IgM-associated gut bacteria in obesity and type 2 diabetes in C57BL/6 mice and humans.

AIMS/HYPOTHESIS: IgM is the primary antibody produced by B cells and we hypothesise that IgM antibodies to gut microbiota may play a role in immunometabolism in obesity and type 2 diabetes. To test our hypothesis, we used B6 mice deficient in activation-induced cytidine deaminase (Aid-/- [also known as Aicda-/-]) which secrete only IgM antibodies, and human faecal samples. METHODS: We studied the immunometabolic effects and gut microbial changes in high-fat-diet-induced obesity (HFDIO) in Aid-/- B6 mice compared with wild-type mice. To determine similarities between mice and humans, human stool samples were collected from children and adolescents who were obese with normal glucose tolerance (NGT), obese with glucose intolerance (IGT), or obese and newly diagnosed with type 2 diabetes, for faecal microbiota transplant (FMT) into germ-free (GF) B6 mice and we assessed IgM-bound bacteria and immune responses. RESULTS: Compared with wild-type mice, Aid-/- B6 mice developed exacerbated HFDIO due to abundant levels of IgM. FMT from Aid-/- B6 to GF B6 mice promoted greater weight gain in recipient mice compared with FMT using wild-type mouse faecal microbiota. Obese youth with type 2 diabetes had more IgM-bound gut bacteria. Using the stools from the obese youth with type 2 diabetes for FMT to GF B6 mice, we observed that the gut microbiota promoted body weight gain and impaired glucose tolerance in the recipient GF B6 mice. Importantly, some clinical features of these obese young individuals were mirrored in the GF B6 mice following FMT. CONCLUSIONS/INTERPRETATION: Our results suggest that IgM-bound gut microbiota may play an important role in the immuno-pathogenesis of obesity and type 2 diabetes, and provide a novel link between IgM in obesity and type 2 diabetes in both mice and humans. DATA AVAILABILITY: The 16s rRNA sequencing datasets supporting the current study have been deposited in the NCBI SRA public repository ( https://www.ncbi.nlm.nih.gov/sra ; accession no. SAMN18796639).

A low n-6 to n-3 polyunsaturated fatty acid ratio diet improves hyperinsulinaemia by restoring insulin clearance in obese youth.

AIM: To examine the determinants and metabolic impact of the reduction in fasting and postload insulin levels after a low n-6 to n-3 polyunsaturated fatty acid (PUFA) ratio diet in obese youth. MATERIALS AND METHODS: Insulin secretion and clearance were assessed by measuring and modelling plasma insulin and C-peptide in 17 obese youth who underwent a nine-point, 180-minute oral glucose tolerance test (OGTT) before and after a 12-week, eucaloric low n-6:n-3 polyunsaturated fatty acid (PUFA) ratio diet. Hepatic fat content was assessed by repeated abdominal magnetic resonance imaging. RESULTS: Insulin clearance at fasting and during the OGTT was significantly increased after the diet, while body weight, glucose levels, absolute and glucose-dependent insulin secretion, and model-derived variables of ß-cell function were not affected. Dietary-induced changes in insulin clearance positively correlated with changes in whole-body insulin sensitivity and ß-cell glucose sensitivity, but not with changes in hepatic fat. Subjects with greater increases in insulin clearance showed a worse metabolic profile at enrolment, characterized by impaired insulin clearance, ß-cell glucose sensitivity, and glucose tolerance, and benefitted the most from the diet, achieving greater improvements in glucose-stimulated hyperinsulinaemia, insulin resistance, and ß-cell function. CONCLUSIONS: We showed that a 12-week low n-6:n-3 PUFA ratio diet improves hyperinsulinaemia by increasing fasting and postload insulin clearance in obese youth, independently of weight loss, glucose concentrations, and insulin secretion.

Colonic Fermentation and Acetate Production in Youth with and without Obesity.

BACKGROUND: In the last few years, there has been a growing interest in the role of gut microbiota in the development of obesity and its complications. OBJECTIVES: In this study, we tested the following hypotheses: 1) lean youth and youth with obesity experience a different capability of their gut microbiota to ferment carbohydrates and produce acetate; and 2) colonic acetate may serve as a substrate for hepatic de novo lipogenesis (DNL). METHODS: Nineteen lean youth [mean ± SE BMI (in kg/m2): 21.8 ± 0.521] and 19 youth with obesity (BMI: 35.7 ± 1.66), ages 15-21 y, frequency-matched by age and sex, underwent a fasting 10-h sodium [d3]-acetate intravenous infusion to determine the rate of appearance of acetate (Raacet) into the peripheral circulation before and after an oral dose of 20 g of lactulose. Pre- and post-lactulose Raacet values were determined at a quasi-steady state and changes between groups were compared using a quantile regression model. Acetate-derived hepatic DNL was measured in 11 subjects (6 youth with obesity) and its association with Raacet was assessed using Spearman correlation. RESULTS: Mean ± SE Raacet was not different before lactulose ingestion between the 2 groups (7.69 ± 1.02 µmol · kg-1 · min-1 in lean youth and 7.40 ± 1.73 µmol · kg-1 · min-1 in youth with obesity, P = 0.343). The increase in mean ± SE Raacet after lactulose ingestion was greater in lean youth than in youth with obesity (14.7 ± 2.33 µmol · kg-1 · min-1 and 9.29 ± 1.44 µmol · kg-1 · min-1, respectively, P = 0.001). DNL correlated with Raacet, calculated as changes from the pre- to the post-lactulose steady state (ρ = 0.621; P = 0.046). CONCLUSIONS: These data suggest that youth with obesity ferment lactulose to a lesser degree than youth without obesity and that colonic acetate serves as a substrate for hepatic DNL.This trial was registered at clinicaltrials.gov as NCT03454828.

A Low ω-6 to ω-3 PUFA Ratio (n-6:n-3 PUFA) Diet to Treat Fatty Liver Disease in Obese Youth.

BACKGROUND: Recent literature suggests that the Western diet's imbalance between high ω-6 (n-6) and low ω-3 (n-3) PUFA intake contributes to fatty liver disease in obese youth. OBJECTIVES: We tested whether 12 wk of a low n-6:n-3 PUFA ratio (4:1) normocaloric diet mitigates fatty liver and whether the patatin-like containing domain phospholipase 3 (PNPLA3) rs738409 variant affects the response. METHODS: In a single-arm unblinded study, obese youth 9-19 y of age with nonalcoholic fatty liver disease were treated with a normocaloric low n-6:n-3 PUFA ratio diet for 12 wk. The primary outcome was change in hepatic fat fraction (HFF%), measured by abdominal MRI. Metabolic parameters included alanine aminotransferase (ALT), lipids, measures of insulin sensitivity, and plasma oxidized linoleic acid metabolites (OXLAMs). Outcomes were also analyzed by PNPLA3 rs738409 genotype. Wilcoxon's signed rank test, the Mann-Whitney U test, and covariance pattern modeling were used. RESULTS: Twenty obese adolescents (median age: 13.3 y; IQR: 10.5-16.4 y) were enrolled and 17 completed the study. After 12 wk of dietary intervention, HFF% decreased by 25.8% (P = 0.009) despite stable weight. We observed a 34.4% reduction in ALT (P = 0.001), 21.9% reduction in triglycerides (P = 0.046), 3.28% reduction in LDL cholesterol (P = 0.071), and a 26.3% improvement in whole body insulin sensitivity (P = 0.032). The OXLAMs 9-hydroxy-octadecandienoic acid (9-HODE) (P = 0.011), 13-HODE (P = 0.007), and 9-oxo-octadecadienoic acid (9-oxoODE) (P = 0.024) decreased after 12 wk. HFF% declined in both the not-at-risk (CC/CG) and at-risk (GG) PNPLA3 rs738409 genotype groups, with significant (P = 0.016) HFF% reduction in the GG group. Changes in 9-HODE (P = 0.023), 9-oxoODE (P = 0.009), and 13-oxoODE (P = 0.003) differed between the 2 genotype groups over time. CONCLUSIONS: These data suggest that, independently of weight loss, a low n-6:n-3 PUFA diet ameliorates the metabolic phenotype of adolescents with fatty liver disease and that response to this diet is modulated by the PNPLA3 rs738409 genotype.This trial was registered at clinicaltrials.gov as NCT01556113.

Intrahepatic fat, irrespective of ethnicity, is associated with reduced endogenous insulin clearance and hepatic insulin resistance in obese youths: A cross-sectional and longitudinal study from the Yale Pediatric NAFLD cohort.

AIM: To evaluate whether intrahepatic fat accumulation contributes to impaired insulin clearance and hepatic insulin resistance across different ethnic groups. METHODS: The intrahepatic fat content (HFF%) was quantified by magnetic resonance imaging in a multi-ethnic cohort of 632 obese youths aged 7-18 years at baseline and after a 2-year follow-up. Insulin secretion rate (ISR), endogenous insulin clearance (EIC) and hepatic insulin resistance index (HIRI) were estimated by modelling glucose, insulin and C-peptide data during 3-hour, 9-point oral glucose tolerance tests. RESULTS: African American youths exhibited the lowest HFF% and a prevalence of non-alcoholic fatty liver disease (NAFLD) less than half of that shown by Caucasians and Hispanics. Furthermore, African Americans had lower EIC and glucose-stimulated ISR, despite similar HIRI and plasma insulin levels, compared with Caucasians and Hispanics. EIC and HIRI were markedly reduced in individuals with NAFLD and declined across group-specific HFF% tertiles in all ethnic groups. Consistently, the HFF% correlated with EIC and HIRI, irrespective of the ethnic background, after adjustment for age, sex, ethnicity, adiposity, waist-hip ratio, pubertal status and plasma glucose levels. An increased HFF% at follow-up was associated with decreased EIC and increased HIRI across all groups. CONCLUSIONS: Intrahepatic lipid accumulation is associated with reduced insulin clearance and hepatic insulin sensitivity in obese youths, irrespective of their ethnic background.

Comparison of Metabolic Response to Colonic Fermentation in Lean Youth vs Youth With Obesity.

Importance: Pediatric obesity is a growing health care burden. Understanding how the metabolic phenotype of youth with obesity may modify the effect of intestinal fermentation on human metabolism is key to designing early intervention. Objective: To assess whether adiposity and insulin resistance in youth may be associated with colonic fermentation of dietary fibers and its production of acetate, gut-derived hormone secretion, and adipose tissue lipolysis. Design, Setting, and Participants: Cross-sectional study of youths aged 15 to 22 years with body mass index in the 25th to 75th percentile or higher than the 85th percentile for age and sex throughout the New Haven County community in Connecticut. Recruitment, studies, and data collection occurred from June 2018 to September 2021. Youths were assigned to a lean, obese insulin sensitive (OIS), or obese insulin resistant (OIR) group. Data were analyzed from April 2022 to September 2022. Exposure: Participants consumed 20 g of lactulose during a continuous 10-hour sodium d3-acetate intravenous infusion to measure the rate of appearance of acetate in plasma. Main Outcomes and Measures: Plasma was obtained hourly to measure acetate turnover, peptide tyrosine tyrosine (PYY), ghrelin, active glucagon-like peptide 1 (GLP-1), and free fatty acids (FFA). Results: A total of 44 youths participated in the study (median [IQR] age, 17.5 [16.0-19.3] years; 25 [56.8%] were female; 23 [52.3%] were White). Consequent to lactulose ingestion, there was a reduction of plasma FFA, an improvement of adipose tissue insulin sensitivity index, an increase in colonic acetate synthesis, and an anorexigenic response characterized by an increased plasma concentration of PYY and active GLP-1 and a reduction of ghrelin in the subgroups. Compared with the lean and OIS groups, the OIR group showed a less marked median (IQR) rate of acetate appearance (OIR: 2.00 [-0.86 to 2.69] µmol × kg-1 × min-1; lean: 5.69 [3.04 to 9.77] µmol × kg-1 × min-1; lean vs OIR P = .004; OIS: 2.63 [1.22 to 4.52] µmol × kg-1 × min-1; OIS vs OIR P = .09), a blunted median (IQR) improvement of adipose insulin sensitivity index (OIR: 0.043 [ 0.006 to 0.155]; lean: 0.277 [0.220 to 0.446]; lean vs OIR P = .002; OIS: 0.340 [0.048 to 0.491]; OIS vs OIR P = .08), and a reduced median (IQR) PYY response (OIR: 25.4 [14.8 to 36.4] pg/mL; lean: 51.3 [31.6 to 83.3] pg/mL; lean vs OIR P = .002; OIS: 54.3 [39.3 to 77.2] pg/mL; OIS vs OIR P = .011). Conclusions and Relevance: In this cross-sectional study, lean, OIS, and OIR youth demonstrated different associations between colonic fermentation of indigestible dietary carbohydrates and the metabolic response, with OIR youth showing minimal metabolic modifications as compared with the other 2 groups. Trial Registration: ClinicalTrials.gov Identifier: NCT03454828.

A New Oral Model to Assess Postprandial Lactate Production Rate.

OBJECTIVE: Pediatric obesity predisposes children and adolescents to early onset insulin resistance and dysglycemia. In the last 20 years this has led to a rise in the prevalence of prediabetes, diabetes and fatty liver in youngsters, due to the high degree of insulin resistance experienced by these patients and the consequent high availability of glucose. As glucose accesses the liver, it is partly metabolized through glycolysis, whose main product is pyruvate that is then converted into Acetyl CoA and lactate. Therefore, lactate production rate (LPR) represents the best proxy for the assessment of glycolysis. Since to date there are not methods to estimate postprandial LPR, here we proposed a novel oral glucose-lactate model to estimate LPR during an oral glucose tolerance test and tested it in 24 youth with and without obesity. METHODS: The model is based on the oral glucose minimal model and assumes that LPR is a fraction (fr) of glucose disposal rate, proportional to glucose concentration and controlled by insulin action. RESULTS: The model well fitted the glucose and lactate data, and provided both precise parameter estimates (e.g., fr = 22.5 [12.6-54.1]%, median [IQR]), CV = 18 [13-25]%) and LPR time course. CONCLUSIONS: The proposed model is a valid tool to assess LPR, and thus glycolysis, during OGTT in nondiabetic subjects. SIGNIFICANCE: The proposed methodology will allow to assess postprandial LPR in simple and cost-effective way.

Insights from shotgun metagenomics into bacterial species and metabolic pathways associated with NAFLD in obese youth.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease and is often the precursor for more serious liver conditions such as nonalcoholic steatohepatitis and cirrhosis. Although the gut microbiome has been implicated in the development of NAFLD, the strong association of obesity with NAFLD and its effect on microbiome structure has made interpreting study outcomes difficult. In the present study, we examined the taxonomic and functional differences between the microbiomes of youth with obesity and with and without NAFLD. Shotgun metagenome sequencing was performed to profile the microbiomes of 36 subjects, half of whom were diagnosed with NAFLD using abdominal magnetic resonance imaging. Beta diversity analysis showed community-wide differences between the groups (p = 0.002). Specific taxonomic differences included increased relative abundances of the species Fusicatenibacter saccharivorans (p = 0.042), Romboutsia ilealis (p = 0.046), and Actinomyces sp. ICM47 (p = 0.0009), and a decrease of Bacteroides thetaiotamicron (p = 0.0002), in the NAFLD group as compared with the non-NAFLD group. At the phylum level, Bacteroidetes (p < 0.0001) was decreased in the NAFLD group. Functionally, branched-chain amino acid (p = 0.01343) and aromatic amino acid (p = 0.01343) synthesis pathways had increased relative abundances in the NAFLD group along with numerous energy use pathways, including pyruvate fermentation to acetate (p = 0.01318). Conclusion: Community-wide differences were noted based on NAFLD status, and individual bacterial species along with specific metabolic pathways were identified as potential drivers of these differences. The results of the present study support the idea that the NAFLD phenotype displays a differentiated microbial and functional signature from the obesity phenotype.

Growth differentiation factor 15 (GDF15) is associated with non-alcoholic fatty liver disease (NAFLD) in youth with overweight or obesity.

OBJECTIVE: Growth differentiation factor 15 (GDF15) has been associated with food intake and weight regulation in response to metabolic stress. In animal models, it has been noted that it may play a role in the progression of non-alcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease in children. DESIGN: In the current study, we explored the association of circulating plasma concentrations of GDF15 with NAFLD in youth with overweight/obesity, and whether changes in plasma concentrations in GDF15 parallel the changes in intrahepatic fat content (HFF%) over time. METHODS: Plasma GDF15 concentrations were measured by ELISA in 175 youth with overweight/obesity who underwent an oral glucose tolerance test (OGTT) and magnetic resonance imaging (MRI) to assess intrahepatic, visceral, and subcutaneous fat. Baseline fasting GDF15 concentrations were measured in twenty-two overweight/obese youth who progressed (n = 11) or regressed (n = 11) in HFF% by more than 30% of original over a 2-year period. RESULTS: Youth with NAFLD had significantly higher plasma concentrations of GDF15 than those without NAFLD, independent of age, sex, ethnicity, BMI z-score (BMIz), and visceral fat (P = 0.002). During the OGTT, there was a decline in plasma GDF15 concentrations from 0 to 60 min, but GDF15 concentrations returned to basal levels by the end of the study. There was a statistically significant association between change in HFF% and change in GDF15 (P = 0.008; r2 = 0.288) over ~2 years of follow-up. CONCLUSIONS: These data suggest that plasma GDF15 concentrations change with change in intrahepatic fat content in youth with overweight/obesity and may serve as a biomarker for NAFLD in children.

A Reduced Incretin Effect Mediated by the rs7903146 Variant in the TCF7L2 Gene Is an Early Marker of ß-Cell Dysfunction in Obese Youth.

OBJECTIVE: The risk genotype for the common variant rs7903146 of the transcription factor 7-like-2 (TCF7L2) gene has been found to affect the incretin response in healthy and obese adults; however, whether a similar functional defect is also present in obese adolescents remains unexplored. Herein, we examined the functional effect of the rs7903146 variant in the TCF7L2 gene on the incretin effect and determined its translational metabolic manifestation by performing deep phenotyping of the incretin system, ß-cell function relative to insulin sensitivity, the gastrointestinal-induced glucose disposal (GIGD) in obese youth with normal and impaired glucose tolerance. RESEARCH DESIGN AND METHODS: Thirty-nine obese adolescents without diabetes (median age 15 [25th, 75th percentile 14, 18] years; BMI 37 [33, 43] kg/m2) were genotyped for the rs7903146 variant of TCF7L2 and underwent a 3-h oral glucose tolerance test (OGTT) followed by an isoglycemic intravenous glucose infusion (iso-intravenous glucose tolerance test [IVGTT]) to match the plasma glucose concentrations during the OGTT and a hyperglycemic clamp with arginine stimulation. The incretin effect was measured as 100 * (AUC-SROGTT - AUC-SRiso-IVGTT) / AUC-SROGTT, where AUC-SR = area under the curve of C-peptide secretion rate. Participants were grouped into tertiles according to the percentage incretin effect (high, moderate, and low) to describe their metabolic phenotype. RESULTS: The presence of T risk allele for TCF7L2 was associated with a markedly reduced incretin effect compared with the wild-type genotype (0.3% [-7.2, 14] vs. 37.8% [12.5, 52.4], P < 0.002). When the cohort was stratified by incretin effect, the high, moderate, and low incretin effect groups did not differ with respect to anthropometric features, while the low incretin effect group exhibited higher 1-h glucose (P = 0.015) and a reduced disposition index, insulin sensitivity, and insulin clearance compared with the high incretin effect group. GIGD was reduced in the low incretin effect group (P = 0.001). The three groups did not differ with respect to intravenous glucose-induced insulin secretion and arginine response during the hyperglycemic clamp. CONCLUSIONS: A reduced incretin effect and its association with the TCF7L2 variant rs7903146 identify an early metabolic phenotype in obese youth without diabetes, featuring a higher plasma glucose peak at 1 h; lower insulin secretion, sensitivity, and clearance; and GIGD.

Effect of Gut Microbiota and PNPLA3 rs738409 Variant on Nonalcoholic Fatty Liver Disease (NAFLD) in Obese Youth.

CONTEXT: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease, affecting approximately 3 in 10 obese children worldwide. OBJECTIVE: We aimed to investigate the potential relationship between gut microbiota and NAFLD in obese youth, while considering the role of PNPLA3 rs738409, a strong genetic contributor to NAFLD. DESIGN: In this cross-sectional study, participants completed an abdominal magnetic resonance imaging to measure hepatic fat fraction (HFF), oral glucose tolerance test, and PNPLA3 rs738409 genotyping. Fecal samples were collected to analyze the V4 region of the 16S rRNA gene for intestinal bacteria characterization. SETTING: Yale Pediatric Obesity Clinic. PARTICIPANTS: Obese youth (body mass index >95th percentile) with NAFLD (HFF ≥5.5%; n = 44) and without NAFLD (HFF <5.5%; n = 29). MAIN OUTCOME MEASURE: Shannon-Wiener diversity index values and proportional bacterial abundance by NAFLD status and PNPLA3 genotype. RESULTS: Subjects with NAFLD had decreased bacterial alpha-diversity compared with those without NAFLD (P = 0.013). Subjects with NAFLD showed a higher Firmicutes to Bacteroidetes (F/B) ratio (P = 0.019) and lower abundance of Bacteroidetes (P = 0.010), Prevotella (P = 0.019), Gemmiger (P = 0.003), and Oscillospira (P = 0.036). F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were associated with HFF when controlling for group variations. We also observed an additive effect on HFF by PNPLA3 rs738409 and Gemmiger, and PNPLA3 rs738409 and Oscillospira. CONCLUSIONS: Obese youth with NAFLD have a different gut microbiota composition than those without NAFLD. These differences were still statistically significant when controlling for factors associated with NAFLD, including PNPLA3 rs738409.

Altered In Vivo Lipid Fluxes and Cell Dynamics in Subcutaneous Adipose Tissues Are Associated With the Unfavorable Pattern of Fat Distribution in Obese Adolescent Girls.

Patterns of abdominal fat distribution (for example, a high vs. low visceral adipose tissue [VAT]/[VAT + subcutaneous adipose tissue (SAT)] ratio), independent of obesity, during adolescence carry a high risk for insulin resistance and type 2 diabetes. Longitudinal follow-up of a cohort of obese adolescents has recently revealed that a high ratio (high VAT/[VAT + SAT]) is a major determinant of fatty liver and metabolic impairment over time, with these effects being more pronounced in girls than in boys. To unravel the underlying metabolic alterations associated with the unfavorable VAT/(VAT + SAT) phenotype, we used the 2H2O labeling method to measure the turnover of adipose lipids and cells in the subcutaneous abdominal and gluteal/femoral adipose tissue (SAT) of weight-stable obese adolescent girls with a similar level of obesity but discordant VAT/(VAT + SAT) ratios. Girls with the unfavorable (high VAT/[VAT + SAT]) phenotype exhibited higher in vivo rates of triglyceride (TG) turnover (representing both lipolysis and synthesis at steady state), without significant differences in de novo lipogenesis in both abdominal and gluteal depots, compared with obese girls with the favorable phenotype. Moreover, mature adipocytes had higher turnover, with no difference in stromal vascular cell proliferation in both depots in the metabolically unfavorable phenotype. The higher TG turnover rates were significantly correlated with higher intrahepatic fat stores. These findings are contrary to the hypothesis that impaired capacity to deposit TGs or proliferation of new mature adipocytes are potential mechanisms for ectopic fat distribution in this setting. In summary, these results suggest that increased turnover of TGs (lipolysis) and of mature adipocytes in both abdominal and gluteal SAT may contribute to metabolic impairment and the development of fatty liver, even at this very early stage of disease.