Marianne Lunde

Variations in Postprandial Blood Glucose Responses and Satiety after Intake of Three Types of Bread

Background. The magnitude and duration of postprandial blood glucose (PPG) elevations are important risk factors of diabetes and coronary heart diseases. Aim. To study PPG after ingestion of breads with and without pea fibre and rapeseed oil. Methods. After fasting overnight, 10 Pakistani immigrant women participated in three experiments having a crossover design and involving ingestion of various types of bread: regular coarse bread or fibre enriched-bread with two levels of rapeseed oil, all providing 25 g available carbohydrates (CHO). Blood glucose and satiety were determined before the meal and every 15 min over the next 2 hours. Results. Intake of an amount of pea fibre-enriched bread containing 25 g CHO attenuated, the postprandial peak glucose value, the incremental area under the glucose versus time curve during 15 to 75 min, and the glycemic profile, and increased duration of satiety (P < .05), as compared with intake of regular bread with 25 g carbohydrate. Conclusion. Pea fibre-enriched breads can reduce PPG and prolong satiety.

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Background: Sodium-calcium exchanger 1 (NCX1) and calpain are up-regulated in heart failure (HF). Molecular mechanisms and functional consequences of NCX1 cleavage by calpain are not known. Results: Calpain anchors to two NCX1 regions and cleaves at methionine-369, leading to inactivation. Conclusion: NCX1 inhibition by calpain might improve cardiac function. Significance: Calpain might play a pivotal role in NCX1 regulation during HF. Cardiac sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is central to the maintenance of normal Ca2+ homeostasis and contraction. Studies indicate that the Ca2+-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca2+ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met369 antibody identified a novel calpain cleavage site at Met369. Engineering NCX1-Met369 into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met369 inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met369 cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Cheese can reduce indexes that estimate fatty acid desaturation. Results from the Oslo Health Study and from experiments with human hepatoma cells

Previously, cheese intake was shown to be inversely related to serum triglycerides, raising the possibility that cheese might inhibit triglyceride synthesis, which is governed by fatty acid desaturases. Therefore, analyses were done to study whether cheese intake was associated with indexes that reflect fatty acid desaturation in 121 healthy ethnic Norwegians aged 40-45 years, a subsample from the Oslo Health Study (N = 18 777). Experiments with human hepatoma cells (HepG2) were done to clarify whether cheese might have a causal effect on desaturases. Fatty acid distribution in lipids of human sera and HepG2 cells was determined by gas chromatography. Δ9-Desaturase was estimated by the (16:1,n-7)/(16:0) and (18:1,n-9)/(18:0) ratios, abbreviated ds9_1 and ds9_2, and Δ5-desaturase (ds5) by the (20:4,n-6)/(18:2,n-6) ratio. Correlation, ANOVA, and multiple linear regression models were used to study associations. Oslo Health Study: Subjects with cheese intake >4-6 times per week had 33% lower ds9_1 and 16% lower ds5 than subjects with intake ≤ 4-6 times per week. The cheese intake vs. ds5 association prevailed when adjusting for sex, time since last meal, fatty fish, vegetables, fruit-berries, fruit juice, cod liver oil, coffee, alcohol, body mass index, physical activity, length of education, and smoking. HepG2 cells: An ethanol extract of Jarlsberg cheese lowered the desaturase indexes. Inhibition of ds9_1 increased with increasing amount cheese extract added. Thus, cheese may contain inhibitors of desaturases, thereby providing an explanation for the previously reported negative association between cheese intake and triglycerides.

Slowed relaxation and preserved maximal force in soleus muscles of mice with targeted disruption of the Serca2 gene in skeletal muscle

Non‐technical summary  Muscle function depends on tightly regulated Ca2+ movement between the intracellular sarcoplasmic reticulum (SR) Ca2+ store and cytoplasm in muscle cells. Disturbances in these processes have been linked to impaired muscle function and muscle disease. We disrupted the gene for the SERCA2 SR Ca2+ pump in mouse skeletal muscle to study how decreased transport of Ca2+ into the SR would affect soleus muscle function. We found that the SERCA2 content was strongly reduced in the 40% fraction of soleus muscle fibres normally expressing SERCA2. Muscle relaxation was slowed, supporting the hypothesis that reduced SERCA2 would reduce Ca2+ transport into the SR and prolong muscle relaxation time. Surprisingly, the muscles maintained maximal force, despite the fact that less SERCA2 in these fibres would be expected to lower the amount of Ca2+ released during contraction, and thereby lower the maximal force. Our findings raise important questions regarding the roles of SERCA2 and SR in muscle function.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na+/K+-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca2+ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5–8Φ1Φ2-X8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

Mapping the in vitro interactome of cardiac sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca2+ homeostasis, serving as the primary mechanism for Ca2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.

Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

NCX1 (Na+/Ca2+ exchanger 1) is an important regulator of intracellular Ca2+ and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer68-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer68-PLM–NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1–PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer68-PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser68.

Short communication: Daily intake of 125 g of cheese for 2 weeks did not alter amount or distribution of serum lipids or desaturase indexes in healthy adults in an exploratory pilot study

Regular cheese contains saturated fat, consumption of which may negatively influence the amount of serum lipids. The American Dietary Guidelines (https://health.gov/dietaryguidelines/2015/guidelines/) recommend consumption of low-fat food. However, we observed a negative association between cheese intake and serum triglycerides and a positive association with high-density lipoprotein cholesterol. Cheese intake was also inversely related to metabolic syndrome and blunted the harmful association of intake of soft drinks with serum lipids. Cheese contains calcium and factors that may inhibit desaturases, thereby partly explaining why cheese might not have negative effects on serum lipids. Thus, opposing forces seem to govern the cheese effect but will any of these prevail? In an exploratory pilot study, 17 healthy subjects participated in a 4-wk crossover trial without washout. During the first 2 wk, 9 subjects were randomly assigned to add 125 g/d of regular cheese to their habitual diet. After 2 wk, cheese intake was discontinued and the subjects were instructed to return to their habitual diet. The other 8 subjects followed their habitual diet during the first 2 wk, and then added 125 g/d of cheese for the next 2 wk. Mean values (mmol/L) before and after 2 wk on habitual (cheese) diet were as follows: serum triglycerides: 0.91 (0.89) and 0.95 (0.91); total cholesterol: 5.25 (5.16) and 5.08 (5.24); low-density lipoprotein cholesterol: 3.18 (3.17) and 3.09 (3.22); and high-density lipoprotein cholesterol: 1.71 (1.64) and 1.61 (1.66). The fatty acid pattern in total serum lipids and desaturase indexes did not change significantly in response to high cheese intake. Thus, an appreciable increase in daily cheese intake for 2 wk may not alter concentrations of serum lipids, estimates of desaturases, or the distribution of serum fatty acids.