Metformin action in human hepatocytes: coactivation of atypical protein kinase C alters 5′-AMP-activated protein kinase effects on lipogenic and gluconeogenic enzyme expression

Aims/hypothesis

Atypical protein kinase C (aPKC) levels and activity are elevated in hepatocytes of individuals with type 2 diabetes and cause excessive increases in the levels of lipogenic and gluconeogenic enzymes; aPKC inhibitors largely correct these aberrations. Metformin improves hepatic gluconeogenesis by activating 5′-AMP-activated protein kinase (AMPK). However, metformin also activates aPKC in certain tissues; in the liver, this activation could amplify diabetic aberrations and offset the positive effects of AMPK. In this study, we examined whether metformin activates aPKC in human hepatocytes and the metabolic consequences of any such activation.

Methods

We compared protein kinase activities and alterations in lipogenic and gluconeogenic enzyme levels during activity of the AMPK activators metformin and AICAR, relative to those of an aPKC-ι inhibitor, in hepatocytes from non-diabetic and type 2 diabetic human organ donors.

Results

Metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) activated aPKC at concentrations comparable with those required for AMPK activation. Moreover, both agents increased lipogenic enzyme levels by an aPKC-dependent mechanism. Thus, whereas insulin- and diabetes-dependent increases in lipogenic enzyme levels were reversed by aPKC inhibition, such levels were increased in hepatocytes from non-diabetic donors and remained elevated in hepatocytes from diabetic donors following metformin and AICAR treatment. In addition, whereas aPKC inhibition diminished gluconeogenic enzyme levels in the absence and presence of insulin in hepatocytes from both non-diabetic and diabetic donors, metformin and AICAR increased gluconeogenic enzyme levels in hepatocytes from non-diabetic individuals, but nevertheless diminished gluconeogenic enzyme levels in insulin-treated hepatocytes from diabetic donors.

Conclusions/interpretation

Metformin and AICAR activate aPKC together with AMPK in human hepatocytes. Activation of aPKC increases lipogenic enzyme levels and alters gluconeogenic enzyme levels, and therefore appears to offset the positive effects of AMPK.     

Berberine Attenuates Ischemia-Reperfusion Injury Via Regulation of Adenosine-5′-monophosphate Kinase Activity in Both Non-ischemic and Ischemic Areas of the Rat Heart

Background

Berberine exhibits numerous pharmacological effects, but the mechanism for its protective effects against ischemia-reperfusion cardiac injury is unknown.

Methods

Male Wistar rats were treated with berberine (100?mg/Kg/day, ig) for 14?days and controls treated with water. Hearts were isolated in vitro and perfused in the Langendorff mode and subjected to 30?min of global ischemia followed by 30?min of reperfusion and hemodynamic data examined. In a separate set of experiments, hearts were subjected in vivo to left anterior descending coronary artery ligation for 30?min followed by 120?min reperfusion and hemodynamic data, type and duration of arrhythmias, and myocardial infarct size determined. AMP-activated protein kinase (AMPK) level, ADP/ATP and AMP/ATP ratios were examined in non-ischemic areas and risk areas of the heart.

Results

Subsequent to ischemia-reperfusion injury, left ventricular developed pressure, left ventricular end diastolic pressure and maximum rate of intraventricular pressure contractility and relaxation were significantly improved in the berberine treatment groups compared to controls. Berberine treatment decreased infarct size and diminished the duration and incidence of arrhythmias compared to controls. Berberine treatment significantly decreased AMPK protein concentration, and the ratio of ADP/ATP and AMP/ATP in the myocardial risk areas. In contrast, berberine treatment significantly increased AMPK protein concentration, and the ratio of ADP/ATP and AMP/ATP in the non-ischemia areas compared to controls.

Conclusion

These findings suggest that berberine may exert its cardioprotective effect on ischemia-reperfusion injury via regulation of AMPK activity in both non-ischemic areas and risk areas of the heart.     

Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice

Aims/hypothesis

Metformin, the major target of which is liver, is commonly used to treat type 2 diabetes. Although metformin activates AMP-activated protein kinase (AMPK) in hepatocytes, the mechanism of activation is still not well known. To investigate AMPK activation by metformin in liver, we examined the role of reactive nitrogen species (RNS) in suppression of hepatic gluconeogenesis.

Methods

To determine RNS, we performed fluorescence examination and immunocytochemical staining in mouse hepatocytes. Since metformin is a mild mitochondrial complex I inhibitor, we compared its effects on suppression of gluconeogenesis, AMPK activation and generation of the RNS peroxynitrite (ONOO^?) with those of rotenone, a representative complex I inhibitor. To determine whether endogenous nitric oxide production is required for ONOO^? generation and metformin action, we used mice lacking endothelial nitric oxide synthase (eNOS).

Results

Metformin and rotenone significantly decreased gluconeogenesis and increased phosphorylation of AMPK in wild-type mouse hepatocytes. However, unlike rotenone, metformin did not increase the AMP/ATP ratio. It did, however, increase ONOO^? generation, whereas rotenone did not. Exposure of eNOS-deficient hepatocytes to metformin did not suppress gluconeogenesis, activate AMPK or increase ONOO^? generation. Furthermore, metformin lowered fasting blood glucose levels in wild-type diabetic mice, but not in eNOS-deficient diabetic mice.

Conclusions/interpretation

Activation of AMPK by metformin is dependent on ONOO^?. For metformin action in liver, intra-hepatocellular eNOS is required.     

METformin in DIastolic Dysfunction of MEtabolic Syndrome (MET-DIME) Trial: Rationale and Study Design

Purpose

Insulin resistance plays a central role in the pathophysiology of metabolic syndrome (MS). Its cardiac deleterious effects are characterized by an increase in fibrous tissue that increases myocardial stiffness and contributes to subclinical left ventricular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction in patients with MS. In addition to lifestyle counseling (LC), metformin treatment may attenuate or even reverse diastolic dysfunction in these patients. This trial aims to evaluate if treating non-diabetic patients with MS and LVDD with metformin in addition to LC improves diastolic function and assess its impact in functional capacity and health-related quality of life (HRQoL).

Design

MET-DIME is a phase II prospective, randomized, open-label, blinded-endpoint trial with a scheduled follow-up of 24 months. Fifty-four patients (adults 40–65 years old with AHA/NHLBI criteria of MS and rest LVDD) will be randomized by minimization to LC only or LC plus metformin (target dose of 1,000 mg twice daily). The primary endpoint will be change in mean of early diastolic mitral annular velocity, an echocardiographic parameter highly correlated with myocardial fibrosis (serial measurements will be performed at 6, 12 and 24 months). The secondary endpoints will include change in diastolic parameters at rest; metabolic, inflammatory and remodeling biomarkers; functional capacity; adipose tissue volumes and HRQoL.

Conclusion

MET-DIME is a pragmatic trial designed to evaluate if adding metformin to the standard treatment of patients with MS improves diastolic dysfunction, assessing its impact in metabolic homeostasis, proinflammatory state, functional capacity and HRQoL.     

Chronic Metformin Treatment is Associated with Reduced Myocardial Infarct Size in Diabetic Patients with ST-segment Elevation Myocardial Infarction

Purpose

Increased myocardial infarct (MI) size is associated with higher risk of developing left ventricular dysfunction, heart failure and mortality. Experimental studies have suggested that metformin treatment reduces MI size after induced ischaemia but human data is lacking. We aimed to investigate the effect of metformin on MI size in patients presenting with an acute MI.

Methods

All consecutive patients (n?=?3,288) presenting to our hospital with ST-segment elevation myocardial infarction (STEMI) undergoing primary PCI between January 2004 and December 2010 were included in this retrospective analysis. Patients with diabetes were divided according to metformin versus non-metformin based pharmacotherapy. MI size was estimated using peak values of serum creatine kinase (CK), myocardial band of CK (CK-MB), and troponin-T.

Results

We identified 677 (20.6 %) patients with diabetes, of whom 189 (27.9 %) were treated with metformin. Chronic metformin treatment was associated with lower peak levels of CK (1,101 vs. 1,422 U/L, P?=?0.005), CK-MB (152 vs. 182 U/L, P?=?0.018) and troponin-T (2.5 vs. 4.0 ng/L, P?=?0.021) compared to non-metformin using diabetics. After adjustment for age, sex, TIMI flow post PCI, and previous MI, the use of metformin treatment remained an independent predictor of smaller MI size. Patient with diabetes treated with metformin had even smaller MI size than patients without diabetes.

Conclusions

Chronic metformin treatment is associated with reduced MI size compared to non-metformin based strategies in diabetic patients presenting with STEMI. Metformin might have additional beneficial effects beyond glucose lowering efficacy.     

Novel Synthesized Radical-Containing Nanoparticles Limit Infarct Size Following Ischemia and Reperfusion in Canine Hearts

Purpose

Although nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) scavenge free radicals, their short half-life and considerable side effects such as systemic hypotension and bradycardia have limited their clinical application. Since a radical-containing nanoparticle (RNP) delivers nitroxyl radicals with a prolonged half-life specific to ischemic hearts, we investigated whether RNPs reduce infarct size without the occurrence of substantial side effects and whether nitric oxide (NO) contributes to the cardioprotective effects of RNPs.

Methods

The left anterior descending coronary arteries of dogs were occluded for 90 min, followed by reperfusion for 6 h. Either RNPs, micelles (not containing TEMPO) (control), or 4-hydroxy-TEMPO (TEMPOL) was injected into a systemic vein for 5 min before reperfusion. We evaluated the infarct size, myocardial apoptosis, plasma NO levels in coronary venous blood, and the RNP spectra using an electron paramagnetic resonance assay.

Results

RNPs reduced infarct size compared with the control group and TEMPOL group (19.5 ± 3.3 vs. 42.2 ± 3.7 vs. 30.2 ± 3.4%). RNPs also reduced myocardial apoptosis compared with the control and TEMPOL group. Coronary venous NO levels increased in the RNP group.

Conclusions

In conclusion, the administration of 2,2,6,6-tetramethylpiperidine-1-oxyl as a RNP exerted cardioprotective effects against ischemia and reperfusion injury in canine hearts without exerting unfavorable hemodynamic effects. RNPs may represent a promising new therapy for patients with acute myocardial infarction.

     

Pterostilbene Decreases Cardiac Oxidative Stress and Inflammation via Activation of AMPK/Nrf2/HO-1 Pathway in Fructose-Fed Diabetic Rats

Purpose

Oxidative stress has a pivotal role in the pathogenesis of diabetes-associated cardiovascular problems, which has remained a primary cause of the increased morbidity and mortality in diabetic patients. It is of paramount importance to prevent the diabetes-associated cardiac complications by reducing oxidative stress with the help of nutritional or pharmacological agents. Pterostilbene (PT), the primary antioxidant in blueberries, has recently gained attention for its promising health benefits in metabolic and cardiac diseases. However, the mechanism whereby PT reduces diabetic cardiac complications is currently unknown.

Methods

Sprague-Dawley rats were fed with 65% fructose diet with or without PT (20 mg kg^?1 day^?1) for 8 weeks. Heart rate and blood pressure were measured by tail-cuff apparatus. Real-time PCR and western blot experiments were executed to quantify the expression levels of mRNA and protein, respectively.

Results

Fructose-fed rats demonstrated cardiac hypertrophy, hypertension, enhanced myocardial oxidative stress, inflammation and increased NF-κB expression. Administration of PT significantly decreased cardiac hypertrophy, hypertension, oxidative stress, inflammation, NF-κB expression and NLRP3 inflammasome. We demonstrated that PT improved mitochondrial biogenesis as evidenced by increased protein expression of PGC-1α, complex III and complex V in fructose-fed diabetic rats. Further, PT increased protein expressions of AMPK, Nrf2, HO-1 in cardiac tissues, which may account for the prevention of cardiac oxidative stress and inflammation in fructose-fed rats.

Conclusions

Collectively, PT reduced cardiac oxidative stress and inflammation in diabetic rats through stimulation of AMPK/Nrf2/HO-1 signalling.

     

Aleglitazar,a Balanced Dual PPARα and -γ Agonist,Protects the Heart Against Ischemia-Reperfusion Injury

Purpose

To evaluate whether aleglitazar (Ale), a dual PPARα/γ agonist, has additive effects on myocardial protection against ischemia-reperfusion injury.

Methods

Human cardiomyocytes (HCMs), cardiomyocytes from cardiac-specific PPARγ knockout (MCM-PPARγ ^CKO ) or wild type (MCM-WT) mice were incubated with different concentrations of Ale, and subjected to simulated ischemia-reperfusion (SIR) or normoxic conditions (NSIR). Cell viability, apoptosis and caspase-3 activity were determined. HCMs were transfected with siRNA against PPARα (siPPARα) or PPARγ (siPPARγ) followed by incubation with Ale. PPARα/γ DNA binding capacity was measured. Cell viability, apoptosis and levels of P-AKT and P-eNOS were assessed. Infarct size following 30 min coronary artery occlusion and 24 h reperfusion were assessed in WT and db/db diabetic mice following 3-day pretreatment with vehicle, Ale or glimeperide.

Results

Ale (at concentrations of 150–600 nM) increased cell viability and reduced apoptosis in HCMs, MCM-WT and MCM-PPAR ^CKO exposed to SIR. In HCM, the protective effect was partially blocked by siPPARα alone or siPPARγ alone, and completely blocked by siPPARα+siPPARγ. Ale increased P-Akt/P-eNOS in HCMs. P-Akt or P-eNOS levels were decreased when PPARα alone, PPARγ alone and especially when both were knocked down. Peritoneal GTTs revealed that db/db mice had developed impaired glucose tolerance and insulin sensitivity, which were normalized by Ale or glimepiride treatment. Ale, but not glimepiride, limited infarct size in both WT and diabetic mice after ischemia-reperfusion.

Conclusions

Ale protects against myocardial apoptosis caused by hypoxia-reoxygenation in vitro and reduces infarct size in vivo.

     

Granulocyte-Colony Stimulating Factor Reduces Cardiomyocyte Apoptosis and Ameliorates Diastolic Dysfunction in Otsuka Long-Evans Tokushima Fatty Rats

Background

In recent studies, granulocyte-colony stimulating factor (G-CSF) was shown to improve cardiac function in myocardial infarction and non-ischemic cardiomyopathies. The mechanisms of these beneficial effects of G-CSF in diabetic cardiomyopathy are not yet fully understood. Therefore, we investigated the mechanisms of action of G-CSF on diabetic cardiomyopathy in a rat model of type 2 diabetes.

Methods

Seventeen-week-old OLETF (Otsuka Long Evans Tokushima Fatty) diabetic rats and LETO (Long Evans Tokushima Otuska) rats were randomized to treatment with 5 days of G-CSF (100 μg/kg/day) or with saline. Cardiac function was evaluated by serial echocardiography performed before and 4 weeks after treatment. We measured expression of the G-CSF receptor (GCSFR) and Bcl-2, as well as the extent of apoptosis in the myocardium.

Results

G-CSF treatment significantly improved cardiac diastolic function in the serial echocardiography assessments. Expression of G-CSFR was down-regulated in the diabetic myocardium (0.03?±?0.12 % vs. 1?±?0.15 %, p?<?0.05), and its expression was stimulated by G-CSF treatment (0.03?±?0.12 % vs. 0.42?±?0.06 %, p?<?0.05). In addition, G-CSF treatment increased the expression of Bcl-2 in the diabetic myocardium (0.69?±?0.06 % vs. 0.26?±?0.11 %, p?<?0.05), consistent with the reduced cardiomyocyte apoptosis (9.38?±?0.67 % vs. 17.28?±?2.16 %, p?<?0.05).

Conclusions

Our results suggest that G-CSF might have a cardioprotective effect in diabetic cardiomyopathy through up-regulation of G-CSFR, attenuation of apoptosis by up-regulation of Bcl-2 expression, and glucose-lowering effect. Our findings support the therapeutic potential of G-CSF in diabetic cardiomyopathy.     

Two Classes of Anti-Platelet Drugs Reduce Anatomical Infarct Size in Monkey Hearts

Background

Recent studies in rabbits have demonstrated that platelet P2Y₁₂ receptor antagonists are cardioprotective, and that the mechanism is surprisingly not related to blockade of platelet aggregation but rather to triggering of the same signal transduction pathway seen in pre- and postconditioning. We wanted to determine whether this same cardioprotection could be documented in a primate model and whether the protection was limited to P2Y₁₂ receptor antagonists or was a class effect.

Methods

Thirty-one macaque monkeys underwent 90-min LAD occlusion/4-h reperfusion.

Results

The platelet P2Y₁₂ receptor blocker cangrelor started just prior to reperfusion significantly decreased infarction by an amount equivalent to that seen with ischemic postconditioning (p?<?0.001). For any size of risk zone, infarct size in treated hearts was significantly smaller than that in control hearts. OM2, an investigational murine antibody against the primate collagen receptor glycoprotein (GP) VI, produced similar protection (p?<?0.01) suggesting a class effect. Both cangrelor and OM2 were quite effective at blocking platelet aggregation (94 % and 97 %, respectively).

Conclusions

Thus in a primate model in which infarct size could be determined directly platelet anti-aggregatory agents are cardioprotective. The important implication of these investigations is that patients with acute myocardial infarction who are treated with platelet anti-aggregatory agents prior to revascularization may already be in a postconditioned state. This hypothesis may explain why in recent clinical trials postconditioning-mimetic interventions which were so protective in animal models had at best only a modest effect.     

The effect of metformin on the recurrence of colorectal adenoma in diabetic patients with previous colorectal adenoma

Purpose

Existing studies suggest that metformin lowers the risk and mortality of colorectal cancer. However, the effect of metformin on the suppression and prevention of colorectal adenomas is not clear. The aim of this study was to evaluate the effect of metformin on the recurrence of colorectal adenoma in diabetic patients with previous colorectal adenoma.

Methods

Among 423 diabetic patients who underwent surveillance colonoscopy after resection of colorectal adenoma between 2005 and 2011, 257 patients were retrospectively reviewed. The patients were divided into two groups: one group comprising 106 patients who took metformin and another group comprising 151 patients who did not take metformin. The clinical characteristics, colorectal adenoma recurrence, and valuable factors for adenoma recurrence were analyzed.

Results

At surveillance colonoscopy after colonoscopic polypectomy for adenoma, 38 patients (35.8%) exhibited colorectal adenoma among 106 patients who took metformin, compared with 85 patients (56.3%) with colorectal adenoma among 151 patients who did not take metformin (odds ratio 0.434, 95% confidence interval 0.260–0.723, P = 0.001). Multivariate Cox analysis showed that metformin was associated with decreased recurrence of colorectal adenoma (hazard ratio 0.572, 95% confidence interval 0.385–0.852, P = 0.006) in diabetic patients with previous colorectal adenoma. The cumulative probability of colorectal adenoma recurrence was significantly lower in the metformin group than in the non-metformin group (P = 0.001).

Conclusion

Metformin use in diabetic patients with previous colorectal adenoma is associated with a lower risk of colorectal adenoma recurrence.

     

Angiotensin II Type 2 Receptor (AT2R) is Associated with Increased Tolerance of the Hyperthyroid Heart to Ischemia-Reperfusion

Background

Thyroid hormone induces cardiac hypertrophy and preconditions the myocardium against Ischemia/Reperfusion (I/R) injury. Type 2 Angiotensin II receptors (AT2R) are shown to be upregulated in cardiac hypertrophy observed in hyperthyroidism and this receptor has been reported to mediate cardioprotection against ischemic injury.

Methods

The aim of the present study was to evaluate the role of AT2R in the recovery of myocardium after I/R in isolated hearts from T3 treated rats. Male Wistar rats were treated with triiodothyronine (T3; 7 μg/100 g BW/day, i.p.) in the presence or not of a specific AT2R blocker (PD123,319; 10 mg/Kg) for 14 days, while normal rats served as control. After treatment, isolated hearts were perfused in Langendorff mode; after 30 min of stabilization, hearts were subjected to 20 min of zero-flow global ischemia followed by 25 min, 35 min and 45 min of reperfusion.

Results

T3 treatment induced cardiac hypertrophy, which was not changed by PD treatment. Post-ischemic recovery of cardiac function was increased in T3-treated hearts after 35 min and 45 min of reperfusion as compared to control and the ischemic contracture was accelerated and intensified. AT2R blockade was able to return the evaluated functional parameters of cardiac performance (LVDP, +dP/dt_máx and ?dP/dt_min) to the control condition. Furthermore, AT2R blockade prevented the increase in AMPK expression levels induced by T3, suggesting its possible involvement in this process.

Conclusion

AT2R plays a significant role in T3-induced cardioprotection.     

Cardioprotection by Farnesol: Role of the Mevalonate Pathway

Purpose

Farnesol is a key metabolite of the mevalonate pathway and known as an antioxidant. We examined whether farnesol treatment protects the ischemic heart.

Methods

Male Wistar rats were treated orally with 0.2, 1, 5, and 50 mg/kg/day farnesol/vehicle for 12 days, respectively. On day 13, the effect of farnesol treatment on cardiac ischemic tolerance and biochemical changes was tested. Therefore, hearts were isolated and subjected either to 30 min coronary occlusion followed by 120 min reperfusion to measure infarct size or to 10 min aerobic perfusion to measure cardiac mevalonate pathway end-products (protein prenylation, cholesterol, coenzyme Q9, coenzyme Q10, dolichol), and 3-nitrotyrosine (oxidative/nitrosative stress marker), respectively. The cytoprotective effect of farnesol was also tested in cardiomyocytes subjected to simulated ischemia/reperfusion.

Results

Farnesol pretreatment decreased infarct size in a U-shaped dose–response manner where 1 mg/kg/day dose reached a statistically significant reduction (22.3?±?3.9 % vs. 40.9?±?6.1 % of the area at risk, p?<?0.05). Farnesol showed a similar cytoprotection in cardiomyocytes. The cardioprotective dose of farnesol (1 mg/kg/day) significantly increased the marker of protein geranylgeranylation, but did not influence protein farnesylation, cardiac tissue cholesterol, coenzyme Q9, coenzyme Q10, and dolichol. While the cardioprotective dose of farnesol did not influence 3-nitrotyrosine, the highest dose of farnesol (50 mg/kg/day) tested did not show cardioprotection, however, it significantly decreased cardiac 3-nitrotyrosine.

Conclusions

This is the first demonstration that oral farnesol treatment reduces infarct size. The cardioprotective effect of farnesol likely involves increased protein geranylgeranylation and seems to be independent of the antioxidant effect of farnesol.     

Triple Therapy Greatly Increases Myocardial Salvage During Ischemia/Reperfusion in the in situ Rat Heart

Background

Cangrelor, a P2Y₁₂ receptor blocker, administered just prior to reperfusion reduced but did not eliminate myocardial infarction in rabbits. Combining cangrelor with ischemic postconditioning offered no additional protection suggesting they protected by a similar mechanism. To determine if cangrelor’s protection might be additive to other cardioprotective interventions we tested cangrelor in combination with ischemic preconditioning, cariporide, a sodium-hydrogen exchange blocker, and mild hypothermia.

Methods

Open-chest rats underwent 30-min coronary occlusion/2-h reperfusion.

Results

Cangrelor, administered as a bolus (60 μg/kg) 10 min before reperfusion and continued as an infusion (6 μg/kg/min) for the duration of the experiment, decreased infarction from 45.3 % of risk zone in control hearts to 25.0 %. Combining cangrelor and ischemic preconditioning offered no additional protection. Mild hypothermia (32–33 °C) instituted by peritoneal lavage with cold saline just prior to coronary occlusion resulted in 25.2 % infarction, and combining cangrelor and hypothermia nearly halved infarction to 14.1 % of risk zone. Cariporide (0.5 mg/kg) just prior to occlusion resulted in 27.2 % infarction and 15.8 % when combined with cangrelor. Combining cangrelor, hypothermia and cariporide further halved infarction to only 6.3 %. We also tested another P2Y₁₂ inhibitor ticagrelor which is chemically similar to cangrelor. Ticagrelor (20 mg/kg) fed 1 h prior to surgery reduced infarct size by an amount similar to that obtained with cangrelor (25.6 % infarction), and this protective effect was abolished by chelerythrine and wortmannin, thus implicating participation of PKC and PI3-kinase, resp., in signaling.

Conclusions

Cardioprotection from a P2Y₁₂ receptor antagonist can be combined with at least 2 other strategies to magnify the protection. Combining multiple interventions that use different cardioprotective mechanisms could provide powerful protection against infarction in patients with acute coronary thrombosis.     

Remote Postconditioning Induced by Trauma Protects the Mouse Heart against Ischemia Reperfusion Injury. Involvement of the Neural Pathway and Molecular Mechanisms

Purpose

Abdominal superficial surgical incision elicits cardioprotection against myocardial ischemia reperfusion (I/R) injury in mice. This cardioprotective phenomenon, termed remote preconditioning of trauma (RPCT), results in an 80 to 85 % reduction in cardiac infarct size. We evaluated cardioprotection and the molecular mechanisms of remote postconditioning of trauma (RPostCT) in a murine I/R injury model.

Methods

Mice were analyzed using a previously established I/R injury model. An abdominal superficial surgical incision was made 45 min after myocardial ischemia at the end of coronary occlusion, and infarct size was determined 24 h after reperfusion.

Results

The results indicated that a strong cardioprotective effect occurred during RPostCT (56.94 ± 2.71 % sham vs. 15.58 ± 2.16 % RPostCT; the mean area of the infarct divided by the mean area of the region at risk; p ≤ 0.05; n = 10). Furthermore, pharmacological intervention revealed neurogenic signaling involvement in the beneficial effects of RPostCT via sensory and sympathetic thoracic nerves. Pharmacological experiments in transgenic mice demonstrated that bradykinin receptors, β-adrenergic receptors (AR), and protein kinase C were implicated in the cardioprotective effects of RPostCT.

Conclusions

RPostCT significantly decreased myocardial infarction size via neurogenic transmission and various signaling pathways. This study describes a new cardiac I/R injury prevention method that might lead to the development of therapies that are more clinically relevant for myocardial I/R injury.

     

Exposure of mouse embryonic pancreas to metformin enhances the number of pancreatic progenitors

Aims/hypothesis

Developing beta cells are vulnerable to nutrient environmental signals. Early developmental processes that alter the number of pancreatic progenitors can determine the number of beta cells present at birth. Metformin, the most widely used oral agent for treating diabetes, alters intracellular energy status in part by increasing AMP-activated protein kinase (AMPK) signalling. This study examined the effect of metformin on developing pancreas and beta cells.

Methods

Pancreatic rudiments from CD-1 mice at embryonic day 13.0 (E13.0) were cultured with metformin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, an AMPK activator) or vehicle control in vitro. In another set of studies, pregnant C57BL/6 mice were treated with metformin throughout gestation. Embryonic (E14.0) and neonatal pancreases were then analysed for their morphometry.

Results

In vitro metformin treatment led to an increase in the proliferation and number of pancreatic duodenal homeobox 1-positive (PDX1⁺) progenitors. These results were reproduced by in vitro culture of embryonic pancreas rudiments with AICAR, suggesting that AMPK activation was involved. Similarly, metformin administration to pregnant dams induced an increase in both PDX1⁺ and neurogenin 3-positive progenitors in the embryonic pancreas at E14.0 and these changes resulted in an increased beta cell fraction in neonates.

Conclusions/interpretation

These results indicate that exposure to metformin during gestation modulates the early steps of beta cell development (prior to E14.0) towards an increase in the number of pancreatic and endocrine progenitors. These changes ultimately result in a higher beta cell fraction at birth. These findings are of clinical importance given that metformin is currently used for the treatment of gestational diabetes.     

Is l-Glutathione More Effective Than l-Glutamine in Preventing Enteric Diabetic Neuropathy?

Background

Diabetes and its complications appear to be multifactorial. Substances with antioxidant potential have been used to protect enteric neurons in experimental diabetes.

Aim

This study evaluated the effects of supplementation with l-glutamine and l-glutathione on enteric neurons in the jejunum in diabetic rats.

Methods

Rats at 90 days of age were distributed into six groups: normoglycemic, normoglycemic supplemented with 2 % l-glutamine, normoglycemic supplemented with 1 % l-glutathione, diabetic (D), diabetic supplemented with 2 % l-glutamine (DG), and diabetic supplemented with 1 % l-glutathione (DGT). After 120 days, the jejunums were immunohistochemically stained for HuC/D+ neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP). Western blot was performed to evaluate nNOS and VIP. Submucosal and myenteric neurons were quantitatively and morphometrically analyzed.

Results

Diabetic neuropathy was observed in myenteric HuC/D, nNOS, and VIP neurons (p < 0.05). In the submucosal plexus, diabetes did not change nitrergic innervation but increased VIPergic neuronal density and body size (p < 0.05). Supplementation with l-glutathione prevented changes in HuC/D neurons in the enteric plexus (p < 0.05), showing that supplementation with l-glutathione was more effective than with l-glutamine. Myenteric nNOS neurons in the DGT group exhibited a reduced density (34.5 %) and reduced area (p < 0.05). Submucosal neurons did not exhibit changes. The increase in VIP-expressing neurons was prevented in the submucosal plexus in the DG and DGT groups (p < 0.05).

Conclusion

Supplementation with l-glutathione exerted a better neuroprotective effect than l-glutamine and may prevent the development of enteric diabetic neuropathy.     

Aberrant gut microbiota composition at the onset of type 1 diabetes in young children

Aims/hypothesis

Recent studies indicate that an aberrant gut microbiota is associated with the development of type 1 diabetes, yet little is known about the microbiota in children who have diabetes at an early age. To this end, the microbiota of children aged 1–5 years with new-onset type 1 diabetes was compared with the microbiota of age-matched healthy controls.

Methods

A deep global analysis of the gut microbiota composition was established by phylogenetic microarray analysis using a Human Intestinal Tract Chip (HITChip).

Results

Principal component analyses highlighted the importance of age when comparing age-matched pairs. In pairs younger than 2.9 years, the combined abundance of the class Bacilli (notably streptococci) and the phylum Bacteroidetes was higher in diabetic children, whereas the combined abundance of members of Clostridium clusters IV and XIVa was higher in the healthy controls. Controls older than 2.9 years were characterised by a higher fraction of butyrate-producing species within Clostridium clusters IV and XIVa than was seen in the corresponding diabetic children or in children from the younger age groups, while the diabetic children older than 2.9 years could be differentiated by having an increased microbial diversity.

Conclusions/interpretation

The results from both age groups suggest that non-diabetic children have a more balanced microbiota in which butyrate-producing species appear to hold a pivotal position.