Lise Madsen

Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life.

The gut microbiota is central to human health, but its establishment in early life has not been quantitatively and functionally examined. Applying metagenomic analysis on fecal samples from a large cohort of Swedish infants and their mothers, we characterized the gut microbiome during the first year of life and assessed the impact of mode of delivery and feeding on its establishment. In contrast to vaginally delivered infants, the gut microbiota of infants delivered by C-section showed significantly less resemblance to their mothers. Nutrition had a major impact on early microbiota composition and function, with cessation of breast-feeding, rather than introduction of solid food, being required for maturation into an adult-like microbiota. Microbiota composition and ecological network had distinctive features at each sampled stage, in accordance with functional maturation of the microbiome. Our findings establish a framework for understanding the interplay between the gut microbiome and the human body in early life.

Persistent organic pollutant exposure leads to insulin resistance syndrome.

Background The incidence of the insulin resistance syndrome has increased at an alarming rate worldwide, creating a serious challenge to public health care in the 21st century. Recently, epidemiological studies have associated the prevalence of type 2 diabetes with elevated body burdens of persistent organic pollutants (POPs). However, experimental evidence demonstrating a causal link between POPs and the development of insulin resistance is lacking. Objective We investigated whether exposure to POPs contributes to insulin resistance and metabolic disorders. Methods Sprague-Dawley rats were exposed for 28 days to lipophilic POPs through the consumption of a high-fat diet containing either refined or crude fish oil obtained from farmed Atlantic salmon. In addition, differentiated adipocytes were exposed to several POP mixtures that mimicked the relative abundance of organic pollutants present in crude salmon oil. We measured body weight, whole-body insulin sensitivity, POP accumulation, lipid and glucose homeostasis, and gene expression and we performed microarray analysis. Results Adult male rats exposed to crude, but not refined, salmon oil developed insulin resistance, abdominal obesity, and hepatosteatosis. The contribution of POPs to insulin resistance was confirmed in cultured adipocytes where POPs, especially organochlorine pesticides, led to robust inhibition of insulin action. Moreover, POPs induced down-regulation of insulin-induced gene-1 (Insig-1) and Lpin1, two master regulators of lipid homeostasis. Conclusion Our findings provide evidence that exposure to POPs commonly present in food chains leads to insulin resistance and associated metabolic disorders.

Eicosapentaenoic and Docosahexaenoic Acid Affect Mitochondrial and Peroxisomal Fatty Acid Oxidation in Relation to Substrate Preference

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[3H]glycerol/diacyl[3H]glycerol ratio was observed. The distribution of [1-14C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-14C]EPA was oxidized to a much greater extent than [1-14C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.

Dietary Linoleic Acid Elevates Endogenous 2‐AG and Anandamide and Induces Obesity

Suppressing hyperactive endocannabinoid tone is a critical target for reducing obesity. The backbone of both endocannabinoids 2‐arachidonoylglycerol (2‐AG) and anandamide (AEA) is the ω‐6 fatty acid arachidonic acid (AA). Here we posited that excessive dietary intake of linoleic acid (LA), the precursor of AA, would induce endocannabinoid hyperactivity and promote obesity. LA was isolated as an independent variable to reflect the dietary increase in LA from 1 percent of energy (en%) to 8 en% occurring in the United States during the 20th century. Mice were fed diets containing 1 en% LA, 8 en% LA, and 8 en% LA + 1 en% eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in medium‐fat diets (35 en% fat) and high‐fat diets (60 en%) for 14 weeks from weaning. Increasing LA from 1 en% to 8 en% elevated AA‐phospholipids (PL) in liver and erythrocytes, tripled 2‐AG + 1‐AG and AEA associated with increased food intake, feed efficiency, and adiposity in mice. Reducing AA‐PL by adding 1 en% long‐chain ω‐3 fats to 8 en% LA diets resulted in metabolic patterns resembling 1 en% LA diets. Selectively reducing LA to 1 en% reversed the obesogenic properties of a 60 en% fat diet. These animal diets modeled 20th century increases of human LA consumption, changes that closely correlate with increasing prevalence rates of obesity. In summary, dietary LA increased tissue AA, and subsequently elevated 2‐AG + 1‐AG and AEA resulting in the development of diet‐induced obesity. The adipogenic effect of LA can be prevented by consuming sufficient EPA and DHA to reduce the AA‐PL pool and normalize endocannabinoid tone.

UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase Activity

Background The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis. Methodology/Principal Findings Here we report that cyclooxygenase (COX) activity and prostaglandin E2 (PGE2) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE2 receptor antagonists implicated EP4 as a main PGE2 receptor, and injection of the stable PGE2 analog (EP3/4 agonist) 16,16 dm PGE2 induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality. Conclusions/Significance Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.

Cyclic AMP (cAMP)-Mediated Stimulation of Adipocyte Differentiation Requires the Synergistic Action of Epac- and cAMP-Dependent Protein Kinase-Dependent Processes

ABSTRACT Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.

Chronic Consumption of Farmed Salmon Containing Persistent Organic Pollutants Causes Insulin Resistance and Obesity in Mice

Background Dietary interventions are critical in the prevention of metabolic diseases. Yet, the effects of fatty fish consumption on type 2 diabetes remain unclear. The aim of this study was to investigate whether a diet containing farmed salmon prevents or contributes to insulin resistance in mice. Methodology/Principal Findings Adult male C57BL/6J mice were fed control diet (C), a very high-fat diet without or with farmed Atlantic salmon fillet (VHF and VHF/S, respectively), and Western diet without or with farmed Atlantic salmon fillet (WD and WD/S, respectively). Other mice were fed VHF containing farmed salmon fillet with reduced concentrations of persistent organic pollutants (VHF/S-POPs). We assessed body weight gain, fat mass, insulin sensitivity, glucose tolerance, ex vivo muscle glucose uptake, performed histology and immunohistochemistry analysis, and investigated gene and protein expression. In comparison with animals fed VHF and WD, consumption of both VHF/S and WD/S exaggerated insulin resistance, visceral obesity, and glucose intolerance. In addition, the ability of insulin to stimulate Akt phosphorylation and muscle glucose uptake was impaired in mice fed farmed salmon. Relative to VHF/S-fed mice, animals fed VHF/S-POPs had less body burdens of POPs, accumulated less visceral fat, and had reduced mRNA levels of TNFα as well as macrophage infiltration in adipose tissue. VHF/S-POPs-fed mice further exhibited better insulin sensitivity and glucose tolerance than mice fed VHF/S. Conclusions/Significance Our data indicate that intake of farmed salmon fillet contributes to several metabolic disorders linked to type 2 diabetes and obesity, and suggest a role of POPs in these deleterious effects. Overall, these findings may participate to improve nutritional strategies for the prevention and therapy of insulin resistance.

The hypotriglyceridemic effect of dietary n−3 FA is associated with increased β-oxidation and reduced leptin expression

To study the mechanisms responsible for the hypotriglyceridemic effect of marine oils, we monitored the effects of high dietary intake of n−3 PUFA on hepatic and muscular β-oxidation, plasma leptin concentration, leptin receptor gene expression, and in vivo insulin action. Two groups of male Wistar rats were fed either a high-fat diet [28% (w/w) of saturated fat] or a high-fat diet containing 10% n−3 PUFA and 18% saturated fat for 3 wk. The hypotriglyceridemic effect of n−3 PUFA was accompanied by increased hepatic oxidation of palmitoyl-CoA (125%, P<0.005) and palmitoyl-l-carnitine (480%, P<0.005). These findings were corroborated by raised carnitine palmitoyltransferase-2 activity (154%, P<0.001) and mRNA levels (91%, P<0.01) as well as by simultaneous elevation of hepatic peroxisomal acyl-CoA oxidase activity (144%, P<0.01) and mRNA content (82%, P<0.05). In contrast, hepatic carnitine palmitoyltransferase-1 activity remained unchanged despite a twofold increased mRNA level after n−3 PUFA feeding. Skeletal muscle FA oxidation was less affected by dietary n−3 PUFA, and the stimulatory effect was found only in peroxisomes. Dietary intake of n−3 PUFA was followed by increased acyl-CoA oxidase activity (48%, P<0.05) and mRNA level (83%, P<0.05) in skeletal muscle. The increased FA oxidation after n−3 PUFA supplementation of the high-fat diet was accompanied by lower plasma leptin concentration (−38%, P<0.05) and leptin mRNA expression (−66%, P<0.05) in retroperitoneal adipose tissue, and elevated hepatic mRNA level for the leptin receptor Ob-Ra (140%, P<0.05). Supplementation of the high-fat diet with n−3 PUFA enhanced in vivo insulin sensitivity, as shown by normalization of the glucose infusion rate during euglycemic hyperinsulinemic clamp.Our results indicate that the hypotriglyceridemic effect of dietary n−3 PUFA is associated with stimulation of FA oxidation in the liver and to a smaller extent in skeletal muscle. This may ameliorate dyslipidemia, tissue lipid accumulation, and insulin action, in spite of decreased plasma leptin level and leptin mRNA in adipose tissue.

A catalog of the mouse gut metagenome.

We established a catalog of the mouse gut metagenome comprising ∼2.6 million nonredundant genes by sequencing DNA from fecal samples of 184 mice. To secure high microbiome diversity, we used mouse strains of diverse genetic backgrounds, from different providers, kept in different housing laboratories and fed either a low-fat or high-fat diet. Similar to the human gut microbiome, >99% of the cataloged genes are bacterial. We identified 541 metagenomic species and defined a core set of 26 metagenomic species found in 95% of the mice. The mouse gut microbiome is functionally similar to its human counterpart, with 95.2% of its Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologous groups in common. However, only 4.0% of the mouse gut microbial genes were shared (95% identity, 90% coverage) with those of the human gut microbiome. This catalog provides a useful reference for future studies.

Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer

Objective To evaluate the potential for diagnosing colorectal cancer (CRC) from faecal metagenomes. Design We performed metagenome-wide association studies on faecal samples from 74 patients with CRC and 54 controls from China, and validated the results in 16 patients and 24 controls from Denmark. We further validated the biomarkers in two published cohorts from France and Austria. Finally, we employed targeted quantitative PCR (qPCR) assays to evaluate diagnostic potential of selected biomarkers in an independent Chinese cohort of 47 patients and 109 controls. Results Besides confirming known associations of Fusobacterium nucleatum and Peptostreptococcus stomatis with CRC, we found significant associations with several species, including Parvimonas micra and Solobacterium moorei. We identified 20 microbial gene markers that differentiated CRC and control microbiomes, and validated 4 markers in the Danish cohort. In the French and Austrian cohorts, these four genes distinguished CRC metagenomes from controls with areas under the receiver-operating curve (AUC) of 0.72 and 0.77, respectively. qPCR measurements of two of these genes accurately classified patients with CRC in the independent Chinese cohort with AUC=0.84 and OR of 23. These genes were enriched in early-stage (I–II) patient microbiomes, highlighting the potential for using faecal metagenomic biomarkers for early diagnosis of CRC. Conclusions We present the first metagenomic profiling study of CRC faecal microbiomes to discover and validate microbial biomarkers in ethnically different cohorts, and to independently validate selected biomarkers using an affordable clinically relevant technology. Our study thus takes a step further towards affordable non-invasive early diagnostic biomarkers for CRC from faecal samples.