Leukotriene B4 receptor 2 governs macrophage migration during tissue inflammation.

Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies in vitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation in vivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.

Characterization of lipoproteins in human placenta and fetal circulation as well as gestational changes in lipoprotein assembly and secretion in human and mouse placentas.

In the maternal circulation, apoB-containing low-density lipoproteins (LDL) and apoA1-containing high-density lipoproteins (HDL) transport lipids. The production of lipoproteins in the placenta has been suggested, but the directionality of release has not been resolved. We compared apolipoprotein concentrations and size-exclusion chromatography elution profiles of lipoproteins in maternal/fetal circulations, and in umbilical arteries/veins; identified placental lipoprotein-producing cells; and studied temporal induction of lipoprotein-synthesizing machinery during pregnancy. We observed that maternal and fetal lipoproteins are different with respect to concentrations and elution profiles. Surprisingly, concentrations and elution profiles of lipoproteins in umbilical arteries and veins were similar indicating their homeostatic control. Human placental cultures synthesized apoB100-containing LDL-sized and apoA1-containing HDL-sized particles. Immunolocalization techniques revealed that ApoA1 was present mainly in syncytiotrophoblasts. MTP, a critical protein for lipoprotein assembly, was in these trophoblasts. ApoB was in the placental stroma indicating that trophoblasts secrete apoB-containing lipoproteins into the stroma. ApoB and MTP expressions increased in placentas from the 2nd trimester to term, whereas apoA1 expression was unchanged. Thus, our studies provide new information regarding the timing of lipoprotein gene induction during gestation, the cells involved in lipoprotein assembly and the gel filtration profiles of human placental lipoproteins. Next, we observed that mouse placenta produces MTP, apoB100, apoB48 and apoA1. The expression of genes gradually increased and peaked in late gestation. This information may be useful in identifying transcription factors regulating the induction of these genes in gestation and the importance of placental lipoprotein assembly in fetal development.

Protocol to isolate immune cells from mouse pancreatic lymph nodes and whole pancreas for mass cytometric analyses.

Investigating the immune attack on ß cells is critical to understanding autoimmune diabetes. Here, we present a protocol to isolate immune cells from mouse pancreatic lymph nodes and whole pancreas, followed by mass cytometric analyses. This protocol can be used to analyze subsets of innate and adaptive immune cells that play critical roles in autoimmune diabetes, with as few as 5 × 105 cells. This protocol can also be adapted to study resident immune cells from other tissues. For complete details on the use and execution of this protocol, please refer to Piñeros et al. (2022).1.

An Observational Study showing Dipeptidyl Peptidase-4 (DPP-4) Activity and Gene Expression Variation in Chronic Liver Disease (CLD) Patients from a Tertiary Care Hospital of Eastern India.

Introduction: The studies in animal models of cirrhosis suggest that dipeptidyl peptidase type 4 (DPP-4) enzymes play a crucial role in disease pathogenesis. In this clinical observational study, activity of DPP-4 and related gene expression were analysed in chronic liver disease patients. Objectives: To understand the DPP-4 enzyme activity variation in the common types of chronic liver disease by assessing plasma and peripheral blood mononuclear cell (PBMC) DPP-4 activity and comparing with healthy controls and to explore DPP-4 gene expression in PBMC. Methods: We recruited 130 study subjects in four cohorts-46 nonalcoholic fatty liver disease (NAFLD), 23 non-alcoholic cirrhosis (NAC) excluding viral aetiology, 21 alcoholic liver disease (ALC), and 40 control subjects. Blood samples were analysed for relevant biochemical parameters and plasma DPP-4 activity. PBMC fraction was used for the DPP-4 activity assay and gene expression analysis. Results: We found that lower plasma DPP-4 activity among patient cohorts but this was not statistically significant. The PBMC DPP-4 activity was significantly lower in NAFLD cohort. In the same cohort, DPP-4 gene expression in PBMC fraction was significantly increased (P < 0.05). There was significant correlation between plasma DPP-4 activity and liver injury marker alanine aminotransferase (ALT) among NAFLD (rho = 0.459, P < 0.01), NAC (rho = 0.475, P < 0.05), and ALC (rho = -0.572, P < 0.01) patients. Plasma DPP-4 activity modestly predicted ALT plasma level (beta coefficient = 0.489, P < 0.01). Conclusions: The PBMC DPP-4 activity and DPP-4 gene expression gets significantly altered in NAFLD patients. Plasma DPP-4 activity also shows correlation with ALT levels in CLD patients. The role of DPP-4 in disease pathology in NAFLD and other forms of CLD needs to be explored.

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling.

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.

Incretins in fibrocalculous pancreatic diabetes: A unique subtype of pancreatogenic diabetes.

BACKGROUND: Studies evaluating endocrine and exocrine functions in fibrocalculous pancreatic diabetes (FCPD) are scarce. METHODS: Insulin, C-peptide, glucagon, incretin hormones (glucagon-like peptide 1 [GLP-1] and gastric inhibitory peptide [GIP]), and dipeptidyl peptidase IV (DPP-IV) were estimated in patients with FCPD (n = 20), type 2 diabetes mellitus (T2DM) (n = 20), and controls (n = 20) in fasting and 60 minutes after 75 g glucose. RESULTS: Fasting and post-glucose C-peptide and insulin in FCPD were lower than that of T2DM and controls. Plasma glucagon decreased after glucose load in controls (3.72, 2.29), but increased in T2DM (4.01, 5.73), and remained unchanged in FCPD (3.44, 3.44). Active GLP-1 (pmol/L) after glucose load increased in FCPD (6.14 to 9.72, P = <.001), in T2DM (2.87 to 4.62, P < .001), and in controls (3.91 to 6.13, P < .001). Median active GLP-1 in FCPD, both in fasting and post-glucose state (6.14, 9.72), was twice that of T2DM (2.87, 4.62) and 1.5 times that of controls (3.91, 6.13) (P < .001 for all). Post-glucose GIP (pmol/L) increased in all: FCPD (15.83 to 94.14), T2DM (21.85 to 88.29), and control (13.00 to 74.65) (P < .001 for all). GIP was not different between groups. DPP-IV concentration (ng/mL) increased in controls (1578.54, 3012.00) and FCPD (1609.95, 1995.42), but not in T2DM (1204.50, 1939.50) (P = .131). DPP-IV between the three groups was not different. Fecal elastase was low in FCPD compared with T2DM controls. CONCLUSIONS: In FCPD, basal C-peptide and glucagon are low, and glucagon does not increase after glucose load. GLP-1, but not GIP, in FCPD increases 1.5 to 2 times as compared with T2DM and controls (fasting and post glucose) without differences in DPP-IV.

Interplay between ß-carotene and lipoprotein metabolism at the maternal-fetal barrier.

Vitamin A is an essential nutrient, critical for proper embryonic development in mammals. Both embryonic vitamin A-deficiency or -excess lead to congenital malformations or lethality in mammals, including humans. This is due to the defective transcriptional action of retinoic acid, the active form of vitamin A, that regulates in a spatial- and temporal-dependent manner the expression of genes essential for organogenesis. Thus, an adequate supply of vitamin A from the maternal circulation is vital for normal mammalian fetal development. Provitamin A carotenoids circulate in the maternal bloodstream and are available to the embryo. Of all the dietary carotenoids, ß-carotene is the main vitamin A precursor, contributing at least 30% of the vitamin A intake in the industrialized countries and often constituting the sole source of retinoids (vitamin A and its derivatives) in the developing world. In humans, up to 40% of the absorbed dietary ß-carotene is incorporated in its intact form in chylomicrons for distribution to other organs within the body, including the developing tissues. Here, it can serve as a source of vitamin A upon conversion into apocarotenoids by its cleavage enzymes. Given that ß-carotene is carried in the bloodstream by lipoproteins, and that the placenta acquires, assembles and secretes lipoproteins, it is becoming evident that the maternal-fetal transfer of ß-carotene relies on lipoprotein metabolism. Here, we will explore the current knowledge about this important biological process, the cross-talk between carotenoid and lipid metabolism in the context of the maternal-fetal transfer of this provitamin A precursor, and the mechanisms whereby ß-carotene is metabolized by the developing tissues. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

Impaired compensatory hyperinsulinemia among nonobese type 2 diabetes patients: a cross-sectional study.

AIMS: Obesity associated prolonged hyperinsulinemia followed by ß-cell failure is well established as the pathology behind type 2 diabetes mellitus (T2DM). However, studies on nonobese T2DM have reported it to be a distinct clinical entity with predominant insulin secretory defect. We, therefore, hypothesized that compensatory hyperinsulinemia in response to weight gain is impaired in nonobese subjects. METHODS: This was a cross-sectional study from a community-based metabolic health screening program. Adiposity parameters including body mass index (BMI), waist circumference (WC), body fat percentage, plasma leptin concentration and metabolic parameters namely fasting insulin, glucose, total cholesterol, and triglycerides were measured in 650 individuals (73% healthy, 62% nonobese with a BMI <25). RESULTS: In contrast to obese T2DM, nonobese T2DM patients did not exhibit significant hyperinsulinemia compared with the nonobese healthy group. Age, sex, and fasting glucose adjusted insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR) and HOMA-beta cell function (HOMA-B) were increased in obese T2DM compared with nonobese T2DM. Although adiposity parameters showed strong correlation with fasting insulin in obese healthy (r = 0.38, 0.38, and 0.42, respectively; all p values < 0.001) and T2DM (r = 0.54, 0.54, and 0.66, respectively; all p < 0.001), only BMI and leptin showed a weak correlation with insulin in the nonobese healthy group (0.13 and 0.13, respectively; all p < 0.05) which were completely lost in the nonobese T2DM. CONCLUSIONS: Compensatory hyperinsulinemia in response to weight gain is impaired in the nonobese population making insulin secretory defect rather than IR the major pathology behind nonobese T2DM.

Increased Plasma Dipeptidyl Peptidase-4 (DPP4) Activity Is an Obesity-Independent Parameter for Glycemic Deregulation in Type 2 Diabetes Patients.

Background: Increase in circulating dipeptidyl peptidase-4 (DPP4) activity and levels has been reported to associate both with hyperglycemia and obesity. Here we aim to decipher the role of enhanced plasma DPP4 activity in obese type 2 diabetes (T2DM) patients. Materials and methods: Plasma DPP4 levels and activity were measured in obese and non-obese newly diagnosed T2DM patients (n = 123). Visceral and subcutaneous adipose tissue DPP4 expression and activity were determined in 43 obese subjects (T2DM = 21 and non-T2DM = 22). 20 subjects undergoing Mini-Gastric Bypass (MGB) surgery were followed up over 4-6 weeks for plasma DPP4. Results: Plasma DPP4 levels and activity both were increased in T2DM patients compared to control group. However, DPP4 levels and not DPP4 activity were increased in obese T2DM patients compared to non-obese T2DM (62.49 ± 26.27 µg/ml vs. 48.4 ± 30.98 µg/ml, respectively, p = 0.028). DPP4 activity in visceral adipose tissue (VAT) from obese T2DM and obese non-T2DM groups were similar (5.05 ± 3.96 nmol/min/ml vs. 5.83 ± 4.13 nmol/min/ml respectively, p = 0.548) in spite of having increased DPP4 expression in the obese T2DM group. Moreover, in obese patients, plasma DPP4 levels and activity did not show any significant change after weight reduction and glycemic control following MGB surgery. Conclusion: Enhanced plasma DPP4 activity in T2DM occurs independently of obesity. Thus, adipose derived DPP4 may not be playing any significant role in glycemic deregulation in obese T2DM patients.

Significance of circulatory DPP4 activity in metabolic diseases.

Dipeptidyl peptidase 4 (DPP4), also known as CD26 is a type II transmembrane protein that is released from the cell membrane in a nonclassical secretory mechanism. This exopeptidase selectively degrades varieties of substrates including incretin hormones, growth factors, and cytokines. A significant detectable amount of DPP4 activity can be measured in plasma as well as in different tissues such as intestinal epithelium, vascular endothelium, lymphocytes, monocytes, kidney, liver, adipose, lung, thymus, spleen, prostate, etc. Enzymatically active circulatory DPP4 is shed from the plasma membrane via proteolytic cleavage, a process responsible for the enhanced plasma DPP4 levels and activity. Elevated circulatory DPP4 activity as well as levels has been found in wide spectrum of metabolic diseases including diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver diseases. Moreover, recent preclinical studies have further expanded the repertoire for the usage of DPP4 inhibitors in the treatment of other metabolic diseases and in their consequent complications. In the present review we highlight the reason behind the elevated circulatory DPP4 levels in metabolic diseases with a focus on the tissue of origin. We also underscore the discrepancy of protein levels with enzyme activity of circulatory DPP4 in metabolic diseases. © 2018 IUBMB Life, 70(2):112-119, 2018.

KLK5 induces shedding of DPP4 from circulatory Th17 cells in type 2 diabetes.

OBJECTIVE: Increasing plasma levels and activity of dipeptidyl peptidase-4 (DPP4 or CD26) are associated with rapid progression of metabolic syndrome to overt type 2 diabetes mellitus (T2DM). While DPP4 inhibitors are increasingly used as anti-hyperglycemic agents, the reason for the increase in plasma DPP4 activity in T2DM patients remains elusive. METHODS: We looked into the source of plasma DPP4 activity in a cohort of 135 treatment naive nonobese (BMI < 30) T2DM patients. A wide array of ex vivo, in vitro, and in silico methods were employed to study enzyme activity, gene expression, subcellular localization, protease identification, surface expression, and protein-protein interactions. RESULTS: We show that circulating immune cells, particularly CD4+ T cells, served as an important source for the increase in plasma DPP4 activity in T2DM. Moreover, we found kallikrein-related peptidase 5 (KLK5) as the enzyme responsible for cleaving DPP4 from the cell surface by directly interacting with the extracellular loop. Expression and secretion of KLK5 is induced in CD4+ T cells of T2DM patients. In addition, KLK5 shed DPP4 from circulating CD4+ T helper (Th)17 cells and shed it into the plasma of T2DM patients. Similar cleavage and shedding activities were not seen in controls. CONCLUSIONS: Our study provides mechanistic insights into the molecular interaction between KLK5 and DPP4 as well as CD4+ T cell derived KLK5 mediated enzymatic cleavage of DPP4 from cell surface. Thus, our study uncovers a hitherto unknown cellular source and mechanism behind enhanced plasma DPP4 activity in T2DM.

Neck height ratio is an important predictor of metabolic syndrome among Asian Indians.

BACKGROUND AND AIMS: The predictive potential of neck circumference (NC) based indices (a measure of upper body fat distribution) for predicting metabolic syndrome (MetS) and its components among Indians is not known. This study aimed to evaluate the role of NC and neck height ratio (NHtR) as independent predictors of MetS and its components as compared to traditional anthropometric indices. MATERIALS AND METHODS: A total of 451 individuals from 867 screened individuals, 30-80 years age, without any co-morbid state who gave informed written consent underwent clinical, anthropometric, and biochemical assessment. RESULTS: Patients with MetS in both the sexes had significantly higher NC, NHtR, glycated hemoglobin, fasting glucose, and dyslipidemia (higher triglycerides, total cholesterol/high-density lipoprotein cholesterol (HDL-C) ratio, low-density lipoprotein cholesterol/HDL-C ratio, and lower HDL-C). In both sexes, individuals in the highest tertile of NC had significantly greater central and generalized obesity, lower HDL-C, and significantly higher MetS. Receiver operating characteristic analysis revealed waist circumference (WC) to have the largest area under the curve for predicting MetS in both sexes, followed by NHtR, NC, and body mass index. NC and NHtR of >34.9 cm (sensitivity 78.6%; specificity 59.3%) and >21.17 cm/m (sensitivity 80.7% and specificity 64.6%) respectively for men and >31.25 cm (sensitivity 72.3%; specificity 64.4%) and >20.48 cm/m (sensitivity 80.4% and specificity 60%) respectively for women were the best values for identifying MetS. Increased NC and NHtR had odds ratio of 1.52 (95% confidence interval [CI]: 1.37-1.68; P < 0.001) and 1.96 (95% CI: 1.67-2.29; P < 0.001) respectively in identifying MetS. CONCLUSION: NC and NHtR are good predictors of MetS and cardiovascular risk factors in Asian Indians. NHtR is reliable and perhaps an even better index than NC with regards to cardiovascular risk prediction.

Adipose Recruitment and Activation of Plasmacytoid Dendritic Cells Fuel Metaflammation.

In obese individuals, visceral adipose tissue (VAT) is the seat of chronic low-grade inflammation (metaflammation), but the mechanistic link between increased adiposity and metaflammation largely remains unclear. In obese individuals, deregulation of a specific adipokine, chemerin, contributes to innate initiation of metaflammation by recruiting circulating plasmacytoid dendritic cells (pDCs) into VAT through chemokine-like receptor 1 (CMKLR1). Adipose tissue-derived high-mobility group B1 (HMGB1) protein activates Toll-like receptor 9 (TLR9) in the adipose-recruited pDCs by transporting extracellular DNA through receptor for advanced glycation end products (RAGE) and induces production of type I interferons (IFNs). Type I IFNs in turn help in proinflammatory polarization of adipose-resident macrophages. IFN signature gene expression in VAT correlates with both adipose tissue and systemic insulin resistance (IR) in obese individuals, which is represented by ADIPO-IR and HOMA2-IR, respectively, and defines two subgroups with different susceptibility to IR. Thus, this study reveals a pathway that drives adipose tissue inflammation and consequent IR in obesity.