Type 2 diabetes mellitus risk and exercise: is resistin involved?

Obesity and associated disorders such as type 2 diabetes mellitus (T2DM) pose an increasing risk to the health of both individuals and society. Adipose tissue is an active endocrine organ, secreting many hormones, known as adipokines. Evidence suggests that one suggest adipokine, resistin, may be elevated in the plasma of individuals with T2DM, and early reports indicated that this may contribute to the impaired glucose tolerance and insulin resistance observed in T2DM, hence its name, resistin, however subsequent evidence suggests it may have a proinflammatory role. Performing regular exercise and dietary interventions improve insulin sensitivity and glucose tolerance in the majority of individuals with T2DM, and we hypothesized that plasma resistin levels may drop in individuals following a long term aerobic and/or resistance exercise intervention program. Articles analyzing the effects of exercise on serum resistin levels were therefore analyzed. Articles were selected and analyzed based on a minimum aerobic and/or resistance training program of 2 sessions per week for 12 weeks. Plasma resistin levels decreased following either endurance aerobic exercise and/or resistance training plans in individuals with obesity and/or insulin resistance, although this was not related to BMI change. Exercise intervention programs generally showed potentially beneficial changes in plasma resistin concentrations. It is possible these effects are mediated through exercise-induced decreases in inflammation via anti-inflammatory cytokine release rather than alterations in glucose metabolism and reductions in BMI per se. A possible link between resistin, exercise and T2DM is discussed.

Heterogeneous responses of personalised high intensity interval training on type 2 diabetes mellitus and cardiovascular disease risk in young healthy adults.

Hypertension, decreased glucose tolerance, adverse lipid profiles and low physical activity levels are associated with increased type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) risk. High intensity interval training (HIIT), a low volume, reduced time, high intensity programme, may be a useful alternative to current government guidelines which specify a minimum of 150 minutes of physical activity per week. We describe a personalised programme of high intensity exercise which provides significant improvements in CVD risk markers. Healthy volunteers undertook 6 weeks of HIIT. T2DM and CVD risk predictors including glucose tolerance, VO2max, blood pressure (BP), and lipids were measured before and after HIIT. HIIT training was associated with beneficial changes in a range of predictors of blood flow and cardiovascular risk. There was a heterogeneous response to HIIT, with some subjects responding with favourable changes and others being non-responders to HIIT. In responders, HIIT was associated with a statistically significant (p = 0.023) increase in VO2max, from 45.4 (38.4,52.5) to 56.9 (51.2,65.7) (median (interquartile range)(ml/min/kg)). In responders HIIT resulted in a decrease in systolic BP from 127 (126,129) to 116 (106,122) (mmHg) with p = 0.026 and a decrease is diastolic blood pressure from 72 (69,74) to 57 (56,66) with p = 0.026. There was also some evidence of a beneficial change in blood lipid and glucose concentrations with HIIT. In conclusion, personalised HIIT has potential as an intervention to improve blood flow and cardiovascular health.

A Comparison of the Effects of Aerobic and Intense Exercise on the Type 2 Diabetes Mellitus Risk Marker Adipokines, Adiponectin and Retinol Binding Protein-4.

With a more sedentary population comes growing rates of obesity and increased type 2 diabetes mellitus (T2DM) risk. Exercise generally induces positive changes in traditional T2DM risk markers such as lipids, glucose tolerance, and insulin sensitivity; however alterations in concentrations of many circulating cytokines and their respective receptors are also becoming apparent. These cytokines may be early-response health risk factors otherwise overlooked in traditional T2DM risk marker analysis. Plasma levels of two adipocyte-originating cytokines, adiponectin and retinol binding protein 4 (RBP-4), alter following exercise. Adiponectin has anti-inflammatory, anti-atherosclerotic, and anti-insulin resistance roles and its secretion increases with physical activity, whilst elevated RBP-4 leads to increased insulin resistance, and secretion decreases with increasing physical activity; thus these plasma adipokine levels alter favourably following exercise. Although current data are limited, they do suggest that the more intense the exercise, the greater the positive effect on plasma RBP-4 levels, whilst lower intensity aerobic exercise may positively improve adiponectin concentrations. Therefore short-duration, high intensity training may provide a time-efficient alternative to the recommended 150 min moderate aerobic exercise per week in providing positive changes in RBP-4 and other traditional T2DM risk markers and due to increased compliance give greater health benefits over the longer term.

Creatine supplementation: can it improve quality of life in the elderly without associated resistance training?

INTRODUCTION: Ageing is associated with decreased muscle mass, strength, power and function, and reduction in bone density and mineral content, leading to reduced independence and increased risk of falls. Creatine supplementation is reported to improve muscular strength and performance with training in younger athletes, and therefore could benefit older individuals. AIMS: This review critically appraises the current literature on whether creatine supplementation enhances muscular performance and function, body composition, bone mineral density and content in older adults without the addition of resistance training, and thus determines whether creatine supplementation can lead to an improved lifestyle for the sedentary elderly population. RESULTS: There is conflicting evidence regarding the usefulness of creatine supplementation in older subjects. Generally, however, creatine supplementation, without associated resistance training, seems to enhance muscular strength, power and endurance, increase lean body mass (LBM) and improve the functional capacity of the elderly. Furthermore, it has been demonstrated that increased muscle mass due to creatine supplementation can result in increased local bone density. It appears that the effect of creatine supplementation is more beneficial in larger muscles and less effective in smaller muscles, however there are exceptions. The mechanism by which creatine supplementation works requires further research, however it is likely that the effects of creatine are related to creatine kinase activity, providing enhanced energy production for greater muscular contraction. CONCLUSIONS: These data indicate that creatine supplementation without associated training in the elderly could potentially delay atrophy of muscle mass, improve endurance and strength, and increase bone strength, and thus may be a safe therapeutic strategy to help decrease loss in functional performance of everyday tasks.

The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans.

The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-alpha by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.

Reduced redox potential of the cytosol is important for African swine fever virus capsid assembly and maturation.

Assembly of African swine fever virus (ASFV) involves the transfer of the major capsid protein, p73, from the cytosol onto the cytoplasmic face of endoplasmic reticulum-derived membranes. During this process, the folding of p73 is dependent upon transient association with a specific viral chaperone, CAP80. The cell cytoplasm maintains high concentrations of reduced glutathione, leading to a reducing environment. Here, the effects of redox environment on the assembly of ASFV have been studied. Diamide, which oxidizes the cell cytosol, slowed the folding of p73 and prevented release from CAP80 and subsequent binding of p73 to membranes. Similarly, cell oxidation slowed the assembly of p73 molecules already bound to membranes into virus capsid precursors. Interestingly, addition of oxidized glutathione to newly assembled virus capsid precursors in vitro led to disassembly; however, virus particles released from cells were resistant to oxidized glutathione. These data show that assembly of ASFV requires the reducing environment that prevails in the cytosol, but as the virus matures, it becomes resistant to oxidation, possibly indicating preparation for release from the cell.

Cell biology of membrane trafficking in human disease.

Understanding the molecular and cellular mechanisms underlying membrane traffic pathways is crucial to the treatment and cure of human disease. Various human diseases caused by changes in cellular homeostasis arise through a single gene mutation(s) resulting in compromised membrane trafficking. Many pathogenic agents such as viruses, bacteria, or parasites have evolved mechanisms to subvert the host cell response to infection, or have hijacked cellular mechanisms to proliferate and ensure pathogen survival. Understanding the consequence of genetic mutations or pathogenic infection on membrane traffic has also enabled greater understanding of the interactions between organisms and the surrounding environment. This review focuses on human genetic defects and molecular mechanisms that underlie eukaryote exocytosis and endocytosis and current and future prospects for alleviation of a variety of human diseases.

Erythropoietic protoporphyria: a functional analysis of the leader sequence of human ferrochelatase.

Erythropoietic protoporphyria (EPP) results from an inherited partial deficiency of ferrochelatase, the terminal enzyme of haem biosynthesis. Excess protoporphyrin IX accumulates in erythrocytes, plasma, liver, and skin, which mediates a distinctive form of cutaneous photosensitivity that manifests during childhood. Ferrochelatase is synthesised on cytosolic ribosomes as a preprotein with a cleavable presequence at its amino-terminus. This leader sequence is thought to target ferrochelatase to mitochondria where it is cleaved to produce the active mature protein. In this study, we show that the 62 amino acid leader sequence is sufficient for targeting of a leader sequence-YFP fusion protein to mitochondria. A truncated fusion protein lacking the first 62 amino acids did not target to mitochondria, and formed punctate aggregates in the cytoplasm of cells. This suggests that all the information required for mitochondrial localisation resides within the first 62 amino acid presequence. A missense mutation, P62R, predicted to be located within the ferrochelatase presequence has been identified in a patient with EPP. We hypothesised that this mutation may exert its effect through defective targeting to mitochondria. Our data showed that this mutated full-length ferrochelatase successfully targeted to mitochondria. Interestingly, there was inhibited cleavage of YFP from wild-type and mutant leader sequence fusion proteins. Generation of leader sequence-YFP fusion proteins containing an additional 11 amino acids from the mature protein allowed proteolytic processing to occur. These data suggest that the first 62 amino acids allow targeting to mitochondria but do not contain sufficient information for efficient processing of the protein.

Actin and microtubule regulation of trans-Golgi network architecture, and copper-dependent protein transport to the cell surface.

The Menkes disease ATPase (MNK) is a copper transporter that localizes to the mammalian trans-Golgi network (TGN) and shows substantial co-localization wih a ubiquitous TGN resident protein and marker, TGN46. We tested our hypothesis that these two TGN residents and integral membrane proteins are localized to biochemically distinct TGN sub-compartments using constitutively active mutant proteins and drugs that disrupt membrane traffic, lumenal pH and the cellular cytoskeleton. The pH-disrupting agent, monensin, causes MNK to be more diffusely distributed with partial separation of staining patterns for these two TGN residents. Expression of a constitutively active Rho-kinase (ROCK-KIN), which causes formation of juxta-nuclear astral actin arrays, also effects separation of MNK and TGN46 staining patterns. Treatment of ROCK-KIN expressing cells with latrunculin B, an actin-depolymerizing agent, causes complete overlap of MNK and TGN46 staining patterns with concomitant disappearance of polymerized actin. When microtubules are depolymerized in ROCK-KIN expressing cells by nocodazole, both MNK and TGN46 are found in puncate structures throughout the cell. However, a substantial proportion of MNK is still found in a juxta-nuclear location in contrast to TGN46. Actin distribution in these cells reveals that juxta-nuclear MNK is distinct to the astral actin clusters in ROCK-KIN expressing cells where the microtubules were depolymerized. The TGN to cell-surface transport of MNK requires both actin and microtubules networks, whilst the constitutive trafficking of proteins is independent of actin. Taken together, our findings indicate that at least two TGN sub-domains are regulated by separate cytoskeletal dynamics involving actin and tubulin.

Impaired trafficking of the desmoplakins in cultured Darier's disease keratinocytes.

Darier's disease is an autosomal dominantly inherited skin disorder characterized by loss of adhesion between epidermal cells, breakdown of desmosome-keratin filaments, and abnormal keratinization. ATP2A2 has been identified as the causative gene of Darier's disease. This gene encodes the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) isoform 2 pump, which transports Ca2+ from the cytosol into the endoplasmic reticulum lumen to maintain a low cytosolic Ca2+ concentration. Using indirect immunofluorescence and biochemical analysis, we investigated the distribution of key desmosomal proteins in normal human and Darier's disease keratinocytes under various calcium conditions. We show that inhibition of SERCA by thapsigargin in normal human keratinocytes impairs the trafficking of the desmoplakins, desmoglein, and desmocollin to the cell surface; these proteins show a diffuse cytoplasmic distribution and, together with plakoglobin, form detergent-insoluble aggregates. In Darier's disease keratinocytes, only the trafficking of desmoplakin is significantly inhibited; in these cells, desmoplakin forms insoluble aggregates when extracted with mild detergent. In contrast, the transmembrane proteins desmoglein and desmocollin are efficiently transported to the cell surface. These proteins, along with plakoglobin, remain equally distributed between detergent-soluble and -insoluble fractions. We also demonstrate an interaction between SERCA2 and desmoplakin during differentiation. Our results provide further insights into the critical role of calcium ATPases in maintaining epidermal integrity.

Aberrant trafficking of transmembrane proteins in human disease.

Transmembrane domain (TMD) proteins comprise a major group of proteins that perform a wide range of functions and act to translate extracellular signals to intracellular responses. They include G-protein coupled receptors (GPCRs), growth factor receptors, ion channels, transporters and metabolic enzymes. In this review, we focus on the current understanding of trafficking of mutant membrane proteins in human disease and speculate on therapeutic strategies.

LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome.

SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevance.

The Menkes disease ATPase (ATP7A) is internalized via a Rac1-regulated, clathrin- and caveolae-independent pathway.

The Menkes disease gene encodes a P-type transmembrane ATPase (ATP7A) that translocates cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. ATP7A moves from the trans-Golgi network (TGN) to the cell surface in response to exogenously added copper ions and recycles back to the TGN upon copper removal. The protein contains a C-terminal di-leucine motif necessary for internalization from the cell surface. In this study we show that ATP7A is internalized by a novel pathway that is independent of clathrin-mediated endocytosis. Expression of dominant-negative mutants of the dynamin-I, dynamin-II and Eps15 proteins that block clathrin-dependent endocytosis of the transferrin receptor do not inhibit internalization of endogenous ATP7A, or an ATP7A reporter molecule (CD8-MCF1). Similarly, inhibitors of caveolae-mediated uptake do not affect ATP7A internalization whilst preventing uptake of PODIPY-ganglioside GM(1), a caveolae marker. In contrast, expression of a constitutively active mutant of the Rac1 GTPase inhibits plasma membrane internalization of both the ATP7A and transferrin receptor transmembrane proteins. These findings define a novel route required for ATP7A internalization and delivery to endosomes.

Novel membrane traffic steps regulate the exocytosis of the Menkes disease ATPase.

The Menkes disease protein (ATP7A or MNK) is a P-type transmembrane ATPase that regulates translocation of cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. In this study, we show that endogenous MNK in cultured cell lines is localized to the distal Golgi apparatus and translocates to the plasma membrane in response to exogenous copper ions. This transport event is not blocked by expression of a dominant-negative mutant protein kinase D, an enzyme implicated in regulating constitutive trafficking from the trans-Golgi network (TGN) to the plasma membrane, whereas constitutive transport of CD4 is inhibited. In contrast, protein kinase A inhibitors block copper-stimulated MNK delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as Cdc42, Rac1 and RhoA reveals a requirement for Cdc42 in the trafficking of MNK, to the cell surface. Furthermore, overexpression of WASp inhibits anterograde transport of MNK, further supporting regulation by the Cdc42 GTPase. These findings define a novel step in TGN-to-plasma membrane traffic required to export MNK to the cell surface.