage region for diabetic nephropathy (DN). The present study aims to evaluate the genetic and functional effects of MME in the development of DN. METHODS: A case-control genetic study of the MME gene in type 1 diabetes (T1D) patients with and without DN (n = 578/599) was performed. All subjects were selected from the Genetics of Kidneys in Diabetes study. Genotyping was performed with TagMan allelic discrimination. Mme mRNA and protein expression levels in kidney tissues of db/db mice at the ages of 5, 12 and 26 weeks were analyzed with TaqMan real-time RT-PCR and Western blot. RESULTS: The haplotype A-C constructed with single nucleotide polymorphisms (SNPs) rs3796268A/G and rs3773885C/T in the MME gene was found to be associated with DN (p = 0.015, OR = 1.33, 95% CI 1.05-1.68) in female T1D patients. Further analyses of renal traits in T1D patients with DN and end-stage renal disease according to the genotypes of SNP rs3773885 indicated that the C allele carriers had higher serum creatinine levels compared to the subjects carrying T allele in both females and males. Mme expression at mRNA and protein levels was upregulated in kidneys of db/db mice at the ages of 12 and 26 weeks (p = 0.017 and <0.001) but not at the age of 5 weeks compared to the controls. CONCLUSIONS: The present study provides the first evidence that MME has genetic and biological effects on the development of DN, and suggests that the inhibition of MME expression in the kidney with the agent of vasopeptidase may be a useful therapeutic approach for this disease.

Epigenetic DNA methylation in the promoters of the Igf1 receptor and insulin receptor genes in db/db mice.

We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state.