The lymphatic vessels constitute an extensive network of vessels distributed throughout the body that contribute to fluid homeostasis and antigen and lipid transport. Nearly half of the body lymph originates in the gut and virtually all the lymph within the mesenteric lymphatics is of intestinal origin (1). The collecting lymphatic vessels have inherent permeability, allowing lymph contents to leak into the surrounding adipose tissue for uptake by dendritic cells and macrophages that are located in close proximity to these vessels (2). Augmented lymphatic leakage has been shown to promote adipose tissue inflammation and accumulation (3). Considering that the gut lymphatics transporting lipids, antigens, and immune cells traverse mesenteric fat (MFAT), leak of their contents leads to what we propose is “lymphatic-adipose cross talk” potentially affecting MFAT metabolic function.
Adipose tissue inflammation is closely associated with insulin resistance (IR). IR in adipose tissue has been reported to precede that of other insulin target tissues, such as skeletal muscle and liver (4). Not all adipose tissue depots display similar phenotypes, and depot-specific adipose tissue characteristics have been reported by several investigators (5, 6). For example, impaired insulin-stimulated glucose disposal is associated with increased visceral fat but not increased subcutaneous fat (SFAT) (7). In contrast, lower visceral fat content is associated with greater insulin sensitivity, and “metabolically healthy obese individuals” have a 6-fold lower risk for type 2 diabetes compared with the “at risk” obese population (8). An important difference between visceral and subcutaneous adipose tissue is its exposure to lymphatic-derived molecules from lymphatics draining intestinal contents (9).
Alcohol has been shown to alter adipose tissue biology resulting in decreased adiponectin levels, decreased adipose tissue mass, macrophage infiltration, inflammatory cytokine secretion, reduced insulin-stimulated glucose uptake, and IR (9–11). However, the depot-specific effects of alcohol as they may reflect alterations in inflammatory milieu, adipokine expression, or insulin sensitivity have not been previously examined. Previously, we showed that acute alcohol intoxication following a “binge-like” intragastric alcohol administration increases mesenteric collecting lymphatic vessel compliance and permeability, leads to an inflammatory milieu in the mesenteric perilymphatic adipose tissue (MFAT), and alteration in systemic adipokine profile (11, 12). We believe these results suggest an alcohol-induced “lymphatic-MFAT crosstalk” initiating a cascade of events that we predict can culminate in alcohol-induced metabolic derangements. While acutely alcohol does not alter (13), chronic alcohol decreases insulin-stimulated glucose uptake in adipose tissue (10). Thus, the aim of the present study was to test the hypothesis that repeated binge-like alcohol intoxication (RBAI) would increase mesenteric lymphatic permeability leading to “lymphatic-MFAT crosstalk,” resulting in altered MFAT adipokine profile, inflammatory milieu, and insulin signaling. Moreover, we hypothesized that these alterations would be depot specific affecting only adipose tissue in close proximity to mesenteric lymphatics.
Animal studies were approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center and were performed in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Male Sprague–Dawley rats (270 g–350 g body wt) were housed in a controlled temperature (22°C) and controlled illumination (12:12 h light–dark cycle) environment. After arrival, the rats were allowed a 1-week acclimation period and were provided standard rat chow (2018 Teklad Global 18% Protein Rodent Diet, Harlan) and water ad libitum.
Gastric catheter placement and alcohol administration protocol
The gastric catheter placement was performed as previously described (12, 14). Seventy-two hours post catheter implantation surgery, conscious, unrestrained male Sprague–Dawley rats (270 g–350 g) received an intragastric bolus of 2.5 g/kg/day of alcohol in a nutritionally complete diet (Vanilla Ensure) (Abbott, Ill) or diet plus dextrose (isocaloric for alcohol diet) for 3 days through the indwelling gastric catheter. Intragastric alcohol administration through an indwelling catheter was selected because it is less stressful than gavage (15). The model for repeated binge-like intoxication was adapted from Lindtner et al. (16), who administered 3 g/kg of alcohol daily on three consecutive days. To control and ensure precise food intake, we further modified the above approach (16) by supplementing the alcohol administration with a complete liquid diet (Vanilla Ensure) (17). Chow was removed during the 3-day binge alcohol administration protocol and water was available ad libitum. Overnight fasted rats received either 12.5% alcohol w/v or isocaloric dextrose in Ensure on the first morning (between 7 AM and 8 AM) followed by Ensure only at the end of the day (5–6 PM). A total of 116 kcal/kg/day was administered to alcohol- and vehicle animals. This was repeated for two more times with a total of three administration days. On the third day, at 30 min after the last alcohol or dextrose/Ensure binge administration, animals were anesthetized with ketamine/xylazine (90/9 mg/kg), the mesentery isolated and excised and pinned in a dissection chamber containing 4°C albumin physiological salt solution for MFAT isolation. MFAT was completely isolated off connective tissue, blood vessels, and lymphatic vessels. In addition to MFAT, SFAT, and blood were also collected. The tissues were weighed, immediately frozen in liquid nitrogen, and stored at − 80°C for further analysis. The blood was centrifuged at 23,447 × g for 15 min and the supernatant was collected, frozen in liquid nitrogen, and stored at − 80°C for further analysis (Fig. 1A).
Determining lymphatic permeability
Lymphatic permeability was determined by Evans blue extravasation into MFAT, as previously described (11). Briefly, Evans Blue dye (1%) was diluted in the last dose of alcohol/dextrose (n = 5/group) + diet. Thirty minutes later the animals were sacrificed and MFAT was carefully removed from the mesenterium, freed of connective tissue, large blood vessels and lymphatic vessels, and was stored at –80°C until Evans blue concentration measurements were made (Fig. 1A) (11). A Evans blue concentration in MFAT was measured using spectrophotometry at 620 nm and normalized to MFAT weight. Evans blue standard curve was generated from a 1 mg/mL stock (10, 1, 0.1, 0.01, and 0.001 μg/mL).
Cytokine and adipokine measurements
MFAT (300 μL/100 mg of tissue) and SFAT (500 μL/100 mg of tissue) samples were homogenized in Tissue Protein Extraction Reagent buffer containing protease and phosphatase inhibitors (ThermoScientific, Waltham, Mass). Homogenate concentrations of cytokines in alcohol (n = 3) and vehicle (n = 3) treated animals were determined with a Milliplex Rat Cytokine/Chemokine Magnetic Bead Panel; that measures seven different cytokines (IL-1α, IL-1β, IL-6, IL-10, IFN-γ, TNF-α, and GM-CSF) (EMD Millipore, Billerica, Mass). Results for IL-1β and IL-6 (n = 6/cytokine/group) from MFAT were confirmed with single cytokine ELISA kits (Ray Biotech Inc, Norcross, Ga). Circulating Adiponectin (n = 4/group) and Visfatin (n = 3/group) were measured from plasma samples using a commercially available Rat Adiponectin ELISA (EMD Millipore) and rat Visfatin EIA (Ray Biotech Inc, Norcross, Ga), respectively, according to manufacturer's instructions and repeated at least one more time.
Total RNA isolation and real-time quantitative PCR (qPCR)
Total RNA was extracted using the RNeasy Mini Lipid Kit (Qiagen, Germantown, Md), as per the manufacturer's instructions. cDNA was synthesized from 1 μg of the resulting total RNA using the Quantitect Reverse Transcriptase Kit (Qiagen), in accordance with the manufacturer's instructions. Primers were designed to span exon-exon junctions and purchased from Integrated DNA Technologies (IDT, Coralville, Iowa; Table) and used at a concentration of 500 nmoles. The final reactions were made to a total volume of 20 μL with Quantitect SyBr Green PCR kit and DNase RNase-free water (Qiagen). All reactions were carried out in duplicate on a CFX96 system (Bio-Rad Laboratories, Hercules, Calif) for qPCR detection. qPCR data were analyzed using the comparative Ct (delta-delta-Ct, DDCT) method. Target genes were compared with the endogenous control, ribosomal protein S13 (RPS13), and alcohol values normalized to control values. The total RNA was extracted from the isolated MFAT of three animals per group. After the first run we repeated the qPCR from the same samples three more times to confirm our findings.
MFAT mast cell staining
Mesenteric branches (n = 3/group; 3–4 branches/group) containing adipose tissue, lymphatic vessels, and blood vessels were isolated and fixed in formamide, processed for paraffin embedding, and sectioned at a thickness of 5 μm. Toluidine blue (Sigma-Aldrich, St Louis, Mo) was used for histological staining of mast cells (11). Representative color micrographs were obtained from tissue sections (8–10/group) using ×20 objectives for mast cell analyses. Slides were imaged in a blinded manner using an Olympus DP72 Digital Camera System mounted to an Olympus BX51 TRF Microscope (Olympus, Center Valley, Pa) and the mast cells were counted per micrograph and averaged. Images were analyzed by a blinded investigator.
Determination of insulin signaling
After the last alcohol or dextrose administration (third day of the study), animals received Ensure only (no alcohol) and were then fasted for 16 h prior to receiving an intraperitoneal injection of insulin (0.25 U/kg BW) (18). Thirty minutes after the insulin injection, the animals were sacrificed and MFAT and SFAT were isolated as described above (Fig. 2A).
Tissue lysates from MFAT (n = 3 vehicle and n = 5 alcohol) and SFAT (n = 3/group) were prepared on Tissue Protein Extraction Reagent buffer containing protease and phosphatase inhibitors (ThermoScientific, Waltham, Mass). Protein concentration was determined using the BCA Protein Assay Kit (ThermoScientific, Ill). Equal amounts of protein from each sample (20 μg) were separated on 10% Mini-Protean TGX Gels (Biorad Laboratories, Philadelphia, Pa). PVDF Immobilon-P Transfer Membranes (EMD Millipore) were then blocked with 5% BSA in TBST and incubated overnight at 4°C with the following primary antisera: anti-pAKT (ser473—1:2000), anti-AKT (1:1,000), and anti-GRB2 (1:1,000) as a loading control and anti-Rabbit IgG HRP-linked (1:25,000) as secondary antisera (Cell Signaling Technology, Beverly, Mass). Blots were developed using Luminata Crescendo Western HRP substrate (EDM Millipore, Mass) and exposed on x-ray film. Immunoreactive bands were quantified by densitometry using an Image J imaging system and data were normalized to GRB2 levels. Western blots were repeated two more times using the same samples to confirm targeted protein expression.
Statistical data analysis
Summarized data are presented as mean ± SE and the n indicated for each group in the respective figure legends. T test was used to detect significant differences between alcohol- and vehicle groups. GraphPad Prism 4.0 Software (GraphPad, La Jolla, Calif) was used, and data were considered statistically significant at P < 0.05.
Repeated binge-like alcohol intoxication (RBAI) increases mesenteric lymphatic permeability
RBAI resulted in a significant increase in Evans blue concentration in MFAT compared with that of MFAT isolated from vehicle-treated animals (Fig. 1, B and C). As shown in Figure 1C, Evans blue was visible within the mesenteric lymphatics 30 min after its administration. The arrows indicate the mesenteric collecting lymphatic vessels with Evans blue among the MFAT, the red on the picture indicates blood vessels.
Repeated binge-like alcohol intoxication increases pro-inflammatory cytokine expression in MFAT but not in SFAT
Cytokine levels were determined in MFAT and SFAT. Our results showed significantly greater expression of IL-1α, IL-1β, IL-6, and GM-CSF in the MFAT of RBAI animals compared with vehicle (Fig. 3A). There were no statistically significant differences in cytokine expression in SFAT of RBAI compared with that of vehicle-treated animals (Fig. 3B).
Because of the high variability in cytokine expression observed in the samples, and to confirm the differences in expression of IL-1β and IL-6 detected with the Multiplex assay, single ELISA kits were used to measure and compare IL-1β and IL-6 expression across the two adipose depots. Confirming our Multiplex results, we observed a significant increase in IL-1β and IL-6 expression in the MFAT isolated from RBAI animals compared with vehicle (Fig. 3, C and E) and no significant differences in cytokine expression between groups in SFAT (Fig. 3D). IL-6 was not detectable in SFAT samples. The percentage expression of IL-1β was significantly higher in MFAT than in SFAT (MFAT 100 ± 4.3 and SFAT 20.4 ± 2.6, t test P < 0.0001).
Repeated binge-like alcohol intoxication increases gene expression of t lymphocytes, macrophages, and dendritic cell markers in MFAT
To examine if the alcohol-induced cytokine increase in MFAT was associated with adaptive immune cell infiltration, we measured the gene expression of CD4 (T-cell marker), CD11c (macrophage marker), and MHCII (dendritic cell marker) in MFAT. Alcohol significantly increased the mRNA expression of CD4 (Fig. 4A), CD11c (Fig. 4B), and MHCII (Fig. 4C) compared with vehicle.
Repeated binge-like alcohol intoxication increases mast cells in MFAT
The number of mast cells, quantified per micrograph of MFAT, was significantly higher in MFAT from RBAI animals than that of vehicle-treated animal (Fig. 5).
Repeated binge-like alcohol intoxication decreases circulating adiponectin and visfatin levels
To determine whether localized MFAT inflammation was associated with altered adipokine profiles, adiponectin and visfatin, both insulin-sensitizing adipokines were measured in circulation. RBAI resulted in a significant decrease in circulating plasma levels of adiponectin (Fig. 6A) and visfatin (Fig. 6B) when compared with vehicle.
Repeated binge-like alcohol intoxication decreases AKT phosphorylation in MFAT
We determined whether RBAI-induced lymphatic leak and the associated localized inflammation and decrease in insulin-sensitizing adipokines (adiponectin and visfatin) levels were associated with alterations in insulin signaling. MFAT and SFAT protein expression of phosphorylated serine/threonine-specific protein kinase (AKT-Ser473), a key mediator of insulin-stimulated glucose uptake (19), were determined. Insulin injection produced a marked 50% decrease in plasma glucose concentrations to an average of 76 mg/dL in both groups 30 min after injection. MFAT of RBAI showed significantly lower AKT phosphorylation than MFAT from vehicle-treated animals (Fig. 2B). No significant changes in SFAT AKT phosphorylation were detected between the two groups (Fig. 2C).
This study tested the hypothesis that the mechanisms underlying whole-body metabolic dysregulation resulting from RBAI involve mesenteric lymphatic hyperpermeability, adipose tissue inflammation, and associated dysregulated adipokine expression and insulin signaling. RBAI increased mesenteric lymphatic permeability, promoted an inflammatory milieu in MFAT but not in SFAT, decreased circulating adiponectin and visfatin levels, and impaired MFAT insulin signaling pathway. These results demonstrate that RBAI produces depot specific alterations in adipose tissue homeostasis.
Our results show adipose inflammation including increased recruitment of T cells, macrophages, and dendritic cells and enhanced pro-inflammatory cytokine expression limited to MFAT and not evident in SFAT. We believe this is a consequence of enhanced mesenteric collecting lymphatic permeability, allowing lymphatic gut-derived content to leak to the immediately surrounding MFAT. We propose this mechanism of lymphatic vessel-mesenteric perilymphatic adipose tissue crosstalk to be an initial event contributing to MFAT metabolic dysregulation, and possibly preceding whole-body metabolic dysregulation increasing the risk for systemic insulin resistance.
Human visceral adipose tissue is comprised of mesenteric and omental adipose tissues. In rodents, the presence of omental adipose tissue is negligible. Therefore, mesenteric adipose constitutes the rodent visceral adipose depot (20). The principal difference between visceral and non-visceral intra- and extra-abdominal adipose depots is their venous drainage. Visceral adipose tissue is drained by the portal vein, while the rest of the adipose depots are drained systemically. Thus MFAT venous drainage would be a direct route of transport of any mediators produced and released from MFAT directly to the liver. Because in contrast to other depots the MFAT contains mesenteric collecting lymphatic vessels draining from the intestine (21); MFAT is a direct target for gut-derived lymphatic content-mediated effects. These two factors make understanding the impact of alcohol on lymphatic content, permeability, and transport of critical importance as we unravel the mechanisms of alcohol-induced metabolic tissue injury.
Our results show that repeated binge-like alcohol intoxication leads to mesenteric lymphatic hyperpermeability and are in agreement with our previous findings where a single alcohol binge increased mesenteric lymphatic permeability and systemic circulating lipopolysaccharide (LPS) levels (11). LPS, derived from the cell wall of gram-negative bacteria, has been proposed as an important trigger of alcohol-induced organ and tissue damage (22). Overgrowth of gram-negative bacteria in the small intestine has been reported in alcoholics and it is predicted to result in increased production of LPS (23). Exogenous LPS administration leads to mesenteric collecting lymphatic leakage of macromolecules into surrounding tissues, suggesting a role for LPS in mesenteric collecting lymphatic hyperpermeability (24). Although we did not measure lymph bacterial and LPS content, we believe that alcohol-induced gram-negative bacterial growth and gut leakage (25) allows bacterial and LPS translocation through lacteals (intestinal initial lymphatics) into mesenteric collecting lymphatics. A consequence of increased LPS transport through these mesenteric collecting lymphatic vessels would be hyperpermeability and leakage of lymph contents into MFAT promoting inflammation and in turn metabolic dysregulation. Consistent with this notion, hyperpermeable lymphatic vasculature, induced by haploinsufficiency of Prox1, was shown to increase MFAT accumulation and MFAT inflammatory cells in mice (3).
Visceral adiposity is strongly associated with IR and systemic inflammation (7). The “portal theory” suggests that the development of hepatic IR and liver steatosis is a consequence of the direct exposure of the liver to increased amounts of FFA and pro-inflammatory factors released from visceral adipose tissue (26). In addition to inducing lipolysis, which would most likely lead to hepatic adipose triglyceride deposition, enhanced inflammatory cytokine release following RBAI could promote hepatic insulin resistance (27).
Our results showed increased gene expression of T cell, macrophages, and dendritic cell markers as well as an increased amount of mast cells on the MFAT from alcohol treated animals compared with vehicle, suggesting that alcohol indeed promotes immune cell recruitment. Whether these pro-inflammatory cells are being recruited or leaked from lymphatic vessels remains to be determined. However, recent studies in a rat model of metabolic syndrome found that macrophages and dendritic cells leak out of collecting lymphatic vessels and into surrounding adipose tissue, modulating adipose tissue inflammation and immunity (2). Consistent with our findings, episodic binge alcohol also drives macrophage infiltration into adipose tissue and increases adipose pro-inflammatory cytokines in burn-injured animals (28). Dendritic cells are antigen-presenting cells, and their accumulation in adipose tissue of obese rodents and humans directly correlates with insulin resistance (29). These cells are also capable of presenting antigens to naive CD4+ T cells in an antigen-specific manner, making adipose dendritic cells potential important regulators of inflammation (29). Taken together, our findings suggest an active immune response to lymphatic leak that most likely contributes to metabolic dysregulation in MFAT.
Several lines of evidence support the link between adipose tissue metabolic dysfunction and inflammation. Decreased insulin sensitivity is associated with adipose tissue metabolic dysfunction, characterized by enlarged adipocyte cell size, altered adipokine profile, and low expression of GLUT4 (30). Adipose cells from normoglycemic individuals with genetic predisposition for type 2 diabetes present marked impairments in insulin signaling with insulin-stimulated glucose transport and GLUT4 expression reduced to similar extent as in diabetic cells, suggesting that insulin resistance localized to adipose tissue may be an initial pathophysiological change preceding diabetes (31). Pro-inflammatory cytokines and adipokines can interfere with insulin signal transduction (32), and alcohol-induced adipose tissue inflammation has been shown to decrease insulin-stimulated glucose uptake in isolated rat adipocytes, partially due to decreased GLUT4 membrane translocation (33). Our results show that repeated binge-like alcohol intoxication induces MFAT inflammation, decreases circulating adiponectin and visfatin levels, and impairs insulin signaling pathway in MFAT, suggesting a potential role of alcohol on the onset of visceral fat insulin resistance. While visfatin exerts dose-dependent insulin-mimetic effects similar to those of insulin (34), adiponectin has been shown to increase insulin sensitivity (35). In our earlier studies, we saw that one bolus of alcohol decreased adiponectin levels prior to an increase in pro-inflammatory cytokines in MFAT. Here, after 3 days of alcohol administration, adiponectin was decreased and MFAT inflammatory cytokines were increased at the same time. Overall, we believe that alcohol initiates a cascade of events culminating in alteration of adipokine profile and promotion of mesenteric adipose tissue inflammation and a consequence of this inflammation is insulin signaling impairment.
In summary, repeated binge-like alcohol intoxication promotes MFAT adaptive immune cell infiltration and inflammation and decreases adiponectin and visfatin levels, impairing MFAT insulin signaling. SFAT does not present an alcohol-induced inflammation, most likely because it is not in direct contact to gut lymphatic vessels and their contents. We speculate that alcohol-induced gut and lymphatic permeability is an important driving force for LPS dissemination and leakage into MFAT, promoting MFAT inflammation and metabolic dysregulation. We propose a link between visceral adipose tissue inflammation and increased risk for metabolic disorders stemming from alcohol-induced gut lymphatic leakage and the associated “lymphatic- mesenteric perilymphatic adipose tissue crosstalk.” We predict that restoring lymphatic integrity, preventing leak of gut-derived lymphatic contents to the visceral adipose tissue should decrease risk for the development of metabolic dysregulation resulting from risky alcohol consumption.
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