No effect of PCSK9 inhibitor alirocumab on the incidence of diabetes in a pooled analysis from 10 ODYSSEY Phase 3 studies

AIMS: Statins have modest adverse effects on glycaemic control. Alirocumab, a proprotein convertase subtilisin/kexin type 9 inhibitor, lowers low-density lipoprotein cholesterol. This study assessed the effects of alirocumab on new-onset diabetes and pre-diabetes incidence in individuals without diabetes at baseline. METHODS AND RESULTS: Pooled analysis of 10 ODYSSEY Phase 3 trials (n = 4974) of 24–104 weeks duration. Six trials (n = 4211) were ≥52 weeks in length. Most patients received background maximally tolerated statin. Alirocumab effect on the rate of diabetes-related treatment-emergent adverse events (TEAEs), and/or fasting plasma glucose (FPG) and glycated haemoglobin A(1C) (HbA(1C)) was measured at baseline and every 12–24 weeks. Transition to diabetes analysis combined TEAE and FPG/HbA(1C) laboratory data. At baseline, 30.7% of individuals had diabetes and were excluded from the current analysis. The remaining 3448 individuals without diabetes had pre-diabetes (39.6%) or were normoglycaemic (29.7%). The hazard ratio (HR; 95% confidence interval) for diabetes-related TEAEs in alirocumab was 0.64 (0.36–1.14) vs. placebo and 0.55 (0.22–1.41) vs. ezetimibe. The HR associated for transition from pre-diabetes to new-onset diabetes for alirocumab was 0.90 (0.63–1.29) vs. placebo and 1.10 (0.57–2.12) vs. ezetimibe. Mean change in FPG/HbA(1C) over time showed no difference between treatment groups in patients without diabetes. CONCLUSIONS: There was no evidence of an effect of alirocumab on transition to new-onset diabetes in 3448 individuals without diabetes at baseline with a follow-up period of 6–18 months, compared to either placebo or ezetimibe. Longer follow-up with larger number of individuals is needed to conclusively rule out an effect

Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo

Liver-Specific Expression of Transcriptionally Active SREBP-1c Is Associated with Fatty Liver and Increased Visceral Fat Mass

The pathogenesis of fatty liver is not understood in detail, but lipid overflow as well as de novo lipogenesis (DNL) seem to be the key points of hepatocyte accumulation of lipids. One key transcription factor in DNL is sterol regulatory element-binding protein (SREBP)-1c. We generated mice with liver-specific over-expression of mature human SREBP-1c under control of the albumin promoter and a liver-specific enhancer (alb-SREBP-1c) to analyze systemic perturbations caused by this distinct alteration. SREBP-1c targets specific genes and causes key enzymes in DNL and lipid metabolism to be up-regulated. The alb-SREBP-1c mice developed hepatic lipid accumulation featuring a fatty liver by the age of 24 weeks under normocaloric nutrition. On a molecular level, clinical parameters and lipid-profiles varied according to the fatty liver phenotype. The desaturation index was increased compared to wild type mice. In liver, fatty acids (FA) were increased by 50% (p<0.01) and lipid composition was shifted to mono unsaturated FA, whereas lipid profile in adipose tissue or serum was not altered. Serum analyses revealed a ∼2-fold (p<0.01) increase in triglycerides and free fatty acids, and a ∼3-fold (p<0.01) increase in insulin levels, indicating insulin resistance; however, no significant cytokine profile alterations have been determined. Interestingly and unexpectedly, mice also developed adipositas with considerably increased visceral adipose tissue, although calorie intake was not different compared to control mice. In conclusion, the alb-SREBP-1c mouse model allowed the elucidation of the systemic impact of SREBP-1c as a central regulator of lipid metabolism in vivo and also demonstrated that the liver is a more active player in metabolic diseases such as visceral obesity and insulin resistance

Divergent phenotypes in siblings with identical novel mutations in the HNF-1α gene leading to maturity onset diabetes of the young type 3

BACKGROUND: Maturity onset diabetes of the young (MODY) is an autosomal dominant form of non–insulin-dependent diabetes mellitus caused by mutations in at least 13 different genes. The hepatocyte nuclear factor (HNF)-1α gene is affected in the most common form (HNF1A-MODY [MODY3]). CASE PRESENTATION: We describe the co-inheritance of a novel heterozygous missense mutation c.1761C &gt; G (p.Pro588Ala) with a novel complex deletion insertion mutation (c.1765_1766delinsGCCCGfs86*) in the HNF-1α gene among affected members of one family. Both mutations were present in the affected patients and neither was present in unaffected family members. The family had not only inheritance of MODY but also increased susceptibility to type 2 diabetes. Therefore one family member had classical type 2 diabetes including metabolic syndrome aggravated by a genetic predisposition in the form of HNF1A-MODY. CONCLUSION: The presence of common type 2 diabetes features should not detract from the possibility of MODY in patients with a striking autosomal-dominant family history

Weight gain and Food intake of C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a transgenic animals.

<p>Male C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a mice (n = 20 per genotype) were housed as groups of four under standard conditions with unlimited access to water and regular chow (13.0 MJ/kg: 53% carbohydrates, 11% fat, 36% protein). Weight gain (<b>A</b>) and Food intake (<b>B</b>) were measured once a week starting at weaning and monitored for an observation period of 18 weeks. Body weight (<b>C</b>), liver weight ((<b>D</b>) and WAT weight (<b>E</b>) were determined at sacrification. WAT contend per body weight (<b>F</b>), food uptake per body weight and (<b>G</b>) weight gain per food uptake (<b>H</b>) were determined in each group of mice. Data are given as means including standard deviation (±SD).</p

Macroscopic comparisons of C57Bl6 and transgenic alb-SREBP-1c animals.

<p>(A) First, sections of male mice at 24 weeks of age are shown. (B) Histology of visceral adipose tissue indicated hyperplasia but no signs of infiltration. All photographs were taken with the same magnification.</p

Effect of phosphorylation on DNA binding and acitvity of SREBP-1a <i>in vitro</i>.

<p>(<b>A</b>) His-SREBP-1a-NT fusion protein was incubated with or without JNK1 (40 ng/µg protein) as indicated. An EMSA of His-SREBP-1a-NT incubated with sre-1 fragment (upper panel) or E-box fragment (lower panel) is shown. To confirm equal loading, western-blot analyses with monoclonal anti-HisG-HRP antibody was performed. To control phosphorylation efficiency a JNK1 kinase assay with (γ³²P) ATP was performed and dried SDS-PAGE was exposed to X-ray film. For further details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032609#s4" target="_blank">Materials and Methods</a>”. (<b>B</b>) His-SREBP-1a-NT fusion protein was incubated with or without p38α (40 ng/µg protein) as indicated. An EMSA of His-SREBP-1a-NT incubated with sre-1 fragment (upper panel)or E-box fragment (lower panel) is shown. To confirm equal loading, western-blot analysis with monoclonal anti-HisG-HRP antibody were performed. To control phosphorylation efficiency a p38α kinase assay with (γ³²P) ATP was performed and dried SDS-PAGE was exposed to X-ray film. For further details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032609#s4" target="_blank">Materials and Methods</a>”.</p

Macroscopic and histological comparison of livers from C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a mice.

<p>Male mice of each genotype (C57Bl6, alb-SREBP1aΔP, alb-SREBP-1a (n = 20 per genotype)) were housed as groups of 4 under standard conditions with unlimited access to water and regular chow (13.0 MJ/kg: 53% carbohydrates, 11% fat, 36% protein). (<b>A</b>) Livers of a C57Bl6 mouse (left), alb-SREBP-1aΔP (middle) or alb-SREBP-1a (right). All photographs were taken with the same magnification. (<b>B</b>) Liver tissue of the Lobus caudatus, Lobus sinister- and Lobus dexter lateralis were fixed in 4% paraformaldehyd/PBS and embedded in paraffin with automated standard histological procedures. (I) Standard hematoxylin and eosin staining was performed on 3 µm deparaffinized sections. (II) PAS staining was performed to determine glycogen contend. (III) The tissues were also used for cryofixation and Oil-red-O staining was used for lipid visualization. (IV) Fibers and extra cellular matrix were visualized using the “van Gierson kit” to determine tissue integrity. The overview magnification is 1∶10 and details are shown in 1∶80 magnification.</p

Verification of p38 specific phosphorylation sites in SREBP-1a-NT.

<p>(<b>A</b>) Coomassie brilliant blue stained SDS-PAGE of GST-SREBP-1a-NT and each of mutated SREBP-1a-NT phosphorylated by activated recombinant p38α (40 ng/µg protein). (<b>B</b>) Autoradiography of SDS-PAGE of GST-SREBP-1a-NT fusion protein, single mutated forms S63A, T426V or double mutant GST-SREBP-1a-NT S63A/T426V phosphorylated by activated recombinant p38α. Excised radioactive slices of p38α phosphorylated recombinant proteins were trypsin-digested and the resulting peptides GST-SREBP-1a-NT, S63A, T426V or double mutant S63A/T426V, as indicated in the figure, were subjected to anion exchange chromatography. Elution was performed with a KH₂PO₄ pH 4 buffer gradient. Reactions performed are described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032609#s4" target="_blank">Materials and Methods</a>”. (<b>C</b>) Autoradiography of SDS-PAGE of GST-SREBP-1a-NT fusion protein, single mutated forms S63A, T426V or double mutant GST-SREBP-1a-NT S63A/T426V phosphorylated by activated recombinant p38β or p38γ (40 ng/µg protein).</p