Am J Physiol Regul Integr Comp Physiol 315: R1210–R1219, 2018.

First published October 10, 2018; doi:10.1152/ajpregu.00240.2018.

RESEARCH ARTICLE Obesity, Diabetes and Energy Homeostasis

The effect of a short-term low-carbohydrate, high-fat diet with or without

postmeal walks on glycemic control and inflammation in type 2 diabetes: a
randomized trial
Étienne Myette-Côté,1 Cody Durrer,1 Helena Neudorf,1 Tyler D. Bammert,2 José Diego Botezelli,3
James D. Johnson,3 Christopher A. DeSouza,2 and Jonathan P. Little1
1
School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC, Canada; 2Integrative
Vascular Biology Laboratory, Department of Integrative Physiology, University of Colorado, Boulder, Colorado; and
3
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada
Submitted 30 July 2018; accepted in final form 5 October 2018

Myette-Côté É, Durrer C, Neudorf H, Bammert TD, Botezelli Immune cells become activated and infiltrate various tissues,
JD, Johnson JD, DeSouza CA, Little JP. The effect of a short- contributing to a state of chronic low-grade inflammation (8).
term low-carbohydrate, high-fat diet with or without postmeal High glucose promotes proinflammatory cytokine release, el-
walks on glycemic control and inflammation in type 2 diabetes: a evates surface protein expression of the key innate immune cell
randomized trial. Am J Physiol Regul Integr Comp Physiol 315:
activator toll-like receptor (TLR) 4, potentiates proinflamma-
R1210 –R1219, 2018. First published October 10, 2018; doi:
10.1152/ajpregu.00240.2018.—Lowering carbohydrate consump- tory signaling pathways [e.g., c-Jun NH2-terminal kinase
tion effectively lowers glucose, but impacts on inflammation are (JNK)], and causes the release of characteristic proinflamma-
unclear. The objectives of this study were to: 1) determine whether tory microparticles (MPs) in monocytes/macrophages (10, 20,
reducing hyperglycemia by following a low-carbohydrate, high-fat 44). In T2D, elevated levels of circulating proinflammatory
(LC) diet could lower markers of innate immune cell activation in cytokines (52), increased monocyte TLR4 expression (9), over-
type 2 diabetes (T2D) and 2) examine if the combination of an LC diet activation of the JNK pathway in peripheral blood mononu-
with strategically timed postmeal walking was superior to an LC diet clear cells (PBMCs) (58), and elevated circulating monocyte-
alone. Participants with T2D (n ⫽ 11) completed a randomized derived MPs (MMPs) (35) have been repeatedly demonstrated,
crossover study involving three 4-day diet interventions: 1) low-fat linking mechanistic work with clinical features of T2D. Thus,
low-glycemic index (GL), 2) and 3) LC with 15-min postmeal walks high glucose appears to trigger the activation of innate immune
(LC⫹Ex). Four-day mean glucose was significantly lower in the
LC⫹Ex group as compared with LC (⫺5%, P ⬍ 0.05), whereas both
cells, suggesting that hyperglycemia itself drives a vicious
LC⫹Ex (⫺16%, P ⬍ 0.001) and LC (⫺12%, P ⬍ 0.001) conditions cycle of inflammation and insulin resistance in T2D.
were lower than GL. A significant main effect of time was observed Lifestyle therapy is a frontline treatment for improving glucose
for peripheral blood mononuclear cells phosphorylated c-Jun N-ter- control in people with T2D. Most current dietary guidelines
minal kinase (P ⬍ 0.001), with decreases in all three conditions (GL: advocate a “healthy” diet low in saturated fat and sugar, with
⫺32%, LC: ⫺45%, and LC⫹Ex: ⫺44%). A significant condition by emphasis on low glycemic index (GI) foods. However, accumu-
time interaction was observed for monocyte microparticles (P ⫽ lating evidence shows that consuming a low-carbohydrate high-
0.040) with a significant decrease in GL (⫺76%, P ⫽ 0.035) and a fat (LCHF) diet is more effective for lowering glucose (60). In a
tendency for a reduction in LC (⫺70%, P ⫽ 0.064), whereas there companion paper, we demonstrated that a short-term LCHF diet
was no significant change in LC⫹Ex (0.5%, P ⫽ 0.990). Both LC quickly stabilizes glucose in patients with T2D (17). However,
(⫺27%,
P ⫽ 0.001) and LC⫹Ex (⫺35%, P ⫽ 0.005) also led to significant
whether the rapid improvement in glucose control is accompanied
reductions in circulating proinsulin. An LC diet improved 4-day by lowered inflammation has, to our knowledge, not been tested.
glycemic control and fasting proinsulin levels when compared with It is possible that factors related to T2D pathophysiology, other
GL, with added glucose-lowering benefits when LC was combined than hyperglycemia, are more important in driving chronic in-
with postmeal walking. flammation. It is therefore unknown whether lowering glucose by
following an LCHF diet can reduce innate immune cell activation
cytokines; exercise; glucose; ketogenic diet
and inflammation in T2D.
Consuming an LCHF diet with less than 130 g of carbohy-
drates is contraindicated by the American Diabetes Associa-
INTRODUCTION
tion, partly because of the perceived negative impacts of
consuming high amounts of fat (1a). Acute feeding studies
Inflammation is associated with the pathogenesis of insulin indicate that an isolated “high-fat meal” increases inflamma-
resistance, type 2 diabetes (T2D), and related complications. tion and triggers proinflammatory activation of immune cells,
particularly in individuals with insulin resistance or T2D (5,
48). Thus, attempting to lower hyperglycemia using an LCHF
Address for reprint requests and other correspondence: J. P. Little, Univ. of
British Columbia Okanagan, School of Health and Exercise Sciences, 3333
diet may have unintended consequences. Strategically timed
University Way, Kelowna, BC V1V 1V7, Canada (e-mail: jonathan.little exercise may be one way to counteract this. Padilla et al. (47)
@ubc.ca). have shown that exercising in the postprandial period reversed
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 LOW-CARBOHYDRATE DIET AND INFLAMMATION R1211
the detrimental impacts of a high-fat meal on endothelial
function. Additionally, several recent studies have highlighted 28 people phone or
the benefits of postmeal walking exercise on glycemic control email screened
(55). Combining an LCHF diet with postmeal exercise may be
an optimal combination for improving glucose control and
10 exclusions
reducing inflammation. 3 Not interested
The objective of the present study was to determine whether 1 age over 75 years
reducing hyperglycemia with an LCHF diet could lower mark- 2 exogenous insulin
ers of innate immune cell activation and systemic inflammation 3 CVD
in people with T2D. A secondary objective was to examine if 1 too far to travel
the combination of an LCHF diet with strategically timed post-
meal walking was superior to an LCHF diet alone. To reduce the 18 attended
confounding influence of both weight loss and long-term adapta- screening visit
tion to an LCHF diet (59) we employed a 4-day controlled feeding
protocol in attempts to isolate the direct effects of lowering
1 not interested
glucose using a whole food LCHF diet, with or without exercise.
1 too busy
A control condition involving low GI, low-fat whole foods was
employed to directly compare the LCHF conditions to “gold-
standard” dietary guidelines (10a). The primary outcome was 16 eligible
glucose control, defined as average glucose assessed by continu- participants
randomized crossover
ous glucose monitoring (CGM). Secondary outcomes included a
comprehensive array of systemic, cellular, and molecular indices
of inflammation from plasma and PBMCs, along with standard
markers of metabolic control. 11 completed protocol
METHODS 5 drop-out
1 family reason
Ethical approval. The study was approved by the University of 3 inability to follow study diets
British Columbia Clinical Research Ethics Board (ID H15-01952) and 1 change of medications
was registered on clinicaltrials.gov (NCT02683135). The study con-
formed to the standards set by the Declaration of Helsinki. Partici- Fig. 1. Consolidated standards of reporting trials flow diagram. CVD, cardio-
pants provided written informed consent before study commencement vascular disease.
during the initial screening visit.
Overview. Individuals with physician-diagnosed T2D [glycated
hemoglobin (HbA1c) ⬎6.5%, fasting plasma glucose (FPG) ⬎7.0 ously demonstrated an ~15% reduction in postprandial glucose area
mmol/l, or 2-h glucose oral glucose tolerance test ⬎11.1 mmol/l (32)] under the curve with postmeal walking. With the use of means and SD
were recruited to complete three short-term controlled-intervention for CGM from the literature and our own T2D studies (15), a sample
periods in a randomized crossover design: 1) low-fat low-GI diet size of 11 would be needed to detect a 15% difference in mean
(GL), 2) low-carbohydrate high-fat diet (LC), and 3) LC with 15-min glucose with 80% power at an alpha of 0.05, assuming a correlation
postmeal walks (LC⫹Ex). The randomization sequence was gener- among repeated measures of r ⫽ 0.7 (G*Power v3.1.9.3). To account
ated by a computer random number generator and was revealed to for drop-outs and/or missing data, we aimed to recruit 16 patients with
study staff from a master list after the participant provided informed T2D.
consent. Neither participants nor staff were blinded. All food was Experimental protocol. During the initial screening visit, anthro-
provided to participants using a local service (https://mealprepforyou. pometric measurements (height, body weight, body mass index, and
ca/), and diets were matched for energy and protein content. Fasting waist circumference) were taken, and both a physical activity readi-
blood samples following an overnight fast were collected before and ness (PARQ⫹) and Godin leisure-time exercise questionnaires were
after each experimental intervention. Accelerometers (Actigraph filled out, followed by the three randomized 4-day diet interventions.
wGTX3⫹) were worn to monitor activity for all conditions and The 24-h period preceding each diet intervention was standardized for
confirm the completion of postmeal walks. physical activity and included a standardized mixed meal in the
Participants. Participants (n ⫽ 16; 8 men and 8 women) aged evening followed by a ⱕ10-h fast. A washout period of 9 –14 days
between 48 and 72 yr, not on exogenous insulin, and without diagnosed was introduced between each intervention where participants were
cardiovascular, kidney, or any other diabetes complications were re- asked to return to their regular diet and physical activity habits.
cruited from the local community in Kelowna, BC, Canada from No- Fasting blood samples were collected at the same time in the morning
vember 2015 to March 2017. Individuals currently involved in a regular before and after each 4-day intervention for future analysis. During
exercise routine (⬎3 days of structured exercise per week), following an each intervention, a CGM (iPro2 professional CGM, Medtronic,
LCHF diet, or unwilling to consume the provided meat-containing diets Northridge, CA and Enlite sensor) was worn to measure glycemic
were excluded. Five of the sixteen participants did not complete all three control. Average blood glucose across 4 days was the primary out-
conditions because of family reasons (n ⫽ 1), inability or unwillingness come. Area under the curves were calculated using the trapezoid
to follow study diets (n ⫽ 3), and change of medications (n ⫽ 1, addition method (34), and the mean amplitude of glycemic excursion (MAGE)
of sodium-glucose cotransporter-2 inhibitor after completing one condi- and continuous overall net glycemic action (CONGA) were computed
tion) (Fig. 1). Participants were instructed to take their medications as using EasyGV (version 9.0.R2., University of Oxford). All interven-
usual during the three experimental conditions, and their compliance was tion diets were isoenergetic, with calories estimated using the Harris
assessed with a medication log. Benedict equation (27) and habitual intake (i.e., matched from first
This study was designed as a pilot trial to generate effect sizes to trial). An example meal plan for 1 day of each diet is provided in
guide a larger randomized controlled trial. Nygaard et al. (46) previ- Table 1.

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R1212 LOW-CARBOHYDRATE DIET AND INFLAMMATION

Table 1. Composition of study diets

Diet GL LC LC⫹Ex

Carbohydrate/protein/fat, % 55/25/20 10/25/65 10/25/65

Sat./poly./mono. fat, % 5/5/10 15/11/39 15/11/39
Mean glycemic index 40 N/A N/A
Breakfast
150 g whole egg 150 g whole egg
95 g oats 110 g egg whites 110 g egg whites
25 g whey 55 g avocado 55 g avocado
30 g blueberries 30 g peppers 30 g peppers
30 g raspberries 40 g onions 40 g onions
40 g carrots 40 g carrots
10 g almonds 10 g almonds
Lunch
105 g ground turkey 105 g ground turkey
105 g chicken breast 38 g cashews 38 g cashew
230 g yams 15 g olive oil 15 g olive oil
40 g green beans 35 g spinach 35 g spinach
17 g cashews 30 g carrots 30 g carrots
30 g cucumber 30 g cucumber
Dinner
100 g steak (rib eye) 100 g steak (rib eye)
100 g turkey 30 g cashews 30 g cashews
85 g brown rice 13 g olive oil 13 g olive oil
14 g cashews 30 g apple 30 g apple
40 g broccoli 30 g spinach 30 g spinach
50 g cucumber 50 g cucumber
Snack
1 ⫻ Solo bar 50 g cheddar cheese 50 g cheddar cheese
(Solo GI Nutrition) 20 g almonds
Menu shown is an individual example based on a daily energy intake of ~1,700 kcal, including 3 meals of 500 kcal each and 200 kcal snacks for a 68-yr-old
female participant with an estimated energy expenditure of 1,700 kcal per day (body mass of 68.2 kg, height 1.51 m, physical activity level of 1.3). In the
low-carbohydrate high-fat diet (LC) and exercise (LC⫹Ex) condition, 135 kcal were added to compensate for the daily postmeal walks. GL, low-fat low-glycemic
index guidelines diet; Mono., monounsaturated fat; Poly., polyunsaturated fat; Sat., saturated fat.

GL diet. The GL diet followed the current dietary guidelines for ELISA, Crystal Chem) was analyzed on an iMark Microplate Absor-
adults with T2D, comprising low-fat, low-GI whole foods (12). Each bance Reader (Bio-Rad). Active proinsulin (ALPCO, STELLUX
meal comprised ~55% of total energy from carbohydrates (predomi- Chemi Human Total Proinsulin ELISA) was analyzed on a POLARstar
nately from low-GI and high-fiber carbohydrate sources), 20% energy Omega plate reader (BMG Labtech, Durham, NC). Plasma C-peptide
from fat (aiming for ⬍7% saturated fatty acids), and 25% protein was measured by Meso Scale Discovery C-peptide Singleplex (MD).
(primarily from lean meats). All assays were run in duplicate. The coefficient of variation for
LC diet. The LC diet provided the same energy content as the GL duplicate samples was 2.9% (TGs), 3.0% (glucose), 5.0% (insulin),
diet but with carbohydrates limited to ~10% of total energy. The 4.5% (proinsulin), and 6.6% (C-peptide).
percent protein was matched at ~25%, with the remainder of the Inflammatory markers. Fasting plasma concentrations of tumor ne-
energy coming from fat (~65% of total kcal). crosis factor-␣ (TNF-␣), monocyte chemoattractant protein-1 (MCP-1),
LC⫹Ex diet. Participants performed 15 min of walking beginning interleukin-6 (IL-6), IL-10, and IL-18 were analyzed by multiplex
~30 min after breakfast, lunch, and dinner. The exercise intensity of immunoassay (U-PLEX Human Panel, Meso Scale Discovery) and
the postmeal walking was light-to-moderate, which was confirmed on read on a MESO Quickplex SQ 120. Plasma was centrifuged at 1,500
day 1 of the intervention by having participants walk on a horizontal g for 15 min at 4°C to remove debris and analyzed in duplicate. The
treadmill in the laboratory at a comfortable pace that elicited a rating coefficient of variation was 7.6% (TNF-␣), 4.6% (MCP-1), 5.7%
of perceived exertion (CR-10 scale) of 3 ⫾ 1 (equating to ~60% of (IL-6), 7.9% (IL-10), and 4.1% (IL-18).
maximal heart rate, 93 ⫾ 13 beats/min). Participants were instructed Flow cytometry. Fc receptor-blocking reagent (130 – 059 –901;
to replicate this pace at home for each postmeal walk. Accelerometers Miltenyi Biotec, Bergisch Gladbach, Germany) was added to 90 ␮l of
were worn to confirm compliance and intensity. Participants con- whole blood and incubated for 10 min at 4°C in the dark. Conjugated
sumed the same diet as the LC intervention but with the addition of antibodies (all Miltenyi Biotec) for human CD14 (Vioblue, 130-094-
the estimated individualized calories expended over the 3 daily 364), TLR2 (phycoerythrin, 130-099-016), and TLR4 (allophycocya-
15-min postmeal walks [(2.5 METs ⫻ 3.5 ⫻ weight (kg) ⫻ 45 nin, 130-096-236) were added, followed by a 10-min incubation at
(min)/200)] (1). 4°C in the dark. Next, 1 ml of red blood cell lysis buffer (120-001-
Hormones and metabolites. Morning fasting blood samples were 339, Miltenyi Biotec) was added, followed by a 15-min incubation at
collected by venipuncture using a 21-gauge needle into EDTA tubes room temperature (RT) in the dark. Two microliters of propidium
and were centrifuged at 1,550 g for 15 min at 4°C. Following iodide (130 – 093–233; Miltenyi Biotec) were added for dead cell
centrifugation, plasma samples were stored at ⫺80°C for batch exclusion, and samples were analyzed on a MACSQuant Analyzer 10
analyses. Plasma triglycerides (TGs) (Pointe Scientific) and glucose flow cytometer. Ten thousand monocytes were counted in each
(Pointe Scientific) were analyzed on a Chemwell 2910 automated sample, and data were analyzed with MACSQuantify version 2.6
analyzer (Awareness Technologies). Plasma insulin (Human Insulin (Miltenyi Biotec). CD14⫹ monocytes were identified via a hierarchi-

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 LOW-CARBOHYDRATE DIET AND INFLAMMATION R1213
Table 2. Characteristics of participants Western blotting analysis. PBMCs were extracted from ~8 ml of
whole blood using Leucoep tubes (163288, Greiner Bio-One In-
Characteristic Value ternational, Kremsmunster, Austria), according to the manufactur-
Number of participants (M/F) 11 (4/7) er’s instructions, and stored at ⫺80°C for batch analyses. Samples
Age, yr 64 (8) were lysed by sonication in ice-cold radioimmunoprecipitation
Body mass index, kg/m2 34.0 (8.0) assay buffer (50 mM Tris·HCl, 1% Triton X-100, 0.5% sodium
Waist circumference, cm 105 (13) deoxycholate, 0.1% SDS, 150 mM NaCl) containing protease (cat.
Systolic blood pressure, mmHg 124 (9) no. 04693159001; complete, Protease Inhibitor Cocktail, Roche) and
Diastolic blood pressure, mmHg 77 (5) phosphatase (100 mM NaVO3, 20 mM NaF, 10 mM EDTA) inhibitors
Glycated hemoglobin, % 7.0 (1.0) and centrifuged (14,000 g for 20 min). Protein was quantified in
Time since diagnosis, yr 6.4 (4.3)
supernatants with the bicinchoninic acid assay (cat. no. 23225, Pierce
Medications
MET, n 5 BCA Protein Assay Kit), and 20-␮g samples were prepared in
MET ⫹ SU, n 2 Laemmli Buffer (cat. no. 7722, Blue Loading Buffer, 1.25 M DTT),
MET ⫹ GLP-1, n 1 heated at 95°C for 5 min, and separated using 12% SDS-polyacryl-
MET ⫹ SU ⫹ DPP4, n 1 amide gel electrophoresis (cat. no. 170 –3930, Mini Trans-Blot, Bio-
Statin, n 3 Rad). Proteins were electrotransferred to polyvinylidene difluoride
Antihypertensive, n 3 membranes using Towbin buffer (25 mM Tris·HCl, 192 mM glycine,
Data are presented as mean (SD), except for number of participants (n)
20% methanol, pH 8.3) for 3 h at 70 V. Membranes were stained with
(count; male/postmenopausal female) and medications (count). DPP4, dipep- Ponceau S Solution (P7170, Sigma-Aldrich) to verify the efficiency of
tidyl peptidase-4; F, female; GLP-1, glucagon-like-peptide-1; M, male; MET, protein transfer. Membranes were blocked in Tris-buffered saline-
metformin; SU, sulfonylurea. Tween 20 (TBS-T) ⫹ iBlock solution (20 mM Tris·HCl, 140 mM
NaCl, 0.1% Tween-20, 0.2% iBlock) for 1 h at RT and then incubated
in primary phospho-stress-activated protein kinase (SAPK)/Jun NH2-
cal gating strategy. Specifically, cells that stained positive for pro- terminal kinase (JNK) (Thr183/Tyr185; p-JNK) antibody (1:1,000,
pidium iodide were excluded, and then cells were characterized as cat. no. 4668, Cell Signaling Technology) in TBS-T ⫹ iBlock solution
CD14⫹ and confirmed to be monocytes via a characteristic scatter overnight at 4°C, followed by secondary anti-rabit IgG horseradish
profile. TLR2 and TLR4 median fluorescence intensity were then peroxidase-linked antibody (1:5,000, cat. no. 7074, Cell Signaling
determined on CD14⫹ monocytes with fluorescence minus one con- Technology) in TBS-T ⫹ iBlock solution for 2 h at RT. Proteins were
trol used to determine positive and negative populations. Total gran- detected using Luminata Forte HRP Substrate (cat. no. WBLUF0100,
ulocyte, monocyte, and lymphocyte numbers were determined based Millipore Sigma), and the images were acquired using Amersham
on characteristic scatter profiles. Hyperfilm ECL (cat. no. 28906835, GE Life Sciences). Membranes
Monocyte and leukocyte-derived MPs. MMPs and leukocyte-de- were stripped (cat. no. 21059, Thermo Fisher Scientific) for 20 min at
rived MPs (LMPs) were characterized using flow cytometry, as RT and blocked again in TBS-T ⫹ iBlock. The same procedure was
previously described (4, 16). Plasma samples were centrifuged at adopted to quantify total SAPK/JNK (1:1,000, cat. no. 9252, Cell
13,000 g for 2 min, and 200 ␮l of platelet-free plasma were trans- Signaling Technology). Results were quantified using Adobe Photo-
ferred to TruCount tubes (BD Biosciences). MP size threshold was shop CC 2017 and relativized to the protein content using the Ponceau
established using Megamix-Plus SSC calibrator beads (Biocytex, staining as described by Gilda et al. (21). Samples from each partic-
Marseille, France), and only events ⬍1 ␮m in size were counted. MPs ipant were run on the same gel, and bands were expressed relative to
were defined as events falling within the established size ranges (0.16, an internal standard containing pooled human PMBC lysate included
0.20, 0.24, and 0.5 ␮m). Monocyte- (CD14⫹) and leukocyte- in each blot. PBMC analyses were performed on n ⫽ 6 participants
(CD45⫹) specific antibodies (BioLegend, San Diego, CA) identified because of sample availability.
MMPs and LMPs for events falling within the respective MP size Statistical analysis. Data were analyzed using SPSS v.21 (SPSS,
range. Samples were incubated with antibodies for 20 min in the dark Chicago, IL). Normality was assessed using Q-Q plots and Shapiro-
at RT, fixed with 2% paraformaldehyde (ChemCruz Biochemicals, Wilk tests within each experimental condition. Four-day CGM data
Santa Cruz, CA), diluted with PBS, and analyzed using BD Biosci- were analyzed using a one-way repeated-measures ANOVA with
ences FACSAria I High Speed Cell sorter and flow cytometer (Uni- Fisher’s least-significant difference post hoc testing. A linear mixed-
versity of Colorado Anschutz Medical Campus, Allergy and Clinical effects model (condition and time as fixed factors, subject as random
Immunology/Infectious Disease Flow Core). The concentration of factor) was used to determine the treatment effects for all inflamma-
MMPs and LMPs was determined using the formula: [(number of tory and metabolic markers. Significant interactions and time effects
events in region containing MPs/number of events in absolute count were followed up with preplanned contrasts comparing pre- versus
bead region) ⫻ (total number of beads per test/total volume of postintervention within each condition. Cohen’s d effect size was
sample)]. MP analyses were performed on n ⫽ 8 subjects. calculated for these preplanned comparisons within each condition.

Table 3. Four-day continuous glucose monitoring

GL LC LC⫹Ex ANOVA GL vs. LC GL vs. LC⫹Ex LC vs. LC⫹Ex

Mean, mmol/l 7.4 (1.6) 6.5 (1.2) 6.2 (1.1) 0.001 0.000 0.001 0.049
SD, mmol/l 1.7 (0.9) 0.8 (0.3) 0.8 (0.4) 0.001 0.002 0.003 0.476
MAGE, mmol/l 4.3 (2.2) 2.0 (1.0) 1.7 (0.9) 0.000 0.000 0.001 0.109
CONGA, mmol/l 6.6 (1.4) 6.2 (1.1) 5.9 (1.0) 0.004 0.005 0.005 0.037
Time ⬎ 10 mmol/l, % 13.5 (18.1) 2.3 (5.5) 1.3 (3.9) 0.020 0.018 0.022 0.218
Time ⬍ 4 mmol/l, % 0.3 (0.7) 0.5 (1.1) 1.1 (2.4) 0.332
Data are presented as mean (SD). P values are shown for the overall one-way repeated-measures ANOVA and for the Fisher least-significant difference post
hoc tests. Please see text for effect sizes. CONGA, continuous overall net glycemic action; GL, low-fat low-glycemic index guidelines diet; LC, low-carbohydrate
high-fat diet; LC⫹Ex, low-carbohydrate high-fat diet and exercise; MAGE, mean amplitude of glycemic excursion. Values in bold denote P values with statistical
significance.

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R1214 LOW-CARBOHYDRATE DIET AND INFLAMMATION

Table 4. Fasting hormones and metabolites before (pre) and after (post) each four-day diet intervention
GL LC LC⫹Ex Linear Mixed-Model

Pre Post Pre Post Pre Post Time Condition Time ⫻ Condition

Glucose, mmol/l 8.3 (2.1) 8.1 (2.0) 8.4 (1.9) 7.6 (1.7)* 7.8 (1.8) 7.0 (1.5)* 0.000 0.059 0.144
Triglycerides, mmol/l 2.0 (1.1) 1.9 (0.7) 1.9 (1.0) 2.1 (1.2) 1.9 (0.9) 1.9 (1.3) 0.723 0.506 0.540
Insulin, pmol/l 63.9 (33.5) 58.0 (29.2) 64.8 (29.7) 62.1 (46.4) 59.6 (38.7) 50.1 (38.9) 0.077 0.655 0.558
Proinsulin, pmol/l 33.5 (14.7) 30.7 (16.1) 35.5 (15.1) 26.0 (12.8)† 35.3 (21.1) 22.8 (14.0)* 0.173 0.034 0.000
C-peptide, nmol/l 1.11 (0.39) 1.18 (0.52) 1.18 (0.38) 1.07 (0.45) 1.20 (0.64) 1.05 (0.51) 0.558 0.341 0.268
Proinsulin-insulin ratio 0.7 (0.5) 0.6 (0.3) 0.7 (0.5) 0.6 (0.3) 0.6 (0.3) 0.6 (0.5) 0.141 0.711 0.516
Proinsulin-C-peptide ratio 0.031 (0.013) 0.027 (0.012) 0.030 (0.013) 0.025 (0.008)* 0.032 (0.016) 0.022 (0.011)‡ 0.000 0.563 0.331
Data are presented as mean (SD). GL, low-fat low-glycemic index guidelines diet; LC, low-carbohydrate high-fat diet; LC⫹Ex, low-carbohydrate high-fat diet
and exercise. *P ⱕ 0.01, †P ⫽ 0.001, ‡P ⬍ 0.05 for preplanned contrast vs. pre within condition. Values in bold denote P values with statistical significance.

Significance was set at P ⬍ 0.05. Data in Tables 2–5 and Figs. 2– 4 are CGM. Four-day CGM data are presented in Table 3. One-
presented as mean (SD). way repeated measures ANOVA showed a significant effect
for 4-day mean, standard deviation, MAGE, CONGA, and time
RESULTS over 10 mmol/l (all P ⬍ 0.02). Four-day mean glucose was
significantly lower in the LC⫹Ex as compared with LC (⫺5%,
Baseline characteristics and body weight changes. Charac-
P ⬍ 0.05, d ⫽ 0.8), whereas both LC⫹Ex and LC conditions
teristics of the participants who completed the study are shown
were lower than GL (⫺16%, P ⬍ 0.001, d ⫽ 1.9 and ⫺12%,
in Table 2. Leisure-time exercise per week was 0.1 ⫾ 0.3 for
strenuous exercise, 1.7 ⫾ 0.9 for moderate exercise, and P ⬍ 0.001, d ⫽ 2.3, respectively) (Fig. 2A). CONGA was also
2.3 ⫾ 0.7 for light exercise. The average total score [arbitrary significantly lower in LC⫹Ex as compared with LC (⫺5%,
units calculation: (9 ⫻ strenuous) ⫹ (5 ⫻ moderate) ⫹ (3 ⫻ P ⬍ 0.05, d ⫽ 0.9), whereas both LC⫹Ex and LC conditions
light)] at baseline was 16.3 ⫾ 4.5. A significant overall time were lower than GL (⫺11%, P ⬍ 0.01, d ⫽ 1.2 and ⫺6%, P ⬍
effect was observed for body weight, which decreased by 0.01, d ⫽ 1.2, respectively). LC⫹Ex and LC were not signif-
⫺2.0 ⫾ 1.0 kg, ⫺2.0 ⫾ 0.8 kg, and ⫺1.9 ⫾ 1.1 kg for LC, icantly different but were both respectively lower than GL for
LC⫹Ex, and GL, respectively (P ⬍ 0.001), with no condition standard deviation (both ⫺53%, P ⬍ 0.01, respectively,
by time interaction (P ⫽ 0.969). d ⫽ 1.6 and d ⫽ 2.4), MAGE (⫺61%, P ⱕ 0.001, d ⫽ 2.1 and
Accelerometer. One-way repeated measures ANOVA showed ⫺54%, P ⬍ 0.001, d ⫽ 2.8, respectively) (Fig. 2B), and time
a significant effect for length of time at moderate intensity over over 10 mmol/l (⫺90%, P ⬍ 0.05, d ⫽ 2.0 and ⫺83%, P ⬍
the 4-day interventions (235 ⫾ 136) in LC⫹Ex compared with 0.05, d ⫽ 2.5). No significant difference between conditions
LC (75 ⫾ 92) and GL (117 ⫾ 137) (P ⬍ 0.001). As expected, was observed for time under 4 mmol/l.
total moderate intensity minutes in LC⫹Ex were higher when Metabolites and hormones. Metabolites and hormone data
compared with LC (161 ⫾ 107 min, P ⫽ 0.01, d ⫽ 4.5) and GL are presented in Table 4, and the change from baseline for
(119 ⫾ 88 minutes, P ⫽ 0.02, d ⫽ 3.8) with no significant fasting glucose, C-peptide, insulin, and proinsulin is presented
difference between LC and GL. Time spent in sedentary, light, in Fig. 3. A significant condition by time interaction was found
and vigorous intensity activity was similar between all condi- for proinsulin (P ⬍ 0.001), with a significant decrease in LC
tions (data not shown). (⫺27%, P ⫽ 0.001, d ⫽ 1.5) and LC⫹Ex (⫺35%, P ⫽ 0.005,

Table 5. Fasting inflammatory markers before (pre) and after (post) each four-day diet intervention
GL LC LC⫹Ex Linear Mixed-Model

Pre Post Pre Post Pre Post Time Condition Time ⫻ condition

TNF-␣, pg/ml 14.6 (4.6) 15.7 (4.6) 14.1 (3.6) 14.8 (4.6) 14.8 (4.1) 15.7 (4.8) 0.200 0.660 0.968
MCP1, pg/ml 725 (201) 710 (222) 727 (217) 686 (229) 769 (173) 680 (156)* 0.045 0.859 0.449
IL-6, pg/ml 8.1 (2.8) 9.1 (4.7) 8.2 (3.8) 8.8 (3.6) 9.9 (3.1) 9.2 (3.2) 0.608 0.272 0.559
IL-18, pg/ml 2,810 (1,217) 2,760 (1,152) 2,838 (1,199) 2,818 (1,527) 2,691 (997) 2,658 (1,081) 0.755 0.646 0.994
IL-10, pg/ml 3.6 (2.9) 3.3 (2.8) 3.5 (3.4) 3.3 (2.4) 3.8 (2.9) 3.8 (2.9) 0.461 0.830 0.887
p-JNK, A.U. 100 (30) 68 (20)† 105 (40) 58 (19)† 73 (39) 41 (16)† 0.000 0.008 0.616
TLR2, MFI 7.5 (1.3) 6.9 (0.9) 7.4 (1.9) 6.9 (0.7) 7.8 (1.4) 7.7 (2.2) 0.211 0.228 0.852
TLR4, MFI 4.7 (0.3) 4.8 (0.3) 5.0 (0.4) 5.0 (0.4) 5.0 (0.4) 5.2 (0.5) 0.149 0.014 0.617
MMPs, count/ml 404 (330) 97 (55) † 245 (203) 73 (58) 238 (183) 239 (192) 0.002 0.236 0.040
LMPs, count/ml 1,313 (999) 490 (311) 1,055 (1,317) 890 (1,380) 620 (281) 565 (260) 0.067 0.837 0.206
Granulocytes, count/ml ⫻106 2.4 (0.6) 2.9 (0.6) 2.6 (0.5) 2.6 (0.4) 2.9 (1.1) 2.5 (0.5) 0.539 0.640 0.204
Lymphocytes, count/ml ⫻10 6
1.2 (0.2) 1.4 (0.4) 1.3 (0.3) 1.3 (0.3) 1.3 (0.2) 1.2 (0.4) 0.591 0.586 0.215
Monocytes, count/ml ⫻105 2.7 (0.6) 3.0 (0.7) 2.6 (0.8) 2.7 (0.6) 3.0 (1.1) 2.6 (0.4) 0.980 0.578 0.273
Data are presented as mean (SD). A.U., arbitrary units; GL, low-fat low-glycemic index guidelines diet; LC, low-carbohydrate high-fat diet; LC⫹Ex,
low-carbohydrate high-fat diet and exercise; LMPs, leukocyte-derived microparticles; MFI, median fluorescence intensity; MMPs, monocyte-derived micro-
particles; p-JNK, phosphorylated c-Jun NH2-terminal kinase; TLR, toll-like receptor; TNF-␣, tumor necrosis factor-␣. *P ⬍ 0.01, †P ⬍ 0.05 for preplanned
contrast vs. pre within condition. Values in bold denote P values with statistical significance.

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 LOW-CARBOHYDRATE DIET AND INFLAMMATION R1215

A † †, ‡ 0.064, d ⫽ 0.9), whereas there was no significant change in

11
LC⫹Ex (0.5%, P ⫽ 0.990, d ⫽ 0.0). For LMPs, there was a
tendency for a significant main effect of time (P ⫽ 0.067), with
4-day average glucose

9 exploratory pairwise comparisons within conditions showing

tendencies for reductions in GL (⫺63%, P ⫽ 0.055, d ⫽ 1.2)
(mmol/L)

7 and LC (⫺16%, P ⫽ 0.057, d ⫽ 0.9) but not LC⫹Ex (⫺9%,

P ⫽ 0.516, d ⫽ 0.3).
5
DISCUSSION

0 The main objective of this study was to determine whether

GL LC LC+Ex reducing hyperglycemia by following an LCHF diet alone, or
Conditions in combination with postmeal walking, could lower markers of
B innate immune cell activation and systemic inflammation in
10
people with T2D. Our study showed that while LC and LC⫹Ex
led to superior improvements in glucose control and fasting
4-day average MAGE

8 † † proinsulin levels as compared with GL, all three diets appeared

to lower PBMC p-JNK (a marker of cellular inflammation)
(mmol/L)

6
over the short-term.
4 LCHF diets improve glucose control. Recently, LCHF diets
have been recommended as a first-line treatment for improving
2 glucose control in T2D (14). In studies lasting between 6 and
12 mo, LCHF diets have been shown to reduce HbA1c and
0 fasting glucose to a greater extent than a traditional western
GL LC LC+Ex diet or a low-glycemic diet (25, 33). In line with our results, the
Conditions glucose-lowering effect of carbohydrate restriction can be
Fig. 2. Mean glucose and mean amplitude of glycemic excursions (MAGEs) observed quickly with no or very minimal weight-loss (18, 45).
from continuous glucose monitoring during each 4-day diet intervention. Mean In addition to restricting carbohydrates, light walks performed
glucose (A) and MAGE (B) calculated from continuous glucose monitoring around meal times have been shown to lower glucose excur-
throughout each intervention. Fisher least-significant difference post hoc tests
following significant one-way repeated-measures ANOVA; GL, low-fat low-
sions (7, 11). To the best of our knowledge, our study is the
glycemic index guidelines diet; LC, low-carbohydrate high-fat diet; LC⫹Ex, first to test the combined strategy of an LCHF diet with
low-carbohydrate high-fat diet and exercise. †P ⱕ 0.001 vs. CON. ‡P ⬍ 0.05 postmeal walks (LC⫹Ex) with findings showing that this
vs. LC. approach can improve 24-h glucose control to a greater extent
than both an LC diet alone or a GL diet. The LC diet alone was
also clearly effective at lowering 24-h glucose when compared
d ⫽ 1.5) but not for GL (⫺8%, P ⫽ 0.065, d ⫽ 0.7). A with GL. Change in fasting glucose was not different between
significant main effect of time was observed for fasting glucose conditions, but a significant decrease within LC and LC⫹Ex of
and proinsulin-C-peptide ratio (both P ⬍ 0.001). In preplanned a similar magnitude as a previous 7-day low-carbohydrate diet
contrasts, only LC⫹Ex and LC decreased fasting glucose study in individuals who were overweight/obese was observed
(⫺10%, P ⫽ 0.007, d ⫽ 1.3 and ⫺10%, P ⫽ 0.011, d ⫽ 1.0, (49). The short duration of the current study might explain the
respectively) and proinsulin-C-peptide ratio (⫺31%, P ⫽ absence of change in fasting glucose, as significant weight loss
0.045, d ⫽ 0.7 and ⫺17%, P ⫽ 0.004, d ⫽ 1.2, respectively). or longer-term metabolic adaptations may be needed to reduce
Inflammatory markers. Inflammatory markers are presented hepatic glucose output and/or insulin resistance (50, 60). The
in Table 5. Western blot images of p-JNK and total JNK in dysglycemia of diabetes is characterized by sustained chronic
PBMCs, as well as change from baseline for p-JNK, are shown hyperglycemia, postprandial glucose fluctuations, and in-
in Fig. 4, and MMPs and LMPs are presented in Fig. 5. A creased glucose variability (40). As compared with GL, both
significant overall time effect was observed for MCP-1 (P ⫽ LC and LC⫹Ex improved all of these glycemic outcomes,
0.045) and p-JNK (P ⬍ 0.001). In preplanned contrasts, only including MAGE, which has been shown to be strongly and
LC⫹Ex decreased MCP-1 (⫺12%, P ⫽ 0.003, d ⫽ 1.3), positively correlated to oxidative stress and inflammation
whereas p-JNK decreased in all three conditions (all P ⬍ 0.05, markers (41). The activation of oxidative stress and inflamma-
GL: ⫺32%, d ⫽ 1.6; LC: ⫺45%, d ⫽ 1.7; LC⫹Ex, ⫺44%, tory pathways contributes to insulin resistance and diabetes
d ⫽ 1.4). Despite the randomized crossover design, a main complications (19, 28). Therefore, lowering overall hypergly-
effect of condition was observed for p-JNK and TLR4 (both cemia and glycemic variability in LC and LC⫹Ex may be
P ⱕ 0.01). Levels of p-JNK were overall lower in LC⫹Ex as advantageous in T2D.
compared with GL and LC, whereas overall TLR4 was lower Impact of short-term dietary interventions on inflammation.
in GL as compared with LC and LC⫹Ex. There were no The phosphorylation of JNK is regarded as a key signaling
differences in total granulocyte, monocyte, or lymphocyte cell node controlling inflammation by activating transcription fac-
counts with any of the interventions (Table 5). A significant tors that result in the production of proinflammatory cytokines
condition by time interaction was observed for MMPs (P ⫽ and chemokines (6, 61). Murine studies support a causal role of
0.040) with a significant decrease in GL (⫺76%, P ⫽ 0.035, myeloid JNK activation in the progression of insulin resis-
d ⫽ 1.8) and a tendency for a reduction in LC (⫺70%, P ⫽ tance, adipose tissue macrophage accumulation, and systemic

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R1216 LOW-CARBOHYDRATE DIET AND INFLAMMATION

A B
2

Fasting c-peptide (pmol/L)

† † 600

Fasting glucose (mmol/L)

1 300

0 0

-1 -300
Fig. 3. Changes in (⌬) fasting glucose, C-
peptide, insulin, and proinsulin following -600
-2
each 4-day diet intervention. Fasting glucose
(A), fasting C-peptide (B), fasting insulin -900
-3
(C), and fasting proinsulin (D) presented as GL LC LC+Ex GL LC+Ex LC
change scores (post ⫺ pre within each con-
dition). Linear mixed models revealed a sig- Conditions Conditions
nificant main effect of time for fasting glucose
(P ⬍ 0.001) and a significant condition ⫻ time C D ‡ †
interaction for proinsulin (P ⬍ 0.001). GL,

Fasting proinsulin (pmol/L)

low-fat low-glycemic index guidelines diet; 50 10
Fasting insulin (pmol/L)

LC, low-carbohydrate high-fat diet; LC⫹Ex,

low-carbohydrate high-fat diet and exercise. 25 0
†P ⱕ 0.01 and ‡P ⫽ 0.001 for preplanned
contrast of post vs. pre within condition. 0 -10

-25 -20

-50 -30

GL LC LC+Ex GL LC LC+Ex
Conditions Conditions

inflammation (26). JNK activation is increased in T2D and can diet on LMPs was not as great as MMPs, the GL and LC diets
be triggered by several metabolic stressors, such as elevated tended to reduce circulating LMP concentrations. Reductions
glucose, circulating free fatty acids, insulin, cytokines, and in circulating MMPs and LMPs support anti-inflammatory
oxidative stress (54, 56, 57). Thus, activation of JNK in effects of each diet, potentially via reduced monocyte and
PBMCs may represent both a systemic marker of inflammation leukocyte activation. Interestingly, there were no significant
and an underlying signaling pathway involved in T2D patho- reductions in either MMPs or LMPs in response to a combined
physiology. In the present study, all three short-term dietary LC diet with postmeal light-to-moderate exercise. It is plausi-
interventions led to significant reductions in PBMCs p-JNK. It ble that exercise induced a moderate, transient increase in
is possible that the small (~2 kg) weight loss experienced in monocyte and leukocyte activation and, in turn, MP vesicula-
each intervention could contribute to the reduction in JNK tion, which counteracted the diet effects. Indeed, acute exercise
activation. Since body weight went back to preintervention has been reported to increase circulating MPs, whereas chronic
during washout periods, it is likely that participants were aerobic exercise is associated with reduced MP formation (30).
consuming slightly fewer calories during the three 4-day peri- It is likely that a longer intervention period, resulting in a more
ods as compared with baseline. Alternatively, all three dietary chronic exercise stimulus, may yield different results.
conditions may have been less proinflammatory than the typ- Among the five cytokines assessed in our study, only MCP-1
ical dietary pattern of participants, given that we provided showed a significant decrease over the 4-day interventions. In
minimally processed foods with low sugar and refined carbo- a 7-day study in individuals who were overweight, an LCHF
hydrate content. diet with and without antioxidants (vitamin C/E) led to a
Cell-derived MPs are small (~100 –1,000 ␮m) plasma mem- similar reduction in MCP-1, whereas, in line with our results,
brane vesicles that are released by most eukaryotic cells in C-reactive protein and IL-6 levels were not affected (49). Thus,
response to activation and/or apoptosis. Depending on the cell despite the reduction in PBMC JNK activation, plasma cyto-
of origin, circulating MPs exert distinct biological effects (38, kines were largely unchanged by short-term diet interventions.
53). MPs derived from monocytes and leukocytes are known to Longer term low-carbohydrate diets typically result in substan-
play a role in inflammation (2, 24). For example, MMPs have tial reductions in body and fat mass (43), which can confound
been shown to induce an upregulation in endothelial cell interpretation of obesity-related inflammatory parameters. In
expression of intracellular cell adhesion molecule-1 and pro- our short-term study, the lack of change in proinflammatory
mote T cell infiltration into the vessel wall, enhancing plaque cytokines could be viewed as support for the utilization of
formation (32). In addition, LMPs impact the endothelial LCHF diets in the treatment of T2D. These findings suggest
monolayer, stimulating inflammatory processes, such as the that previous findings of inflammatory activation after hyper-
JNK signaling pathway (39), and the recruitment of chemot- caloric high-fat meals (~950 kcal) may not translate to height-
actic cytokines (2). In the present study, circulating concentra- ened inflammation when eucaloric/hypocaloric LCHF foods
tions of MMPs were markedly lower in response to the GL are consumed over several days (13, 22). However, the use of
(~75%) and LC (~70%) diets. While the main effect of each plasma-borne inflammatory markers, such as cytokines, has

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 LOW-CARBOHYDRATE DIET AND INFLAMMATION R1217

A conditions. Therefore, the small weight loss may be related to

GL LC LC+Ex
an overall increase in diet quality and/or strictly controlling
pre post pre post pre post pool food intake over the 4 days. The clinical relevance of this small
p-JNK reduction is unclear, but we acknowledge that weight loss
(thr183/185) could have played a role in the modulation of inflammation.
All food consumed by participants was provided and con-
t-JNK sisted of healthy whole foods. Thus, it is likely that most
participants improved the quality of their diet as compared with
B what they usually eat by removing processed foods. This might
50 ‡ ‡ ‡
have contributed to the absence of significant differences
p-JNK (arbitrary units)

between conditions for some outcomes since each diet seems to

0 have provided a certain degree of improvement over habitual
patterns of participants. Furthermore, despite providing fresh
whole foods, the strict protocol may have reduced generaliz-
-50
ability as three participants were unable to complete all con-
ditions, citing inability to comply with study procedures.
-100 Like most people with T2D, the participants in the current
investigation were on medications. We made sure to instruct
-150 each participant to continue to take their regular medications
GL LC LC+Ex throughout the entire study and used a daily log to confirm this,
Conditions but the interaction between medications, diets, and inflamma-
Fig. 4. Changes in (⌬) peripheral blood mononuclear cells (PBMCs) phos-
tory outcomes are unknown. The relatively small sample size
phorylated c-Jun NH2-terminal kinase (p-JNK) following each 4-day diet did not allow for an examination of interactions with medica-
intervention. A: representative Western blot image showing total and p-JNK tions or by sex, but the randomized crossover design was a
before (pre) and after (post) each intervention. Band intensities were expressed strength in this regard.
relative to a pooled sample of human PBMCs included in every blot (pool). B: In conclusion, an LCHF diet with or without daily postmeal
data for p-JNK/total JNK presented as change scores (post ⫺ pre within each
condition). Linear mixed models revealed a significant main effect of time for walks improved four-day glycemic control and fasting proin-
p-JNK (P ⬍ 0.05). GL, low-fat low-glycemic index guidelines diet; LC,
low-carbohydrate high-fat diet; LC⫹Ex, low-carbohydrate high-fat diet and
exercise. ‡P ⬍ 0.05 for preplanned contrast of post vs. pre within condition. A 400 ‡
200
MMPs (count/ml)

0
recently been questioned, as they may not reflect inflammatory
processes occurring in cells (29). Our findings are in agreement -200
with this notion, as changes were seen in PBMCs p-JNK and -400
MPs with minimal effects of the dietary interventions on key -600
cytokines, such as IL-6 and TNF-␣.
LCHF diets lower proinsulin. Elevated proinsulin, as well as -800
proinsulin-insulin or the proinsulin-C-peptide ratio, is a strong -1000
predictor of insulin resistance and is associated with beta cell GL LC LC+Ex
dysfunction (37, 51). In our study, LC and LC⫹Ex decreased Conditions
fasting proinsulin, whereas no changes were seen following the B 1000
GL diet. Although the change in proinsulin-C-peptide was not
500
significantly different between conditions (i.e., nonsignificant
LMPs (count/ml)

condition ⫻ time interaction), within-condition comparisons 0

showed that only LC and LC⫹Ex decreased this ratio. Here, -500
we speculate that by reducing carbohydrate intake, and thus the
demand for insulin, LCHF diets can potentially provide the -1000
beta cells with some transient rest, decrease endoplasmic -1500
reticulum stress, and improve proinsulin processing within beta
cells (36). The long-term effects of such diets on proinsulin -2000
levels and beta cell function are unknown, but since both -2500
proinsulin and glucose levels are independent cardiovascular GL LC LC+Ex
risk factors (23, 42), an LCHF diet with and without postmeal Conditions
walks could contribute to improving the metabolic profile and Fig. 5. Changes in (⌬) monocyte-derived microparticles (MMPs) and leuko-
cardiovascular risk of individuals with T2D. cyte-derived microparticles (LMPs) following each 4-day diet intervention.
Limitations and perspectives. Despite our efforts to maintain MMPs (A) and LMPs (B) presented as change scores (post ⫺ pre within each
energy balance, a small (~1–2 kg) but significant weight loss condition). Linear mixed models revealed a significant condition ⫻ time
interaction for MMPs (P ⬍ 0.05). GL, low-fat low-glycemic index guidelines
occurred in all conditions. The short duration of each interven- diet; LC, low-carbohydrate high-fat diet; LC⫹Ex, low-carbohydrate high-fat
tion did not permit adjusting the caloric intake in attempts to diet and exercise. ‡P ⬍ 0.05 for preplanned contrast of post vs. pre within
prevent weight loss, but energy intake was matched between condition.

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R1218 LOW-CARBOHYDRATE DIET AND INFLAMMATION

sulin levels compared with a GL diet. The addition of postmeal 4. Bain AR, Ainslie PN, Bammert TD, Hijmans JG, Sekhon M, Hoiland
walks to an LCHF diet further improved glycemic control. RL, Flück D, Donnelly J, DeSouza CA. Passive heat stress reduces
circulating endothelial and platelet microparticles. Exp Physiol 102: 663–
There were no consistent effects of the individual interventions 669, 2017. doi:10.1113/EP086336.
on inflammatory markers, although inflammatory activation of 5. Ceriello A, Taboga C, Tonutti L, Quagliaro L, Piconi L, Bais B, Da
circulating PBMCs appeared to be reduced following each Ros R, Motz E. Evidence for an independent and cumulative effect of
intervention, with no appreciable changes in characteristic postprandial hypertriglyceridemia and hyperglycemia on endothelial dys-
proinflammatory plasma cytokines. Longer-term studies using function and oxidative stress generation: effects of short- and long-term
simvastatin treatment. Circulation 106: 1211–1218, 2002. doi:10.1161/01.
LCHF diets and exercise looking at cardiometabolic and in- CIR.0000027569.76671.A8.
flammatory markers are necessary to confirm the beneficial 6. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature
effects of such diets in individuals with T2D. 410: 37–40, 2001. doi:10.1038/35065000.
7. Colberg SR, Zarrabi L, Bennington L, Nakave A, Thomas Somma C,
Perspectives and Significance Swain DP, Sechrist SR. Postprandial walking is better for lowering the
glycemic effect of dinner than pre-dinner exercise in type 2 diabetic
LCHF diets have recently regained popularity for the man- individuals. J Am Med Dir Assoc 10: 394 –397, 2009. doi:10.1016/j.jamda.
agement of T2D. The macronutrient distribution characteristic 2009.03.015.
of this kind of diet is in contradiction with the current dietary 8. Dandona P, Aljada A, Bandyopadhyay A. Inflammation: the link
between insulin resistance, obesity and diabetes. Trends Immunol 25: 4 –7,
guidelines for diabetes. As demonstrated in this study, an 2004. doi:10.1016/j.it.2003.10.013.
LCHF diet with and without postmeal walks significantly and 9. Dasu MR, Devaraj S, Park S, Jialal I. Increased toll-like receptor (TLR)
rapidly normalized blood glucose levels. The elevated amount activation and TLR ligands in recently diagnosed type 2 diabetic subjects.
of fat consumed on this diet did not negatively affect inflam- Diabetes Care 33: 861–868, 2010. doi:10.2337/dc09-1799.
mation or circulating TGs, which combined with the glucose- 10. Dasu MR, Devaraj S, Zhao L, Hwang DH, Jialal I. High glucose
induces toll-like receptor expression in human monocytes: mechanism of
lowering effect and suggests that LCHF can be a beneficial activation. Diabetes 57: 3090 –3098, 2008. doi:10.2337/db08-0564.
treatment strategy for T2D. Metabolic adaptations to LCHF 10a.Diabetes Canada Clinical Practice Guidelines Expert Committee;
can take several weeks to months, and deepening our knowl- Houlden RL. Introduction. Can J Diabetes 42, Suppl 1: S1–S5, 2018.
edge on their chronic effect on inflammatory and metabolic doi:10.1016/j.jcjd.2017.10.001.
status will likely help us formulate better therapeutic dietary 11. DiPietro L, Gribok A, Stevens MS, Hamm LF, Rumpler W. Three
15-min bouts of moderate postmeal walking significantly improves 24-h
approaches for individuals with impaired glycemic regulation. glycemic control in older people at risk for impaired glucose tolerance.
Diabetes Care 36: 3262–3268, 2013. doi:10.2337/dc13-0084.
GRANTS
12. Dworatzek PD, Arcudi K, Gougeon R, Husein N, Sievenpiper JL,
This work was supported by the Canadian Institutes of Health Research Williams SL; Canadian Diabetes Association Clinical Practice Guide-
New Investigator Salary Award (MSH-141980), the Michael Smith Founda- lines Expert Committee. Nutrition therapy. Can J Diabetes 37, Suppl 1:
tion for Health Research Scholar Award (16890), and Natural Sciences and S45–S55, 2013. doi:10.1016/j.jcjd.2013.01.019.
Engineering Research Council of Canada Discovery Grant (RGPIN 435807- 13. Erridge C, Attina T, Spickett CM, Webb DJ. A high-fat meal induces
13) to J. P. Little. Continuous glucose monitors and sensors were provided in low-grade endotoxemia: evidence of a novel mechanism of postprandial
kind from an Investigator-Initiated Grant (NERP15-016) from Medtronic. This inflammation. Am J Clin Nutr 86: 1286 –1292, 2007. doi:10.1093/ajcn/86.
work was also supported by the São Paulo Research Foundation fellowship 5.1286.
(2016/23251-7) (to J. D. Botezelli) and National Institutes of Health Awards 14. Feinman RD, Pogozelski WK, Astrup A, Bernstein RK, Fine EJ,
HL-131458 and HL-135598 (to C. A. DeSouza). Westman EC, Accurso A, Frassetto L, Gower BA, McFarlane SI,
Nielsen JV, Krarup T, Saslow L, Roth KS, Vernon MC, Volek JS,
DISCLOSURES Wilshire GB, Dahlqvist A, Sundberg R, Childers A, Morrison K,
J. P. Little and J. D. Johnson are co-Chief Scientific Officers for the Institute Manninen AH, Dashti HM, Wood RJ, Wortman J, Worm N. Dietary
for Personalized Therapeutic Nutrition, a not-for-profit organization that sup- carbohydrate restriction as the first approach in diabetes management:
ports a food-first approach to treating and preventing chronic disease. J. P. critical review and evidence base. Nutrition 31: 1–13, 2015. doi:10.1016/
Little holds shares in Metabolic Insights Inc., a for-profit company that is j.nut.2014.06.011.
developing techniques for noninvasive metabolic monitoring. 15. Francois ME, Durrer C, Pistawka KJ, Halperin FA, Chang C, Little
JP. Combined interval training and post-exercise nutrition in type 2
AUTHOR CONTRIBUTIONS diabetes: a randomized control trial. Front Physiol 8: 528, 2017. doi:10.
3389/fphys.2017.00528.
É.M.-C. and J.P.L., conceived and designed research; É.M.-C., C.D., H.N., 16. Francois ME, Little JP. The impact of acute high-intensity interval
T.D.B., J.D.B., J.D.J., and C.A.D. performed experiments; É.M.-C., C.D., exercise on biomarkers of cardiovascular health in type 2 diabetes. Eur J
H.N., T.D.B., J.D.B., J.D.J., C.A.D., and J.P.L. analyzed data; É.M.-C., C.D., Appl Physiol 117: 1607–1616, 2017. doi:10.1007/s00421-017-3649-2.
H.N., T.D.B., J.D.B., J.D.J., C.A.D., and J.P.L. interpreted results of experi- 17. Francois ME, Myette-Cote E, Bammert TD, Durrer C, Neudorf H,
ments; É.M.-C., C.D., and J.P.L. prepared figures; É.M.-C., C.D., T.D.B., DeSouza CA, Little JP. Carbohydrate restriction with postmeal walking
J.D.B., and J.P.L. drafted manuscript; É.M.-C., C.D., H.N., T.D.B., J.D.B., effectively mitigates postprandial hyperglycemia and improves endothe-
J.D.J., C.A.D., and J.P.L. edited and revised manuscript; É.M.-C., C.D., H.N.,
lial function in type 2 diabetes. Am J Physiol Heart Circ Physiol 314:
T.D.B., J.D.B., J.D.J., C.A.D., and J.P.L. approved final version of manuscript.
H105–H113, 2018. doi:10.1152/ajpheart.00524.2017.
18. Gannon MC, Nuttall FQ. Control of blood glucose in type 2 diabetes
REFERENCES
without weight loss by modification of diet composition. Nutr Metab
1. Ainsworth BE, Haskell WL, Herrmann SD, Meckes N, Bassett DR Jr, (Lond) 3: 16, 2006. doi:10.1186/1743-7075-3-16.
Tudor-Locke C, Greer JL, Vezina J, Whitt-Glover MC, Leon AS. 19. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ
2011 Compendium of Physical Activities: a second update of codes and Res 107: 1058 –1070, 2010. doi:10.1161/CIRCRESAHA.110.223545.
MET values. Med Sci Sports Exerc 43: 1575–1581, 2011. doi:10.1249/ 20. Giannella A, Radu CM, Franco L, Campello E, Simioni P, Avogaro A,
MSS.0b013e31821ece12. de Kreutzenberg SV, Ceolotto G. Circulating levels and characterization
1a.American Diabetes Association. Nutrition recommendations and inter- of microparticles in patients with different degrees of glucose tolerance.
ventions for diabetes—2013. Diabetes Care 36: S12–S32, 2013. Cardiovasc Diabetol 16: 118, 2017. doi:10.1186/s12933-017-0600-0.
2. Angelillo-Scherrer A. Leukocyte-derived microparticles in vascular ho- 21. Gilda JE, Gomes AV. Stain-Free total protein staining is a superior
meostasis. Circ Res 110: 356 –369, 2012. doi:10.1161/CIRCRESAHA. loading control to ␤-actin for Western blots. Anal Biochem 440: 186 –188,
110.233403. 2013. doi:10.1016/j.ab.2013.05.027.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00240.2018 • www.ajpregu.org

Downloaded from journals.physiology.org/journal/ajpregu (213.000.053.141) on March 16, 2025.
 LOW-CARBOHYDRATE DIET AND INFLAMMATION R1219
22. Gregersen S, Samocha-Bonet D, Heilbronn LK, Campbell LV. Inflam- trolled trials. Arch Intern Med 166: 285–293, 2006. doi:10.1001/archinte.
matory and oxidative stress responses to high-carbohydrate and high-fat 166.3.285.
meals in healthy humans. J Nutr Metab 2012: 238056, 2012. doi:10.1155/ 44. Norseen J, Hosooka T, Hammarstedt A, Yore MM, Kant S, Aryal P,
2012/238056. Kiernan UA, Phillips DA, Maruyama H, Kraus BJ, Usheva A, Davis
23. Haffner SJ, Cassells H. Hyperglycemia as a cardiovascular risk factor. RJ, Smith U, Kahn BB. Retinol-binding protein 4 inhibits insulin
Am J Med 115, Suppl 1: 6 –11, 2003. doi:10.1016/j.amjmed.2003.09.009. signaling in adipocytes by inducing proinflammatory cytokines in macro-
24. Halim AT, Ariffin NA, Azlan M. Review: the multiple roles of mono- phages through a c-Jun N-terminal kinase- and toll-like receptor 4-depen-
cytic microparticles. Inflammation 39: 1277–1284, 2016. doi:10.1007/ dent and retinol-independent mechanism. Mol Cell Biol 32: 2010 –2019,
s10753-016-0381-8. 2012. doi:10.1128/MCB.06193-11.
25. Hallberg SJ, McKenzie AL, Williams PT, Bhanpuri NH, Peters AL, 45. Nuttall FQ, Schweim K, Hoover H, Gannon MC. Effect of the Lo-
Campbell WW, Hazbun TL, Volk BM, McCarter JP, Phinney SD, BAG30 diet on blood glucose control in people with type 2 diabetes. Br J
Volek JS. Effectiveness and safety of a novel care model for the man- Nutr 99: 511–519, 2008. doi:10.1017/S0007114507819155.
agement of type 2 diabetes at 1 year: an open-label, non-randomized, 46. Nygaard H, Tomten SE, Høstmark AT. Slow postmeal walking reduces
controlled study. Diabetes Ther 9: 583–612, 2018. doi:10.1007/s13300- postprandial glycemia in middle-aged women. Appl Physiol Nutr Metab
018-0373-9. 34: 1087–1092, 2009. doi:10.1139/H09-110.
26. Han MS, Jung DY, Morel C, Lakhani SA, Kim JK, Flavell RA, Davis 47. Padilla J, Harris RA, Fly AD, Rink LD, Wallace JP. The effect of acute
RJ. JNK expression by macrophages promotes obesity-induced insulin exercise on endothelial function following a high-fat meal. Eur J Appl
resistance and inflammation. Science 339: 218 –222, 2013. doi:10.1126/ Physiol 98: 256 –262, 2006. doi:10.1007/s00421-006-0272-z.
science.1227568. 48. Patel C, Ghanim H, Ravishankar S, Sia CL, Viswanathan P, Mohanty
27. Harris JA, Benedict FG. A biometric study of human basal metabolism. P, Dandona P. Prolonged reactive oxygen species generation and nuclear
Proc Natl Acad Sci USA 4: 370 –373, 1918. doi:10.1073/pnas.4.12.370. factor-kappaB activation after a high-fat, high-carbohydrate meal in the
28. Henriksen EJ, Diamond-Stanic MK, Marchionne EM. Oxidative stress obese. J Clin Endocrinol Metab 92: 4476 –4479, 2007. doi:10.1210/jc.
and the etiology of insulin resistance and type 2 diabetes. Free Radic Biol 2007-0778.
Med 51: 993–999, 2011. doi:10.1016/j.freeradbiomed.2010.12.005. 49. Peairs AT, Rankin JW. Inflammatory response to a high-fat, low-
29. Herieka M, Erridge C. High-fat meal induced postprandial inflammation. carbohydrate weight loss diet: effect of antioxidants. Obesity (Silver
Mol Nutr Food Res 58: 136 –146, 2014. doi:10.1002/mnfr.201300104. Spring) 16: 1573–1578, 2008. doi:10.1038/oby.2008.252.
30. Highton PJ, Martin N, Smith AC, Burton JO, Bishop NC. Micropar- 50. Petersen KF, Dufour S, Befroy D, Lehrke M, Hendler RE, Shulman
ticles and exercise in clinical populations. Exerc Immunol Rev 24: 46 –58, GI. Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance,
2018. and hyperglycemia by moderate weight reduction in patients with type 2
32. Hoyer FF, Giesen MK, Nunes França C, Lütjohann D, Nickenig G, diabetes. Diabetes 54: 603–608, 2005. doi:10.2337/diabetes.54.3.603.
Werner N. Monocytic microparticles promote atherogenesis by modulat- 51. Pfützner A, Kunt T, Hohberg C, Mondok A, Pahler S, Konrad T,
ing inflammatory cells in mice. J Cell Mol Med 16: 2777–2788, 2012. Lübben G, Forst T. Fasting intact proinsulin is a highly specific predictor
doi:10.1111/j.1582-4934.2012.01595.x. of insulin resistance in type 2 diabetes. Diabetes Care 27: 682–687, 2004.
33. Hussain TA, Mathew TC, Dashti AA, Asfar S, Al-Zaid N, Dashti HM. doi:10.2337/diacare.27.3.682.
Effect of low-calorie versus low-carbohydrate ketogenic diet in type 2 52. Pickup JC, Chusney GD, Thomas SM, Burt D. Plasma interleukin-6,
diabetes. Nutrition 28: 1016 –1021, 2012. doi:10.1016/j.nut.2012.01.016. tumour necrosis factor ␣ and blood cytokine production in type 2 diabetes.
34. Le Floch JP, Escuyer P, Baudin E, Baudon D, Perlemuter L. Blood Life Sci 67: 291–300, 2000. doi:10.1016/S0024-3205(00)00622-6.
glucose area under the curve. Methodological aspects. Diabetes Care 13: 53. Rautou P-E, Vion A-C, Amabile N, Chironi G, Simon A, Tedgui A,
172–175, 1990. doi:10.2337/diacare.13.2.172. Boulanger CM. Microparticles, vascular function, and atherothrombosis.
35. Li S, Wei J, Zhang C, Li X, Meng W, Mo X, Zhang Q, Liu Q, Ren K, Circ Res 109: 593–606, 2011. doi:10.1161/CIRCRESAHA.110.233163.
Du R, Tian H, Li J. Cell-derived microparticles in patients with type 2 54. Reyna SM, Ghosh S, Tantiwong P, Meka CS, Eagan P, Jenkinson CP,
diabetes mellitus: a systematic review and meta-analysis. Cell Physiol Cersosimo E, Defronzo RA, Coletta DK, Sriwijitkamol A, Musi N.
Biochem 39: 2439 –2450, 2016. doi:10.1159/000452512. Elevated toll-like receptor 4 expression and signaling in muscle from
36. Little JP, Myette-Côté É. Comment on Alarcon et al. pancreatic ␤-cell insulin-resistant subjects. Diabetes 57: 2595–2602, 2008. doi:10.2337/
adaptive plasticity in obesity increases insulin production but adversely db08-0038.
affects secretory function. Diabetes 2016;65:438-450. Diabetes 65: e28, 55. Reynolds AN, Mann JI, Williams S, Venn BJ. Advice to walk after
2016. doi:10.2337/db16-0492. meals is more effective for lowering postprandial glycaemia in type 2
37. Loopstra-Masters RC, Haffner SM, Lorenzo C, Wagenknecht LE, diabetes mellitus than advice that does not specify timing: a randomised
Hanley AJ. Proinsulin-to-C-peptide ratio versus proinsulin-to-insulin ra- crossover study. Diabetologia 59: 2572–2578, 2016. doi:10.1007/s00125-
tio in the prediction of incident diabetes: the Insulin Resistance Athero- 016-4085-2.
sclerosis Study (IRAS). Diabetologia 54: 3047–3054, 2011. doi:10.1007/ 56. Solinas G, Karin M. JNK1 and IKKbeta: molecular links between obesity
s00125-011-2322-2. and metabolic dysfunction. FASEB J 24: 2596 –2611, 2010. doi:10.1096/
38. Lovren F, Verma S. Evolving role of microparticles in the pathophysi- fj.09-151340. doi:10.1096/fj.09-151340.
ology of endothelial dysfunction. Clin Chem 59: 1166 –1174, 2013. 57. Sriwijitkamol A, Christ-Roberts C, Berria R, Eagan P, Pratipanawatr
doi:10.1373/clinchem.2012.199711. T, DeFronzo RA, Mandarino LJ, Musi N. Reduced skeletal muscle
39. Mesri M, Altieri DC. Leukocyte microparticles stimulate endothelial cell inhibitor of kappaB ␤ content is associated with insulin resistance in
cytokine release and tissue factor induction in a JNK1 signaling pathway. subjects with type 2 diabetes: reversal by exercise training. Diabetes 55:
J Biol Chem 274: 23111–23118, 1999. doi:10.1074/jbc.274.33.23111. 760 –767, 2006. doi:10.2337/diabetes.55.03.06.db05-0677.
40. Monnier L, Colette C, Owens DR. Glycemic variability: the third 58. Takamura T, Honda M, Sakai Y, Ando H, Shimizu A, Ota T, Sakurai
component of the dysglycemia in diabetes. Is it important? How to M, Misu H, Kurita S, Matsuzawa-Nagata N, Uchikata M, Nakamura
measure it? J Diabetes Sci Technol 2: 1094 –1100, 2008. doi:10.1177/ S, Matoba R, Tanino M, Matsubara K, Kaneko S. Gene expression
193229680800200618. profiles in peripheral blood mononuclear cells reflect the pathophysiology
41. Monnier L, Mas E, Ginet C, Michel F, Villon L, Cristol J-P, Colette of type 2 diabetes. Biochem Biophys Res Commun 361: 379 –384, 2007.
C. Activation of oxidative stress by acute glucose fluctuations compared doi:10.1016/j.bbrc.2007.07.006.
with sustained chronic hyperglycemia in patients with type 2 diabetes. 59. Tay J, Brinkworth GD, Noakes M, Keogh J, Clifton PM. Metabolic
JAMA 295: 1681–1687, 2006. doi:10.1001/jama.295.14.1681. effects of weight loss on a very-low-carbohydrate diet compared with an
42. Nagi DK, Hendra TJ, Ryle AJ, Cooper TM, Temple RC, Clark PM, isocaloric high-carbohydrate diet in abdominally obese subjects. J Am Coll
Schneider AE, Hales CN, Yudkin JS. The relationships of concentra- Cardiol 51: 59 –67, 2008. doi:10.1016/j.jacc.2007.08.050.
tions of insulin, intact proinsulin and 32-33 split proinsulin with cardio- 60. Westman EC, Yancy WS Jr, Mavropoulos JC, Marquart M, McDuffie
vascular risk factors in type 2 (non-insulin-dependent) diabetic subjects. JR. The effect of a low-carbohydrate, ketogenic diet versus a low-
Diabetologia 33: 532–537, 1990. doi:10.1007/BF00404140. glycemic index diet on glycemic control in type 2 diabetes mellitus. Nutr
43. Nordmann AJ, Nordmann A, Briel M, Keller U, Yancy WS Jr, Brehm Metab (Lond) 5: 36, 2008. doi:10.1186/1743-7075-5-36.
BJ, Bucher HC. Effects of low-carbohydrate vs low-fat diets on weight 61. Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin
loss and cardiovascular risk factors: a meta-analysis of randomized con- Cell Biol 19: 142–149, 2007. doi:10.1016/j.ceb.2007.02.001.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00240.2018 • www.ajpregu.org

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