Mauricio Krause

Reactive oxygen and nitrogen species generation, antioxidant defenses, and β-cell function: a critical role for amino acids

Growing evidence indicates that the regulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels is essential for maintaining normal β-cell glucose responsiveness. While long-term exposure to high glucose induces oxidative stress in β cells, conflicting results have been published regarding the impact of ROS on acute glucose exposure and their role in glucose stimulated insulin secretion (GSIS). Although β cells are considered to be particularly vulnerable to oxidative damage, as they express relatively low levels of some peroxide-metabolizing enzymes such as catalase and glutathione (GSH) peroxidase, other less known GSH-based antioxidant systems are expressed in β cells at higher levels. Herein, we discuss the key mechanisms of ROS/RNS production and their physiological function in pancreatic β cells. We also hypothesize that specific interactions between RNS and ROS may be the cause of the vulnerability of pancreatic β cells to oxidative damage. In addition, using a hypothetical metabolic model based on the data available in the literature, we emphasize the importance of amino acid availability for GSH synthesis and for the maintenance of β-cell function and viability during periods of metabolic disturbance before the clinical onset of diabetes.

Exercise and possible molecular mechanisms of protection from vascular disease and diabetes: the central role of ROS and nitric oxide

It is now widely accepted that hypertension and endothelial dysfunction are associated with an insulin-resistant state and thus with the development of T2DM (Type 2 diabetes mellitus). Insulin signalling is impaired in target cells and tissues, indicating that common molecular signals are involved. The free radical NO* regulates cell metabolism, insulin signalling and secretion, vascular tone, neurotransmission and immune system function. NO* synthesis is essential for vasodilation, the maintenance of blood pressure and glucose uptake and, thus, if levels of NO* are decreased, insulin resistance and hypertension will result. Decreased blood levels of insulin, increased AngII (angiotensin II), hyperhomocysteinaemia, increased ADMA (asymmetric omega-NG,NG-dimethylarginine) and low plasma L-arginine are all conditions likely to decrease NO* production and which are associated with diabetes and cardiovascular disease. We suggest in the present article that the widely reported beneficial effects of exercise in the improvement of metabolic and cardiovascular health are mediated by enhancing the flux of muscle- and kidney-derived amino acids to pancreatic and vascular endothelial cells aiding the intracellular production of NO*, therefore resulting in normalization of insulin secretion, vascular tone and insulin sensitivity. Exercise may also have an impact on AngII and ADMA signalling and the production of pro- and anti-inflammatory cytokines in muscle, so reducing the progression and development of vascular disease and diabetes. NO* synthesis will be increased during exercise in the vascular endothelial cells so promoting blood flow. We suggest that exercise may promote improvements in health due to positive metabolic and cytokine-mediated effects.

l-Arginine is essential for pancreatic β-cell functional integrity, metabolism and defense from inflammatory challenge

In this work, our aim was to determine whether L-arginine (a known insulinotropic amino acid) can promote a shift of β-cell intermediary metabolism favoring glutathione (GSH) and glutathione disulfide (GSSG) antioxidant responses, stimulus-secretion coupling and functional integrity. Clonal BRIN-BD11 β-cells and mouse islets were cultured for 24 h at various L-arginine concentrations (0-1.15  mmol/l) in the absence or presence of a proinflammatory cytokine cocktail (interleukin 1β, tumour necrosis factor α and interferon γ). Cells were assessed for viability, insulin secretion, GSH, GSSG, glutamate, nitric oxide (NO), superoxide, urea, lactate and for the consumption of glucose and glutamine. Protein levels of NO synthase-2, AMP-activated protein kinase (AMPK) and the heat shock protein 72 (HSP72) were also evaluated. We found that L-arginine at 1.15  mmol/l attenuated the loss of β-cell viability observed in the presence of proinflammatory cytokines. L-arginine increased total cellular GSH and glutamate levels but reduced the GSSG/GSH ratio and glutamate release. The amino acid stimulated glucose consumption in the presence of cytokines while also stimulating AMPK phosphorylation and HSP72 expression. Proinflammatory cytokines reduced, by at least 50%, chronic (24 h) insulin secretion, an effect partially attenuated by L-arginine. Acute insulin secretion was robustly stimulated by L-arginine but this effect was abolished in the presence of cytokines. We conclude that L-arginine can stimulate β-cell insulin secretion, antioxidant and protective responses, enabling increased functional integrity of β-cells and islets in the presence of proinflammatory cytokines. Glucose consumption and intermediary metabolism were increased by L-arginine. These results highlight the importance of L-arginine availability for β-cells during inflammatory challenge.

Divergence of intracellular and extracellular HSP72 in type 2 diabetes: does fat matter?

Mammalian cells have developed a range of adaptations to survive against acute and prolonged (but not lethal) stresses (Beckmann et al. 1992). Among these adaptations, the heat shock response is the most conserved, being found in all prokaryotes and eukaryotes (Locke and Noble 1995). Heat shock proteins (HSPs) are considered part of a family of proteins known as “stress proteins” since their expression is induced by a wide range of stressors, such as oxidative stress (Krause et al. 2007), thermal stress (Yang et al. 1996), ischaemia (Richard et al. 1996), exercise (Krause et al. 2007), metabolic stress (Beckmann et al. 1992) and many others. The genes encoding Hsps are highly conserved, and many of these genes and their protein products can be assigned to different families on the basis of their typical molecular weight (kDa): HSP110 (or officially named HSPH), HSP90 (or HSPC), HSP70 (or HSPA), HSP60 (or HSPD1), HSP40 (or DNAJ) and small hsp families (HSPB) (Kampinga et al. 2009). In eukaryotes, many families comprise multiple members that differ in inducibility, intracellular localization and function (Feder and Hofmann 1999). HSP72 (or HSPA1A) is the inducible and the most abundant of all HSPs, accounting for 1–2% of cellular protein and being rapidly induced during cell stress (Noble et al. 2008), especially in the skeletal muscle cells (Madden et al. 2008). As molecular chaperones, the intracellular HSP70 protein (iHSP72) can interact with other proteins (unfolded, in non-native state and/or stress-denatured conformations) to avoid inappropriate interactions, formation of protein aggregates and degradation of damaged proteins, as well as helping the correct refolding of proteins (Madden et al. 2008). Other HSP functions include protein translocation (Chirico et al. 1988), anti-apoptosis (Garrido et al. 2001) and anti-inflammatory responses (Homem de Bittencourt et al. 2007). The anti-inflammatory role of the iHSP72 is mediated by its interaction with the proteins involved in the activation of the nuclear factor κ-B (NF-κB), blocking its translocation to the nucleus and inducing the cessation of the inflammatory process (Homem de Bittencourt et al. 2007; Silveira et al. 2007). More recently, the HSP roles have been expanded to include control of cell signalling (Calderwood et al. 2007), modulation of immune response (Johnson and Fleshner 2006) and for chronic diseases conditions (Kampinga et al. 2007), such as diabetes, control of obesity and insulin resistance (Chung et al. 2008; Krause and Rodrigues-Krause Jda 2011). Recent data has indicated that a lack of iHSP72 response to stress can be linked to the levels of insulin resistance in skeletal muscle cells (Chung et al. 2008; Kurucz et al. 2002). Patients with T2DM have been shown to have reduced iHSP72 gene expression, which has been correlated with reduced insulin sensitivity (Kurucz et al. 2002). Furthermore, earlier studies in patients with T2DM reported that hot tub therapy improved glycaemic control (Bernstein 2000; Bathaie et al. 2010; Hooper 1999), additionally showing an inverse correlation between iHSP72 messenger RNA (mRNA) expression and the degree of T2DM (Kurucz et al. 2002). The underlying mechanisms behind the lower induction of iHSP72 expression in diabetic patients is not fully understood, but appears to be connected to the activation of proteins involved on inflammatory response (and sensitive to changes in redox state), such as the c-jun amino terminal kinase (JNK), the inhibitor of IκB (IKK), the tumor necrosis factor alpha (TNF-α) and nuclear factor kappa b (NF-κB) (Chung et al. 2008). Activation of these proteins appears to inhibit the major inductor of HSP72, the heat shock factor 1 (HSF-1), leading to a low iHSP72 expression and induction of the stress response (Chung et al. 2008). Currently, several HSP-inducing drugs are under investigation or in clinical trials for diabetic neuropathy, neurodegenerative diseases (Westerheide and Morimoto 2005; Kurthy et al. 2002) and for the prevention of insulin resistance and treatment of impaired glycaemic control (Kurucz et al. 2002; Gupte et al. 2009a; Literati-Nagy et al. 2009; Vitai et al. 2009; Kavanagh et al. 2009). Considering that skeletal muscle is the major tissue responsible for whole body insulin-mediated glucose uptake, disturbances in skeletal muscle, such as oxidative stress and inflammatory processes, can easily progress to insulin resistance and diabetes (Newsholme et al. 2009). Physical exercise is a known inducer of glucose uptake and is also a substantial inductor of iHSP72 expression (Krause et al. 2007). This may explain, in part, the beneficial effects of exercise in diabetic patients. Therefore, strategies to increase skeletal muscle iHSP72 expression (or over-expression) could result in improved glycaemic control, reduced insulin resistance and avoidance of T2DM. Heat shock proteins were long thought to be exclusive cytoplasmic proteins with functions restricted to the intracellular compartment. However, an increasing number of observations have shown that they may be released into the extracellular space (eHSP72) and have a wide variety of effects on other cells (Tytell 2005). The eHSP72 function is in general associated with the activation of the immune system (Whitham and Fortes 2008). For example, eHSP72 has been reported as an inductor of neutrophils microbicidal capacity (Ortega et al. 2006) and chemotaxis (Ortega et al. 2009), recruitment of NK (natural killer) cells (Horn et al. 2007) as well as cytokine production in immune cells (Asea et al. 2000; Johnson and Fleshner 2006). Besides that, eHSP72 was recently shown to be involved in the inducement of neural cell protection under stress conditions (Krause and Rodrigues-Krause Jda 2011). In addition, hyperglycaemia is known to be involved in inflammation and vascular complications associated with diabetes, arising from reactive oxygen species generation and action (Wei et al. 2009; Wright et al. 2006). As oxidative stress is a powerful inductor of iHSP72 (Krause et al. 2007), it is expected that during inflammatory and oxidative stress states in diabetes, the levels of these proteins in the extracellular medium (plasma and serum) may be higher in diabetic than non-diabetic participants. Indeed, type 1 (Oglesbee et al. 2005) and T2DM patients have higher levels of eHSP72, and this response has been related to the duration of the disease (Nakhjavani et al. 2010). In addition, serum eHSP72 concentrations are positively correlated with markers of inflammation, such as C-reactive proteins, monocyte count, and TNF-α (Mayer and Bukau 2005; Njemini et al. 2004). In summary, while intracellular levels of iHSP72 are decreased in T2DM and correlated with insulin resistance, extracellular eHSP72 levels are increased and correlated with oxidative damage and stress. To date, no study has investigated both intracellular and extracellular levels of HSP72 in T2DM patients simultaneously. Herein, we aimed to determine the levels of HSP72, intracellularly (skeletal muscle) and extracellularly (blood plasma) in three groups of patients: obese without T2DM and obese with T2DM and non-obese with T2DM. We also determined HSF-1 skeletal muscle expression for all groups.

A Whey Protein Hydrolysate Promotes Insulinotropic Activity in a Clonal Pancreatic β-Cell Line and Enhances Glycemic Function in ob/ob Mice

Whey protein hydrolysates (WPHs) represent novel antidiabetic agents that affect glycemia in animals and humans, but little is known about their insulinotropic effects. The effects of a WPH were analyzed in vitro on acute glucose-induced insulin secretion in pancreatic BRIN-BD11 β cells. WPH permeability across Caco-2 cell monolayers was determined in a 2-tiered intestinal model. WPH effects on insulin resistance were studied in vivo following an 8-wk oral ingestion (100 mg/kg body weight) by ob/ob (OB-WPH) and wild-type mice (WT-WPH) compared with vehicle control (OB and WT groups) using a 2 × 2 factorial design, genotype × treatment. BRIN-BD11 cells showed a robust and reproducible dose-dependent insulinotropic effect of WPH (from 0.01 to 5.00 g/L). WPH bioactive constituents were permeable across Caco-2 cell monolayers. In the OB-WPH and WT-WPH groups, WPH administration improved glucose clearance after a glucose challenge (2 g/kg body weight), as indicated by differences in the area under curves (AUCs) (P ≤ 0.05). The basal plasma glucose concentration was not affected by WPH treatment in either genotype. The plasma insulin concentration was lower in the OB-WPH than in the OB group (P ≤ 0.005) but was similar between the WT and WT-WPH groups; the interaction genotype × treatment was significant (P ≤ 0.005). Insulin release from pancreatic islets isolated from the OB-WPH group was greater (P ≤ 0.005) than that from the OB group but did not differ between the WT-WPH and WT groups; the interaction genotype × treatment was not significant. In conclusion, an 8-wk oral administration of WPH improved blood glucose clearance, reduced hyperinsulinemia, and restored the pancreatic islet capacity to secrete insulin in response to glucose in ob/ob mice. Hence, it may be useful in diabetes management.

Type 1 diabetes: can exercise impair the autoimmune event? The L-arginine/glutamine coupling hypothesis

Prevention of type 1 diabetes mellitus (T1DM) requires early intervention in the autoimmune process directed against β‐cells of the pancreatic islets of Langerhans, which is believed to result from a disorder of immunoregulation. According to this concept, a T‐helper lymphocyte of type 1 (Th1) subset of T‐lymphocytes and their cytokine products, the type 1 cytokines [e.g. interleukin 2 (IL‐2), interferon gamma (IFN‐γ) and tumour necrosis factor beta (TNF‐β)] prevail over immunoregulatory (anti‐inflammatory) Th2 subset and its cytokine products, i.e. type 2 cytokines (e.g. IL‐4, IL‐6 and IL‐10). This allows type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in β‐cell destruction. Activation of sympathetic‐corticotropin‐releasing hormone (CRH) axis by psychological stress induces specifically Th1 cell overactivity that determines enhanced glutamine utilization and consequent poor L‐arginine supply for nitric oxide (NO)‐assisted insulin secretion. This determines the shift of intraislet glutamate metabolism from the synthesis of glutathione (GSH) to that of L‐arginine, leading to a redox imbalance that activates nuclear factor κB exacerbating inflammation and NO‐mediated cytotoxicity. Physical exercise is capable of inducing changes in the pattern of cytokine production and release towards type 2 class and to normalize the glutamine supply to the circulation, which reduces the need for glutamate, whose metabolic fate may be restored in the direction of GSH synthesis and antioxidant defence. Also, the 70‐kDa heat shock protein (hsp70), which is immunoregulatory, may modulate exercise‐induced anti‐inflammation. In this work, we envisage how exercise can intervene in the mechanisms involved in the autoimmune process against β‐cells and how novel therapeutic approaches may be inferred from these observations. Copyright

The Chaperone Balance Hypothesis: The Importance of the Extracellular to Intracellular HSP70 Ratio to Inflammation-Driven Type 2 Diabetes, the Effect of Exercise, and the Implications for Clinical Management

Recent evidence shows divergence between the concentrations of extracellular 70 kDa heat shock protein [eHSP70] and its intracellular concentrations [iHSP70] in people with type 2 diabetes (T2DM). A vital aspect regarding HSP70 physiology is its versatility to induce antagonistic actions, depending on the location of the protein. For example, iHSP70 exerts a powerful anti-inflammatory effect, while eHSP70 activates proinflammatory pathways. Increased eHSP70 is associated with inflammatory and oxidative stress conditions, whereas decreased iHSP70 levels are related to insulin resistance in skeletal muscle. Serum eHSP70 concentrations are positively correlated with markers of inflammation, such as C-reactive protein, monocyte count, and TNF-α, while strategies to enhance iHSP70 (e.g., heat treatment, chemical HSP70 inducers or coinducers, and physical exercise) are capable of reducing the inflammatory profile and the insulin resistance state. Here, we present recent findings suggesting that imbalances in the HSP70 status, described by the [eHSP70]/[iHSP70] ratio, may be determinant to trigger a chronic proinflammatory state that leads to insulin resistance and T2DM development. This led us to hypothesize that changes in this ratio value could be used as a biomarker for the management of the inflammatory response in insulin resistance and diabetes.

Amino acid supplementation and impact on immune function in the context of exercise

Moderate and chronic bouts of exercise may lead to positive metabolic, molecular, and morphological adaptations, improving health. Although exercise training stimulates the production of reactive oxygen species (ROS), their overall intracellular concentration may not reach damaging levels due to enhancement of antioxidant responses. However, inadequate exercise training (i.e., single bout of high-intensity or excessive exercise) may result in oxidative stress, muscle fatigue and muscle injury. Moreover, during the recovery period, impaired immunity has been reported, for example; excessive-inflammation and compensatory immunosuppression. Nutritional supplements, sometimes referred to as immuno-nutrients, may be required to reduce immunosuppression and excessive inflammation. Herein, we discuss the action and the possible targets of key immuno-nutrients such as L-glutamine, L-arginine, branched chain amino acids (BCAA) and whey protein.

Differential nitric oxide levels in the blood and skeletal muscle of Type 2 diabetic subjects may be consequence of adiposity: a preliminary study.

BACKGROUND AND AIMS Nitric oxide (NO·) exerts key regulatory functions including vasodilation and glucose uptake. Thus reduced NO· levels are associated with insulin resistance and hypertension. In this preliminary work we aimed to measure the levels of NO· metabolites in serum and skeletal muscle of obese and non-obese subjects, with or without type 2 diabetes mellitus (T2DM). METHODS Fifteen sedentary male participants [7 obese controls (C) vs 5 obese and 3 non-obese T2DM; age 54±9 years] were selected according to their BMI (>30 kg/m(2) for obese and 23-27 kg/m(2) for non-obese participants) and evaluated for fasted values of blood glucose, HbA1c, lipid profile, serum CRP (C-reactive protein), erythrocyte glutathione (GSH) metabolism, plasma adiponectin, leptin and cytokines (TNF-α and INFγ), serum and skeletal muscle nitric oxide metabolites (nitrite and nitrates; tNOx) and skeletal muscle nNOS and iNOS expression. Body composition was measured by whole body DEXA and muscle microbiopsy was performed in the vastus lateralis. RESULTS We found that serum tNOx (total nitrite/nitrate; μmol/L) was lower in obese T2DM group (12.7±3.5) when compared with their controls (21.1±2.4), although the non-obese group presented higher concentration of tNOx (33.8±7.2). Skeletal muscle nNOS was higher in obese controls, lower in non-obese T2DM and undetected in obese T2DM. On the other hand, expression of iNOS had an inverse relationship with nNOS, showing higher expression in obese T2DM, decrease in non-obese T2DM and absence in obese control group. tNOx levels (μmol/mg protein) were decreased in the non-obese T2DM group (12.07±0.59) when compared with the obese control (21.68±6.2) and the obese T2DM group (26.3±7.26). CONCLUSION We conclude that the decreased serum NO∙ production in obese T2DM patients seems to be associated with adipose mass as lower adiposity was associated with normal NO∙ which was reduced in the skeletal muscle of the non-obese T2DM patients. We suggest that the lower adiposity (and higher adiponectin) in non-obese T2DM could be responsible for differential levels of NO∙ production and insulin resistance.

Physiological concentrations of interleukin-6 directly promote insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal pancreatic β-cell line and mouse islets.

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic β-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal β-cell line and mouse islets are reported. Chronic insulin secretion (in μg/mg protein per 24  h) was increased from 128·7±7·3 (no IL6) to 178·4±7·7 (at 100  pg/ml IL6) in clonal β-cells and increased significantly in islets incubated in the presence of 5·5  mM glucose for 2  h, from 0·148 to 0·167±0·003  ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal β-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal β-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in β-cells and islets). Our data have demonstrated that IL6 can stimulate β-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on β-cell signaling, metabolism, antioxidant status, and insulin secretion.