Effect of carrot intake on glucose tolerance, microbiota, and gene expression in a type 2 diabetes mouse model

Abstract

Type 2 diabetes (T2D) pathophysiology involves insulin resistance (IR) and inadequate insulin secretion. Current T2D management includes dietary adjustments and/or oral medications such as thiazolidinediones (TZDs). Carrots have shown to contain bioactive acetylenic oxylipins that are partial agonists of the peroxisome proliferator‐activated receptor γ (Pparg) that mimic the antidiabetic effect of TZDs without any adverse effects. TZDs exert hypoglycemic effects through activation of Pparg and through the regulation of the gut microbiota (GM) producing short‐chain fatty acids (SCFAs), which impact glucose and energy homeostasis, promote intestinal gluconeogenesis, and influence insulin signaling pathways. This study investigated the metabolic effects of carrot intake in a T2D mouse model, elucidating underlying mechanisms. Mice were fed a low‐fat diet (LFD), high‐fat diet (HFD), or adjusted HFD supplemented with 10% carrot powder for 16 weeks. Oral glucose tolerance tests were conducted at weeks 0 and 16. Fecal, cecum, and colon samples, as well as tissue samples, were collected at week 16 during the autopsy. Results showed improved oral glucose tolerance in the HFD carrot group compared to HFD alone after 16 weeks. GM analysis demonstrated increased diversity and compositional changes in the cecum of mice fed HFD with carrot relative to HFD. These findings suggest the potential effect of carrots in T2D management, possibly through modulation of GM. Gene expression analysis revealed no significant alterations in adipose or muscle tissue between diet groups. Further research into carrot‐derived bioactive compounds and their mechanisms of action is warranted for developing effective dietary strategies against T2D.

Abbreviations

AUC

area under the curve

FaDOH

(3R,8S)‐falcarindiol

FaOH

(3R)‐falcarinol

Glut4

glucose transporter type 4

GM

gut microbiota

GU

glucose uptake

HFD

high‐fat diet

IR

insulin resistance

LFD

low‐fat diet

OGTT

oral glucose tolerance tests

Pparg

peroxisome proliferator‐activated receptor γ

qPCR

quantitative polymerase chain reaction

SCFAs

short‐chain fatty acids

T2D

type 2 diabetes

TNF‐α

tumor necrosis factor‐α

TZDs

thiazolidinediones

UCP

uncoupling protein

Study Highlights.

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

T2D involves IR and inadequate insulin secretion. Management typically includes dietary adjustments and medications like TZDs. Carrots contain bioactive polyacetylenes that act as partial agonists of Pparg, mimicking TZDs' antidiabetic effects without adverse outcomes.

• WHAT QUESTION DID THIS STUDY ADDRESS?

The study investigated the metabolic effects of carrot intake in a T2D mouse model and aimed to elucidate the underlying mechanisms involved.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

The study found that a HFD supplemented with 10% carrot powder improved oral glucose tolerance and increased GM diversity and composition in T2D mice. These effects suggest the potential of carrots in managing T2D through GM modulation. Gene expression analysis showed no significant changes in adipose or muscle tissues between diet groups.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

The findings of this study highlight the potential of carrots as a complementary strategy for T2D management, leveraging GM modulation without side effects. This opens avenues for further research into the bioactive compounds of carrots with potential antidiabetic effects leading to new dietary interventions and advancements in the prevention and treatment of T2D.

INTRODUCTION

In 2015, 415 million adult patients had type 2 diabetes (T2D) worldwide and this number is expected to increase by 42 million in 2040. ¹ The pathophysiological basis of T2D is insulin resistance (IR) and insufficient insulin secretion. ² , ³ IR is observed in many prediabetic conditions and includes reduced insulin action in insulin‐sensitive tissues leading to hyperinsulinemia. Eventually, the pancreatic β‐cells are unable to produce sufficient insulin to maintain glucose homeostasis resulting in hyperglycemia and thereby T2D. ⁴ IR is often associated with high circulating levels of triglycerides, gut microbiota (GM) imbalance and the release of certain hormones, cytokines, and fatty acids from adipose tissue leading to systemic low‐grade inflammation and ectopic lipid deposition in muscles and liver. ⁵ , ⁶

T2D is currently treated by diet adjustments, increased physical activity, and glucose‐lowering drugs that exert their hypoglycemic effects through different mechanisms of action, which include regulation of GM. ⁷ , ⁸ , ⁹ , ¹⁰ The GM produces a wide range of metabolites, including short‐chain fatty acids (SCFAs) that are involved in the production of inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) that are important in the regulation of low‐grade inflammation in the gut. ¹¹ , ¹² TNF‐α interferes with the signaling of insulin and may induce IR in adipose and muscle tissues. ¹³ Increased levels of TNF‐α also lead to a decrease in the expression of the glucose transporter type 4 (Glut4) gene in muscle and adipose tissues affecting the entrance of glucose into the cell for energy production. ¹⁴ Furthermore, SCFAs act as ligands of the peroxisome proliferator‐activated receptor γ (Pparg) that is mainly expressed in brown and white adipose tissue, the colon, and immune cells and is pivotal for the modulation of T2D. ¹⁵ , ¹⁶ Activation of Pparg leads to the alteration of the transcription of several target genes involved in carbohydrate and lipid metabolism resulting in facilitation of glucose uptake (GU) and deposition of lipids in adipose, muscle, and liver tissue, decrease in free fatty acid levels, regulation of adipocytokine secretion and amelioration of IR. ¹⁵ , ¹⁶ , ¹⁷ Further, the activation of Pparg depends on whether agonists act as full or partial agonists. Partial agonists are compounds with diminished agonist efficacy that maintain the insulin‐sensitizing effect but without inducing the same magnitude of side effects as full agonists such as the TDZs. ¹⁶ , ¹⁸ Molecular docking studies have revealed that full and partial agonists have different binding modes in the ligand‐binding domain of Pparg, ¹⁶ , ¹⁹ , ²⁰ which may explain why full and partial agonists recruit different sets of co‐activators, and exhibit different pharmacological activities. ¹⁹ , ²¹ In addition, Pparg agonists strongly stimulate the formation of mitochondrial uncoupling protein (UCP)1, which is primarily found in brown adipose tissue, and plays a crucial role in thermogenesis and energy expenditure. ²² However, mice deficient in UCP1 do not develop obesity despite being fed a high calory diet indicating that inactivation of this major thermogenic mechanism may activate alternative UCP1 independent mechanisms required to regulate body temperature that are more costly energetically resulting in an increased energy expenditure. ²³

Terrestrial plants have traditionally been used in the treatment of various diseases including diabetes and could be important in the prevention and treatment of T2D. ²⁴ , ²⁵ , ²⁶ , ²⁷ In this context carrots are promising because it has been shown that a lipophilic extract of this vegetable stimulates insulin‐dependent GU in 3T3‐L1 adipocytes in a dose‐dependent manner. The bioactive constituents responsible for this effect were identified as the acetylenic oxylipins (3R)‐falcarinol (FaOH) and (3R,8S)‐falcarindiol (FaDOH) exhibiting the characteristics of Pparg partial agonists. ²⁶ , ²⁸ , ²⁹ , ³⁰ FaOH and FaDOH are besides carrots found in many other Apiaceae vegetables such as parsley, celery, and parsnip, ³¹ which are expected to have similar antidiabetic properties as carrots. In addition, FaOH and FaDOH are known to have anti‐inflammatory, anticancer, and antibacterial effects in vitro and in vivo. ³¹ , ³² , ³³ , ³⁴ Regarding the latter, FaOH and FaDOH have shown to have a beneficial influence on GM composition in a rat model of colorectal cancer. ³⁵ Furthermore, FaOH and FaDOH have in the colorectal cancer rat model shown anticancer effect in a dose‐dependent manner and that their antineoplastic effect is most likely due to the inhibition of pro‐inflammatory markers in the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling pathway such as TNFα, interleukin 6, and cyclooxygenase‐2. ³² This is in accordance with other in vivo studies demonstrating that the chemopreventive effects of FaOH and FaDOH are linked to the formation of anti‐inflammatory and cytoprotective phase 2 enzymes via activation of the Kelch‐like ECH‐associated protein 1‐nuclear factor erythroid‐derived 2‐related factor 2 (Keap1–Nrf2) signaling pathway, inhibition of pro‐inflammatory peptides and proteins, induction of endoplasmic reticulum stress and/or activation of Pparg. ³³ Besides representing a potential therapeutic target of T2D, Pparg also plays a central role in growth, differentiation, and apoptosis of cancer cells. ³⁶ The potential antidiabetic effects of carrots and their bioactive constituents, however, remain to be demonstrated in vivo in animal and/or human trials.

The objectives of this study were to investigate the effect of intake of carrots in a mouse model of T2D on oral glucose tolerance tests (OGTT) and to demonstrate the antidiabetic effects of vegetables containing bioactive polyacetylenes as well as to investigate possible mechanisms of action by studying the gut microbiome and expression of selected target genes in brown adipose tissue and muscle tissue.

MATERIALS AND METHODS

Diets

Diets were obtained from Research Diets, New Brunswick, NJ 08901 USA (Table S1). Low‐fat diet (LFD) D12450J contained 10 kcal% fat diet (Sucrose matched to D12492), high‐fat diet (HFD) powder D12492 contained 60 kcal% fat diet, and HFD premix (HFD carrot) D22031408px contained 60 kcal% Fat Diet Premix ready for the addition of w/w 10% dried carrot powder. The macronutrients, energy, and fiber content were adjusted between the HFD and HFD premix. By adding 10% carrot powder to the HFD premix, the two diets will have nearly identical compositions. Dried carrot powder was produced from the carrot cultivar Daucus carota ‘Night Bird’ grown organically at Danroots A/S, Bjerringbro, Denmark. Tops and bottoms were removed from fresh, washed carrots, which were then shredded and freeze‐dried (European Freeze‐Dry, Kirke Hyllinge, Denmark). The carrot powder was packed in sealed aluminum foil pouches and stored at−30°C until use. The content of FaOH and FaDOH in the carrot powder was determined to be 60 mg and 20 mg/100 g Dry Weight, respectively (Lars Porskjær Christensen L.P.C.) by LC‐DAD‐MS analysis as described previously. ³⁴

Animal housing and study design

Fifty‐four C57BL/6NTac male mice were obtained from Taconic Biosciences A/S, (Lille Skensved, Denmark). At arrival, the mice were 5 weeks of age and allowed acclimatization for 1 week. They were housed in individually ventilated cages (Tecniplast, Buguggiate, Italy) with 9 mice per cage at standard laboratory conditions, including a 12 h light/dark cycle and free access to demineralized water and food at the Biomedical Laboratory, University of Southern Denmark, Odense, Denmark. The room was controlled at 55% relative humidity at 22 ± 3°C, and it was health monitored according to FELASA guidelines. ³⁷ Entry into the animal facility went via changing rooms where protective clothing, disposable hats, and dedicated shoes were provided. Gloves were obligatory when touching or handling animals.

The mice were divided into three groups of 18 mice: LFD (as lean control), HFD, and HFD +10% (w/w) carrot powder. Only male C57BL/6NTac mice were included since female mice are protected against dietary‐induced obesity (DIO). ³⁸ For 16 weeks, mice were fed ad libitum adding freshly prepared diet to the cages twice a week.

OGTT

For OGTT, mice were fasted for 11 h from 9 p.m. Blood was obtained from a tail cut and was assessed for baseline blood glucose levels using a glucometer (Contour next 9651E). The mice then received glucose solution (μL) = 10 × body weight (g) (Glucose solution, 45% in water, sterile‐filtered, BioXtra, Sigma, # G8769‐100ML) delivered by oral gavage. At 0, 15, 30, 60, and 120 min after the administration of glucose, dried blood and tissue were quickly removed from the tail wound and blood was collected again to measure the blood glucose concentration. This procedure was performed at week 1 and week 16 of experimental diet feeding.

Sample collection

After 16 weeks the mice were sacrificed by cervical dislocation. Tissue samples from the femur muscle and brown fat from the neck were collected from each mouse. Finally, feces samples from the colon and cecum were collected from each mouse. All samples were stored at −80°C until analysis.

GM analysis

The DNA was extracted from fecal pellets using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany, Cat. #47016), following the manufacturer's instructions. The isolated DNA was stored at −80°C until DNA library construction. Gut microbial composition was examined by near full‐length 16S rRNA gene amplicon sequencing using GridION (Oxford Nanopore Technologies, Oxford, UK) as previously described. ³⁵ Briefly, the sequencing library was constructed using a custom two‐step polymerase chain reaction (PCR) for amplification of the 16S rRNA gene and samples barcoding. PCR 1 reactions containing 12 μL of PCRBIO Ultra Mix (PCR Biosystems Ltd., London, UK), 6 μL of nuclease‐free water, 2 μL of primer mix (5 μM), and 5 μL of genomic DNA (∼1 ng/μL) bringing the volume to a total of 25 μL were run on a SureCycler 8800 Thermal Cycler (Agilent Technologies, Santa Clara, CA, USA). The PCR program consisted of initial denaturation at 95°C for 5 min and two cycles of denaturation at 95°C for 20 s, touch‐down at 48°C for 30 s, annealing at 65°C for 10 s, and extension at 72°C for 45 s, followed by final elongation at 72°C for 4 min. After PCR 1, the PCR products were cleaned using AMPure XP binding beads (Beckman Coulter Genomic, Indianapolis, IN, USA). Following cleaning, a PCR step was performed to barcode PCR amplicons. PCR reactions containing 12 μL of PCRBIO Ultra Mix (PCR Biosystems Ltd., London, UK), 10 μL of clean PCR 1 product, and 2 μL of barcoding primers (10 μM) bringing the volume to a total of 25 μL were run on a SureCycler 8800 Thermal Cycler (Agilent Technologies, Santa Clara, CA, USA). The PCR program was as follows: denaturation at 95°C for 2 min, 33 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 40 s, followed by final extension at 72°C for 4 min. After gel electrophoresis, 5 μL of each PCR 2 products were pooled together and subsequently cleaned with Binding Beads (AMPure XP, Indianapolis, IN, USA). The cleaned and pooled PCR products were eluted in 40 μL of nuclease‐free water. The pooled and barcoded amplicons were completed for library preparation for GridION sequencing using the 1D genomic DNA by ligation protocol (SQK‐LSK110) as previously described in Arildsen et al. ³⁹ Approximate 0.2 μg of amplicons were used for the initial step of end‐prep, and 40 ng of prepared amplicon library was loaded on a R9.4.1. flow cell. The data generated using GridION was collected using MinKNOW software v22.10.7 (Oxford Nanopore Technologies). The Guppy v6.2.8 base calling toolkit was used to base call raw FAST5 to FASTQ (Oxford Nanopore Technologies). The abundance table was generated from raw FASTQ files using the Long Amplicon Consensus Analysis pipeline (LACA, GitHub: https://github.com/yanhui09/laca). Taxonomy assignment of quality‐corrected reads was performed against the SILVA database. ⁴⁰

Gene expression studies

Brown adipose tissue (neck) and femoral muscle tissue were collected for RNA analysis and stored at −80°C. Homogenization, RNA isolation with RNeasy® Midi Kit (Qiagen, Hilden, Germany, Cat. #74104), and cDNA synthesis using High‐Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA) were performed as described previously. Quantitative PCR (qPCR) of the genes Glut4 (Mm00436615_m1), Tnf (Mm00443258_m1), Pparg (Mm00440940_m1), and Ucp1 (Mm01244861_m1) were done using TaqMan gene expression assays (Applied Biosystems) on a Bio‐Rad C1000 Touch CFX96 Real‐Time System Thermal Cycler (Bio‐rad, Hercules, CA). YWHAZ (brown adipose tissue and muscle) and HPRT (brown adipose tissue) were used as reference gene. The amplification data were analyzed using the accompanying CFX Maestro software (Bio‐rad) to obtain threshold cycle (C _t) values. GenEx 6 (MultiD Analyses AB, Gothenburg, Sweden) was used for qPCR data transformation. The Genorm and Normfinder methods in GenEx were used to validate the stability of the selected reference genes and the C _t values were normalized to the reference gene. For each gene, normalized expression levels were set relative to the sample with the lowest expression to establish relative quantities and were log2 transformed before statistical testing.

Statistical analysis

For oral glucose areas under the curve (AUCs) were calculated (GraphPad Prism 10, Boston, USA). Oral glucose values during the OGTT were normalized to the start value at week 0 and Δ‐AUC‐values for oral glucose tolerance were calculated by subtracting values at 0 weeks from values at 16 weeks. Bartlett's test was used to test for equal variances (Minitab 21.2, Coventry, UK). As none of the distributions had equal variances all data were ranked within the groups and tested for normality by Anderson‐Darling test (Minitab) before testing by ANOVA with Tukey's posthoc comparison (Minitab). For weight curves, F‐test was used to test for equal variances, and d'Agostino‐Pearssons was used to test for normality (Prism). Hereafter, a repeated measures ANOVA was used to compare the growth curves followed by Tukey's multiple comparisons test. Due to missing data a mixed effects logistic regression model (www.stata.com) was used for statistical analysis of effect of HFD versus HFD carrot at specific time points (0, 15, 30, 60, and 120 min) and at specific lapse of time (0–15, 15–30, 30–60, and 60–120 min). Gene expression analysis was performed using R statistical software (Version 4.3.1) and was assessed using the Shapiro–Wilk test. Normality was considered met if the p‐value of the Shapiro–Wilk test was greater than the chosen significance level of α = 0.05. The equality of variances among different groups within each gene was assessed using Levene's test. Equality of variances was considered met if the p‐value of the Levene's test was greater than the chosen significance level of α = 0.05. For genes where normality was met in all three feeding groups, a one‐way analysis of variance (ANOVA) test was conducted. For genes where normality was not met in all three feeding groups, indicating non‐normal distribution, a non‐parametric Kruskal–Wallis test was additionally conducted. GM data analysis was also conducted in R statistical software. Alpha diversity was evaluated using the Shannon Diversity and Chao1 index, and statistical significance was determined using ANOVA tests. Additionally, beta diversity was assessed using distance‐based redundancy analysis (db‐RDA) with the Bray‐Curtis Distance after model fitting. ⁴¹

Ethics statement

The study was approved by the Danish Competent Authority, The Animal Experimentation Inspectorate, under the Ministry of Food, Fisheries and Agriculture of Denmark, and with license ID 2022‐15‐0201‐01177. Procedures were carried out in accordance with the Directive 2010/63/EU and the Danish law LBK Nr 726 of 09/091993.

RESULTS

Effect of diets on body weight and OGTT

In an ANOVA analysis of the ranked values of AUC, no differences in the gain of body weight of HFD group and HFD + 10% carrot group were demonstrated. However, the LFD group had significantly lower body weight compared to the 2 other groups (Figure 1).

FIGURE 1.

Growth curves (Mean ± Range) of male C57BL/6NTac mice fed either a HFD with (red) or without (green) carrot powder or LFD (blue). repeated measures ANOVA: over all p = 0.0001; high‐fat carrot versus high‐fat p = 0.6116; high‐fat carrot versus low‐fat p < 0.0012; high‐fat versus low‐fat p = 0.0001.

At the start of the study (week 0), there was no difference in AUC glucose between the three different diet groups. After 16 weeks of treatment with different diets, ANOVA on ranked Δ‐values of AUC glucose showed a tendency for a difference between the three groups: p = 0.053 with the following differences between groups based on post hoc analysis; high‐fat carrot versus high‐fat p = 0.332; high‐fat carrot versus low‐fat p = 0.380; high‐fat versus low‐fat p = 0.044 (Figure 2). However, analyzing specific time points (0, 15, 30, 60, and 120 min) after 16 weeks demonstrated that blood glucose 30 min after glucose administration was significantly lower (p = 0.0136) in mice fed with HFD‐containing carrots compared with HFD without carrots (Figure 2). Furthermore, analyzing specific lapse of time (0–15, 15–30, 30–60, and 60–120 min) revealed a higher increase in blood glucose from 0 to 15 min in mice fed with HFD compared with mice fed with HFD + 10% carrots (p = 0.0063) and a larger decrease in blood glucose from 60 to 120 min in mice fed with HFD compared with mice fed with HFD + 10% carrots (p = 0.0017).

FIGURE 2.

Oral glucose tolerance tests (Mean ± Standard error of mean) at 0 weeks and after 16 weeks of feeding either a HFD with (red) or without (green) carrot powder or a LFD (blue) to male C57BL/6NTac mice.

Microbiota

Cecal microbiome diversity expressed as both Chao1 (diversity) and Shannon diversity (combined diversity and evenness) index increases significantly with the inclusion of 10% carrot powder in the feed compared to both LFD and HFD (Figure 3a). Also, the overall cecal microbiome composition is strongly affected by the inclusion of 10% carrot powder in the feed as seen in Figure 3b. At the genus level inclusion of 10% carrot powder in the HFD led to a higher relative abundance of SCFA producers like Anerostipes, Ruminococcus, Eubacterium and family Prevotellaceae members, while the HFD led to an increased relative abundance of especially microaerophilic genera like Enterococcus, Ligilactobacillus, Lactococcus and Helicobacter relative to HFD added carrot powder (Figure 3c,d).

FIGURE 3.

Supplementation of 10% w/w carrot powder to a HFD influences mice cecal microbiome composition (a) Gut microbiome diversity expressed as both Chao1 and Shannon diversity index increases with 16 weeks of inclusion of 10% carrot powder in the feed. (b) Overall gut microbiome composition influenced by the inclusion of 10% carrot powder in the feed as evidenced by Bray–Curtis dissimilarity index distance‐based redundancy analysis (db‐RDA) of cecal samples. (c) Cecum microbiome composition across the three diet groups—LFD, HFD, and HFD with 10% carrot powder. (d) Volcano Plot illustrating genera differential abundance in mice cecum samples when comparing mice on a HFD with 10% carrot powder in the diet (left) to those on HFD (right side of the plot). Each point on the plot represents a distinct genus, with color‐coded indications based on their respective phyla. The x‐axis represents the log2FoldChange, signifying the magnitude of abundance changes, while the y‐axis corresponds to the negative logarithm (base 10) of the adjusted p‐values, emphasizing the statistical significance of observed alterations. Non‐significant genera (p > 0.05) are shown in green.

Gene expressions

There were no significant differences in the expression of the genes Glut4, Tnf, Ucp1, and Pparg in either brown adipose or muscle tissue (Figure 4).

FIGURE 4.

Expression of the genes Glut4 (a, e), Tnf (b, f), Pparg (c, g), and Ucp1 (d, h) in adipose (a–d) and muscle tissue (e–h) in male C57BL/6NTac mice fed either a HFD group (green), HFD carrot group (red) or an LFD group (blue). ANOVA: No significant differences.

DISCUSSION

The positive effects of carrots on both basal and insulin‐stimulated GU in 3 T3‐L1 adipocytes, and myotube cell cultures are most likely due to the content of FaOH and FaDOH. ²⁸ FaOH and especially FaDOH exhibit the characteristics of partial Pparg agonists as demonstrated by a Pparg transactivation assay and molecular docking studies. ²⁸ , ²⁹ Furthermore, the effect of FaOH and FaDOH on the gene expression of Pparg in 3T3‐L1 adipocytes showed no upregulation of this and other key markers of adipogenesis in accordance with FaOH and FaDOH being weak activators of Pparg. ²⁸ Although in vitro studies indicate a clear antidiabetic effect of carrots, this effect has not been demonstrated in vivo. In the present study, we, therefore, focused on investigating the potential antidiabetic effects of carrots in vivo and possible underlying mechanisms. We used the mouse strain C57BL/6NTac that is a well‐characterized animal model for T2D resulting in overweight and T2D characteristics in mice fed with HFD with many calories within 16 weeks. ⁴² , ⁴³

Inclusion of carrot to the HFD did not result in a significant difference in the body weight of mice compared with mice fed only HFD. However, a difference in the body weight was observed in mice fed with LFD compared to mice fed with HFD or HFD including 10% w/w carrots. This indicates that carrots did not result in weight reduction between the HFD groups even though the HFD carrot group was adjusted to match HFD in terms of protein, carbohydrate, fat, and fiber content when 10% carrots were mixed in the feed. Therefore, no difference in the body weight between both HFD groups was expected as the only difference between the diets were micronutritions such as FaOH and FaDOH.

Based on previous in vitro investigations on the potential antidiabetic effects of carrots, it was expected that including carrots in the diet of the animal model of T2D would affect the regulation of blood glucose after an oral boost of glucose. The overall AUC analysis of the OGTT in the HFD and HFD carrot groups indicated that a daily intake of carrots for 16 weeks resulted in better regulation of the blood glucose level in mice after a boost of glucose intake. OGTT blood samples taken at specific time stamps revealed that 30 min after glucose boosts a significant (p = 0.0136) difference between the HFD and the HFD carrot group was shown, and this could be interpreted as a stabilizing effect of carrot intake on the blood glucose level. Also, OGTT samples taken before and 15 min after the oral glucose boost demonstrate a significant difference in the increase of blood glucose in the HFD group compared to the HFD carrot group. In a similar way, the blood glucose in the OGTT decreased significantly in samples taken at 60 and 120 min after the oral glucose boost. This suggests that intake of carrots regulates GU more efficiently in the mice in accordance with previous in vitro studies of carrot extract, FaOH and FaDOH. ²⁷ , ²⁸ Gene expression studies on early stages of adipocyte differentiation of 3T3‐L1 adipocytes have shown that FaOH inhibits adipocyte differentiation whereas this is not the case for FaDOH. ²⁸ The results from this in vitro study indicate that FaOH and FaDOH may have distinct mechanisms of action in adipocytes, although it is not possible to conclude whether the effect of FaOH and FaDOH on insulin sensitivity is linked to their different affinity to Pparg or different expression of genes involved in adipose metabolism. ²⁸ In the present study, we therefore investigated the expression of genes of other potential biomarkers involved in IR and GU such as Glut4, Tnf, and Ucp1 as well as Pparg. In accordance with the previous in vitro study no effect on the expression of Pparg was observed in this study by including carrots in the diet probably because FaOH and FaDOH act as partial Pparg agonists. ²⁸ However, the insignificant results on the expression of Glut4, Tnf and Ucp1 in neither brown adipose nor muscle tissue could indicate that the enhanced insulin‐stimulated GU observed in vitro in adipocytes and muscle cells are not linked to these biomarkers or at least they play a minor role in vivo. Consequently, the gene expression study did not explain the better regulation of the level of blood glucose in the used mice model after a boost of glucose intake of the HFD carrot group compared to the HFD group.

The demonstrated anticancer effect of FaOH and FaDOH in a rat model for colon cancer may be linked to their effect on the GM composition as GM dysbiosis has an impact on the development of cancer. ³⁵ Similarly, the intestinal microbiota may contribute to the development of metabolic diseases like T2D. ⁴⁴ The microbiome affects inflammation, interacts with dietary constituents, affects gut permeability, glucose and lipid metabolism, insulin sensitivity, and overall energy homeostasis in humans and animal models. In our animal model of T2D, we demonstrated that adding 10% carrot powder to the HFD improved the regulation of the blood glucose level. At the same time, we demonstrated that mice fed with HFD carrot for 16 weeks resulted in changes in the microbiota being more diverse in the cecum than mice fed with HFD. Further, mice receiving the HFD diet had significantly higher relative abundance of microaerophilic/facultatively anaerobic bacteria like Enterococcus, Ligilactobacillus, Lactococcus, and Helicobacter (Figure 3c,d) compared to mice receiving the HFD carrot diet, indicating that inclusion of carrot powder in the HFD alleviates oxidative stress following prolonged HFD feeding, ⁴⁵ which has been linked to T2D development. ⁴⁶ However, whether these changes in the GM composition affect the development of T2D directly, for example, via metabolites formed, or if it merely represents the negative influence of oxidative stress on obligate anaerobes (e.g., SCFA producers) thereby affecting the development of T2D indirectly remains to be elucidated. ⁴⁷ Furthermore, a change in microbiota may affect key players of the immune system and inflammation in the mice. Inclusion of carrot powder into the HFD (Figure 3d) indeed increased the relative abundance of various SCFA‐producing bacteria such as Anerostipes (butyrate) and Ruminococcus (butyrate) relative to HFD‐fed mice. The most significant increase in mice fed HFD plus carrot powder was observed for a member of the family Anaerovoracaceae (Family VIII member, UCG‐001, Figure 3d), which is quite widespread in the intestine producing acetate and butyrate via amino acid fermentation. ⁴⁸ Butyrate acts as energy for the intestinal epithelium and reduces permeability, which is positive, as T2D patients will normally have a tendency towards leaky gut barrier that triggers inflammatory responses that are characteristic of diabetes. ⁴⁹ Furthermore, acetate, propionate and butyrate are all involved in enteroendocrine signaling via, for example, GLP‐1 stimulating insulin sensitivity. ⁵⁰

LIMITATIONS

The present study has some limitations that include concerns about bioavailability, the need for dose–response studies, applicability to humans, reliance on unpublished data, and limitations in sample size. There are some bioavailability concerns in this study using freeze‐dried carrot powder instead of isolated and purified FaOH and FaDOH. This could potentially decrease the bioavailability of these bioactive compounds in vivo, affecting their uptake and thereby the observed outcomes. This limitation suggests that the actual impact of FaOH and FaDOH on the study results might be underestimated. Also, a dose–response study is needed. To ascertain the optimal concentration required to affect OGTT, a dose–response study with varying concentrations of purified FaOH and FaDOH with a larger sample size is warranted. Addressing these limitations in future research could further elucidate the role of these compounds and carrots in the management of T2D and improve the translation of findings to clinical practice.

CONCLUSIONS

This study provides valuable insights into the mechanisms underlying the beneficial effects of carrot intake on glucose regulation. Overall, our findings suggest that incorporating carrots into the diet may promote a “healthier” microbiome and contribute to improved blood glucose regulation in individuals with T2D.

AUTHOR CONTRIBUTIONS

All authors wrote the manuscript. M.K.‐L., L.P.C., S.H.K., D.S.N., A.K.H., and K.H. designed the research and M.K.‐L., and S.M. performed the research and analyzed the data.

FUNDING INFORMATION

This study was supported by the Odense University Hospital Fund for Free Research (122‐A5125). DanRoots A/S supplied carrots at no charge.

CONFLICT OF INTEREST STATEMENT

The authors declared no competing interests for this work.

Supporting information

Table S1

Kobaek‐Larsen M, Maschek S, Kolstrup SH, et al. Effect of carrot intake on glucose tolerance, microbiota, and gene expression in a type 2 diabetes mouse model. Clin Transl Sci. 2024;17:e70090. doi: 10.1111/cts.70090

DATA AVAILABILITY STATEMENT

The datasets used and/or analyzed during the current study are available from the co‐author M.K.‐L. upon reasonable request.