Susanne Lindquist

Lipid digestion and absorption in early life: an update.

Purpose of reviewTo highlight our understanding of digestion and absorption of dietary lipids in newborn infants, and specifically how these processes differ from those in children and adults. Recent findingsThe intestinal concentration of pancreatic triglyceride lipase (PTL) and bile salts is lower in newborns compared to later in life. Instead the PTL-related protein 2 and bile salt-stimulated lipase (BSSL) are the key enzymes secreted from pancreas, which in concerted action with gastric lipase operate to achieve efficient fat absorption during infancy. BSSL is also present in human milk which affects fat absorption and growth in breast-fed preterm infants. Under conditions of low luminal bile salt concentrations fat absorption is likely to occur from liquid crystalline product phases, which may result in absorption from an extended part of the small intestinal mucosal surfaces compared to adults. Chylomicron assembly and secretion also seem to adapt to the specific situation of the newborn. SummaryBoth fat digestion and product absorption are different in newborn infants compared to adults; other lipases are used for digestion and different physical–chemical phases may be used for product absorption. Why these differences occur is still an unsolved question of considerable importance to neonatal nutrition.

AmpG, a signal transducer in chromosomal β-lactamase induction

The chromosomal ampCβ‐lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by β‐lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid‐borne β‐lactamase is still inducible. We have isolated mutants, containing alterations in a novel E. coli gene, ampG, in which a cloned C. freundii ampC gene is unable to respond to β‐lactam inducers. The ampG gene was cloned, sequenced and mapped to minute 9.6 on the E. coli chromosome. The deduced amino acid sequence predicted AmpG to be a 53kDa, trans‐membrane protein, which we propose acts as a signal transducer or permease in the β‐lactamase induction system. Immediately upstream of ampG there is another 579‐base‐pair‐long open reading frame (ORF) encoding a putative lipoprotein shown to be non‐essential for β‐lactamase induction. We have found that ampG and this ORF form an operon, whose promoter is located in front of the ORF. Located closely upstream of the putative promoter is the morphogene bolA, which is transcribed in the opposite orientation. However, using transcription fusions, we have found that the ampG transcription is not regulated by bolA. In addition, we show that transcription is probably not regulated by either the starvation specific sigma factor RpoS, which controls bolA, or by AmpD the negative regulator for ampC transcription.

Bile Salt-Stimulated Lipase from Human Milk Binds DC-SIGN and Inhibits Human Immunodeficiency Virus Type 1 Transfer to CD4^+ T Cells

ABSTRACT A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

Signalling proteins in enterobacterial AmpC β‐lactamase regulation

The cloned Citrobacter freundii ampC β‐lactamase is inducible in the presence of its regulatory gene ampR in Escherichia coli (Lindberg et al., 1985). The basal level of expression and inducibility are affected by two E. coli proteins encoded by the closely linked ampD and ampE genes. Deletion of both genes led to constitutive ampR‐dependent overproduction of β‐lactamase, whereas an out‐of‐frame deletion in AmpD caused the basal expression to increase twofold. This ampD1 mutant was inducible at lower β‐lactam concentrations than the wild type. An IS1 insertion in ampD was polar on ampE expression and increased basal β‐lactamase expression 30‐fold while mediating a semi‐constitutive phenotype. AmpE expressed from a recombinant plasmid in an ampD ampE deletion mutant reduced basal β‐lactamase expression to wild‐type levels but did not markedly reduce β‐lactam resistance since the cells became hyperinducible. in the absence of AmpD, increasing levels of AmpE therefore decrease the basal expression of AmpC β‐lactamase in an AmpR‐dependent manner. AmpD modulated the response exerted on β‐lactamase expression by AmpE. The ampD gene encodes a 20.5kD cytoplasmic protein while the 32.1kD ampE gene product is an integral membrane protein with a likely ATP‐binding site between the second and third putative transmembrane region. Since neither AmpD nor AmpE are needed for β‐lactam induction and since these proteins could not be covalently labelled by benzylpenicillin, they are not thought to act as β‐lactam‐binding sensory tranducers. Instead it is suggested that AmpD and AmpE sense the effect of β‐lactam action on peptidoglycan biosynthesis and relay this signal to AmpR.

Lactoferrin is responsible for the fungistatic effect of human milk

Human milk has recognized anti-microbial effects and it has been repeatedly shown that breast-fed infants have fewer and less severe infections than formula-fed infants. While most studies have focused on anti-bacterial and anti-viral activities few have focused on the anti-fungal effect of human milk. Dermal and other infections caused by fungi are common in very low birth weight (VLBW) infants. Using a liquid culturing method and Candida albicans and Rhodotorula rubra as representative fungi, we studied the anti-fungal effect of human milk and certain human milk proteins. In vitro, human milk showed potent inhibitory effect on fungal growth. Most, if not all of this effect was caused by lactoferrin via its iron-binding capacity; increasing the iron content of the incubation medium abolished the inhibitory effect. In contrast, other human milk proteins with known or suggested anti-microbial effects rather increased fungal growth. Viability test and electron microscopy revealed that the growth inhibitory effect of human milk, i.e. mediated by lactoferrin, is fungistatic rather than fungicidal.

Diabetes and Pancreatic Exocrine Dysfunction Due to Mutations in the Carboxyl Ester Lipase Gene-Maturity Onset Diabetes of the Young (CEL-MODY) A PROTEIN MISFOLDING DISEASE

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.

Bile salt-stimulated lipase and pancreatic lipase-related protein 2 are the dominating lipases in neonatal fat digestion in mice and rats.

During infancy, the basic conditions for digestion of dietary fat differ from later in life. The bile salt–stimulated lipase (BSSL) is an enzyme expressed in the exocrine pancreas and in some species (including human) also in the lactating mammary gland and secreted with the milk. The aim of this study was to compare the ontogeny of four pancreatic lipases [BSSL, pancreatic triglyceride lipase (PL), pancreatic lipase-related protein 2 (PLRP2), and phospholipase A2 (PLA2)] in one species that supplies BSSL with milk (the mouse) and one that does not (the rat). We followed expression of the four pancreatic lipases from postnatal d 1 until after weaning in both species. We found that BSSL and PLRP2, two lipases with broad substrate specificity, dominated. It was not until weaning that significant expression of PL and PLA2 were induced. Thus, BSSL and PLRP2 seem to be responsible for fat digestion as long as milk is the main food. Moreover, the early temporal pattern of BSSL expression differed between species. We speculate that the milk-borne BSSL is able to compensate for a slower ontogeny of pancreatic BSSL expression in the mouse.

BSSL and PLRP2: key enzymes for lipid digestion in the newborn examined using the Caco-2 cell line

In rodents, bile salt-stimulated lipase (BSSL) and pancreatic lipase-related protein 2 (PLRP2) are the dominant lipases expressed in the exocrine pancreas in early life when milk is the main food. The aim of the present study was to evaluate whether BSSL and PLRP2 are also key enzymes in neonatal intestinal fat digestion. Using Caco-2 cells as a model for the small intestinal epithelium, purified human enzymes were incubated in the apical compartment with substrates, bile salt composition and concentrations physiologic to newborn infants. Both BSSL and PLRP2 hydrolyzed triglycerides (TG) to free FA and glycerol. Released FA were absorbed by the cells and reesterfied to TG. Together, BSSL and PLRP2 had a synergistic effect, increasing cellular uptake and reesterification 4-fold compared with the sum of each lipase alone. A synergistic effect was also observed with retinyl ester as a substrate. PLRP2 hydrolyzed cholesteryl ester but not as efficiently as BSSL, and the two had an additive rather than synergistic effect. We conclude the key enzymes in intestinal fat digestion are different in newborns than later in life. Further studies are needed to fully understand this difference and its implication for designing optimal neonatal nutrition.

Depot-specific messenger RNA expression of 11|[beta]|-hydroxysteroid dehydrogenase type 1 and leptin in adipose tissue of children and adults

Objective:To compare expression of messenger RNA (mRNA) coding for the cortisol regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), and the adipocytokines leptin and resistin in paired biopsies of subcutaneous adipose tissue (SC) and omental adipose tissue (OM) from children.Design:Paired biopsies (SC and OM) were obtained from 54 children (age 0.17–16 years, body mass index (BMI) 12.5–28.3 kg/m2, BMI standard deviation score (SDS) −2.5–4.5) and 16 adults (age 27–79 years, BMI 19–46 kg/m2) undergoing open abdominal surgery. mRNA levels of 11β-HSD1, leptin and resistin were measured using quantitative real-time polymerase chain reaction (PCR).Results:11β-HSD1 mRNA level was higher in OM than in SC (P<0.05), whereas leptin mRNA was higher in SC than in OM (P<0.001). There was no difference in the resistin mRNA level between SC and OM. These results were consistent in children and adults. In children, 11β-HSD1 mRNA in SC was positively associated with BMI SDS (P<0.05), whereas in OM it was positively associated with age (P<0.05). The association between 11β-HSD1 expression and age remained significant after adjustment for BMI SDS and gender. Leptin mRNA was positively associated with BMI SDS (SC: P<0.001, OM: P<0.001) but not with age in children. In multiple regression analyses, including anthropometric variables and age, BMI SDS was independently associated with mRNA levels of 11β-HSD1 (P<0.05) and leptin (P<0.001) in SC. When normal weight and overweight children were analyzed separately, 11β-HSD1 mRNA levels were positively associated with leptin in OM in the overweight group (P<0.05).Conclusion:There are depot-specific differences in mRNA levels of 11β-HSD1 and leptin in children and adults. The positive association of 11β-HSD1 mRNA in OM with age may reflect a causal role in visceral fat accumulation during growth. Increasing 11β-HSD1 and leptin mRNA in SC with increasing BMI SDS could suggest that the risk of metabolic consequences of obesity may be established early in life.

Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism

Objective Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms. Study Design ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions. Results DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein. Conclusion The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.