Clinical management of Krabbe disease.

Krabbe disease (KD) is a rare neurodegenerative disorder caused by mutations in the gene encoding the galactocerebrosidase enzyme. The early- and late-infantile subtypes, which are the most common forms of the disease, are rapidly progressive and lead to early death, whereas the later-onset types are clinically heterogeneous. The only disease-modifying treatment currently available is hematopoietic stem cell transplantation, which is effective only when performed early in the course of the disease. Because most patients with KD are diagnosed too late for treatment, primary care physicians are faced with the challenge of caring for a child with severe neurologic impairment. This Review describes presenting symptoms, diagnosis, and disease manifestations of KD and provides basic guidelines for its management. Symptomatic treatment and supportive care that address the unique requirements of these patients can greatly improve the quality of life of patients and their families. © 2016 Wiley Periodicals, Inc.

Antiobesity mechanisms of action of conjugated linoleic acid.

Conjugated linoleic acid (CLA), a family of fatty acids found in beef, dairy foods and dietary supplements, reduces adiposity in several animal models of obesity and some human studies. However, the isomer-specific antiobesity mechanisms of action of CLA are unclear, and its use in humans is controversial. This review will summarize in vivo and in vitro findings from the literature regarding potential mechanisms by which CLA reduces adiposity, including its impact on (a) energy metabolism, (b) adipogenesis, (c) inflammation, (d) lipid metabolism and (e) apoptosis.

Conjugated linoleic acid-mediated inflammation and insulin resistance in human adipocytes are attenuated by resveratrol.

Inflammation plays a role in trans-10, cis-12 (10,12)-conjugated linoleic acid (CLA)-mediated delipidation and insulin resistance in adipocytes. Given the anti-inflammatory role of resveratrol (RSV), we hypothesized that RSV would attenuate inflammation and insulin resistance caused by 10,12 CLA in human adipocytes. RSV blocked 10,12 CLA induction of the inflammatory response by preventing activation of extracellular signal-related kinase and induction of inflammatory gene expression (i.e., IL-6, IL-8, IL-1beta) within 12 h. Similarly, RSV suppressed 10,12 CLA-mediated activation of the inflammatory prostaglandin pathway involving phospholipase A(2), cyclooxygenase-2, and PGF(2alpha). In addition, RSV attenuated 10,12 CLA increase of intracellular calcium and reactive oxygen species associated with cellular stress, and activation of stress-related proteins (i.e., activating transcription factor 3, JNK) within 12 h. 10,12 CLA-mediated insulin resistance and suppression of fatty acid uptake and triglyceride content were attenuated by RSV. Finally, 10,12 CLA-mediated decrease of peroxisome proliferator-activated receptor gamma (PPARgamma) protein levels and activation of a peroxisome proliferator response element (PPRE) reporter were prevented by RSV. RSV increased the basal activity of PPRE, suggesting that RSV increases PPARgamma activity. Collectively, these data demonstrate for the first time that RSV prevents 10,12 CLA-mediated insulin resistance and delipidation in human adipocytes by attenuating inflammation and cellular stress and increasing PPARgamma activity.

Trans-10, cis-12 conjugated linoleic acid antagonizes ligand-dependent PPARgamma activity in primary cultures of human adipocytes.

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) causes human adipocyte delipidation, insulin resistance, and inflammation in part by attenuating PPARgamma target gene expression. We hypothesized that CLA antagonizes the activity of PPARgamma in an isomer-specific manner. 10,12 CLA, but not cis-9, trans-11 (9,11) CLA, suppressed ligand-stimulated activation of a peroxisome proliferator response element-luciferase reporter. This decreased activation of PPARgamma by 10,12 CLA was accompanied by an increase in PPARgamma and extracellular signal-related kinase (ERK)1/2 phosphorylation, followed by decreased PPARgamma protein levels. To investigate if 10,12 CLA-mediated delipidation was preventable with a PPARgamma ligand (BRL), cultures were treated for 1 wk with 10,12 CLA or 10,12 CLA + BRL and adipogenic gene and protein expression, glucose uptake, and triglyceride (TG) were measured. BRL cosupplementation completely prevented 10,12 CLA suppression of adipocyte fatty acid-binding protein, lipoprotein lipase, and perilipin mRNA levels without preventing reductions in PPARgamma or insulin-dependent glucose transporter 4 (GLUT4) expression, glucose uptake, or TG. Lastly, we investigated the impact of CLA withdrawal in the absence or presence of BRL for 2 wk. CLA withdrawal did not rescue CLA-mediated reductions in adipogenic gene and protein expression. In contrast, BRL supplementation for 2 wk following CLA withdrawal rescued mRNA levels of PPARgamma target genes. However, the levels of PPARgamma and GLUT4 protein and TG were only partially rescued by BRL. Collectively, we demonstrate for the first time, to our knowledge, that 10,12 CLA antagonizes ligand-dependent PPARgamma activity, possibly via PPARgamma phosphorylation by ERK.

Preadipocytes mediate lipopolysaccharide-induced inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes.

Recent data suggest that proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. However, characterization of the cell types involved in inflammation and how these cells promote insulin resistance in human adipocytes are unclear. We simulated acute inflammation using the endotoxin lipopolysaccharide (LPS) to define the roles of nonadipocytes in primary cultures of human adipocytes. LPS induction of the mRNA levels of proinflammatory cytokines (e.g. IL-6, TNF-alpha, and IL-1beta) and chemokines (e.g. IL-8, monocyte chemoattractant protein-1) occurred primarily in the nonadipocyte fraction of newly differentiated human adipocytes. Nonadipocytes were characterized as preadipocytes based on their abundant mRNA levels of preadipocyte markers preadipocyte factor-1 and adipocyte enhancer protein-1 and only trace levels of markers for macrophages and myocytes. The essential role of preadipocytes in inflammation was confirmed by modulating the degree of differentiation in the cultures from approximately 0-90%. LPS-induced proinflammatory cytokine/chemokine expression and nuclear factor-kappaB and MAPK signaling decreased as differentiation increased. LPS-induced cytokine/chemokine expression in preadipocytes was associated with: 1) decreased adipogenic gene expression, 2) decreased ligand-induced activation of a peroxisome proliferator activated receptor (PPAR)-gamma reporter construct and increased phosphorylation of PPARgamma, and 3) decreased insulin-stimulated glucose uptake. Collectively, these data demonstrate that LPS induces nuclear factor-kappaB- and MAPK-dependent proinflammatory cytokine/chemokine expression primarily in preadipocytes, which triggers the suppression of PPARgamma activity and insulin responsiveness in human adipocytes.

Bacterial expression system with tightly regulated gene expression and plasmid copy number.

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.