Anna Secher

Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

SUMMARY Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

The arcuate nucleus mediates GLP-1 receptor agonist liraglutide-dependent weight loss

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide also reduces body weight. It is not fully understood how liraglutide induces weight loss or to what degree liraglutide acts directly in the brain. Here, we determined that liraglutide does not activate GLP-1-producing neurons in the hindbrain, and liraglutide-dependent body weight reduction in rats was independent of GLP-1 receptors (GLP-1Rs) in the vagus nerve, area postrema, and paraventricular nucleus. Peripheral injection of fluorescently labeled liraglutide in mice revealed the presence of the drug in the circumventricular organs. Moreover, labeled liraglutide bound neurons within the arcuate nucleus (ARC) and other discrete sites in the hypothalamus. GLP-1R was necessary for liraglutide uptake in the brain, as liraglutide binding was not seen in Glp1r(-/-) mice. In the ARC, liraglutide was internalized in neurons expressing proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Electrophysiological measurements of murine brain slices revealed that GLP-1 directly stimulates POMC/CART neurons and indirectly inhibits neurotransmission in neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) via GABA-dependent signaling. Collectively, our findings indicate that the GLP-1R on POMC/CART-expressing ARC neurons likely mediates liraglutide-induced weight loss.

Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues

Deregulated cellular signalling is a common hallmark of disease, and delineating tissue phosphoproteomes is key to unravelling the underlying mechanisms. Here we present the broadest tissue catalogue of phosphoproteins to date, covering 31,480 phosphorylation sites on 7,280 proteins quantified across 14 rat organs and tissues. We provide the data set as an easily accessible resource via a web-based database, the CPR PTM Resource. A major fraction of the presented phosphorylation sites are tissue-specific and modulate protein interaction networks that are essential for the function of individual organs. For skeletal muscle, we find that phosphotyrosines are over-represented, which is mainly due to proteins involved in glycogenolysis and muscle contraction, a finding we validate in human skeletal muscle biopsies. Tyrosine phosphorylation is involved in both skeletal and cardiac muscle contraction, whereas glycogenolytic enzymes are tyrosine phosphorylated in skeletal muscle but not in the liver. The presented phosphoproteomic method is simple and rapid, making it applicable for screening of diseased tissue samples.

A Major Lineage of Enteroendocrine Cells Coexpress CCK, Secretin, GIP, GLP-1, PYY, and Neurotensin but Not Somatostatin

Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.

The Effect of Real-Time Continuous Glucose Monitoring in Pregnant Women With Diabetes: A randomized controlled trial

OBJECTIVE To assess whether intermittent real-time continuous glucose monitoring (CGM) improves glycemic control and pregnancy outcome in unselected women with pregestational diabetes. RESEARCH DESIGN AND METHODS A total of 123 women with type 1 diabetes and 31 women with type 2 diabetes were randomized to use real-time CGM for 6 days at 8, 12, 21, 27, and 33 weeks in addition to routine care, including self-monitored plasma glucose seven times daily, or routine care only. To optimize glycemic control, real-time CGM readings were evaluated by a diabetes caregiver. HbA1c, self-monitored plasma glucose, severe hypoglycemia, and pregnancy outcomes were recorded, with large-for-gestational-age infants as the primary outcome. RESULTS Women assigned to real-time CGM (n = 79) had baseline HbA1c similar to that of women in the control arm (n = 75) (median 6.6 [range 5.3–10.0] vs. 6.8% [5.3–10.7]; P = 0.67) (49 [34–86] vs. 51 mmol/mol [34–93]). Forty-nine (64%) women used real-time CGM per protocol. At 33 weeks, HbA1c (6.1 [5.1–7.8] vs. 6.1% [4.8–8.2]; P = 0.39) (43 [32–62] vs. 43 mmol/mol [29–66]) and self-monitored plasma glucose (6.2 [4.7–7.9] vs. 6.2 mmol/L [4.9–7.9]; P = 0.64) were comparable regardless of real-time CGM use, and a similar fraction of women had experienced severe hypoglycemia (16 vs. 16%; P = 0.91). The prevalence of large-for-gestational-age infants (45 vs. 34%; P = 0.19) and other perinatal outcomes were comparable between the arms. CONCLUSIONS In this randomized trial, intermittent use of real-time CGM in pregnancy, in addition to self-monitored plasma glucose seven times daily, did not improve glycemic control or pregnancy outcome in women with pregestational diabetes.

Real-time trafficking and signaling of the glucagon-like peptide-1 receptor.

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.

Expression and distribution of glucagon-like peptide-1 receptor mRNA, protein and binding in the male nonhuman primate (Macaca mulatta) brain.

Glucagon-like peptide-1 (GLP-1) is released from endocrine L-cells lining the gut in response to food ingestion. However, GLP-1 is also produced in the nucleus of the solitary tract, where it acts as an anorectic neurotransmitter and key regulator of many autonomic and neuroendocrine functions. The expression and projections of GLP-1-producing neurons is highly conserved between rodent and primate brain, although a few key differences have been identified. The GLP-1 receptor (GLP-1R) has been mapped in the rodent brain, but no studies have described the distribution of GLP-1Rs in the nonhuman primate central nervous system. Here, we characterized the distribution of GLP-1R mRNA and protein in the adult macaque brain using in situ hybridization, radioligand receptor autoradiography, and immunohistochemistry with a primate specific GLP-1R antibody. Immunohistochemistry demonstrated that the GLP-1R is localized to cell bodies and fiber terminals in a very selective distribution throughout the brain. Consistent with the functional role of the GLP-1R system, we find the highest concentration of GLP-1R-immunoreactivity present in select hypothalamic and brainstem regions that regulate feeding, including the paraventricular and arcuate hypothalamic nuclei, as well as the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus. Together, our data demonstrate that GLP-1R distribution is highly conserved between rodent and primate, although a few key species differences were identified, including the amygdala, where GLP-1R expression is much higher in primate than in rodent.

GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand.

Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-β superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15–GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.

Analysis of Continuous Glucose Monitoring in Pregnant Women With Diabetes: Distinct Temporal Patterns of Glucose Associated With Large-for-Gestational-Age Infants

OBJECTIVE Continuous glucose monitoring (CGM) is increasingly used to assess glucose control in diabetes. The objective was to examine how analysis of glucose data might improve our understanding of the role temporal glucose variation has on large-for-gestational-age (LGA) infants born to women with diabetes. RESEARCH DESIGN AND METHODS Functional data analysis (FDA) was applied to 1.68 million glucose measurements from 759 measurement episodes, obtained from two previously published randomized controlled trials of CGM in pregnant women with diabetes. A total of 117 women with type 1 diabetes (n = 89) and type 2 diabetes (n = 28) who used repeated CGM during pregnancy were recruited from secondary care multidisciplinary obstetric clinics for diabetes in the U.K. and Denmark. LGA was defined as birth weight ≥90th percentile adjusted for sex and gestational age. RESULTS A total of 54 of 117 (46%) women developed LGA. LGA was associated with lower mean glucose (7.0 vs. 7.1 mmol/L; P < 0.01) in trimester 1, with higher mean glucose in trimester 2 (7.0 vs. 6.7 mmol/L; P < 0.001) and trimester 3 (6.5 vs. 6.4 mmol/L; P < 0.01). FDA showed that glucose was significantly lower midmorning (0900–1100 h) and early evening (1900–2130 h) in trimester 1, significantly higher early morning (0330–0630 h) and throughout the afternoon (1130–1700 h) in trimester 2, and significantly higher during the evening (2030–2330 h) in trimester 3 in women whose infants were LGA. CONCLUSIONS FDA of CGM data identified specific times of day that maternal glucose excursions were associated with LGA. It highlights trimester-specific differences, allowing treatment to be targeted to gestational glucose patterns.

Higher Gestational Weight Gain Is Associated With Increasing Offspring Birth Weight Independent of Maternal Glycemic Control in Women With Type 1 Diabetes

OBJECTIVE We evaluate the association between gestational weight gain and offspring birth weight in singleton term pregnancies of women with type 1 diabetes. RESEARCH DESIGN AND METHODS One hundred fifteen consecutive women referred at <14 weeks were retrospectively classified as underweight (prepregnancy BMI <18.5 kg/m2; n = 1), normal weight (18.5–24.9; n = 65), overweight (25.0–29.9; n = 39), or obese (≥30.0; n = 10). Gestational weight gain was categorized as excessive, appropriate, or insufficient according to the Institute of Medicine recommendations for each BMI class. Women with nephropathy, preeclampsia, and/or preterm delivery were excluded because of restrictive impact on fetal growth and limited time for total weight gain. RESULTS HbA1c was comparable at ∼6.6% (49 mmol/mol) at 8 weeks and ∼6.0% (42 mmol/mol) at 36 weeks between women with excessive (n = 62), appropriate (n = 37), and insufficient (n = 16) gestational weight gain. Diabetes duration was comparable, and median prepregnancy BMI was 25.3 (range 18–41) vs. 23.5 (18–31) vs. 22.7 (20–30) kg/m2 (P = 0.05) in the three weight gain groups. Offspring birth weight and birth weight SD score decreased across the groups (3,681 [2,374–4,500] vs. 3,395 [2,910–4,322] vs. 3,295 [2,766–4,340] g [P = 0.02] and 1.08 [−1.90 to 3.25] vs. 0.45 [−0.83 to 3.18] vs. −0.02 [−1.51 to 2.96] [P = 0.009], respectively). In a multiple linear regression analysis, gestational weight gain (kg) was positively associated with offspring birth weight (g) (β = 19; P = 0.02) and birth weight SD score (β = 0.06; P = 0.008) when adjusted for prepregnancy BMI, HbA1c at 36 weeks, smoking, parity, and ethnicity. CONCLUSIONS Higher gestational weight gain in women with type 1 diabetes was associated with increasing offspring birth weight independent of glycemic control and prepregnancy BMI.