Kevin N. Keane

Inflammation and Oxidative Stress: The Molecular Connectivity between Insulin Resistance, Obesity, and Alzheimer’s Disease

Type 2 diabetes (T2DM), Alzheimers disease (AD), and insulin resistance are age-related conditions and increased prevalence is of public concern. Recent research has provided evidence that insulin resistance and impaired insulin signalling may be a contributory factor to the progression of diabetes, dementia, and other neurological disorders. Alzheimers disease (AD) is the most common subtype of dementia. Reduced release (for T2DM) and decreased action of insulin are central to the development and progression of both T2DM and AD. A literature search was conducted to identify molecular commonalities between obesity, diabetes, and AD. Insulin resistance affects many tissues and organs, either through impaired insulin signalling or through aberrant changes in both glucose and lipid (cholesterol and triacylglycerol) metabolism and concentrations in the blood. Although epidemiological and biological evidence has highlighted an increased incidence of cognitive decline and AD in patients with T2DM, the common molecular basis of cell and tissue dysfunction is rapidly gaining recognition. As a cause or consequence, the chronic inflammatory response and oxidative stress associated with T2DM, amyloid-β (Aβ) protein accumulation, and mitochondrial dysfunction link T2DM and AD.

Nutrient regulation of insulin secretion and action

Pancreatic β-cell function is of critical importance in the regulation of fuel homoeostasis, and metabolic dysregulation is a hallmark of diabetes mellitus (DM). The β-cell is an intricately designed cell type that couples metabolism of dietary sources of carbohydrates, amino acids and lipids to insulin secretory mechanisms, such that insulin release occurs at appropriate times to ensure efficient nutrient uptake and storage by target tissues. However, chronic exposure to high nutrient concentrations results in altered metabolism that impacts negatively on insulin exocytosis, insulin action and may ultimately lead to development of DM. Reduced action of insulin in target tissues is associated with impairment of insulin signalling and contributes to insulin resistance (IR), a condition often associated with obesity and a major risk factor for DM. The altered metabolism of nutrients by insulin-sensitive target tissues (muscle, adipose tissue and liver) can result in high circulating levels of glucose and various lipids, which further impact on pancreatic β-cell function, IR and progression of the metabolic syndrome. Here, we have considered the role played by the major nutrient groups, carbohydrates, amino acids and lipids, in mediating β-cell insulin secretion, while also exploring the interplay between amino acids and insulin action in muscle. We also focus on the effects of altered lipid metabolism in adipose tissue and liver resulting from activation of inflammatory processes commonly observed in DM pathophysiology. The aim of this review is to describe commonalities and differences in metabolism related to insulin secretion and action, pertinent to the development of DM.

The Impact of Vitamin D Levels on Inflammatory Status: A Systematic Review of Immune Cell Studies

Chronic low-grade inflammation accompanies obesity and its related chronic conditions. Both peripheral blood mononuclear cells (PBMCs) and cell lines have been used to study whether vitamin D has immune modulating effects; however, to date a detailed systematic review describing the published evidence has not been completed. We therefore conducted a systematic review on the effect of vitamin D on the protein expression and secretion of inflammatory markers by human-derived immune cells. The review was registered at the International Prospective Register for Systematic Reviews (PROSPERO, Registration number CRD42015023222). A literature search was conducted using Pubmed, Science Direct, Scopus, Web of Science and Medline. The search strategy used the following search terms: Vitamin D or cholecalciferol or 1,25-dihydroxyvitamin or 25-hydroxy-Vitamin D and Inflam* or cytokine* and supplement* or cell*. These terms were searched in the abstract, title and keywords. Inclusion criteria for study selection consisted of human-derived immune cell lines or cellular studies where PBMCs were obtained from humans, reported in the English language, and within the time period of 2000 to 2015. The selection protocol was mapped according to PRISMA guidelines. Twenty three studies (7 cell line and 16 PBMCs studies) met our criteria. All studies selected except one used the active metabolite 1,25(OH)2, with one study using cholecalciferol and two studies also using 25(OH)D. Four out of seven cell line studies showed an anti-inflammatory effect where suppression of key markers such as macrophage chemotactic protein 1, interleukin 6 and interleukin 8 were observed. Fourteen of sixteen PBMC studies also showed a similar anti-inflammatory effect based on common inflammatory endpoints. Mechanisms for such effects included decreased protein expression of toll-like receptor-2 and toll-like receptor-4; lower levels of phosphorylated p38 and p42/42; reduced expression of phosphorylated signal transducer and activator of transcription 5 and decreased reactive oxygen species. This review demonstrates that an anti-inflammatory effect of vitamin D is a consistent observation in studies of cell lines and human derived PBMCs.

Molecular mechanisms of ROS production and oxidative stress in diabetes.

Oxidative stress and chronic inflammation are known to be associated with the development of metabolic diseases, including diabetes. Oxidative stress, an imbalance between oxidative and antioxidative systems of cells and tissues, is a result of over production of oxidative-free radicals and associated reactive oxygen species (ROS). One outcome of excessive levels of ROS is the modification of the structure and function of cellular proteins and lipids, leading to cellular dysfunction including impaired energy metabolism, altered cell signalling and cell cycle control, impaired cell transport mechanisms and overall dysfunctional biological activity, immune activation and inflammation. Nutritional stress, such as that caused by excess high-fat and/or carbohydrate diets, promotes oxidative stress as evident by increased lipid peroxidation products, protein carbonylation and decreased antioxidant status. In obesity, chronic oxidative stress and associated inflammation are the underlying factors that lead to the development of pathologies such as insulin resistance, dysregulated pathways of metabolism, diabetes and cardiovascular disease through impaired signalling and metabolism resulting in dysfunction to insulin secretion, insulin action and immune responses. However, exercise may counter excessive levels of oxidative stress and thus improve metabolic and inflammatory outcomes. In the present article, we review the cellular and molecular origins and significance of ROS production, the molecular targets and responses describing how oxidative stress affects cell function including mechanisms of insulin secretion and action, from the point of view of possible application of novel diabetic therapies based on redox regulation.

The potential regulatory role of vitamin D in the bioenergetics of inflammation.

Purpose of reviewThe extraskeletal health benefits of vitamin D still need scientific endorsement. Obesity and related chronic diseases are pathogenically linked by inflammation, which carries a considerable energetic cost. Recent techniques for the determination of the bioenergetic demand of inflammation, offer an avenue to cement the regulatory role of vitamin D in this process. Recent findingsNuclear vitamin D receptors may be translocated into mitochondria of certain cell types, opening up a pathway for direct action on cellular bioenergetics. Classical M1 (inflammatory)/M2(anti-inflammatory) phenotypes can vary with the clinical context. M2 macrophages do not always depend on oxidative metabolism/fatty acid oxidation. Newer methodologies offer real-time bioenergetic measurements that can be used as an index of metabolic health. SummaryVitamin D may prove to be a therapeutic agent for inflammation of chronic disease and understanding its role in cellular bioenergetics may offer a diagnostic/prognostic indicator of its action.

Alanyl-glutamine improves pancreatic β-cell function following ex vivo inflammatory challenge

Obesity-associated diabetes and concomitant inflammation may compromise pancreatic β-cell integrity and function. l-glutamine and l-alanine are potent insulin secretagogues, with antioxidant and cytoprotective properties. Herein, we studied whether the dipeptide l-alanyl-l-glutamine (Ala-Gln) could exert protective effects via sirtuin 1/HUR (SIRT1/HUR) signalling in β-cells, against detrimental responses following ex vivo stimulation with inflammatory mediators derived from macrophages (IMMs). The macrophages were derived from blood obtained from obese subjects. Macrophages were exposed (or not) to lipopolysaccharide (LPS) to generate a pro-inflammatory cytokine cocktail. The cytokine profile was determined following analysis by flow cytometry. Insulin-secreting BRIN-BD11 β-cells were exposed to IMMs and then cultured with or without Ala-Gln for 24 h. Chronic insulin secretion, the l-glutamine-glutathione (GSH) axis, and the level of insulin receptor β (IR-β), heat shock protein 70 (HSP70), SIRT1/HUR, CCAAT-enhancer-binding protein homologous protein (CHOP) and cytochrome c oxidase IV (COX IV) were evaluated. Concentrations of cytokines, including interleukin 1β (IL1β), IL6, IL10 and tumour necrosis factor alpha (TNFα) in the IMMs, were higher following exposure to LPS. Subsequently, when β-cells were exposed to IMMs, chronic insulin secretion, and IR-β and COX IV levels were decreased, but these effects were partially or fully attenuated by the addition of Ala-Gln. The glutamine-GSH axis and HSP70 levels, which were compromised by IMMs, were also restored by Ala-Gln, possibly due to protection of SIRT1/HUR levels, and a reduction of CHOP expression. Using an ex vivo inflammatory approach, we have demonstrated Ala-Gln-dependent β-cell protection mediated by coordinated effects on the glutamine-GSH axis, and the HSP pathway, maintenance of mitochondrial metabolism and stimulus-secretion coupling essential for insulin release.

The impact of cryopreservation on human peripheral blood leucocyte bioenergetics.

Circulating immune cells are considered a source for biomarkers in health and disease, since they are exposed to nutritional, metabolic and immunological stimuli in the vasculature. Cryopreservation of leucocytes is routinely used for long-term storage and determination of phenotypic/functional changes at a later date. Exploring the role of bioenergetics and mitochondrial (dys)function in leucocytes is often examined by using freshly isolated cells. The aim of the pilot study described herein was to assess leucocyte bioenergetics in cryopreserved cells. Leucocytes were isolated from whole blood, counted and frozen in liquid nitrogen (LN2) for a period of 3 months. Cells were thawed at regular intervals and bioenergetic analysis performed using the Seahorse XFe96 flux analyser. Cryogenic storage reduced cell viability by 20%, but cell bioenergetic responses were largely intact for up to 1 month storage in LN2. However, after 1 month storage, mitochondrial function was impaired as reflected by decreasing basal respiration, ATP production, maximum (MAX) respiration, reserve capacity and coupling efficiency. Conversely, glycolytic activity was increased after 1 month, most notably the enhanced glycolytic response to 25 mM glucose without any change in glycolytic capacity. Finally, calculation of bioenergetic health index (BHI) demonstrated that this potential diagnostic parameter was sensitive to cryopreservation. The present study has demonstrated for the first time that cryopreservation of primary immune cells modified their metabolism in a time-dependent fashion, indicated by attenuated aerobic respiration and enhanced glycolytic activity. Taken together, we recommend caution in the interpretation of bioenergetic responses or BHI in cryopreserved samples.

Higher β-HCG concentrations and higher birthweights ensue from single vitrified embryo transfers

To examine the effect of cryopreservation on developmental potential of human embryos, this study compared quantitative β-HCG concentrations at pregnancy test after IVF-fresh embryo transfer (IVF-ET) with those arising after frozen embryo transfer (FET). It also tracked outcomes of singleton pregnancies resulting from single-embryo transfers that resulted in singleton live births (n = 869; with 417 derived from IVF-ET and 452 from FET). The initial serum β-HCG concentration indicating successful implantation was measured along with the birthweight of the ensuing infants. With testing at equivalent luteal phase lengths, the median pregnancy test β-HCG was significantly higher following FET compared with fresh IVF-ET (844.5 IU/l versus 369 IU/l; P < 0.001). Despite no significant difference in the average period of gestation (38 weeks 5 days for both groups), the mean birthweight of infants born following FET was significantly heavier by 161 g (3370 g versus 3209 g; P < 0.001). Furthermore, more infants exceeded 4000 g (P < 0.001) for FET although there was no significant difference for the macrosomic category (≥4500 g). We concluded that FET programme embryos lead to infants with equivalent (if not better) developmental potential compared with IVF-ET, demonstrated by higher pregnancy β-HCG concentrations and ensuing birthweights.

The bioenergetics of inflammation: insights into obesity and type 2 diabetes

Diabetes mellitus is one of the most common chronic metabolic disorders worldwide, and its incidence in Asian countries is alarmingly high. Type 2 diabetes (T2DM) is closely associated with obesity, and the staggering rise in obesity is one of the primary factors related to the increased frequency of T2DM. Low-grade chronic inflammation is also accepted as an integral metabolic adaption in obesity and T2DM, and is believed to be a major player in the onset of insulin resistance. However, the exact mechanism(s) that cause a persistent chronic low-grade infiltration of leukocytes into insulin-target tissues such as adipose, skeletal muscle and liver are not entirely known. Recent developments in the understanding of leukocyte metabolism have revealed that the inflammatory polarization of immune cells, and consequently their immunological function, are strongly connected to their metabolic profile. Therefore, it is hypothesized that dysfunctional immune cell metabolism is a central cellular mechanism that prevents the resolution of inflammation in chronic metabolic conditions such as that observed in obesity and T2DM. The purpose of this review is to explore the metabolic demands of different immune cell types, and identify the molecular switches that control immune cell metabolism and ultimately function. Understanding of these concepts may allow the development of interventions that can correct immune function and may possibly decrease chronic low-grade inflammation in humans suffering from obesity and T2DM. We also review the latest clinical techniques used to measure metabolic flux in primary leukocytes isolated from obese and T2DM patients.

Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: Metabolic state of cancer cells.

Malignant mesothelioma (MM) is an aggressive cancer, characterized by rapid progression, along with late metastasis and poor patient prognosis. It is resistant to many forms of standard anti-cancer treatment. In this study, we determined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt pathway inhibitor, on cancer cell proliferation and metabolism using the JU77 mesothelioma cell line. Treatment with sFRP4 (250 pg/ml) resulted in a significant reduction of cell proliferation. The addition of the Wnt activator Wnt3a (250 pg/ml) or sFRP4 had no significant effect on ATP production and glucose utilisation in JU77 cells at both the 24 and 48 h time points examined. We also examined their effect on Akt and Glycogen synthase kinase-3 beta (GSK3β) phosphorylation, which are both important components of Wnt signalling and glucose metabolism. We found that protein phosphorylation of Akt and GSK3β varied over the 24h and 48 h time points, with constitutive phosphorylation of Akt at serine 473 (pAkt) decreasing to its most significant level when treated with Wnt3a+sFRP4 at the 24h time point. A significant reduction in the level of Cytochrome c oxidase was observed at the 48 h time point, when sFRP4 and Wnt3a were added in combination. We conclude that sFRP4 may function, in part, to reduce/alter cancer cell metabolism, which may lead to sensitisation of cancer cells to chemotherapeutics, or even cell death.