Renal activity of Akt kinase in obese Zucker rats

Insulin resistance (IR) and consequent hyperinsulinemia are hallmarks of Type 2 diabetes (DM2). Akt kinase (Akt) is an important molecule in insulin signaling, implicated in regulation of glucose uptake, cell growth, cell survival, protein synthesis, and endothelial nitric oxide (NO) production. Impaired Akt activation in insulin-sensitive tissues contributes to IR. However, Akt activity in other tissues, particularly those affected by complications of DM2, has been less studied. We hypothesized that hyperinsulinemia could have an impact on activity of Akt and its effectors involved in regulation of renal morphology and function in DM2. To address this issue, renal cortical Akt was determined in obese Zucker rats (ZO), a model of DM2, and lean controls (ZL). We also studied expression and phosphorylation of the mammalian target of rapamycin (mTOR) and endothelial NO synthase (eNOS), molecules downstream of Akt in the insulin signaling cascade, and documented modulators of renal injury. Akt activity was measured by a kinase assay with GSK-3 as a substrate. Expression of phosphorylated (active) and total proteins was measured by immunoblotting and immunohistochemistry. Renal Akt activity was increased in ZO as compared to ZL rats, in parallel with progressive hyperinsulinemia. No differences in Akt were observed in the skeletal muscle. Corresponding to increases in Akt activity, ZO rats demonstrated enhanced phosphorylation of renal mTOR. Acute PI3K inhibition with wortmannin (100 mug/kg) attenuated renal Akt and mTOR activities in ZO, but not in ZL rats. In contrast to mTOR, eNOS phosphorylation was similar in ZO and ZL rats, despite higher total eNOS expression. In conclusion, ZO rats demonstrated increases in renal Akt and mTOR activity and expression. However, eNOS phosphorylation did not follow this pattern. These data suggest that DM2 is associated with selective IR in the kidney, allowing pro-growth signaling via mTOR, whereas potentially protective effects mediated by eNOS are blunted.     

Molecular Mechanisms of the Relationship between Thyroid Dysfunctions and Diabetes Mellitus

Type 1 and type 2 diabetes mellitus (DM) are known to increase the incidence of thyroid gland (TG) dysfunctions. The review addresses the literature data and our experimental results on the molecular mechanisms that underlie thyroid disorders under DM. Most important of these mechanisms are the attenuation of thyrocyte adenylyl cyclase signaling system sensitivity to thyroid-stimulating hormone, the decrease in the number of thyroid hormone receptors in peripheral tissues, and the decline in activity as well as changes in the ratio of different deiodinase forms in these tissues. Decreased activity of D2 deiodinases, which convert thyroxine into the active form of triiodothyronine, is associated with the development of insulin resistance, while decreased activity of D3 deiodinases, which catalyze inactivation of triiodothyronine in pancreatic β cells, suppresses insulin secretion and leads to insulin deficiency. Thus, both the excess and the deficiency of thyroid hormones can entail diabetic pathology. Identification of thyroid disorders is of utmost importance for elaborating novel approaches to treat and prevent thyroid diseases associated with type 1 and type 2 DM.     

Renal activity of Akt kinase in experimental Type 1 diabetes

Akt kinase regulates numerous cell functions including glucose metabolism, cell growth, survival, protein synthesis, and control of local hemodynamics. mTOR is one of down-stream effectors of Akt involved in the initiation of protein translation. However, renal Akt signaling in Type 1 diabetes (DM) in vivo, in particular under the conditions reflecting differences in metabolic control, has received less attention. Renal cortical activity and expression of Akt and mTOR (kinase assay, western blotting) were determined in streptozotocin-diabetic rats (D) with different levels of glycemic control (blood glucose 22.0+/-1.0, 13.4+/-1.5, 8.1+/-0.4 mmol/l, p<0.05 between the groups), achieved by varying insulin treatment (0, 4 and 12 IU/day), and in control rats with (C4) or without (C) chronic insulin administration. Renal Akt activity was reduced in D rats without insulin treatment and severe hyperglycemia (D-0, -62 %, p<0.01 vs. C), partially restored in moderately hyperglycemic rats (D-4, -30 %, p<0.05 vs. C), and normalized in D rats with intensive insulin and tight metabolic control (D-12). Expression of active mTOR paralleled Akt activity in D-0 (-51 %, p<0.01 vs. C), but not in D-4 and D-12 that demonstrated increases in active mTOR (+55 %, +80 % resp., p<0.05) as compared to C. Moreover, insulin activated renal Akt (+82 %, p<0.01), but not mTOR in C4. In conclusion, glycemic control and intensity of insulin treatment are important modulators of renal Akt and mTOR activity in diabetes. While Akt activity is reversible by tight metabolic control, combination of hyperglycemia and insulin treatment resulted in enhancement of mTOR activity. In addition to Akt, other signaling pathways likely contribute to regulation of renal mTOR activity in diabetes.     

Altered insulin signaling in retinal tissue in diabetic states

Both type 1 and type 2 diabetes can lead to altered retinal microvascular function and diabetic retinopathy. Insulin signaling may also play a role in this process, and mice lacking insulin receptors in endothelial cells are protected from retinal neovascularization. To define the role of diabetes in retinal function, we compared insulin signaling in the retinal vasculature of mouse models of type 1 (streptozotocin) and type 2 diabetes (ob/ob). In streptozotocin mice, in both retina and liver, insulin receptor (IR) and insulin receptor substrate (IRS)-2 protein and tyrosine phosphorylation were increased by insulin, while IRS-1 protein and its phosphorylation were maintained. By contrast, in ob/ob mice, there was marked down-regulation of IR, IRS-1, and IRS-2 protein and phosphorylation in liver; these were maintained or increased in retina. In both mice, Phosphatidylinositol 3,4,5-trisphosphate generation by acute insulin stimulation was enhanced in retinal endothelial cells. On the other hand, protein levels and phosphorylation of PDK1 and Akt were decreased in retina of both mice. Interestingly, phosphorylation of p38 mitogen-activated protein kinase and ERK1 were responsive to insulin in retina of both mice but were unresponsive in liver. HIF-1alpha and vascular endothelial growth factor were increased and endothelial nitric-oxide synthase was decreased in retina. These observations indicate that, in both insulin-resistant and insulin-deficient diabetic states, there are alterations in insulin signaling, such as impaired PDK/Akt responses and enhanced mitogen-activated protein kinases responses that could contribute to the retinopathy. Furthermore, insulin signaling in retinal endothelial cells is differentially altered in diabetes and is also differentially regulated from insulin signaling in classical target tissues such as liver.     

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.     

Improved angiogenic activity of endothelial progenitor cell in diabetic patients treated with insulin plus metformin

Type 2 diabetes (T2DM) is associated with an increased vascular disease. Moreover, endothelial progenitor cell (EPC) function is impaired in diabetic patients. Decreased EPC number plays a critical role in reduced endothelial repair and development of the vascular disorder. To determine the effect of metformin and insulin plus metformin on functional activity of EPCs, 130 participants were divided into three groups (group 1: healthy control; group 2: metformin; group 3: insulin plus metformin). The concentration of EPCs in the circulation was first quantified. Thereafter, circulating EPCs (cEPCs) were harvested and the biological features of these cells including proliferative, clonogenicity, tubulogenic, and migratory properties were analyzed after expansion. The serum protein levels of some proangiogenic factors were also measured. Our results showed greater numbers of cEPCs in control and in diabetic patients treated with insulin plus metformin than in metformin-treated patients. Insulin plus metformin therapy was associated with augmented proliferative, clonogenicity, migratory, and tubulogenic activity of cEPCs in patients with T2DM. Increased serum concentrations of angiogenic factors were also observed in patients treated with insulin plus metformin. Western blot analysis showed increased protein levels of pTie-2/Tie2 and Pakt/AKT in cEPCs harvested from T2DM, treated with insulin metformin plus. This study showed that treatment with insulin plus metformin in diabetic patients is associated with increased mobilization of EPCs into the circulation, with potential beneficial effect in vascular protection in diabetic patients.     

Endothelin antagonism normalizes VEGF signaling and cardiac function in STZ-induced diabetic rat hearts

Abnormal alterations in cardiac expression of vascular endothelial growth factor (VEGF) as well as its receptors and impairment in the development of coronary collaterals have recently been reported in diabetic subjects. However, the presence of pharmacological intervention on these defects in diabetes remains unsettled. Here, we studied the effect of endothelin (ET) receptor blockade on cardiac VEGF signaling pathways and cardiac function in Sprague-Dawley rats 5 wk after induction of type I diabetes with streptozotocin (65 mg/kg ip) in comparison with age-matched control rats. After streptozotocin (1 wk), some diabetic rats were treated with the ET receptor antagonist SB-209670 (1 mg/day) for 4 wk. VEGF, its receptors, and its angiogenic signaling molecules [phosphorylated Akt and endothelial nitric-oxide synthase (eNOS)] were analyzed by Western blot, ELISA, real-time PCR, and immunohistochemistry, and cardiac function was evaluated by echocardiography. Coronary capillary morphology was assessed by lectin and enzymatic double staining. We found significant decreases in cardiac expression of VEGF, its receptors, phosphorylation of Akt and eNOS, and coronary capillary density in diabetic rats compared with controls. Treatment of diabetic rats with SB-209670 reversed these alterations to the control levels and ameliorated impairment of cardiac function. From a molecular point of view, the present study is the first to indicate the potential usefulness of an ET receptor antagonist in the treatment of cardiac dysfunction in type I diabetes.     

Nutrient excess and altered mitochondrial proteome and function contribute to neurodegeneration in diabetes

Diabetic neuropathy is a major complication of diabetes that results in the progressive deterioration of the sensory nervous system. Mitochondrial dysfunction has been proposed to play an important role in the pathogenesis of the neurodegeneration observed in diabetic neuropathy. Our recent work has shown that mitochondrial dysfunction occurs in dorsal root ganglia (DRG) sensory neurons in streptozotocin (STZ) induced diabetic rodents. In neurons, the nutrient excess associated with prolonged diabetes may trigger a switching off of AMP kinase (AMPK) and/or silent information regulator T1 (SIRT1) signaling leading to impaired peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α) expression/activity and diminished mitochondrial activity. This review briefly summarizes the alterations of mitochondrial function and proteome in sensory neurons of STZ-diabetic rodents. We also discuss the possible involvement of AMPK/SIRT/PGC-1α pathway in other diabetic models and different tissues affected by diabetes.     

Effect of selenite treatment on ultrastructural changes in experimental diabetic rat bones

It is known that streptozotocin (STZ)-induced diabetes causes functional and structural alterations in some types of tissue and organ. A number of methods have been used to characterize the properties of diabetic tissues and their diagnosis. Selenium compounds, playing an antioxidant role, can restore some altered metabolic parameters and diminished functions in experimental diabetes. The first aim of the present study was to investigate the effects of STZ-induced diabetes on structural properties of rat long bones. Electron and light microscopic observations showed deleterious alterations in the structure of the diabetic rat long bones, the most prominent effect being in osteocytic cells. Fine cytoplasmic processes of the osteocytes seemed to be shortened, and diabetes affected the normal cytoplasmic processes in a negative manner. The second aim of the present study was to evaluate the effects of sodium selenite treatment for 4 wk on the long bones of the diabetic rats. Electron and light microscopic observations demonstrated that sodium selenite treatment prevented the STZ-induced structural as well as ultrastructural changes in the long bones of the rats. In conclusion, this study first showed that a period of 5-wk diabetes was enough to cause some important and degenerative changes in the structure of the bone tissues, and, second, it demonstrated that sodium selenite treatment of the diabetic rats could normalize these alterations.     

Effect of metformin on testicular expression and localization of leptin receptor and levels of leptin in the diabetic mice

Diabetes mellitus impairs testicular activity and leads to infertility. Leptin is one of the endogenous regulators of the male reproductive functions, but the role of leptin and its receptor (LEPR/Ob‐R) in the control of testosterone production and testicular proliferation has not been investigated so far, especially in the Type 1 diabetes mellitus (DM1). Metformin is an anti‐hyperglycemic drug which is beneficial for treating the both DM2 and DM1. The aim of this work was to study the possible role of leptin and Ob‐R in the regulation of steroidogenesis and proliferation in the testes of mice with streptozotocin‐induced DM1 (75 mg/kg/day, 4 days) and to estimate the restoring effect of metformin treatment (500 mg/kg, 2 weeks) on the diabetic testes. In the diabetic testes, the plasma and intratesticular leptin levels and plasma testosterone levels were reduced and completely restored by metformin treatment. Metformin also restored the expression of the steroidogenic transport protein steroidogenic acute regulatory protein reduced in DM1. In the diabetic testes, the expression of Ob‐R was downregulated and the immunolocalization of Ob‐R showed weak staining in the Leydig cells, the primary spermatocytes and the round spermatids. The germ cell proliferation was also reduced in DM1, as noticed with proliferating cell nuclear antigen (PCNA) expression. Metformin increased the Ob‐R expression and immunostaining in the different cell types and improved the PCNA expression. Thus, DM1 impairs the testicular steroidogenesis and proliferation by inhibiting the leptin signaling, causing a decrease in leptin levels and Ob‐R expression in the testes of diabetic mice, while metformin improves the leptin signaling and restores testosterone production and testicular proliferation.     

Adiponectin Attenuates Streptozotocin-Induced Tau Hyperphosphorylation and Cognitive Deficits by Rescuing PI3K/Akt/GSK-3β Pathway

Clinical studies have demonstrated that decreased adiponectin is associated with the development of Type 2 diabetes mellitus (T2DM) and Alzheimer’s disease (AD). We focused on determining the neuroprotective effect offered by adiponectin against streptozotocin-induced brain damage in ICV-STZ rat model. We found that adiponectin supplements significantly restored the cognitive deficits in ICV-STZ rat model including shorter escape latency, more crossing times and increased time spent in the target quadrant. Adiponectin supplements also increased number of dendritic branches and mushroom percentage. In addition, adiponectin supplements attenuated tau hyperphosphorylation at multiple AD-related sites through activation of protein Ser9-phosphorylated glycogen synthase kinase-3β (Ser9-GSK-3β) with increased the Akt and PI3K activity. Our data suggest that adiponectin supplements have neuroprotective effects on the ICV-STZ rat model, which may be mediated by the activation of the PI3K/Akt/GSK-3β signaling pathway.     

Vascular PTPs: Current developments and challenges for exploitation in Type 2 diabetes-associated vascular dysfunction

Protein Tyrosine Phosphatases (PTPs) are important contributors to vascular cells normal function, by balancing signaling proteins activation exerted by phosphorylating kinases. Type 2 diabetes related insults, such as hyperglycemia, oxidative stress, and insulin resistance disturb the phosphorylation/dephosphorylation equilibrium towards an abnormal augmented phosphorylation of signaling proteins associated with changes in PTPs expression, enzymatic activity and interaction with cellular substrates. We briefly review here: (i) the new findings on receptor and non-receptor PTPs and their role in vascular cells, (ii) several data on oxidation and phosphorylation of these molecules in endothelial and smooth muscle cells, (iii) vascular PTPs intrinsic activity and dysregulation under the insults of diabetic milieu, and (iv) the potential use of PTPs and their inhibitors as therapeutic targets in Type 2 diabetes-associated vascular dysfunction.     

siRNA-based gene silencing reveals specialized roles of IRS-1/Akt2 and IRS-2/Akt1 in glucose and lipid metabolism in human skeletal muscle

Type 2 diabetes is associated with defects in insulin signaling and the resulting abnormal glucose and lipid metabolism. The complexity of insulin signaling cascades is highlighted by the existence of multiple isoforms of target proteins implicated in metabolic and gene-regulatory events. We utilized siRNA to decipher the specific role of predominant insulin receptor substrates and Akt isoforms expressed in human skeletal muscle. Gene silencing revealed specialized roles of insulin signaling cascades to metabolic endpoints. IRS-1 and Akt2 were required for myoblast differentiation and glucose metabolism, whereas IRS-2 and Akt1 were dispensable. A key role of IRS-2 and Akt1 in lipid metabolism was revealed, highlighting reciprocal relationships between metabolic pathways. Unraveling the isoform-specific regulation of glucose and lipid metabolism by key elements along insulin signaling cascades through siRNA-mediated gene silencing in human tissues will facilitate the discovery of novel targets for the treatment of diabetes and related metabolic disorders.     

c-Jun N-terminal kinase pathways in diabetes

Type 2 diabetes develops from insulin resistance and has become a worldwide epidemic. The c-Jun N-terminal kinases have been considered as signaling molecules linking inflammation and insulin resistance. Genetic disruption of c-Jun N-terminal kinase-1 gene prevents the development of insulin resistance in obese and diabetic mice. Inhibition of c-Jun N-terminal kinases by a small cell-permeable peptide improves insulin sensitivity in mice. Hepatic inhibition of c-Jun N-terminal kinases using a dominant-negative protein or knockdown of c-Jun N-terminal kinase-1 gene by RNA interference reduces blood glucose and insulin levels and enhances hepatic insulin signaling in mice. Recent evidence demonstrates that the hepatic c-Jun N-terminal kinase pathway plays an important role in lipid and lipoprotein homeostasis in mice. This review discusses recent advances in our understanding of the role of c-Jun N-terminal kinase pathway in metabolic control and its potential as a target for the treatment of type 2 diabetes.     

Macroangiopathy in adults and children with diabetes: from molecular mechanisms to vascular damage (part 1).

Type 2 diabetes mellitus (T2DM) is an increasing problem in childhood; however type 1 diabetes mellitus (T1DM) remains by far the most common type of diabetes in this age group. In this review we will focus on T1DM, because this will have the greatest implication for patients diagnosed in childhood. During the atherosclerotic process, several molecular, receptorial and cellular factors provide a continous mechanism of vascular damage. In diabetic children this state seems to be enhanced and facilitated so that accelerated atherosclerosis is associated with an increased risk of cardiovascular events in respect to the non diabetic population. Hyperglycemia PER SE and associated with diabetes is an important risk factor for atherosclerosis. At present a substantial part of children with diabetes do not reach satisfactory glycemic control. Other risk factors for the development and progression of atherosclerosis may be inherited or develop in the course of the disease: hypertension, dyslipidemia, insulin resistance, obesity, cigarette smoking, physical inactivity, disturbance of platelet function, coagulation and fibrinolysis. The development and progression of atherosclerosis should be blocked at an early age, if possible. Primary prevention to all risk factors for cardiovascular disease is important and intervention is indicated if necessary. At the moment the best therapeutic strategy is to maintain metabolic control at a physiologic level and perform screening and early intervention for vascular complications.     

Ca handling alterations and vascular dysfunction in diabetes

More than 65% of patients with diabetes mellitus die from cardiovascular disease or stroke. Hyperglycemia, due to either reduced insulin secretion or reduced insulin sensitivity, is the hallmark feature of diabetes mellitus. Vascular dysfunction is a distinctive phenotype found in both types of diabetes and could be responsible for the high incidence of stroke, heart attack, and organ damage in diabetic patients. In addition to well-documented endothelial dysfunction, Ca²⁺ handling alterations in vascular smooth muscle cells (VSMCs) play a key role in the development and progression of vascular complications in diabetes. VSMCs provide not only structural integrity to the vessels but also control myogenic arterial tone and systemic blood pressure through global and local Ca²⁺ signaling. The Ca²⁺ signalosome of VSMCs is integrated by an extensive number of Ca²⁺ handling proteins (i.e. channels, pumps, exchangers) and related signal transduction components, whose function is modulated by endothelial effectors. This review summarizes recent findings concerning alterations in endothelium and VSMC Ca²⁺ signaling proteins that may contribute to the vascular dysfunction found in the diabetic condition.     

Effects of a selective endothelin a receptor antagonist on the expressions of iNOS and eNOS in the heart of early streptozotocin-induced diabetic rats

Vascular tone is regulated through the actions of locally produced agents. Among the vasoconstrictors, the most potent agent is endothelin (ET), which exerts its vasoconstrictor actions principally through ET type A (ET(A)) receptors. Of the vasodilators, nitric oxide (NO) seems to be the most important contributor to the acute regulation of vascular tone. Vasculopathy is an important feature of diabetes mellitus (DM). Endogenous ET-mediated vasoconstrictor tone is augmented in diabetic states, and conflicting results persist concerning the NO system in diabetes. The present study investigated the expressions of inducible NO synthases (iNOS) and endothelial NOS (eNOS) in the heart of diabetic animals and the effects of a selective ET(A) receptor antagonist on these alterations. Type I diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats, while control (Con) rats received only citrate buffer. After 1 week, the streptozotocin-administered rats were randomly divided into two groups: the selective ET(A) receptor antagonist-administered group (DM+TA-0201, 1 mg/kg/day, by osmotic minipump for 2 weeks) and the DM+vehicle group (comprising the diabetic rats that received saline). The random blood glucose level was 405 +/- 103 mg/dl in DM animals, and this level was unchanged by ET antagonism. Body weight was more greatly decreased in DM rats than in Con rats, but the left ventricle to body weight ratio was increased in the DM group and was unaffected by ET antagonism. Protein expressions of eNOS and iNOS were assessed in the left ventricular tissues. eNOS expression was significantly increased in DM heart and was greatly inhibited by the treatment with ET antagonist. The expression of iNOS was also increased in early DM heart but was reversed by the ET antagonist. Thus, endothelin antagonism might be beneficial for DM heart by reversing the upregulated eNOS and iNOS expressions.     

The effects of type 1 diabetes on the hypothalamic, pituitary and testes axis

Type 1 diabetes is an autoimmune disorder characterized by a lack of insulin production by the beta cells of the pancreas. This lack of insulin causes a variety of systemic effects on whole-body metabolism. Poorly managed type 1 diabetes can lead to cardiovascular disease, diabetic neuropathy, and diabetic retinopathy. Increasingly, even well-managed type 1 diabetic patients show damage to peripheral organs related to complications from the disease. The central role of insulin in energy homeostasis also renders it an important signaling factor in the reproductive tract. type 1 diabetes has now been demonstrated to cause defects in sperm and testes. The aim of this review is to present the known effects of insulin's role in the function of the male reproductive tract. These effects might be mediated through hormonal alterations in the hypothalamic pituitary gonadal axis or through the direct interaction of insulin on the testes and sperm cells. Although fertility complications also occur in type 2 diabetic males, this review will focus on the defects specifically linked with the lack of insulin seen in type 1 diabetes.     

Magnesium metabolism in type 2 diabetes mellitus, metabolic syndrome and insulin resistance

Type 2 diabetes is characterized by cellular and extracellular Mg depletion. Epidemiologic studies showed a high prevalence of hypomagnesaemia and lower intracellular Mg concentrations in diabetic subjects. Insulin and glucose are important regulators of Mg metabolism. Intracellular Mg plays a key role in regulating insulin action, insulin-mediated-glucose uptake and vascular tone. Reduced intracellular Mg concentrations result in a defective tyrosine-kinase activity, post-receptorial impairment in insulin action, and worsening of insulin resistance in diabetic patients. Mg deficit has been proposed as a possible underlying common mechanism of the "insulin resistance" of different metabolic conditions. Low dietary Mg intake is also related to the development of type 2 diabetes. Benefits of Mg supplementation on metabolic profile in diabetic subjects have been found in most, but not all clinical studies, and larger prospective studies are needed to support the potential role of dietary Mg supplementation as a possible public health strategy in diabetes risk.