Liraglutide Treatment Reduces Endothelial Endoplasmic Reticulum Stress and Insulin Resistance in Patients With Diabetes Mellitus.

Background Prior studies have shown that nutrient excess induces endoplasmic reticulum ( ER ) stress in nonvascular tissues from patients with diabetes mellitus ( DM ). ER stress and the subsequent unfolded protein response may be protective, but sustained activation may drive vascular injury. Whether ER stress contributes to endothelial dysfunction in patients with DM remains unknown. Methods and Results To characterize vascular ER stress, we isolated endothelial cells from 42 patients with DM and 37 subjects without DM. Endothelial cells from patients with DM displayed higher levels of ER stress markers compared with controls without DM. Both the early adaptive response, evidenced by higher phosphorylated protein kinase-like ER eukaryotic initiation factor-2a kinase and inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.02, P=0.007, respectively), and the chronic ER stress response evidenced by higher C/ EBP α-homologous protein ( P=0.02), were activated in patients with DM . Higher inositol-requiring ER-to-nucleus signaling protein 1 activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction ( r=0.53, P=0.02). Acute treatment with liraglutide, a glucagon-like peptide 1 receptor agonist, reduced p-inositol-requiring ER-to-nucleus signaling protein 1 ( P=0.01), and the activation of its downstream target c-jun N-terminal kinase ( P=0.025) in endothelial cells from patients with DM . Furthermore, liraglutide restored insulin-stimulated endothelial nitric oxide synthase activation in patients with DM ( P=0.019). Conclusions In summary, our data suggest that ER stress contributes to vascular insulin resistance and endothelial dysfunction in patients with DM . Further, we have demonstrated that liraglutide ameliorates ER stress, decreases c-jun N-terminal kinase activation and restores insulin-mediated endothelial nitric oxide synthase activation in endothelial cells from patients with DM .

A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids.

INTRODUCTION: The only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). METHODS: Human breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. RESULTS: RNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n-3 → 18:4n-3 was suppressed as were downstream products 20:4n-3 and 20:5n-3. In contrast, no significant effect on D6D of linoleic acid 18:2n-6 → 18:3n-6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. CONCLUSION: The novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.

Fads3 modulates docosahexaenoic acid in liver and brain.

Fatty acid desaturase 3 (FADS3) is the third member of the FADS gene cluster. FADS1 and FADS2 code for enzymes required for highly unsaturated fatty acid (HUFA) biosynthesis, but FADS3 function remains elusive. We generated the first Fads3 knockout (KO) mouse with an aim to characterize its metabolic phenotype and clues to in vivo function. All mice (wild type (WT) and KO) were fed facility rodent chow devoid of HUFA. No differences in overt phenotypes (survival, fertility, growth rate) were observed. Docosahexaenoic acid (DHA, 22:6n-3) levels in the brain of postnatal day 1 (P1) KO mice were lower than the WT (P < 0.05). The ratio of docosapentaenoic acid (DPA, 22:5n-3) to DHA in P1 KO liver was higher than in WT suggesting lower desaturase activity. Concomitantly, 20:4n-6 was lower but its elongation product 22:4n-6 was greater in the liver of P1 KO mice. P1 KO liver Fads1 and Fads2 mRNA levels were significantly downregulated whereas expression levels of elongation of very long chain 2 (Elovl2) and Elovl5 genes were upregulated compared to age-matched WT. No Δ13-desaturation of vaccenic acid was observed in liver or heart in WT mice expressing FADS3 as was reported in vitro. Taken together, the fatty acid compositional results suggest that Fads3 enhances liver-mediated 22:6n-3 synthesis to support brain 22:6n-3 accretion before and during the brain growth spurt.

Alternative splicing generates novel Fads3 transcript in mice.

Fads3 is the third member of the fatty acid desaturase gene cluster; with at least eight evolutionarily conserved alternative transcripts (AT), having no clearly established function as are known for FADS2 and FADS1. Here we present identification of a novel Fads3 transcript in mice (Fads3AT9), characterize Fads3AT9 expression in mouse tissues and evaluate correlations with metabolite profiles. Total RNA obtained from mouse tissues is reverse-transcribed into cDNA and used as template for PCR reactions. Tissue fatty acids were extracted and quantified by gas chromatography. Sequencing analysis revealed complete absence of exon 2 resulting in an open reading frame of 1239 bp, encoding a putative protein of 412 aa with loss of 37 aa compared to classical Fads3 (Fads3CS). FADS3AT9 retains all the conserved regions characteristic of front end desaturase (cytochrome b5 domain and three histidine repeats). Both Fads3CS and Fads3AT9 are ubiquitously expressed in 11 mouse tissues. Fads3AT9 abundance was greater than Fads3CS in pancreas, liver, spleen, brown adipose tissue and thymus. Fads3CS expression is low in pancreas while Fads3AT9 is over ten-fold greater abundance. The eicosanoid precursor fatty acid 20:4n - 6, the immediate desaturation product of the Fads1 coded Δ5-desaturase, was highest in pancreas where Fads3CS is low. Changes in expression patterns and fatty acid profiles suggest that Fads3AT9 may play a role in the regulation and/or biosynthesis of long chain polyunsaturated fatty acids from precursors.

Positive Selection on a Regulatory Insertion-Deletion Polymorphism in FADS2 Influences Apparent Endogenous Synthesis of Arachidonic Acid.

Long chain polyunsaturated fatty acids (LCPUFA) are bioactive components of membrane phospholipids and serve as substrates for signaling molecules. LCPUFA can be obtained directly from animal foods or synthesized endogenously from 18 carbon precursors via the FADS2 coded enzyme. Vegans rely almost exclusively on endogenous synthesis to generate LCPUFA and we hypothesized that an adaptive genetic polymorphism would confer advantage. The rs66698963 polymorphism, a 22-bp insertion-deletion within FADS2, is associated with basal FADS1 expression, and coordinated induction of FADS1 and FADS2 in vitro. Here, we determined rs66698963 genotype frequencies from 234 individuals of a primarily vegetarian Indian population and 311 individuals from the US. A much higher I/I genotype frequency was found in Indians (68%) than in the US (18%). Analysis using 1000 Genomes Project data confirmed our observation, revealing a global I/I genotype of 70% in South Asians, 53% in Africans, 29% in East Asians, and 17% in Europeans. Tests based on population divergence, site frequency spectrum, and long-range haplotype consistently point to positive selection encompassing rs66698963 in South Asian, African, and some East Asian populations. Basal plasma phospholipid arachidonic acid (ARA) status was 8% greater in I/I compared with D/D individuals. The biochemical pathway product-precursor difference, ARA minus linoleic acid, was 31% and 13% greater for I/I and I/D compared with D/D, respectively. This study is consistent with previous in vitro data suggesting that the insertion allele enhances n-6 LCPUFA synthesis and may confer an adaptive advantage in South Asians because of the traditional plant-based diet practice.

Desaturase and elongase-limiting endogenous long-chain polyunsaturated fatty acid biosynthesis.

PURPOSE OF REVIEW: Endogenous synthesis of the long-chain polyunsaturated fatty acids (LCPUFAs) is mediated by the fatty acid desaturase (FADS) gene cluster (11q12-13.1) and elongation of very long-chain fatty acids 2 (ELOVL2) (6p24.2) and ELOVL5 (6p12.1). Although older biochemical work identified the product of one gene, FADS2, rate limiting for LCPUFA synthesis, recent studies suggest that polymorphisms in any of these genes can limit accumulation of product LCPUFA. RECENT FINDINGS: Genome-wide association study (GWAS) of Greenland Inuit shows strong adaptation signals within FADS gene cluster, attributed to high omega-3 fatty acid intake, while GWAS found ELOVL2 associated with sleep duration, age and DNA methylation. ELOVL5 coding mutations cause spinocerebellar ataxia 38, and epigenetic marks were associated with depression and suicide risk. Two sterol response element binding sites were found on ELOVL5, a SREBP-1c target gene. Minor allele carriers of a 3 single nucleotide polymorphism (SNP) haplotype in ELOVL2 have decreased 22 : 6n-3 levels. Unequivocal molecular evidence shows mammalian FADS2 catalyzes direct Δ4-desaturation to yield 22 : 6n-3 and 22 : 5n-6. An SNP near FADS1 influences the levels of 5-lipoxygenase products and epigenetic alteration. SUMMARY: Genetic polymorphisms within FADS and ELOVL can limit LCPUFA product accumulation at any step of the biosynthetic pathway.

Brown but not white adipose cells synthesize omega-3 docosahexaenoic acid in culture.

Adipose tissue is a complex endocrine organ which coordinates several crucial biological functions including fatty acid metabolism, glucose metabolism, energy homeostasis, and immune function. Brown adipose tissue (BAT) is most abundant in young infants during the brain growth spurt when demands for omega-3 docosahexaenoic acid (DHA, 22:6n-3) is greatest for brain structure. Our aim was to characterize relative biosynthesis of omega-3 long chain polyunsaturated fatty acids (LCPUFA) from precursors in cultured white (WAT) and brown (BAT) cells and study relevant gene expression. Mouse WAT and BAT cells were grown in regular DMEM media to confluence, and differentiation was induced. At days 0 and 8 cells were treated with albumin bound d5-18:3n-3 (d5-ALA) and analyzed 24h later. d5-ALA increased cellular eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) in undifferentiated BAT cells, whereas differentiated BAT cells accumulated 20:4n-3, EPA and DPA. DHA as a fraction of total omega-3 LCPUFA was greatest in differentiated BAT cells compared to undifferentiated cells. Undifferentiated WAT cells accumulated EPA, whereas differentiated cells accumulated DPA. WAT accumulated trace newly synthesized DHA. Zic1 a classical brown marker and Prdm16 a key driver of brown fat cell fate are expressed only in BAT cells. Ppargc1a is 15 fold higher in differentiated BAT cells. We conclude that in differentiated adipose cells accumulating fat, BAT cells but not WAT cells synthesize DHA, supporting the hypothesis that BAT is a net producer of DHA.

Evidence for anti-inflammatory and antioxidative properties of dried plum polyphenols in macrophage RAW 264.7 cells.

This study presents the anti-inflammatory and antioxidative properties of dried plum (Prunus domestica L.) polyphenols in macrophage RAW 264.7 cells. We hypothesized that dried plum polyphenols have strong anti-inflammatory and antioxidant properties against lipopolysaccharide (LPS)-induced production of the pro-inflammatory markers, nitric oxide (NO) and cyclooxygenase-2 (COX-2), and the lipid peroxidation product, malondialdehyde, in activated macrophage RAW 264.7 cells. To test this hypothesis, macrophage RAW 264.7 cells were stimulated with either 1 µg ml(-1) (for measurement of NO production) or 1 ng ml(-1) (for measurement of COX-2 expression) of LPS to induce inflammation and were treated with different doses of dried plum polyphenols (0.0, 0.1, 1, 10, 100 and 1000 µg ml(-1)). Dried plum polyphenols at a dose of 1000 µg ml(-1) was able to significantly (P < 0.05) reduce NO production by 43%. Additionally, LPS-induced expression of COX-2 was significantly (P < 0.05) reduced by 100 and 1000 µg ml(-1) dried plum polyphenols. To investigate the antioxidant activity of dried plum polyphenols, macrophage RAW 264.7 cells were stimulated with 100 µg ml(-1) of FeSO4 + 1 mM ml(-1) of H2O2 to induce lipid peroxidation. Dried plum polyphenols at a dose of 1000 µg ml(-1) showed a 32% reduction in malondialdehyde production. These findings indicate that dried plum polyphenols are potent anti-inflammatory and antioxidative agents in vitro.

Preparation of amorphous cefuroxime axetil nanoparticles by controlled nanoprecipitation method without surfactants.

Amorphous nanoparticles of cefuroxime axetil (CFA), a poorly water-soluble drug, were produced by the controlled nanoprecipitation method without any surfactants at room temperature. The influence of the operation parameters, such as the types of solvent and anti-solvent, the stirring speed, the solvent/anti-solvent (S/AS) volume ratio, the drug concentration and the precipitation temperature, were experimentally investigated. The results indicated that increasing the stirring speed and the S/AS volume, decreasing the drug concentration and the temperature favored to decrease the particle size from 700 to 900 nm to approximately 300 nm. The XRD analyses confirmed that the as-prepared CFA was amorphous nanoparticles. Furthermore, the amorphous CFA nanoparticles exhibited significantly enhanced dissolution property when compared to the commercial spray-dried product. The results demonstrated that the controlled nanoprecipitation method is a direct and feasible technology which could be utilized for preparation of the poorly water-soluble pharmaceutical nanoparticles.