Quantitative proteomic profiling identifies new renal targets of copper(II)‐selective chelation in the reversal of diabetic nephropathy in rats

This study aimed to identify new diabetic nephropathy (DN)‐related proteins and renal targets of the copper(II)‐selective chelator, triethylenetetramine (TETA) in streptozotocin‐diabetic rats. We used the recently developed iTRAQ? technology to compare renal protein profiles among non‐diabetic, diabetic, and TETA‐treated diabetic rats. In diabetic kidneys, tubulointerstitial nephritis antigen (TINag), voltage‐dependent anion‐selective channel (VDAC) 1, and VDAC2 were up‐regulated in parallel with alterations in expression of proteins with functions in oxidative stress and oxidative phosphorylation (OxPhos) pathways. By contrast, mitochondrial HSP 60, Cu/Zn‐superoxide dismutase, glutathione S‐transferase α3 and aquaporin‐1 were down‐regulated in diabetic kidneys. Following TETA treatment, levels of D ‐amino acid oxidase‐1, epoxide hydrolase‐1, aquaporin‐1, and a number of mitochondrial proteins were normalized, with concomitant amelioration of albuminuria. Changes in levels of TINag, collagen VIα1, actinin 4α, apoptosis‐inducing factor 1, cytochrome C, histone H3, VDAC1, and aquaporin‐1 were confirmed by Western blotting or immunohistochemistry. Changes in expression of proteins related to tubulointerstitial function, podocyte structure, and mitochondrial apoptosis are implicated in the mechanism of DN and their reversal by TETA. These findings are consistent with the hypothesis that this new experimental therapy may be useful for treatment of DN.     

Altered REDD1, myostatin, and Akt/mTOR/FoxO/MAPK signaling in streptozotocin-induced diabetic muscle atrophy

Type 1 diabetes, if poorly controlled, leads to skeletal muscle atrophy, decreasing the quality of life. We aimed to search highly responsive genes in diabetic muscle atrophy in a common diabetes model and to further characterize associated signaling pathways. Mice were killed 1, 3, or 5 wk after streptozotocin or control. Gene expression of calf muscles was analyzed using microarray and protein signaling with Western blotting. We identified translational repressor protein REDD1 (regulated in development and DNA damage responses) that increased seven- to eightfold and was associated with muscle atrophy in diabetes. The diabetes-induced increase in REDD1 was confirmed at the protein level. This result was accompanied by the increased gene expression of DNA damage/repair pathways and decreased expression in ATP production pathways. Concomitantly, increased phosphorylation of AMPK and dephosphorylation of the Akt/mTOR/S6K1/FoxO pathway of proteins were observed together with increased protein ubiquitination. These changes were especially evident during the first 3 wk, along with the strong decrease in muscle mass. Diabetes also induced an increase in myostatin protein and decreased MAPK signaling. These, together with decreased serum insulin and increased serum glucose, remained altered throughout the 5-wk period. In conclusion, diabetic myopathy induced by streptozotocin led to alteration of multiple signaling pathways. Of those, increased REDD1 and myostatin together with decreased Akt/mTOR/FoxO signaling are associated with diabetic muscle atrophy. The increased REDD1 and decreased Akt/mTOR/FoxO signaling followed a similar time course and thus may be explained, in part, by increased expression of genes in DNA damage/repair and possibly also decrease in ATP-production pathways.     

Tocotrienol-rich fraction supplementation reduces hyperglycemia-induced skeletal muscle damage through regulation of insulin signaling and oxidative stress in type 2 diabetic mice

Chronic hyperglycemia induces impairment of muscle growth and development of diabetes mellitus (DM). Since skeletal muscle is the major site for disposal of ingested glucose, impaired glucose metabolism causes imbalance between protein synthesis and degradation which adversely affects physical mobility.In this study, we investigated the effect of tocotrienol-rich fraction (TRF) supplementation on skeletal muscle damage in diabetic mice. Diabetes was induced by a high-fat diet with streptozotocin (STZ) injection (100 mg/kg) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose levels≥250 mg/dl), normal control (CON) and diabetic control (DMC) groups were administrated with olive oil, while TRF treatment groups were administrated with TRF (dissolved in olive oil) at low dose (100 mg/kg BW, LT) or high dose (300 mg/kg BW, HT) by oral gavage for 12 weeks.TRF supplementation ameliorated muscle atrophy, plasma insulin concentration and homeostatic model assessment estimated insulin resistance in diabetic mice. Moreover, TRF treatment up-regulated IRS-1 and Akt levels accompanied by increased translocation of GLUT4. Furthermore, TRF increased mitochondrial biogenesis by activating SIRT1, SIRT3 and AMPK in diabetic skeletal muscle. These changes were in part mechanistically explained by reduced levels of skeletal muscle proteins related to oxidative stress (4-hydroxynonenal, protein carbonyls, Nrf2 and HO-1), inflammation (NFkB, MCP-1, IL-6 and TNF-α), and apoptosis (Bax, Bcl₂ and caspase-3) in diabetic mice. Taken together, these results suggest that TRF might be useful as a beneficial nutraceutical to prevent skeletal muscle atrophy associated with diabetes by regulating insulin signaling via AMPK/SIRT1/PGC1α pathways in type 2 diabetic mice.     

Functional changes in the neural retina occur in the absence of mitochondrial dysfunction in a rodent model of diabetic retinopathy

Diabetic retinopathy is a neurovascular diabetes complication resulting in vision loss. A wealth of literature reports retinal molecular changes indicative of neural deficits, inflammation, and vascular leakage with chronic diabetes, but the mechanistic causes of disease initiation and progression are unknown. Microvascular mitochondrial DNA (mtDNA) damage leading to mitochondrial dysfunction has been proposed to drive vascular dysfunction in retinopathy. However, growing evidence suggests that neural retina dysfunction precedes and may cause vascular damage. Therefore, we tested the hypothesis that neural mtDNA damage and mitochondrial dysfunction are an early initiating factor of neural diabetic retinopathy development in a rat streptozotocin‐induced, Type I diabetes model. Mitochondrial function (oxygen consumption rates) was quantified in retinal synaptic terminals from diabetic and non‐diabetic rats with paired retinal structural and function assessment (optical coherence tomography and electroretinography, respectively). Mitochondrial genome damage was assessed by identifying mutations and deletions across the mtDNA genome by high depth sequencing and absolute mtDNA copy number counting through digital PCR. Mitochondrial protein expression was assessed by targeted mass spectrometry. Retinal functional deficits and neural anatomical changes were present after 3 months of diabetes and prevented/normalized by insulin treatment. No marked dysfunction of mitochondrial activity, maladaptive changes in mitochondrial protein expression, alterations in mtDNA copy number, or increase in mtDNA damage was observed in conjunction with retinal functional and anatomical changes. These results demonstrate that neural retinal dysfunction with diabetes begins prior to mtDNA damage and dysfunction, and therefore retinal neurodegeneration initiation with diabetes occurs through other, non‐mitochondrial DNA damage, mechanisms.

     

Attenuation of diabetic cardiomyopathy by relying on kirenol to suppress inflammation in a diabetic rat model

Diabetic cardiomyopathy is characterized by diabetes‐induced myocardial abnormalities, accompanied by inflammatory response and alterations in inflammation‐related signalling pathways. Kirenol, isolated from Herba Siegesbeckiae, has potent anti‐inflammatory properties. In this study, we aimed to investigate the cardioprotective effect of kirenol against DCM and underlying the potential mechanisms in a type 2 diabetes mellitus model. Kirenol treatment significantly decreased high glucose‐induced cardiofibroblasts proliferation and increased the cardiomyocytes viability, prevented the loss of mitochondrial membrane potential and further attenuated cardiomyocytes apoptosis, accompanied by a reduction in apoptosis‐related protein expression. Kirenol gavage could affect the expression of pro‐inflammatory cytokines in a dose‐dependent manner but not lower lipid profiles, and only decrease fasting plasma glucose, fasting plasma insulin and mean HbA1c levels in high‐dose kirenol‐treated group at some time‐points. Left ventricular dysfunction, hypertrophy, fibrosis and cell apoptosis, as structural and functional abnormalities, were ameliorated by kirenol administration. Moreover, in diabetic hearts, oral kirenol significantly attenuated activation of mitogen‐activated protein kinase subfamily and nuclear translocation of NF‐κB and Smad2/3 and decreased phosphorylation of IκBα and both fibrosis‐related and apoptosis‐related proteins. In an Electrophoretic mobility shift assay, the binding activities of NF‐κB, Smad3/4, SP1 and AP‐1 in the nucleus of diabetic myocardium were significantly down‐regulated by kirenol treatment. Additionally, high dose significantly enhanced myocardial Akt phosphorylation without intraperitoneal injection of insulin. Kirenol may have potent cardioprotective effects on treating for the established diabetic cardiomyopathy, which involves the inhibition of inflammation and fibrosis‐related signalling pathways and is independent of lowering hyperglycaemia, hyperinsulinemia and lipid profiles.     

Gender dimorphism in regulation of plasma proteins in streptozotocin‐induced diabetic rats

In the present study, we examined differentially regulated plasma proteins between healthy control and streptozotocin (STZ)‐induced male and female diabetic rats by 2DE‐based proteomic analysis. Animal experiments revealed that significantly lower plasma insulin levels were observed in female diabetic rats, consequently resulting in higher blood glucose levels in female diabetic rats. Importantly, plasma levels of sex hormones were significantly altered in a gender‐dependent manner before and after STZ treatment. Results of the animal experiment indicated the existence of sexual dimorphism in the regulation of plasma proteins between healthy control and diabetic rats. Plasma proteome analysis enabled us to identify a total of 38 proteins showing sexual dimorphic regulation patterns. In addition, for the first time, we identified several differentially regulated plasma proteins between healthy control and diabetic rats, including apolipoprotein E, fetuin B, α‐1‐acid glycoprotein, β‐2‐glycoprotein 1, 3‐hydroxyanthranilate 3,4‐dioxygenase, and serum amyloid P‐component. To the best of our knowledge, this is the first proteomic approach to address sexual dimorphism in diabetic animals. These proteomic data on gender‐dimorphic regulation of plasma proteins provide valuable information that can be used for evidence‐based gender‐specific clinical treatment of diabetes.     

Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy

An understanding of the diabetes-induced alterations in vitreous protein composition in the absence and in the presence of proliferative diabetic retinopathy (PDR) may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of vitreous samples from individuals with diabetes but without diabetic retinopathy (noDR) or with PDR and nondiabetic individuals (NDM). Using preparative one-dimensional SDS-PAGE and nano-LC/MS/MS of 17 independent vitreous samples, we identified 252 proteins from human vitreous. Fifty-six proteins were differentially abundant in noDR and PDR vitreous compared with NDM vitreous, including 32 proteins increased and 10 proteins decreased in PDR vitreous compared with NDM vitreous. Comparison of noDR and PDR groups revealed increased levels of angiotensinogen and decreased levels of calsyntenin-1, interphotoreceptor retinoid-binding protein, and neuroserpin in PDR vitreous. Biological pathway analysis revealed that vitreous contains 30 proteins associated with the kallikrein-kinin, coagulation, and complement systems. Five of them (complement C3, complement factor I, prothrombin, alpha-1-antitrypsin, and antithrombin III) were increased in PDR vitreous compared with NDM vitreous. Factor XII was detected in PDR vitreous but not observed in either NDM or noDR vitreous. PDR vitreous also had increased levels of peroxiredoxin-1 and decreased levels of extracellular superoxide dismutase, compared with noDR or NDM vitreous. These data provide an in depth analysis of the human vitreous proteome and reveal protein alterations that are associated with PDR.     

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats

Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.     

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.     

Differential expression of kidney proteins in streptozotocin-induced diabetic rats in response to hypoglycemic fungal polysaccharides

Diabetic nephropathy remains a major cause of morbidity and mortality in the diabetic population and is the leading cause of end-stage renal failure. Despite current therapeutics including intensified glycemic control and blood pressure lowering agents, renal disease continues to progress relentlessly in diabetic patients, albeit at a lower rate. Since synthetic drugs for diabetes are known to have side effects, fungal mushrooms as a natural product come into preventing the development of diabetes. Our previous report showed the hypoglycemic effect of extracellular fungal polysaccharides (EPS) in streptozotocin (STZ)-induced diabetic rats. In this study, we analyzed the differential expression patterns of rat kidney proteins from normal, STZ-induced diabetic, and EPS-treated diabetic rats, to discover diabetes-associated proteins in rat kidney. The results of proteomic analysis revealed that up to 500 protein spots were visualized, of which 291 spots were differentially expressed in the three experimental groups. Eventually, 51 spots were statistically significant and were identified by peptide mass fingerprinting. Among the differentially expressed renal proteins, 10 were increased and 16 were decreased significantly in diabetic rat kidney. The levels of different proteins, altered after diabetes induction, were returned to approximately those of the healthy rats by EPS treatment. A histopathological examination showed that EPS administration restored the impaired kidney to almost normal architecture. The study of protein expression in the normal and diabetic kidney tissues enabled us to find several diabetic nephropathy-specific proteins, such as phospholipids scramblase 3 and tropomyosin 3, which have not been mentioned yet in connection with diabetes.     

Insulin regulation of glutathione and contractile phenotype in diabetic rat ventricular myocytes

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and Ca(2+) transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was approximately 36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and gamma-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and gamma-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca(2+) transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca(2+) handling proteins and contraction.     

Enhanced permeability transition explains the reduced calcium uptake in cardiac mitochondria from streptozotocin-induced diabetic rats

Cardiac dysfunction is associated with diabetes. It was previously shown that heart mitochondria from diabetic rats have a reduced calcium accumulation capacity. The objective of this work was to determine whether the reduction in calcium accumulation by cardiac mitochondria from diabetic rats is related to an enhanced susceptibility to induction of the mitochondrial permeability transition. Streptozotocin-induced diabetic rats were used as a model to study the alterations caused by diabetes in the permeability transition, 21 days after streptozotocin administration. Heart mitochondria were isolated to evaluate respiratory parameters and susceptibility to the calcium-dependent permeability transition. Our results show that streptozotocin diabetes facilitates the mitochondrial permeability transition in cardiac mitochondria, resulting in decreased mitochondrial calcium accumulation. We also observed that heart mitochondria from diabetic rats had depressed oxygen consumption during the phosphorylative state. The reduced mitochondrial calcium uptake observed in heart mitochondria from diabetic rats is related to an enhanced susceptibility to the permeability transition rather than to damage to the calcium uptake machinery.     

Streptozotocin-induced alterations in the levels of functional mitochondrial anion transport proteins

The effect of streptozotocin-induced diabetes on the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach utilized for these studies consisted of the extraction of each of four mitochondrial anion transport proteins from rat liver mitoplasts (isolated from diabetic and control animals) with the nonionic detergent Triton X-114, followed by the functional reconstitution of each transporter in a liposomal system via the freeze-thaw-sonication technique. This approach permitted the quantification of transporter function without the complications that occur when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental diabetes caused an increase in the extractable and reconstitutable specific (and total) transport activities of the pyruvate and dicarboxylate transporters, a decrease in the activity of the citrate transporter, and no significant change in the activity of the phosphate transporter relative to control values. An examination of the time course of the appearance of changes in the reconstitutable activities of the pyruvate and citrate transporters following the injection of streptozotocin revealed differences. Thus, whereas the activity of the pyruvate transporter displayed the most pronounced increase (193%) 1 week following streptozotocin injection and then subsequently declined from this peak and plateaued at later times (99% and 96% increases at 3 and 8 weeks, respectively), the activity of the citrate transporter progressively decreased with time (31-51% decreases at 1-8 weeks). We suggest that the observed diabetes-induced changes in mitochondrial anion transporter function are predictable on the basis of diabetes-induced alterations in the activities of enzymes that constitute metabolic pathways to which these transporters either supply substrate or remove product. Furthermore, we speculate that mitochondrial anion transport proteins may be regulated in coordination with the enzymes of such associated metabolic pathways.     

Gender‐dimorphic regulation of DJ1 and its interactions with metabolic proteins in streptozotocin‐induced diabetic rats

Regulation of DJ1 is associated with a number of human diseases. To determine the involvement of DJ1 in progression of diabetes in a gender‐dependent manner, we investigated its tissue‐specific expression in streptozotocin (STZ)‐induced diabetic male and female rats in this study. In animal experiments, females showed greater susceptibility towards developing diabetes because of lower insulin secretion and higher blood glucose levels as compared to male diabetic rats upon exposure to STZ. Immunoblotting confirmed sexually dimorphic regulation of DJ1 in various metabolic tissues such as the liver, pancreas and skeletal muscle. Immunofluorescence analysis revealed the location as well as reinforced the gender‐dependent expression of DJ1 in hepatic tissue. Co‐immunoprecipitation assay identified several interacting proteins with DJ1 whose functions were shown to be involved in various metabolic pathways viz. antioxidative and stress defence system, protein and methionine metabolism, nitrogen metabolism, urea metabolism, etc. Using GeneMANIA, a predictive web interface for gene functions, we showed for the first time that DJ1 may regulate T1DM via the JNK1 pathway, suggesting DJ1 interacts with other proteins from various metabolic pathways. We anticipate that the current data will provide insights into the aetiology of T1DM.     

Altered calcium signaling in colonic smooth muscle of type 1 diabetic mice

Seventy-six percent of diabetic patients develop gastrointestinal symptoms, such as constipation. However, the direct effects of diabetes on intestinal smooth muscle are poorly described. This study aimed to identify the role played by smooth muscle in mediating diabetes-induced colonic dysmotility. To induce type 1 diabetes, mice were injected intraperitoneally with low-dose streptozotocin once a day for 5 days. Animals developed hyperglycemia (>200 mg/dl) 1 wk after the last injection and were euthanized 7-8 wk after the last treatment. Computed tomography demonstrated decreased overall gastrointestinal motility in the diabetic mice. In vitro contractility of colonic smooth muscle rings from diabetic mice was also decreased. Fura-2 ratiometric Ca(2+) imaging showed attenuated Ca(2+) increases in response to KCl stimulation that were associated with decreased light chain phosphorylation in diabetic mice. The diabetic mice also exhibited elevated basal Ca(2+) levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca(2+) handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for <1 wk also showed decreased colonic contractile responses that were associated with decreased Ca(2+) increases in response to KCl stimulation, although without an elevation in basal Ca(2+) levels or a significant change in the expression of Ca(2+) signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca(2+) influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility.     

Kidney growth in normal and diabetic mice is not affected by human insulin-like growth factor binding protein-1 administration

Insulin-like growth factor I (IGF-I) accumulates in the kidney following the onset of diabetes, initiating diabetic renal hypertrophy. Increased renal IGF-I protein content, which is not reflected in messenger RNA (mRNA) levels, suggests that renal IGF-I accumulation is due to sequestration of circulating IGF-I rather than to local synthesis. It has been suggested that IGF-I is trapped in the kidney by IGF binding protein 1 (IGFBP-1). We administered purified human IGFBP-1 (hIGFBP-1) to nondiabetic and diabetic mice as three daily sc injections for 14 days, starting 6 days after induction of streptozotocin diabetes when the animals were overtly diabetic. Markers of early diabetic renal changes (i.e., increased kidney weight, glomerular volume, and albuminuria) coincided with accumulation of renal cortical IGF-I despite decreased mRNA levels in 20-day diabetic mice. Human IGFBP-1 administration had no effect on increased kidney weight or albuminuria in early diabetes, although it abolished renal cortical IGF-I accumulation and glomerular hypertrophy in diabetic mice. Increased IGF-I levels in kidneys of normal mice receiving hIGFBP-1 were not reflected on kidney parameters. IGFBP-1 administration in diabetic mice had only minor effects on diabetic renal changes. Accordingly, these results did not support the hypothesis that IGFBP-1 plays a major role in early renal changes in diabetes.     

Asparagus racemosus Willd (Liliaceae) ameliorates early diabetic nephropathy in STZ induced diabetic rats

Diabetic nephropathy is a major "microvascular" complication of diabetes, differs from other causes of chronic kidney diseases in its predictability, with well-defined functional progression from hyperfiltration to micro- to macroalbuminuria to renal failure. The present study was undertaken to investigate the effect of Asparagus racemosus Willd (Liliaceae) on streptozotocin-induced early diabetic nephropathy. Single i.p injection of streptozotocin (55 mg/kg) was administered to induce early diabetic nephropathy in Wistar rats and thereafter treated orally with ethanolic extract of Asparagus racemosus (EEAR) at a dose level of 100 and 250 mg/kg daily for 4 weeks. The efficacy of extract was compared with diabetic control rats. A. racemosus treatment significantly decreased plasma glucose, creatinine, urea nitrogen, total cholesterol and triglyceride levels. Renal hypertrophy, polyuria, hyperfiltration, microalbuminuria and abnormal changes in the renal tissue as well as oxidative stress were effectively attenuated by EEAR treatment. Basement membrane thickening and mesangial proliferation formation without nodules were seen in diabetic rats, whereas these structural changes were reduced in EEAR treated groups. Results of this study suggested that A. racemosus has beneficial effect in the treatment of diabetic     

Regulation of gene expression and biochemical changes in small intestine of newborn diabetic rats by exogenous ghrelin

The aim of this study was to investigate (i) the cholecystokinin, somatostatin and apelin mRNA levels, (ii) the changes in levels and localization of these peptides, (iii) relation between these peptides, (iv) antiapoptotic effects and (v) antioxidant effects of ghrelin. The rats were divided into four groups second day after birth. These groups were respectively treated with physiological saline, ghrelin (100μg/kg/day), streptozotocin (100mg/kg), ghrelin and streptozotocin. After four weeks, small intestine and blood samples were taken from rats. Cholecystokinin mRNA and peptide, somatostatin mRNA, release to duodenal lumen of apelin peptide and apelin mRNA signals decreased in ghrelin-treated diabetic rats compared to the diabetic group. There was no statistically significant difference among the four groups for somatostatin and apelin peptides. Caspase-3 signals were not observed only in diabetic group treated with ghrelin. Caspase-8 signals were increased while PCNA signals were decreased in diabetic group given ghrelin compared to diabetic group. Small intestine CAT, SOD, GP(x) and GST activities and GSH levels were decreased and LPO, PC levels were increased in diabetic rats. Administration of ghrelin to diabetic rats caused an increase in intestinal CAT, SOD, GP(x) and GST activities and GSH levels, while PC levels decreased. As a result, we observed positive changes in diabetic rats treated with ghrelin in both microscopic and biochemical studies. We can suggest that ghrelin may be an important hormone for the treatment of diabetes.     

Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.