Insulin-deficient Rats Research Articles

Apigetrin-enriched Pulmeria alba extract prevents assault of STZ on pancreatic β-cells and neuronal oxidative stress with concomitant attenuation of tissue damage and suppression of inflammation in the brain of diabetic rats

In the present study, in vitro, in vivo, and in silico models were used to evaluate the therapeutic potential of Pulmeria alba methanolic (PAm) extract, and we identified the major phytocompound, apigetrin. Our in vitro studies revealed dose-dependent increased glucose uptake and inhibition of α-amylase (50% inhibitory concentration (IC50)= 217.19 µg/mL), antioxidant (DPPH, ferric-reducing activity of plasma (FRAP), and lipid peroxidation (LPO) [IC50 = 103.23, 58.72, and 114.16 µg/mL respectively]), and anti-inflammatory potential (stabilizes human red blood cell (HRBC) membranes, and inhibits proteinase and protein denaturation [IC50 = 143.73, 131.63, and 198.57 µg/mL]) by the PAm extract. In an in vivo model, PAm treatment reversed hyperglycemia and attenuated insulin deficiency in rats with streptozotocin (STZ)-induced diabetes. A post-treatment tissue analysis revealed that PAm attenuated neuronal oxidative stress, neuronal inflammation, and neuro-cognitive deficiencies. This was evidenced by increased levels of antioxidants enzymes (superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH)), and decreased malondialdehyde (MDA), proinflammatory markers (cyclooxygenase 2 (COX2), nuclear factor (NF)-κB and nitric oxide (NOx)), and acetylcholinesterase (AChE) activities in the brain of PAm-treated rats compared to the STZ-induced diabetic controls. However, no treatment-related changes were observed in levels of neurotransmitters, including serotonin and dopamine. Furthermore, STZ-induced dyslipidemia and alterations in serum biochemical markers of hepatorenal dysfunction were also reversed by PAm treatment. Extract characterization identified apigetrin (retention time: 21,227 s, 30.48%, m/z: 433.15) as the major bioactive compound in the PAm extract. Consequently, we provide in silico insights into the potential of apigetrin to target AChE/COX-2/NOX/NF-κB Altogether the present study provides preclinical evidence of the therapeutic potential of the apigetrin-enriched PAm extract for treating oxidative stress and neuro-inflammation associated with diabetes.

The experimental model of type 2 diabetes mellitus caused by a high-fat diet with low-dose streptozotocin in rats

Aim . To develop a pathogenetically reasonable model of type 2 diabetes with marked peripheral insulin resistance and relative insulin deficiency in rats using a high-fat diet and a single injection of streptozotocin in the low dose. Materials and methods. Experiments were conducted on 16 outbred male rats. Type 2 diabetes model in experimental animals was achieved by feeding them with high-fat diet (55% of energy from fat) for 28 days followed by a single injection of streptozotocin (35 mg/kg). The serum glucose and insulin concentrations in rats were measured before streptozotocin administration and at the end of the experiment. To estimate insulin resistance, insulin tolerance test and glucose tolerance test were performed. Total protein, albumin, total and direct bilirubin, urea, uric acid, total cholesterol, high-density lipoproteins and low-density lipoproteins, and activity of alanine aminotransferase and aspartate aminotransferase were measured in the blood serum. Results . A high-fat diet with a single injection of streptozotocin resulted in lipid and protein metabolism disorders and peripheral tissues insulin resistance in experimental animals. Basal insulin levels did not change against the backdrop of high glucose level. Conclusions . These results indicate that feeding rats with a high-fat diet (55% of calories from fats) and a single administration of streptozotocin at a low dose (35 mg/kg) reproduce general pathological processes of type 2 diabetes. This model can be used to study the pathogenesis of type 2 diabetes as well as to investigate the effect of potential hypoglycemic agents.

Evidence for a circulating factor released by the brain that contributes to chronic antidiabetic actions of leptin

We previously demonstrated that leptin has powerful central nervous system (CNS)‐mediated antidiabetic actions that are independent of the autonomic nervous system (ANS) and the pituitary. In this study, we examined if a circulating factor released by the brain contributes to leptin's chronic antidiabetic actions in insulin deficient rats. Male inbred 4‐week‐old Lewis rats (n=14) were conjoined by attaching their skin to allow shared circulation. Six weeks later, the rats were implanted with an intracerebroventricular (ICV) cannula into the lateral ventricle to infuse leptin in one rat and vehicle in the other rat of each pair. Eight days after surgery, food intake, body weight and blood glucose concentration were measured for 5 consecutive days and then streptozotocin (STZ) was injected (40 mg/kg) to induce insulin‐deficient diabetes in both rats. Five days after STZ injection, leptin (15 mg/day) or vehicle (0.5 ml/h) was delivered ICV continuously for 7 days via osmotic minipumps. STZ injection significantly increased average blood glucose concentration (359±19 vs. 79±6 mg/dL) and food intake (49±2 vs. 39±3 g/day). Despite higher food intake, average body weight decreased after STZ injection (619±23 vs. 664±23 g). Chronic ICV leptin infusion restored normoglycemia (110±31 vs. 359±19 mg/dL) in leptin‐treated rats and reduced blood glucose by ~27% in the paired parabiotic rat infused with vehicle (269±20 vs. 366±41 mg/dL). Leptin treatment did not reduce food intake (53±20 vs. 49±23 g/day) or body weight (619±23 vs. 610±20 g) in the paired rats. After stopping leptin treatment, blood glucose concentration returned to diabetic values in leptin‐treated (305±44 vs. 110±31 mg/dL) as well as in vehicle‐treated rats (330±11 vs. 269±20 mg/dL), and food intake increased 37% (67±5 vs. 49±3 g/day). These results suggest that the chronic antidiabetic effects of leptin may involve release into the systemic circulation of a brain‐derived neuroendocrine factor that enhances glucose homeostasis in insulin deficient rats. (NHLBI‐PO1HL51971, NIGMS P20GM104357)Support or Funding InformationDepartment of Physiology and Biophysics and Mississippi Center for Obesity Research. University of Mississippi Medical Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NORMAL VASCULAR REACTIVITY IS RESTORED BY APIGENIN IN DIABETIC RATS

Objective: Diabetes is a disease whose complications have serious implications for the health of sufferers; one of the most serious such complications is the deterioration of vascular reactivity. Apigenin is a natural flavonoid with PKC inhibiting and antioxidant properties. In this study, the impact of apigenin on vascular reactivity deterioration was investigated.Methods: Insulin resistance (IR) and insulin deficiency (ID) were induced by fructose and streptozotocin respectively. The isolated aortae vasoconstriction response to phenylephrine (PE) and potassium chloride (KCl) in addition to the vasodilation response to acetylcholine (ACh) and sodium nitroprusside (SNP) were tested.Results: IR and ID were associated with significantly exaggerated vasoconstriction to KCl and PE while significantly impaired vasodilation to ACh. Response to SNP was not significantly affected by both IR and ID. In vitro incubation with apigenin (7 7µM) for 20 min restored normal responses to PE, KCl and ACh in aortae isolated from insulin-resistant or insulin-deficient rats. Incubation for one hour with the PKC stimulant, phorbol 12-myristate 13-acetate (PMA, 800 nM) resulted in aortic impairment similar to that seen in aortae isolated from IR and ID animals. Incubation with both apigenin prevented PMA-induced exaggerated vasoconstriction response to both PE and KCl.Conclusion: Apigenin alleviates vascular exaggerated vasoconstriction and impaired dilation associated with diabetes or PKC activated.

Insulin deficiency results in reversible protein kinase A activation and tau phosphorylation

Alzheimer's disease (AD) is a highly prevalent multifactorial disease for which Diabetes Mellitus (DM) is a risk factor. Abnormal phosphorylation and aggregation of tau is a key hallmark of AD. In animal models, DM induces or exacerbates the phosphorylation of tau, suggesting that DM may influence the risk at AD by directly facilitating tau pathology. Previously we reported that tau phosphorylation induced in response to metabolic stress is reversible. Since identification and understanding of early players in tau pathology is pivotal for therapeutic intervention, we here investigated the mechanism underlying tau phosphorylation in the diabetic brain and its potential for reversibility. To model DM we used streptozotocin-treatment to induce insulin deficiency in rats. Insulin depletion leads to increased tau phosphorylation in the brain and we investigated the activation status of known tau kinases and phosphatases in this model. We identified protein kinase A (PKA) as a tau kinase activated by DM in the brain. The potential relevance of this signaling pathway to AD pathogenesis is indicated by the increased level of active PKA in temporal cortex of early stage AD patients. Our data indicate that activation of PKA and tau phosphorylation are associated with insulin deficiency per se, rather than the downstream energy deprivation. In vitro studies confirm that insulin deficiency results in PKA activation and tau phosphorylation. Strikingly, both active PKA and induced tau phosphorylation are reversed upon insulin treatment in the steptozotocin animal model. Our data identify insulin deficiency as a direct trigger that induces the activity of the tau kinase PKA and results in tau phosphorylation. The reversibility upon insulin treatment underscores the potential of insulin as an early disease-modifying intervention in AD and other tauopathies.

Glucagon receptor antibody completely suppresses type 1 diabetes phenotype without insulin by disrupting a novel diabetogenic pathway

Insulin monotherapy can neither maintain normoglycemia in type 1 diabetes (T1D) nor prevent the long-term damage indicated by elevated glycation products in blood, such as glycated hemoglobin (HbA1c). Here we find that hyperglycemia, when unaccompanied by an acute increase in insulin, enhances itself by paradoxically stimulating hyperglucagonemia. Raising glucose from 5 to 25 mM without insulin enhanced glucagon secretion ∼two- to fivefold in InR1-G9 α cells and ∼18-fold in perfused pancreata from insulin-deficient rats with T1D. Mice with T1D receiving insulin treatment paradoxically exhibited threefold higher plasma glucagon during hyperglycemic surges than during normoglycemic intervals. Blockade of glucagon action with mAb Ac, a glucagon receptor (GCGR) antagonizing antibody, maintained glucose below 100 mg/dL and HbA1c levels below 4% in insulin-deficient mice with T1D. In rodents with T1D, hyperglycemia stimulates glucagon secretion, up-regulating phosphoenolpyruvate carboxykinase and enhancing hyperglycemia. GCGR antagonism in mice with T1D normalizes glucose and HbA1c, even without insulin.

Loss of the Anorexic Response to Systemic 5-Aminoimidazole-4-Carboxamide-1-β-D-Ribofuranoside Administration Despite Reducing Hypothalamic AMP-Activated Protein Kinase Phosphorylation in Insulin-Deficient Rats

This study tested whether chronic systemic administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) could attenuate hyperphagia, reduce lean and fat mass losses, and improve whole-body energy homeostasis in insulin-deficient rats. Male Wistar rats were first rendered diabetic through streptozotocin (STZ) administration and then intraperitoneally injected with AICAR for 7 consecutive days. Food and water intake, ambulatory activity, and energy expenditure were assessed at the end of the AICAR-treatment period. Blood was collected for circulating leptin measurement and the hypothalami were extracted for the determination of suppressor of cytokine signaling 3 (SOCS3) content, as well as the content and phosphorylation of AMP-kinase (AMPK), acetyl-CoA carboxylase (ACC), and the signal transducer and activator of transcription 3 (STAT3). Rats were thoroughly dissected for adiposity and lean body mass (LBM) determinations. In non-diabetic rats, despite reducing adiposity, AICAR increased (∼1.7-fold) circulating leptin and reduced hypothalamic SOCS3 content and food intake by 67% and 25%, respectively. The anorexic effect of AICAR was lost in diabetic rats, even though hypothalamic AMPK and ACC phosphorylation markedly decreased in these animals. Importantly, hypothalamic SOCS3 and STAT3 levels remained elevated and reduced, respectively, after treatment of insulin-deficient rats with AICAR. Diabetic rats were lethargic and displayed marked losses of fat and LBM. AICAR treatment increased ambulatory activity and whole-body energy expenditure while also attenuating diabetes-induced fat and LBM losses. In conclusion, AICAR did not reverse hyperphagia, but it promoted anti-catabolic effects on skeletal muscle and fat, enhanced spontaneous physical activity, and improved the ability of rats to cope with the diabetes-induced dysfunctional alterations in glucose metabolism and whole-body energy homeostasis.

Ethanolic extract of Commiphora mukul gum resin attenuates streptozotocin-induced alterations in carbohydrate and lipid metabolism in rats.

The purpose of this study was to investigate the effects of Commiphora mukul gum resin ethanolic extract (CMEEt) administration against altered activities of key enzymes of carbohydrate metabolism, lipid metabolism and changes in glycogen content (liver and muscle) and lipids (liver and heart) in streptozotocin (STZ) induced insulin deficient diabetic Wistar albino rats. Diabetes was induced by intraperitoneal injection of STZ (55 mg/kg body wt) to male Wistar rats. The animals were divided into four groups: Control (C), control-treated (C+CM), diabetic (D) and diabetic-treated group (D+CM). Diabetic-treated and control-treated rats were treated with C. mukul gum resin ethanolic extract (CMEEt) in 2 ml distilled water, orally (200 mg/kg body weight/day for 60 days). At the end of the experimental period, biochemical parameters related to carbohydrate and lipid metabolism were assayed. The significant enhancement in tissue lipids (heart and liver) total cholesterol, triglycerides, phospholipids and free fatty acids of diabetic rats were nearer to normalized in diabetic treated rats (D+CM). Alterations in the activities of enzymes of glucose metabolism (hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-phosphatase, fructose-1,6-bisphosphatase and glucose-6-phosphate dehydrogenase) and lipid metabolism (fatty acid synthase, malic enzyme and lipoprotein lipase) as observed in diabetic (D) rats were prevented with CMEEt administration. In conclusion, our findings indicate improvement of glucose and lipid metabolisms in STZ induced diabetic rats by treatment with Commiphora mukul and suggest that the plant can be used as an adjuvant for the prevention and/or management of insulin deficiency and disorder related to it.

Quercetin Protects against Diabetes-Induced Exaggerated Vasoconstriction in Rats: Effect on Low Grade Inflammation

Vascular complications are the leading cause of morbidity and mortality in patients with diabetes. Quercetin is an important flavonoid with antioxidant and anti-inflammatory activity. Here, the effect of quercetin on diabetes-induced exaggerated vasoconstriction in insulin deficient and insulin resistant rat models was investigated. Insulin deficiency was induced by streptozotocin while, insulin resistance by fructose. Rats were left 8 weeks or 12 weeks after STZ or fructose administration respectively. Quercetin was daily administered in the last 6 weeks. Then, tail blood pressure (BP) was recorded in conscious animals; concentration-response curves for phenylephrine (PE) and KCl were studied in thoracic aorta rings. Non-fasting blood glucose level, serum insulin level, insulin resistance index, serum tumour necrosis factor-α (TNF-α) and serum C-reactive protein (CRP) were determined. Nuclear transcription factor-κB (NF-κB) was assessed by immunofluorescence technique. Histopathological examination was also performed. The results showed that quercetin protected against diabetes-induced exaggerated vasoconstriction and reduced the elevated blood pressure. In addition, quercetin inhibited diabetes associated adventitial leukocyte infiltration, endothelial pyknosis and increased collagen deposition. These effects were accompanied with reduction in serum level of both TNF-α and CRP and inhibition of aortic NF-κB by quercetin in both models of diabetes. On the other hand, quercetin did not affect glucose level in any of the used diabetic models. This suggests that the protective effect of quercetin is mediated by its anti-inflammatory effect rather than its metabolic effects. In summary, quercetin is potential candidate to prevent diabetic vascular complications in both insulin deficiency and resistance via its inhibitory effect on inflammatory pathways especially NF-κB signaling.

Chronic Treatment with the AMP-Kinase Activator AICAR Increases Glycogen Storage and Fatty Acid Oxidation in Skeletal Muscles but Does Not Reduce Hyperglucagonemia and Hyperglycemia in Insulin Deficient Rats

This study tested whether the glycogen-accumulating effect of chronic in vivo pharmacological 5′AMP-activated protein kinase (AMPK) activation could improve glycemic control under conditions of insulin deficiency. Male Wistar rats were rendered diabetic through the administration of streptozotocin (STZ) and then treated for 7 consecutive days with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Subsequently, glycogen content and synthesis, glucose oxidation, and fatty acid oxidation (FAO) were determined in oxidative and glycolytic skeletal muscles. Glycemia, insulinemia, glucagonemia, and circulating triglycerides (TG) and non-esterified fatty acids (NEFAs) were measured after AICAR treatment. Insulin was almost undetectable in STZ rats and these animals were severely hyperglycemic. Glycogen content was markedly low mainly in glycolytic muscles of STZ rats and AICAR treatment restored it to control values. No differences were found among all muscles studied with regards to the content and phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3. Even though glycogen synthase content was reduced in all muscles from STZ rats, insulin-induced dephosphorylation/activation of this enzyme was preserved and unaffected by AICAR treatment. Glucagon and NEFAS were 2- and 7.4-fold fold higher in STZ rats than controls, respectively. AICAR did not affect hyperglycemia and hyperglucagonemia in STZ rats; however, it normalized circulating NEFAs and significantly increased FAO in glycolytic muscles. In conclusion, even though AICAR-induced AMPK activation enhanced glycogen accumulation in glycolytic muscles and normalized circulating NEFAs and TG levels, the hyperglycemic effects of glucagon likely offset the potentially glucose-lowering effects of AICAR, resulting in no improvement of glycemic control in insulin-deficient rats.

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.

Significance of Insulin Signaling in Liver Regeneration Triggered by Portal Vein Ligation

To investigate the role of insulin signaling in liver regeneration following portal vein ligation (PVL). Streptozotocin-induced insulin-deficient rats underwent PVL, and were sacrificed at indicated time points. Liver regeneration indices, including volumetric shifting, BrdU, proliferative cell nuclear antigen (PCNA), and Ki-67 labeling index, were determined. Cell cycle markers, telomerase reverse transcriptase (TERT), and apoptosis-related genes were detected using quantitative real time polymerase chain reaction (PCR). Cell cycle analysis was determined using flow cytometry. Expression of insulin-like growth factor receptor (IGFR)-2 and TGFβ1 were determined using Western blot. Restituted liver mass and redistributed volume ratio of insulin-deficient rats were decreased compared with those of normal rats. Labeling index of BrdU and PCNA of insulin-deficient rats were increased compared with normal rats, evidenced by an increased S-phase fraction detected by flow cytometry. Expression of cyclin A2, cyclin B1, TERT mRNA, and telomerase activity were decreased in insulin-deficient rats. Increased Bax, Daxx, and JNK mRNA expression and decreased Bcl X(L) expression in insulin-deficient rats, led to increased hepatocyte apoptosis than normal rats. Finally, expression of IGFR-2 was increased in insulin-deficient rats. Insulin signaling plays an important role in liver regeneration triggered by portal vein ligation.

GHTD-amide: A naturally occurring beta cell-derived peptide with hypoglycemic activity

In the early 1970s, a peptide fraction with insulin potentiating activity was purified from human urine but the identity and origins of the active constituent remained unknown. Here we identify the active component and characterize its origins. The active peptide was identified as an alpha amidated tetrapeptide with the sequence GHTD-amide. The peptide was synthesized and tested for stimulation of glycogen synthesis and insulin potentiation by insulin tolerance testing in insulin-deficient rats, which confirmed GHTD-amide as the active peptide. Tissue localization using a peptide-specific anti-serum and epifluorescent and confocal microscopy showed decoration of pancreatic islets but not other tissues. Confocal microscopy revealed co-localization with insulin and immunogold and electron microscopy showed localization to dense core secretory granules. Consistent with these observations GHTD-amide was found in media conditioned by MIN6 islet beta cells. Sequence database searching found no annotated protein in the human proteome encoding a potential precursor for GHTD-amide. We conclude that the insulin potentiating activity originally described in human urine is attributable to the tetrapeptide GHTD-amide. GHTD-amide is a novel peptide produced by pancreatic beta cells and no precursor protein is present in the annotated human proteome. Stimulation of glycogen synthesis and co-localization with insulin in beta cells suggest that GHTD-amide may play a role in glucose homeostasis by enhancing insulin action and glucose storage in tissues.

Amelioration of streptozotocin-induced diabetes in the rat by exercise-training: role of muscle glycogenolytic and glycolytic enzyme activity.

Streptozotocin-induced insulin deficient rats allowed to run at will had significantly (P less than 0.001) lower mean (+/- SEM) plasma glucose levels (12.1 +/- 0.9 vs 22.6 +/- 1.7 mM/l) than did equally insulin deficient sedentary rats. Muscle glycogen phosphorylase and synthase activities were similar in exercise-trained and sedentary diabetic rats, and were unchanged from control values. In contrast, muscle phosphofructokinase activity was reduced (P less than 0.001) in sedentary rats with insulin deficiency, and the defect was significantly reversed (P less than 0.01) when such rats were allowed to run spontaneously. These results are consistent with the view that the ability of exercise-training to attenuate the magnitude of hyperglycemia in streptozotocin-induced insulin deficiency is associated with an effect on a key regulatory enzyme in the glycolytic pathway.

Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons

Many organ systems are adversely affected by diabetes, including the brain, which undergoes changes that may increase the risk of cognitive decline. Although diabetes influences the hypothalamic-pituitary-adrenal axis, the role of this neuroendocrine system in diabetes-induced cognitive dysfunction remains unexplored. Here we demonstrate that, in both insulin-deficient rats and insulin-resistant mice, diabetes impairs hippocampus-dependent memory, perforant path synaptic plasticity and adult neurogenesis, and the adrenal steroid corticosterone contributes to these adverse effects. Rats treated with streptozocin have reduced insulin and show hyperglycemia, increased corticosterone, and impairments in hippocampal neurogenesis, synaptic plasticity and learning. Similar deficits are observed in db/db mice, which are characterized by insulin resistance, elevated corticosterone and obesity. Changes in hippocampal plasticity and function in both models are reversed when normal physiological levels of corticosterone are maintained, suggesting that cognitive impairment in diabetes may result from glucocorticoid-mediated deficits in neurogenesis and synaptic plasticity.

Hypoinsulinemia regulates amphetamine-induced reverse transport of dopamine.

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level–dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders.

Insulin is not a prerequisite for rapid regulation of neuropeptide Y gene transcription in the arcuate nucleus in food-restricted rats

Neuropeptide Y (NPY), synthesized in the arcuate nucleus of the hypothalamus, is one of the most potent orexigenic neuropeptides in the brain. The NPY neurons project to other hypothalamic nuclei, such as paraventricular nucleus (PVN), and it is reported that NPY contents in the PVN, but not NPY mRNA levels in the arcuate nucleus, decreased rapidly after food consumption. While many signals reflecting energy balance in the periphery are integrated at the NPY neurons, insulin has been implicated as one of the key regulators for NPY neurons. In the present study, we first examined whether there exist dynamic changes in NPY gene transcription in the arcuate nucleus in association with food intake in rats which had access to food only 4h a day. To detect possible changes in NPY gene transcription, we measured the expression levels of NPY heteronuclear (hn) RNA, a sensitive indicator of gene transcription, with intronic in situ hybridization. Our data showed that NPY hnRNA levels in the arcuate nucleus decreased rapidly after food consumption. We next examined whether postprandial increases in insulin release might contribute to the rapid downregulation of NPY gene transcription. To do so, insulin-deficient rats by streptozotocin injection were subjected to the same paradigm. Our data showed that NPY hnRNA levels also decreased rapidly after food consumption, suggesting that the postprandial increase in insulin release is not a prerequisite for the rapid downregulation of NPY gene transcription in the arcuate nucleus.

Proteolytic Mechanisms, Not Malnutrition, Cause Loss of Muscle Mass in Kidney Failure

Hypoalbuminemia and muscle atrophy are frequently found in patients with chronic kidney disease (CKD) and patients being treated by dialysis. These abnormalities are usually attributed to malnutrition, meaning that they are caused by an inadequate diet. However, the evidence indicates that malnutrition is rarely the mechanism causing loss of protein stores. Instead, low values of serum albumin are closely related to the presence of inflammation and loss of muscle mass is attributable to activation of specific proteases. In uremic rodents and patients, the initial step in the loss of muscle protein is an activation of caspase-3. This cleaves the complex structure of muscle, and its action can be detected by the presence of a characteristic 14-kDa actin fragment in the insoluble fraction of muscle. The second step in uremia-induced loss of muscle protein is an activation of the ubiquitin-proteasome system, which rapidly degrades proteins released by caspase-3 cleavage of muscle proteins. Activation of both caspase-3 and the ubiquitin-proteasome system occur when there is suppression of the cellular signaling pathway activated by insulin/insulinlike growth factor 1, the phosphatidylinositol 3-kinase/Akt pathway. A potential therapeutic target for preventing loss of muscle protein is to stimulate activity of this signaling pathway.

Chronic antidiabetic and cardiovascular actions of leptin: role of CNS and increased adrenergic activity

This study examined the importance of direct central nervous system (CNS) actions and increased adrenergic activity in mediating the chronic antidiabetic and cardiovascular actions of leptin. Insulin-deficient rats (streptozotocin, 50 mg/kg) were used to examine the effects of leptin on glucose homeostasis independent of changes in insulin. Male Sprague-Dawley rats were instrumented with arterial and venous catheters and intracerebroventricular cannula for 24-h/day blood pressure (BP) and heart rate (HR) monitoring and intravenous and intracerebroventricular infusions. Insulin-deficient diabetes was associated with marked hyperglycemia, hyperphagia, decreased BP, and pronounced fall in HR. Leptin treatment, intravenous or intracerebroventricular, completely restored to control values plasma glucose levels (384+/-58 to 102+/-28 and 307+/-38 to 65+/-7 mg/dl, respectively), food intake, BP, and HR (304+/-8 to 364+/-7 and 317+/-13 to 423+/-9 bpm, respectively). Combined blockade of alpha1-, beta1-, and beta2-adrenergic receptors attenuated the rise in HR by 30 to 50% but had no effect on the antidiabetic and dietary actions of leptin. Blockade of beta3-adrenergic receptors did not attenuate the chronic cardiovascular or metabolic effects of leptin. These data demonstrate that leptin, via its direct actions in the CNS, has powerful antidiabetic actions in insulin-deficient rats independent of increased peripheral alpha1, beta1, beta2, and beta3-adrenergic activity. Leptin also exerts important long-term cardiovascular actions that are partially mediated via alpha1- and beta1/beta2-adrenergic activation. These findings provide new insights into novel pathways for long-term control of glucose homeostasis and cardiovascular regulation.

Hypoglycemic potency of novel trivalent chromium in hyperglycemic insulin-deficient rats

Two sources of chromium III, “chromium 454” and “chromium picolinate,” were tested in insulin-deficient Streptozocin-treated diabetic rats. This model was selected in order to evaluate the possibility of any hypoglycemic potency of chromium in a relative absence of blood insulin concentration. Three weeks of the treatment with CRC454 and CrP resulted in a 38% and 11% reduction of blood glucose levels, respectively. Body weight gains were equally improved by both treatments. Blood levels of CK, ALT and AST were significantly reduced by CRC454 and CrP. These results might suggest that any hypoglycemic effect of trivalent chromium under insulin-deficient conditions could be largely dependent upon the type of chromium agent and associated characteristics such as solubility and bioavalibility. In contrast, improvement of body weight gains and blood levels of CK, AST and ALT seems to be less dependent on the type of chromium compound under these experimental conditions. In conclusion, CRC454 showed significant reduction of hyperglycemia under insulin-deficient conditions.