Results of the BfR MEAL Study: Acrylamide in foods from the German market with highest levels in vegetable crisps.

Acrylamide (AA) is formed in foods due to thermal processes. AA was analysed in 230 foods in the first German Total Diet Study and the highest mean levels of AA were found in vegetable crisps (1430 µg/kg), followed by potato pancakes (558) µg/kg) and pan-fried potatoes (450 µg/kg). In various foods, e.g. French fries and sweet potatoes, AA was also tested for different browning degrees and cooking methods. French fries cooked to a browning degree of 3 in all cooking methods exceeded the benchmark level set by the European Union. French fries prepared in the oven and sweet potatoes in the air fryer had the lowest AA levels. In foods from the German market, AA was found also in foods such as popcorn (243 µg/kg), salty sticks (190 µg/kg), and dark chocolate (130 µg/kg). Levels of AA found in our study may support future dietary exposure and food safety assessments.

Alterations of Lipid Profile in Livers with Impaired Lipophagy.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in the liver. Various mechanisms such as an increased uptake in fatty acids or de novo synthesis contribute to the development of steatosis and progression to more severe stages. Furthermore, it has been shown that impaired lipophagy, the degradation of lipids by autophagic processes, contributes to NAFLD. Through an unbiased lipidome analysis of mouse livers in a genetic model of impaired lipophagy, we aimed to determine the resulting alterations in the lipidome. Observed changes overlap with those of the human disease. Overall, the entire lipid content and in particular the triacylglycerol concentration increased under conditions of impaired lipophagy. In addition, we detected a reduction in long-chain polyunsaturated fatty acids (PUFAs) and an increased ratio of n-6 PUFAs to n-3 PUFAs, which was due to the depletion of n-3 PUFAs. Although the abundance of major phospholipid classes was reduced, the ratio of phosphatidylcholines to phosphatidylethanolamines was not affected. In conclusion, this study demonstrates that impaired lipophagy contributes to the pathology of NAFLD and is associated with an altered lipid profile. However, the lipid pattern does not appear to be specific for lipophagic alterations, as it resembles mainly that described in relation to fatty liver disease.

Results of the BfR MEAL Study: The food type has a stronger impact on calcium, potassium and phosphorus levels than factors such as seasonality, regionality and type of production.

The BfR MEAL Study aims to provide representative levels of chemical substances in foods consumed by the population in Germany for dietary exposure assessment. Calcium, potassium and phosphorus (Ca, K, P) are essential to obtain physiological functions in humans. Levels were investigated in 356 foods. Foods were purchased representatively, prepared as typically consumed and pooled before analysis. High mean levels were found in milk, dairy products, legumes, nuts, oilseeds and spices as well as chia seeds (Ca, K, P), chewing gum (Ca) and cocoa powder (K). Different levels comparing organically and conventionally produced foods were determined among others in cereal cracker (puffed), olives and tofu. Higher K levels were found in fried compared to boiled potatoes. Similar P levels were mainly found in regionally and seasonally sampled foods. These data provide a substantially improved basis to address dietary exposure assessment of the population in Germany for Ca, K and P.

MiR-205 is up-regulated in islets of diabetes-susceptible mice and targets the diabetes gene Tcf7l2.

AIM: MicroRNAs play an important role in the maintenance of cellular functions by fine-tuning gene expression levels. The aim of the current study was to identify genetically caused changes in microRNA expression which associate with islet dysfunction in diabetic mice. METHODS: To identify novel microRNAs involved in islet dysfunction, transcriptome and miRNome analyses were performed in islets of obese, diabetes-susceptible NZO and diabetes-resistant B6-ob/ob mice and results combined with quantitative trait loci (QTL) and functional in vitro analysis. RESULTS: In islets of NZO and B6-ob/ob mice, 94 differentially expressed microRNAs were detected, of which 11 are located in diabetes QTL. Focusing on conserved microRNAs exhibiting the strongest expression difference and which have not been linked to islet function, miR-205-5p was selected for further analysis. According to transcriptome data and target prediction analyses, miR-205-5p affects genes involved in Wnt and calcium signalling as well as insulin secretion. Over-expression of miR-205-5p in the insulinoma cell line INS-1 increased insulin expression, left-shifted the glucose-dependence of insulin secretion and supressed the expression of the diabetes gene TCF7L2. The interaction between miR-205-5p and TCF7L2 was confirmed by luciferase reporter assay. CONCLUSION: MiR-205-5p was identified as relevant microRNA involved in islet dysfunction by interacting with TCF7L2.

Overexpression of Gjb4 impairs cell proliferation and insulin secretion in primary islet cells.

OBJECTIVE: Altered gene expression contributes to the development of type 2 diabetes (T2D); thus, the analysis of differentially expressed genes between diabetes-susceptible and diabetes-resistant mouse models is an important tool for the determination of candidate genes that participate in the pathology. Based on RNA-seq and array data comparing pancreatic gene expression of diabetes-prone New Zealand Obese (NZO) mice and diabetes-resistant B6.V-ob/ob (B6-ob/ob) mice, the gap junction protein beta 4 (Gjb4) was identified as a putative novel T2D candidate gene. METHODS: Gjb4 was overexpressed in primary islet cells derived from C57BL/6 (B6) mice and INS-1 cells via adenoviral-mediated infection. The proliferation rate of cells was assessed by BrdU incorporation, and insulin secretion was measured under low (2.8 mM) and high (20 mM) glucose concentration. INS-1 cell apoptosis rate was determined by Western blotting assessing cleaved caspase 3 levels. RESULTS: Overexpression of Gjb4 in primary islet cells significantly inhibited the proliferation by 47%, reduced insulin secretion of primary islets (46%) and INS-1 cells (51%), and enhanced the rate of apoptosis by 63% in INS-1 cells. Moreover, an altered expression of the miR-341-3p contributes to the Gjb4 expression difference between diabetes-prone and diabetes-resistant mice. CONCLUSIONS: The gap junction protein Gjb4 is highly expressed in islets of diabetes-prone NZO mice and may play a role in the development of T2D by altering islet cell function, inducing apoptosis and inhibiting proliferation.

Immunity-related GTPase induces lipophagy to prevent excess hepatic lipid accumulation.

BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association. METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2. RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage. CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets. LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.

Epigenetic regulation of hepatic Dpp4 expression in response to dietary protein.

Dipeptidyl peptidase 4 (DPP4) is known to be elevated in metabolic disturbances such as obesity, type 2 diabetes and fatty liver disease. Lowering DPP4 concentration by pharmacological inhibition improves glucose homeostasis and exhibits beneficial effects to reduce hepatic fat content. As factors regulating the endogenous expression of Dpp4 are unknown, the aim of this study was to examine whether the Dpp4 expression is epigenetically regulated in response to dietary components. Primary hepatocytes were treated with different macronutrients, and Dpp4 mRNA levels and DPP4 activity were evaluated. Moreover, dietary low-protein intervention was conducted in New Zealand obese (NZO) mice, and subsequently, effects on Dpp4 expression, methylation as well as plasma concentration and activity were determined. Our results indicate that Dpp4 mRNA expression is mediated by DNA methylation in several tissues. We therefore consider the Dpp4 southern shore as tissue differentially methylated region. Amino acids increased Dpp4 expression in primary hepatocytes, whereas glucose and fatty acids were without effect. Dietary protein restriction in NZO mice increased Dpp4 DNA methylation in liver leading to diminished Dpp4 expression and consequently to lowered plasma DPP4 activity. We conclude that protein restriction in the adolescent and adult states is a sufficient strategy to reduce DPP4 which in turn contributes to improve glucose homeostasis.

Effect of adipocyte-derived IGF-I on adipose tissue mass and glucose metabolism in the Berlin Fat Mouse.

Besides liver, IGF-I is expressed in adipose tissue. However, the effects of this local IGF-I on adipose tissue and metabolism are unclear. We generated adipocyte-specific knock-out mice on the background of the Berlin Fat Mouse Inbred (BFMI) line to evaluate the contribution of adipocyte-IGF-I on glucose metabolism and adipose tissue development. BFMI mice are obese, non-diabetic with elevated plasma insulin and IGF-I concentration. The knock-out in adipocytes led to a total white adipose tissue expression of 50-60% due to unaltered Igf-1 expression in stromavascular cells. The lack of IGF-I from adipocytes did not alter plasma IGF-I concentration. BFMIChr3-Igf-I-KOQ-AT mice had reduced adipose tissue mass in most depots. During oral glucose tolerance tests, BFMIChr3-Igf-I-KOQ-AT mice showed an impaired glucose clearance (p = .03). Interestingly, insulin action was enhanced during insulin tolerance tests (p = .05). In conclusion, adipocyte-specific IGF-I ablation in obese BFMI mice results in reduced adipose tissue mass and thereby alters glucose metabolism.

Increased Ifi202b/IFI16 expression stimulates adipogenesis in mice and humans.

AIMS/HYPOTHESIS: Obesity results from a constant and complex interplay between environmental stimuli and predisposing genes. Recently, we identified the IFN-activated gene Ifi202b as the most likely gene responsible for the obesity quantitative trait locus Nob3 (New Zealand Obese [NZO] obesity 3). The aim of this study was to evaluate the effects of Ifi202b on body weight and adipose tissue biology, and to clarify the functional role of its human orthologue IFI16. METHODS: The impact of Ifi202b and its human orthologue IFI16 on adipogenesis was investigated by modulating their respective expression in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes. Furthermore, transgenic mice overexpressing IFI202b were generated and characterised with respect to metabolic traits. In humans, expression levels of IFI16 in adipose tissue were correlated with several variables of adipocyte function. RESULTS: In mice, IFI202b overexpression caused obesity (Δ body weight at the age of 30 weeks: 10.2 ± 1.9 g vs wild-type mice) marked by hypertrophic fat mass expansion, increased expression of Zfp423 (encoding the transcription factor zinc finger protein [ZFP] 423) and white-selective genes (Tcf21, Tle3), and decreased expression of thermogenic genes (e.g. Cidea, Ucp1). Compared with their wild-type littermates, Ifi202b transgenic mice displayed lower body temperature, hepatosteatosis and systemic insulin resistance. Suppression of IFI202b/IFI16 in pre-adipocytes impaired adipocyte differentiation and triacylglycerol storage. Humans with high levels of IFI16 exhibited larger adipocytes, an enhanced inflammatory state and impaired insulin-stimulated glucose uptake in white adipose tissue. CONCLUSIONS/INTERPRETATION: Our findings reveal novel functions of Ifi202b and IFI16, demonstrating their role as obesity genes. These genes promote white adipogenesis and fat storage, thereby facilitating the development of obesity-associated insulin resistance.