High Fat Intake Leads to Acute Postprandial Exposure to Circulating Endotoxin in Type 2 Diabetic Subjects

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High Fat Intake Leads to AcutePostprandial Exposure to CirculatingEndotoxin in Type 2 Diabetic SubjectsALISON L. HARTE, PHD1MADHUSUDHAN C. VARMA, MRCP1

GYANENDRA TRIPATHI, PHD1

KIRSTY C. MCGEE, PHD1

NASSER M. AL-DAGHRI, PHD2

OMAR S. AL-ATTAS, PHD2

SHAUN SABICO, MD2JOSEPH P. OHARE, MD1

ANTONIO CERIELLO, MD3

PONNUSAMY SARAVANAN, PHD4

SUDHESH KUMAR, MD1

PHILIP G. MCTERNAN, PHD1

OBJECTIVEdTo evaluate thechanges in circulatingendotoxin after a highsaturated fat mealto determine whether these effects depend on metabolic disease state.

RESEARCH DESIGN AND METHODSdSubjects (n = 54) were given a high-fat meal(75 g fat, 5 g carbohydrate, 6 g protein) after an overnight fast (nonobese control [NOC]: age39.96 11.8 years [mean6 SD], BMI 24.96 3.2 kg/m2, n = 9; obese: age 43.86 9.5 years, BMI

33.36

2.5 kg/m

2

,n

= 15; impaired glucose tolerance [IGT]: age 41.76

11.3 years, BMI 32.06

4.5 kg/m2, n = 12; type 2 diabetic: age 45.46 10.1 years, BMI 30.36 4.5 kg/m2, n = 18). Bloodwas collected before (0 h) and after the meal (14 h) for analysis.

RESULTSdBaseline endotoxin was significantlyhigher in thetype 2 diabetic andIGT subjectsthan in NOC subjects, with baseline circulating endotoxin levels 60.6% higher in type 2 diabeticsubjects than in NOC subjects (P, 0.05). Ingestion of a high-fat meal led to a significant rise inendotoxin levels in type 2 diabetic, IGT, and obese subjects over the 4-h time period (P, 0.05).Thesefindingsalsoshowed that, at 4 h after a meal, type 2 diabetic subjects hadhigher circulatingendotoxin levels (125.4%) than NOC subjects (P, 0.05).

CONCLUSIONSdThese studies have highlighted that exposure to a high-fat meal elevatescirculating endotoxin irrespective of metabolic state, as early as 1 h after a meal. However, thisincrease is substantial in IGT and type 2 diabetic subjects, suggesting that metabolic endotoxinemiais exacerbated after high fatintake.In conclusion, ourdata suggest that, in a compromisedmetabolicstate such as type 2 diabetes, a continual snacking routine willcumulatively promote their condition

more rapidly than in other individuals because of the greater exposure to endotoxin.

Diabetes Care 35:375382, 2012

Studies examining the interrelation-ships between adipose tissue, inflam-mation, and insulin resistance appear

key to understanding type 2 diabetes risk(1,2). It is known that low-grade chronicsystemic inflammation contributes to thisrisk, which appears altered by several fac-tors such as increasing age, sex, ethnicity,genetics, and dietary influences. However,

systemic inflammation appears to persist

in type 2 diabetic subjects, despite medica-tion, while the mechanisms and mediatorsof this continual inflammation appear lessclear. Evidently, adipose tissue accumula-tion has a significant impact on disease riskand inflammation in type 2 diabetes butmay merely act in response to systemic pri-mary insults (39).

One potential cellular mechanism for

increased inflammation may arise through

activation of the innate immune systemin human adipose tissue (1013). Previ-ous studies have shown that increasedactivation of the innate immune pathwaymay arise through excess circulatinggut-derived bacteria, known as lipopoly-saccharide (LPS) or endotoxin, whichrepresents the outer cell wall membraneof gram-negative bacteria (10,11,1417).Our previous work has shown that endo-toxin has an immediate impact on the in-nate immune pathway in human adiposetissue, acting via key receptors known as

theToll-like receptors, which recognizean-tigens, such as the LPS component, to ini-tiate an acute-phase response to infection(8,10). Stimulation of the Toll-like recep-tors leads to intracellular activation of nu-clearfactor-kB (NF-kB),a keytranscriptionfactor in the inflammatory cascade that reg-ulates the transcription of numerous proin-flammatory adipokines (9,10). Therefore,in vitro endotoxin may act as a mediatorof inflammation through activation ofNF-kB, leading to a rapid response withinadipose tissue that may be exacerbated by

increased adipose tissue mass (18

22).However, clinical studies have alsoimplicated gut-derived endotoxin as aprimary insult to activate the inflamma-tory state, contributing to metabolic dis-ease, with current cross-sectional datashowing elevated systemic endotoxin lev-els in conditions of obesity, type 2 diabe-tes, coronary artery disease, and fatty liverdisease (8,10,11,1417). Within thesestudies, circulating endotoxin is observedto be positively associated with waist cir-cumference, waist-to-hip ratio, insulinlevels, inflammatory cytokines and lipids,

including total cholesterol, triglycerides(TGs), and LDL cholesterol, and nega-tively associated with HDL cholesterol(8,10,11,1417). The combined impor-tance of dietary lipids and LPS in deter-mining inflammatory risk may arise,since endotoxin has a strong affinity forchylomicrons (lipoproteins that trans-port dietary long-chain saturated fattyacids [SFAs] through the gut wall) as en-dotoxin crosses the gastrointestinal mu-cosa (2325). As such, atherogenic andinflammatory risk may arise through a

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From the 1Division of Metabolic and Vascular Health, University of Warwick, Coventry, U.K.; the 2College ofScience, Biomarkers Research Programme and Center of Excellence in Biotechnology Research, King SaudUniversity, Riyadh, Saudi Arabia; the 3Institut dInvestigacions Biomdiques August Pi i Sunyer (IDIBAPS)and Centro de Investigacin Biomdica en Red de Diabetes y Enfermedades Metablicas Asociadas(CIBERDEM), Barcelona, Spain; and the 4University of Warwick and George Eliot Hospital, Clinical Sci-ences Research Laboratories, Warwick Medical School (University Hospital Coventry and WarwickshireCampus) Coventry, U.K.

Corresponding author: Philip G. McTernan, [email protected] 18 August 2011 and accepted 4 November 2011.DOI: 10.2337/dc11-1593 2012 by the American Diabetes Association. Readers may use this article as long as the work is properly

cited,theuse iseducationaland notforprofit, andthe work is notaltered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

care.diabetesjournals.org DIABETES CARE, VOLUME 35, FEBRUARY 2012 375

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As anticipated, the baseline NOCgroup had significantly lower fastingplasma glucose levels than the type 2 di-abetic group, while showing similar glu-cose levels to the obese and IGT cohorts(NOC: 4.7 6 0.69 mmol/L vs. type 2 di-abetic: 8.16 1.8 mmol/L***; IGT: 5.661.2 mmol/L; obese IGT: 4.9 6 0.93mmol/L; ***P, 0.001). HbA1c was sim-ilar in the obese and NOC groups but

was significantly higher in the IGT andtype 2 diabetic groups (NOC: 5.9 60.31% vs. type 2 diabetic: 7.5 6 1.12%***; IGT: 6.3 6 0.47%*; obese IGT:5.9 6 0.49%; ***P , 0.001, *P ,0.05). Within each cohort, glucose levelswere not significantly altered over the 4-hpostprandial time period.

The baseline lipid profile across thegroups was comparable. Serum endotoxinlevels were significantly lower in the baselineNOCgroupcomparedwith theIGT and type2 diabetic groups (NOC: 3.3 6 0.15 endo-toxin unit/mL [EU/mL] vs.obese: 5.160.94EU/mL; IGT 5.76 0.10 EU/mL**; type 2diabetic: 5.36 0.54 EU/mL*; **P, 0.01,*P, 0.05; Fig. 1A, Table 2).

Postprandial change in endotoxinlevels over time in individual groupsPostprandial exposure to a high-fat mealled to a significant rise in endotoxin levelsin obese subjects (baseline: 5.1 6 0.94EU/mL; 1 h: 4.2 6 0.71 EU/mL; 2 h:6.2 6 0.49* EU/mL; 3 h: 7.8 6 0.76**EU/mL; 4 h: 7.7 6 0.58** EU/mL;**P, 0.01, *P, 0.05; Fig. 1A, Table 2);

IGT (IGT: baseline: 5.760.10 EU/mL; 1 h:5.86 0.22 EU/mL; 2 h: 5.56 1.0 EU/mL;3 h: 7.46 0.26* EU/mL; 4 h: 7.56 0.20*EU/mL; *P, 0.05, Fig. 1A, Table 2), andtype 2 diabetic subjects (baseline: 5.3 60.54 EU/mL; 1 h: 5.5 6 0.44 EU/mL;2 h: 5.8 6 0.34 EU/mL; 3 h: 9.86 1.2**EU/mL; 4 h: 14.26 3.0** EU/mL; **P,0.01, Fig. 1A, Table 2) over the 4-h timeperiod. In the NOC group, whereas there

was a rise in circulating endotoxin overthe 4-h period, this trend did not reachsignificance past 1 h (Fig. 1A, Table 2).Fasting endotoxin levels showed a posi-tive correlation with fasting TG levels inthe whole cohort (r= 0.303, P= 0.026).Further examination of this relationshippostprandially identified the positive corre-lation strengthened over time, with thestrongest relationship between endotoxinand TG noted at 2 and 3 h, respectively(2-h time point: r= 0.531, P, 0.001; 3-htime point: r = 0.498, P, 0.001) with adecline by 4 h after feeding (r= 0.434, P=0.001). No further correlations with anyother parameters were observed, and thosenoted were not influenced by age or sex.

Postprandial change in endotoxinlevels between groupsFasting endotoxin levels were significantlyhigher in IGT and type 2 diabetic subjectsthan in NOCsubjects (72.7%** and60.6%*increase, respectively; **P, 0.01, *P,0.05, Fig. 2B and C). However, during thepostprandial 4-h time period, the circulat-ing endotoxin levels in both obese and IGT

subjects diminished to ;20% higher thanthat of the NOC (4 h, Fig. 2A and B),whereas circulating endotoxin levels weresustainedat significantlyhigherlevelsin thetype 2 diabetic subjects than in the NOCsubjects (4 h, Fig. 2C, P, 0.05).

Postprandial changes in lipids overtime in individual groupsPostprandial exposure to a high-fat meal led

to a significant rise in TG levels in NOC,IGT, and type 2 diabetic subjects after 1 h(P, 0.05, Fig. 1B, Table 2). Although theobese subjects followed the same trend,TGs levels were only significantly altered at2-h post-feeding (P,0.05; Fig. 3, Table 2).

Total cholesterol remained relevantlyunaltered over the 4-h period within allfour groups (Table 2). In addition, nochange was noted in LDL cholesterol andHDL cholesterol for the NOC subjects overthe 4-h period (Table 2). LDL cholesteroland HDL cholesterol in the other threegroups did show significant individualgroup changes over time. For all metabolicstates, LDL and HDL cholesterol signifi-cantly changed (increased and reduced, re-spectively; P, 0.05; Table 2), whereaslevels in NOC subjects were not altered.

Postprandial changes in lipidlevels between groupsFasting total cholesterol, TG, LDL choles-terol, and HDL cholesterol levels were com-parable at baseline within the four groupsand did not differ significantly throughoutthe 4-h duration (Figs. 1B and 3; Table 2).

Table 1dAnthropometric data for the different cohorts

Normal group Obese group IGT group

Type 2 diabetic

group P

n 9 15 12 18

Age (years) 39.9 6 11.8 43.8 6 9.5 41.7 6 11.3 45.4 6 10.1 NS

Sex (M:F) 6:3 10:5 7:5 11:7

Ethnicity Asian, n = 8 Afro-Caribbean, n = 1

Asian, n = 15 Asian, n = 12 Asian, n = 17Afro-Caribbean, n = 1

BMI# (kg/m2) 24.9 6 3.2 33.3 6 2.5 32.0 6 4.5 30.3 6 4.5 Normal vs. obese: P, 0.001;

normal vs. IGT: P= 0.001;

normal vs. T2DM: P= 0.003;

IGT vs. obese: NS; IGT vs. T2DM:

NS; obese vs. T2DM: P= 0.019

Waist circumference (cm) 86.9 6 8.25 108.9 6 17.9 106.4 6 10.37 100.1 6 10.2 Norma l vs. obese: P= 0.001;

normal vs. IGT: P, 0.001;

normal vs. T2DM: P= 0.002;

IGT vs. obese: NS; IGT vs. T2DM:

NS; obese vs. T2DM: NS

Estimated glomerular

filtration rate# 105.62 6 19.71 91.076 17.40 105.92 6 19.77 92.28 6 24.36 NS

Data are means 6 SD unless otherwise indicated. T2DM, type 2 diabetes. #Data with a non-Gaussian distribution that were transformed before statistical analysis.


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CONCLUSIONSdThis is the firststudy to examine the comparative and dif-ferential changes in circulating endotoxinafter a SFA meal from subjects with andwithout type 2 diabetes, obesity, or IGT.The novel data highlight that a SFA mealincreases circulating endotoxin levels inall subjects irrespective of their metabolicstatus, although circulating endotoxinshows dramatic postprandial changes inthe highmetabolic risk groups. More spe-cific comparative analysis of NOC subjectsversus subjects with type 2 diabetes at 4-hpostprandial identified that the latter hada mean endotoxin level 125.4% higherthan that of NOC subjects. Cumulativedata derived from the fasting state andthe SFA postprandial state indicate thattype 2 diabetic subjects are subjected to336% more circulating endotoxin thanNOC subjects over the 4-h duration. Incomparison to other metabolic states, the

obese andIGT subjects were still subjectedto 167 and 198.5% more circulating en-dotoxin than NOC subjects. As such, en-dotoxin, which is considered a potentialmediator of chronic low-grade inflamma-tion, is considerably higher in the state oftype 2 diabetes, with implications for acontinual inflammatory state, as other ar-ticles have observed (15,16,28,29).

While our previous studies haveshown significant associations in thefasted state among circulating endotoxin,lipoprotein patterns, and anthropometricdata (8,10,11,1417), these current stud-ies have sought to establish whether en-dotoxin acutely changes postprandiallyand whether this is altered by differingmetabolic states. By undertaking this,our current studies have highlightedsubtle but significant differences in howendotoxin levels change in the pos tpran -dial period. After a SFA meal, the NOC

endotoxin levels rose over the 4-h dura-tion, but circulating levels did not increasesignificantly. In contrast, in the obese andIGT groups, there was a significant rise inendotoxin, which appeared to plateau by4 h. However, at the 4-h time point, boththe IGT and obese groups endotoxin lev-els were much lower than those of the type2 diabetic subjects, since the levels of en-dotoxin in the type 2 diabetic subjects ap-pearedto still be rising4 h after a SFA meal.Circulating endotoxin in the type 2 diabeticgroup, after 4 h, did not appear to normal-ize, which suggests the cumulative expo-sure to endotoxin after a high-SFA meal isdisproportionately highcompared with anyother group. Furthermore, in the type 2 di-abetic subjects, the rising endotoxin levelsmay be further compounded by the refeed-ing stage. These data appear to indicatethat a person eating three high-SFA mealseach day may encounter endotoxin levels

Table 2dVariable data for the different cohorts

Baseline 1 h 2 h 3 h 4 h

Normal groupTriglycerides# (mmol/L) 1.1 6 0.07 1.3 6 0.08* 1.8 6 0.08*** 2.2 6 0.2** 2.4 6 0.27**

Total cholesterol (mmol/L) 4.9 6 0.95 5.1 6 0.85 5.1 6 0.73 5.1 6 0.94 5.2 6 0.81

LDL cholesterol# (mmol/L) 3.2 6 0.04 3.2 6 0.03 3.1 6 0.03 2.82 6 0.04* 2.79 6 0.04

HDL cholesterol# (mmol/L) 1.1 6 0.004 1.1 6 0.004 0.98 6 0.03 1.06 6 0.004 1.03 6 0.008TNF-a# (pg/mL) 8.0 6 2.3 8.1 6 2.2 7.4 6 2.0 7.4 6 1.8 8.0 6 2.0

Leptin# (ng/mL) 16.8 6 4.8 17.0 6 5.3* 14.6 6 4.0* 13.6 6 3.5* 14.7 6 4.4

Endotoxin# (EU/mL) 3.3 6 0.15 4.0 6 0.17* 4.32 6 0.19 5.5 6 0.64 6.3 6 1.4

Obese group

Triglycerides# (mmol/L) 1.6 6 0.08 1.7 6 0.08 2.3 6 0.10*** 2.7 6 0.12*** 3.0 6 0.14***

Total cholesterol (mmol/L) 5.3 6 0.80 5.1 6 0.67 5.1 6 0.99 5.3 6 1.0 5.2 6 0.76

LDL cholesterol# (mmol/L) 3.566 0.04 3.34 6 0.02 3.0 6 0.05*** 3.0 6 0.06*** 2.9 6 0.04***

HDL cholesterol# (mmol/L) 0.926 0.01 0.90 6 0.01 0.84 6 0.01* 0.85 6 0.008** 0.81 6 0.01***

TNF-a# (pg/mL) 8.4 6 1.3 8.4 6 1.4 7.8 6 1.6 7.5 6 1.4* 7.5 6 1.4*

Leptin# (ng/mL) 25.6 6 3.1 23.3 6 2.7*** 20.7 6 2.3 21.7 6 2.6 21.4 6 2.9***

Endotoxin# (EU/mL) 5.1 6 0.94 4.2 6 0.71 6.2 6 0.49 7.8 6 0.76** 7.7 6 0.58**

IGT group

Triglycerides# (mmol/L) 1.36

0.03 1.56

0.04*** 1.96

0.07*** 2.56

0.15*** 2.56

0.15***Total cholesterol (mmol/L) 4.9 6 0.80 4.7 6 0.74 4.8 6 0.74 4.8 6 0.77 4.8 6 0.77

LDL cholesterol# (mmol/L) 3.246 0.04 3.08 6 0.03 2.9 6 0.03** 2.6 6 0.04** 2.66 6 0.05**

HDL cholesterol# (mmol/L) 0.886 0.008 0.86 6 0.008 0.82 6 0.006*** 0.81 6 0.008*** 0.77 6 0.01***

TNF-a# (pg/mL) 4.4 6 2.1 4.3 6 2.0 4.3 6 2.0 4.1 6 2.0 4.3 6 2.1

Leptin# (ng/mL) 37.0 6 2.7 32.6 6 2.7*** 32.0 6 2.7* 31.1 6 3.0** 33.0 6 3.3**

Endotoxin# (EU/mL) 5.7 6 0.10 5.8 6 0.22 5.5 6 1.0 7.4 6 0.26* 7.5 6 0.20*

Type 2 diabetic group

Triglycerides# (mmol/L) 1.4 6 0.03 1.6 6 0.05*** 2.2 6 0.08*** 2.8 6 0.12*** 3.1 6 0.13***

Total cholesterol (mmol/L) 5.0 6 1.0 4.8 6 0.95* 4.8 6 0.91 5.0 6 1.0 4.9 6 0.93

LDL cholesterol# (mmol/L) 3.176 0.11 2.9 6 0.07*** 2.6 6 0.10*** 2.4 6 0.16** 2.4 6 0.14***

HDL cholesterol# (mmol/L) 1.0 6 0.02 1.0 6 0.02 0.94 6 0.02*** 0.92 6 0.02*** 0.86 6 0.02***

TNF-a# (pg/mL) 8.6 6 2.0 8.4 6 2.0 8.4 6 2.0 8.3 6 1.8 8.1 6 2.3

Leptin# (ng/mL) 18.1 6 3.5 20.0 6 5.0*** 18.7 6 3.7 18.2 6 3.4 15.2 6 3.3

Endotoxin# (EU/mL) 5.3 6 0.54 5.5 6 0.44 5.8 6 0.34 9.8 6 1.2** 14.2 6 3.0**Data are means 6 SD. TNF, tumor necrosis factor. #Data with a non-Gaussian distribution that were transformed before statistical analysis. Significant differencecompared time points with baseline values. *P, 0.05. **P, 0.01. ***P, 0.001.

378 DIABETES CARE, VOLUME 35, FEBRUARY 2012 care.diabetesjournals.org

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that remain perpetually high, since refeed-ing may increase the levels. As such, fastedendotoxin data, while important, may ap-pear to miss the daily variation, as feedingdata appear to show. The type of meal isclearly important, since previous studieshighlight that dietary changes alter circulat-ing endotoxin and influence inflammation,even in healthy subjects (13,28). In addi-tion, recent studies have reported that thesimultaneous ingestion of certain healthy

food groups with saturatedfat cannegate anincrease in circulating endotoxin and thecustomary inflammatory response (29). Be-cause it is acknowledged that obese andtype 2 diabetic subjects tend to eat highSFA without correspondingly high levelsof fruit or healthy foods, this diet wouldclearly affecttheir endotoxin andinflamma-tory status (28,31,32). Therefore, a high-SFA intake could represent a continualinflammatory insult for type 2 diabeticsubjects, daily.

In the obese and IGT groups, thepostprandial 4-h endotoxin levels appearto plateau, while still remaining high com-pared with NOC subjects. Subsequently,another SFA meal may compound thecirculating endotoxin levels further withinthe obese and IGT groups; therefore, thetype and frequency of meals may signifi-cantly affect the metabolic risk. In additionto the type of meal, the food intake fre-quency is also relevant, although cur-rently, there are few studies examining theimportance of this. Previous studies in-dicate no difference between a diet basedon three meals a day or a diet comprising

smaller meals and snacks, with regard tothe long-term effects on glucose, lipid, orinsulin responses; although the unknownacute postprandial effects on the inflam-matory status may have a more profoundlong-term impact (32,33). In addition,previous studies have often stressed thedivision of food intake should be basedon individual preference, with no clearrecommendations on pattern of food in-take. Within type 2 diabetes clinics, therec-

ommendation for patients is to consumefive smaller meals per day. This step mayreduce the potentially overwhelmingorexi-genic effects patients might experiencewith only three meals a day, as well as thepotential spikes in insulin, although thedata do not necessarily give clear insightinto these benefits (32,33). Based on thesecurrent studies, more frequent saturated fatexposure may exacerbate both endotoxinand inflammation further. Furthermore,smaller more frequent meals have the po-tential to allow endotoxin to spike severaltimes a day, thus activating the innate im-mune system within adipose tissue with-out desensitization (9,10,28,29). As such,the resulting downstream production ofdiabetogenic cytokines would be in con-tinuous production, as previous in vivoand in vitro studies have demonstrated(9,10,17,28,29). The TG levels did notdiffer significantly across the four groupsof subjects at any of the time points; how-ever, the TG levels did increase from base-line to 4 h within each group, in a similarpattern to circulating endotoxin, but mostsignificantly in the metabolic risk subjects

(obese, IGT, and type 2 diabetic), whilealso demonstrating an association with en-dotoxin, over the 4-h period (10,16,17).Thethree different metabolic states showedsignificantly higher fasting TG levels thanNOC subjects, which postprandially be-came further exacerbated in the obese andtype 2 diabetic subjects.

Unsurprisingly, postprandial TG levelsincreased in a similar pattern to circulatingendotoxin, while also demonstrating an as-

sociation over the 4-h period (10,16,17).Thethree different metabolic states showedno significant differences in TG levels com-pared with levels in NOC subjects. How-ever, the significant correlation betweenfasting TG and endotoxin levels confirmsprevious studies in which an associationbetween these two metabolic parametershad been observed (10,16,17). Our dataindicated that the association betweenTGs and circulating endotoxin becamestronger in the postprandial state eachhour over the 4-h duration, substantiatingprevious evidence that lipids mediate thetransfer of endotoxin from the gastrointes-tinal tract into the circulation (9,11).

Concurrent with postprandial changesin TGs, the LDL/HDL ratio reduced com-pared with baseline measurements. Specif-ically, HDL was significantly reduced attime pointspostprandially within all exceptthe NOC group, potentially due to parallelelevations in chylomicrons and VLDL, asnoted in other studies (3436). Whereas itis established that obese type 2 diabetic pa-tientssufferfrom a syndrome of high serumTG and low HDL (37), low levels of HDL

Figure 1dChanges in circulating endotoxin levels (A) and triglyceride levels (B) in NOC, IGT, obese, and type 2 diabetic (T2DM) subjects.Endotoxin and triglyceride levels were measured at baseline and then, after a high-SFA meal, at each hour postprandially over a 4-h duration. Each

point on the graph represents the mean value for each cohort (6

SEM).




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are also associated with low levels of sCD14(soluble CD14) (38). This result corre-sponds with the data that endotoxin hasbeen demonstrated to bind to HDL in thepresence of sCD14 and LPS binding pro-tein(39,40),an enzymeinvolved in presen-tation of endotoxin to sCD14. Thisoutcome supports a role for HDL in theimmunological response to endotoxin.Therefore, a reduction in HDL would re-duce the removal of endotoxin further andexacerbate the inflammatory status, furthercompounded by highercirculating levels ofendotoxin in the obese, IGT, and type 2diabetic subject groups.

Whereas our studies have highlightedthe impact of metabolic disease status oncirculating endotoxin, it is important torecognize the limitations of the study.In all research, it is always preferable toincrease the subject numbers that com-prise each cohort. In the present studies, in-creased numbers might have noted different

postprandial responses to the high-fatmeal within each cohort, if the groupswere further subdivided. However, in lightof this being a cross-sectional study, inwhich intra- and inter-comparisons can bemade, the numbers do not detract from thefindings. Consistent and significant trendswere observed within the subjects overthe 4-h postprandial duration, and differ-ences between thecohortswere duly noted.

We also recognize that the research sub-jects were given a very high-fat meal (75 g),roughly equivalent to their total daily intakeof fat, which some observers might argue isan excessive (nonphysiological) amount offat. However, despite the fat load, therewas no significant change in endotoxin,cholesterol, LDL, or HDL levels postpran-dially in the NOC subjects in contrast tothe other groups examined; the fat loadadministered was based on previous stud-ies (30). Furthermore, administration of75 g glucose could also be considered

high and would far exceed normal intakeof glucose in one sitting, yet this is stan-dard clinical practice for assessment of in-sulin sensitivity, whereas the fat load isonly currently used as a research tool.No ill effects were noted in any of the pa-tients during or after the study.

In summary, our current data shednew light on our understanding of met-abolic endotoxinemia in the postprandialstate in metabolic disease. Our findingssuggest that circulating endotoxin levelschange depending on whether you areprediabetic, are nonobese, are obese, haveIGT, or have type 2 diabetes. Further,circulating endotoxin levels noted in sub-

jects with type 2 diabetes, at 4-h post-prandial high-fat meal, far exceed ourprevious understanding based on otherfeeding studies in healthy subjects orthe fasted state in type 2 diabetic subjects.Therefore, our 4-h data suggest a muchhigher inflammatory risk than previous

Figure 2d

Increase in endotoxin levels between the NOCsubjects and the obese (A),IGT(B), and type2 diabetic(T2DM) (C) subjects from baselineto 4 h after a high-fat meal. Endotoxin is measured in EU/mL, and the percentage increase compared with NOC is also shown.




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studies have indicated. These findingshighlight the point that requesting pa-tients to eat smaller, more frequent mealsmay actually increase their inflammatoryrisk further, especially in subjects withtype 2 diabetes (who tend to favor high-fatfoods) (32). Finally, while the most obvi-ous solution to metabolic endotoxinemiaappears to be to reduce saturated fat in-take, the Western diet is not conduciveto this mode of action, and it is difficultfor patients to comply with this request.Therefore, we need to understand the

complexity of diet, meal frequency, andits acute effects on inflammatory riskand give more guidelines to particularsubject groups, since leaving food intaketo individual preferences appears not torepresent a beneficial solution to reducethe inflammatory state in metabolic at-risk subjects.

AcknowledgmentsdM.C.V. was funded by aBritish Medical Institute fellowship. The au-thors thank Birmingham Science City for sup-porting this research and the British Heart

Foundation for the Intermediate Fellowshipfor funding A.L.H.

No potential conflicts of interest relevant tothis article were reported.

A.L.H. performed design, endotoxin ex-periments, and statistical analysis and draftedthe manuscript. M.C.V. conducted the in vivoexperiments and acquired all of the samplesand anthropometric data. G.T. drafted andrevised the manuscript. K.C.M. carried out theadipokine assays. N.M.A.-D. and O.S.A.-A.performed the lipid analysis. S.S. performedstatistical analysis and interpretation of data.J.P.O., A.C., and P.S. provided the concept,

interpreted data, and provided intellectualinput. S.K. and P.G.M. provided design andconcept, developed the manuscript, and per-formed the final revision of the manuscript.P.G.M. is also the guarantor of the article.

The authors thank Dr. Martin Been (Uni-versity Hospital Coventry and Warwickshire[UHCW]) and the Cardiology Department,Nuclear Physics and Radiology Department,and all the departments and teams based atboth UHCW and St Georges Hospital Londonfor their contributions. The authors acknowl-edge and thank Mr. Saim Ulhaq Quddusi,Biomarkers Research Programme, for his inputto the statistical analysis.

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Figure 3dIncrease in triglyceride levels between the NOC subjects and the obese (A), IGT (B), and type 2 diabetic (T2DM) (C) subjects frombaseline to 4 h after a high-fat meal. Triglyceride levels are measured in mmol/L, and the percentage increase compared with NOC is also shown.




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High Fat Intake Leads to Acute Postprandial Exposure to Circulating Endotoxin in Type 2 Diabetic Subjects