Novel Proteome Targets Marking Insulin Resistance in Metabolic Syndrome.

CONTEXT/OBJECTIVE: In order to better understand which metabolic differences are related to insulin resistance in metabolic syndrome (MetSyn), we used hyperinsulinemic-euglycemic (HE) clamps in individuals with MetSyn and related peripheral insulin resistance to circulating biomarkers. DESIGN/METHODS: In this cross-sectional study, HE-clamps were performed in treatment-naive men (n = 97) with MetSyn. Subjects were defined as insulin-resistant based on the rate of disappearance (Rd). Machine learning models and conventional statistics were used to identify biomarkers of insulin resistance. Findings were replicated in a cohort with n = 282 obese men and women with (n = 156) and without (n = 126) MetSyn. In addition to this, the relation between biomarkers and adipose tissue was assessed by nuclear magnetic resonance imaging. RESULTS: Peripheral insulin resistance is marked by changes in proteins related to inflammatory processes such as IL-1 and TNF-receptor and superfamily members. These proteins can distinguish between insulin-resistant and insulin-sensitive individuals (AUC = 0.72 ± 0.10) with MetSyn. These proteins were also associated with IFG, liver fat (rho 0.36, p = 1.79 × 10-9) and visceral adipose tissue (rho = 0.35, p = 6.80 × 10-9). Interestingly, these proteins had the strongest association in the MetSyn subgroup compared to individuals without MetSyn. CONCLUSIONS: MetSyn associated with insulin resistance is characterized by protein changes related to body fat content, insulin signaling and pro-inflammatory processes. These findings provide novel targets for intervention studies and should be the focus of future in vitro and in vivo studies.

Anaerobic Feces Processing for Fecal Microbiota Transplantation Improves Viability of Obligate Anaerobes.

Fecal microbiota transplantation (FMT) is under investigation for several indications, including ulcerative colitis (UC). The clinical success of FMT depends partly on the engraftment of viable bacteria. Because the vast majority of human gut microbiota consists of anaerobes, the currently used aerobic processing protocols of donor stool may diminish the bacterial viability of transplanted material. This study assessed the effect of four processing techniques for donor stool (i.e., anaerobic and aerobic, both direct processing and after temporary cool storage) on bacterial viability. By combining anaerobic culturing on customized media for anaerobes with 16S rRNA sequencing, we could successfully culture and identify the majority of the bacteria present in raw fecal suspensions. We show that direct anaerobic processing of donor stool is superior to aerobic processing conditions for preserving the bacterial viability of obligate anaerobes and butyrate-producing bacteria related to the clinical response to FMT in ulcerative colitis patients, including Faecalibacterium, Eubacterium hallii, and Blautia. The effect of oxygen exposure during stool processing decreased when the samples were stored long-term. Our results confirm the importance of sample conditioning to preserve the bacterial viability of oxygen-sensitive gut bacteria. Anaerobic processing of donor stool may lead to increased clinical success of FMT, which should further be investigated in clinical trials.

A systems biology approach to study non-alcoholic fatty liver (NAFL) in women with obesity.

Non-alcoholic fatty liver disease (NAFLD) is now the most frequent global chronic liver disease. Individuals with NAFLD exhibited an increased risk of all-cause mortality driven by extrahepatic cancers and liver and cardiovascular disease. Once the disease is established, women have a higher risk of disease progression and worse outcome. It is therefore critical to deepen the current knowledge on the pathophysiology of NAFLD in women. Here, we used a systems biology approach to investigate the contribution of different organs to this disease. We analyzed transcriptomics profiles of liver and adipose tissues, fecal metagenomes, and plasma metabolomes of 55 women with and without NAFLD. We observed differences in metabolites, expression of human genes, and gut microbial features between the groups and revealed that there is substantial crosstalk between these different omics sets. Multi-omics analysis of individuals with NAFLD may provide novel strategies to study the pathophysiology of NAFLD in humans.

Glucose-mediated insulin secretion is improved in FHL2-deficient mice and elevated FHL2 expression in humans is associated with type 2 diabetes.

AIMS/HYPOTHESIS: The general population is ageing, involving an enhanced incidence of chronic diseases such as type 2 diabetes. With ageing, DNA methylation of FHL2 increases, as well as expression of the four and a half LIM domains 2 (FHL2) protein in human pancreatic islets. We hypothesised that FHL2 is actively involved in glucose metabolism. METHODS: Publicly available microarray datasets from human pancreatic islets were analysed for FHL2 expression. In FHL2-deficient mice, we studied glucose clearance and insulin secretion. Gene expression analysis and glucose-stimulated insulin secretion (GSIS) were determined in isolated murine FHL2-deficient islets to evaluate insulin-secretory capacity. Moreover, knockdown and overexpression of FHL2 were accomplished in MIN6 cells to delineate the underlying mechanism of FHL2 function. RESULTS: Transcriptomics of human pancreatic islets revealed that individuals with elevated levels of HbA1c displayed increased FHL2 expression, which correlated negatively with insulin secretion pathways. In line with this observation, FHL2-deficient mice cleared glucose more efficiently than wild-type littermates through increased plasma insulin levels. Insulin sensitivity was comparable between these genotypes. Interestingly, pancreatic islets isolated from FHL2-deficient mice secreted more insulin in GSIS assays than wild-type mouse islets even though insulin content and islet size was similar. To support this observation, we demonstrated increased expression of the transcription factor crucial in insulin secretion, MAF BZIP transcription factor A (MafA), higher expression of GLUT2 and reduced expression of the adverse factor c-Jun in FHL2-deficient islets. The underlying mechanism of FHL2 was further delineated in MIN6 cells. FHL2-knockdown led to enhanced activation of forkhead box protein O1 (FOXO1) and its downstream genes such as Mafa and Pdx1 (encoding pancreatic and duodenal homeobox 1), as well as increased glucose uptake. On the other hand, FHL2 overexpression in MIN6 cells blocked GSIS, increased the formation of reactive oxygen species and increased c-Jun activity. CONCLUSIONS/INTERPRETATION: Our data demonstrate that FHL2 deficiency improves insulin secretion from beta cells and improves glucose tolerance in mice. Given that FHL2 expression in humans increases with age and that high expression levels of FHL2 are associated with beta cell dysfunction, we propose that enhanced FHL2 expression in elderly individuals contributes to glucose intolerance and the development of type 2 diabetes. DATA AVAILABILITY: The human islet microarray datasets used are publicly available and can be found on https://www.ncbi.nlm.nih.gov/geo/ .

Hyperinsulinemia Is Highly Associated With Markers of Hepatocytic Senescence in Two Independent Cohorts.

Cellular senescence is an essentially irreversible growth arrest that occurs in response to various cellular stressors and may contribute to development of type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD). In this article, we investigated whether chronically elevated insulin levels are associated with cellular senescence in the human liver. In 107 individuals undergoing bariatric surgery, hepatic senescence markers were assessed by immunohistochemistry as well as transcriptomics. A subset of 180 participants from the ongoing Finnish Kuopio OBesity Surgery (KOBS) study was used as validation cohort. We found plasma insulin to be highly associated with various markers of cellular senescence in liver tissue. The liver transcriptome of individuals with high insulin revealed significant upregulation of several genes associated with senescence: p21, TGFß, PI3K, HLA-G, IL8, p38, Ras, and E2F. Insulin associated with hepatic senescence independently of NAFLD and plasma glucose. By using transcriptomic data from the KOBS study, we could validate the association of insulin with p21 in the liver. Our results support a potential role for hyperinsulinemia in induction of cellular senescence in the liver. These findings suggest possible benefits of lowering insulin levels in obese individuals with insulin resistance.

Compensatory intestinal antibody response against pro-inflammatory microbiota after bariatric surgery.

Obesity and type 2 diabetes (T2D) are growing burdens for individuals and the health-care system. Bariatric surgery is an efficient, but drastic treatment to reduce body weight, normalize glucose values, and reduce low-grade inflammation. The gut microbiome, which is in part controlled by intestinal antibodies, such as IgA, is involved in the development of both conditions. Knowledge of the effect of bariatric surgery on systemic and intestinal antibody response is limited. Here, we determined the fecal antibody and gut microbiome response in 40 T2D and non-diabetic (ND) obese individuals that underwent bariatric surgery (N = 40). Body weight, fasting glucose concentrations and inflammatory parameters decreased after bariatric surgery, whereas pro-inflammatory bacterial species such as lipopolysaccharide (LPS), and flagellin increased in the feces. Simultaneously, concentrations of LPS- and flagellin-specific intestinal IgA levels increased with the majority of pro-inflammatory bacteria coated with IgA after surgery. Finally, serum antibodies decreased in both groups, along with a lower inflammatory tone. We conclude that intestinal rearrangement by bariatric surgery leads to expansion of typical pro-inflammatory bacteria, which may be compensated by an improved antibody response. Although further evidence and mechanistic insights are needed, we postulate that this apparent compensatory antibody response might help to reduce systemic inflammation by neutralizing intestinal immunogenic components and thereby enhance intestinal barrier function after bariatric surgery.

Fecal microbiota transplantation does not alter bacterial translocation and visceral adipose tissue inflammation in individuals with obesity.

AIMS: Visceral adipose tissue inflammation is a fundamental mechanism of insulin resistance in obesity and type 2 diabetes. Translocation of intestinal bacteria has been suggested as a driving factor for the inflammation. However, although bacterial DNA was detected in visceral adipose tissue of humans with obesity, it is unclear to what extent this is contamination or whether the gut microbiota is causally involved. Effects of fecal microbiota transplantation (FMT) on bacterial translocation and visceral adipose tissue inflammation in individuals with obesity and insulin resistance were assessed. MATERIAL AND METHODS: Eight individuals with clinically severe obesity (body mass index [BMI] >35 kg/m2) and metabolic syndrome received lean donor FMT 4 weeks prior to elective bariatric surgery. The participants were age-, sex-, and BMI-matched to 16 controls that underwent no fecal transplantation. Visceral adipose tissue was collected during surgery. Bacterial translocation was assessed by 16S rRNA gene sequencing of adipose tissue and feces. Pro-inflammatory cytokine expression and histopathological analyses of visceral adipose tissue were performed to assess inflammation. RESULTS: Fecal microbiota transplantation significantly altered gut microbiota composition. Visceral adipose tissue contained a very low quantity of bacterial DNA in both groups. No difference in visceral bacterial DNA content between groups was observed. Also, visceral expression of pro-inflammatory cytokines and macrophage infiltration did not differ between groups. No correlation between inflammatory tone and bacterial translocation was observed. CONCLUSIONS: Visceral bacterial DNA content and level of inflammation were not altered upon FMT. Thus, bacterial translocation may not be the main driver of visceral adipose tissue inflammation in obesity.

Evaluating causality of cellular senescence in non-alcoholic fatty liver disease.

Cellular senescence is a state of irreversible cell cycle arrest that has important physiological functions. However, cellular senescence is also a hallmark of ageing and has been associated with several pathological conditions. A wide range of factors including genotoxic stress, mitogens and inflammatory cytokines can induce senescence. Phenotypically, senescent cells are characterised by short telomeres, an enlarged nuclear area and damaged genomic and mitochondrial DNA. Secretion of proinflammatory proteins, also known as the senescence-associated secretory phenotype, is a characteristic of senescent cells that is thought to be the main contributor to their disease-inducing properties. In the past decade, the role of cellular senescence in the development of non-alcoholic fatty liver disease (NAFLD) and its progression towards non-alcoholic steatohepatitis (NASH) has garnered significant interest. Until recently, it was suggested that hepatocyte cellular senescence is a mere consequence of the metabolic dysregulation and inflammatory phenomena in fatty liver disease. However, recent work in rodents has suggested that senescence may be a causal factor in NAFLD development. Although causality is yet to be established in humans, current evidence suggests that targeting senescent cells has therapeutic potential for NAFLD. We aim to provide insights into the quality of the evidence supporting a causal role of cellular senescence in the development of NAFLD in rodents and humans. We will elaborate on key cellular and molecular features of senescence and discuss the efficacy and safety of novel senolytic drugs for the treatment or prevention of NAFLD.

Donor Fecal Microbiota Transplantation Alters Gut Microbiota and Metabolites in Obese Individuals With Steatohepatitis.

The intestinal microbiota has been linked to the development and prevalence of steatohepatitis in humans. Interestingly, steatohepatitis is significantly lower in individuals taking a plant-based, low-animal-protein diet, which is thought to be mediated by gut microbiota. However, data on causality between these observations in humans is scarce. In this regard, fecal microbiota transplantation (FMT) using healthy donors is safe and is capable of changing microbial composition in human disease. We therefore performed a double-blind randomized controlled proof-of-principle study in which individuals with hepatic steatosis on ultrasound were randomized to two study arms: lean vegan donor (allogenic n = 10) or own (autologous n = 11) FMT. Both were performed three times at 8-week intervals. A liver biopsy was performed at baseline and after 24 weeks in every subject to determine histopathology (Nonalcoholic Steatohepatitis Clinical Research Network) classification and changes in hepatic gene expression based on RNA sequencing. Secondary outcome parameters were changes in intestinal microbiota composition and fasting plasma metabolomics. We observed a trend toward improved necro-inflammatory histology, and found significant changes in expression of hepatic genes involved in inflammation and lipid metabolism following allogenic FMT. Intestinal microbial community structure changed following allogenic FMT, which was associated with changes in plasma metabolites as well as markers of . Conclusion: Allogenic FMT using lean vegan donors in individuals with hepatic steatosis shows an effect on intestinal microbiota composition, which is associated with beneficial changes in plasma metabolites and markers of steatohepatitis.

Donor metabolic characteristics drive effects of faecal microbiota transplantation on recipient insulin sensitivity, energy expenditure and intestinal transit time.

OBJECTIVE: Bariatric surgery improves glucose metabolism. Recent data suggest that faecal microbiota transplantation (FMT) using faeces from postbariatric surgery diet-induced obese mice in germ-free mice improves glucose metabolism and intestinal homeostasis. We here investigated whether allogenic FMT using faeces from post-Roux-en-Y gastric bypass donors (RYGB-D) compared with using faeces from metabolic syndrome donors (METS-D) has short-term effects on glucose metabolism, intestinal transit time and adipose tissue inflammation in treatment-naïve, obese, insulin-resistant male subjects. DESIGN: Subjects with metabolic syndrome (n=22) received allogenic FMT either from RYGB-D or METS-D. Hepatic and peripheral insulin sensitivity as well as lipolysis were measured at baseline and 2 weeks after FMT by hyperinsulinaemic euglycaemic stable isotope (2H2-glucose and 2H5-glycerol) clamp. Secondary outcome parameters were changes in resting energy expenditure, intestinal transit time, faecal short-chain fatty acids (SCFA) and bile acids, and inflammatory markers in subcutaneous adipose tissue related to intestinal microbiota composition. Faecal SCFA, bile acids, glycaemic control and inflammatory parameters were also evaluated at 8 weeks. RESULTS: We observed a significant decrease in insulin sensitivity 2 weeks after allogenic METS-D FMT (median rate of glucose disappearance: from 40.6 to 34.0 µmol/kg/min; p<0.01). Moreover, a trend (p=0.052) towards faster intestinal transit time following RYGB-D FMT was seen. Finally, we observed changes in faecal bile acids (increased lithocholic, deoxycholic and (iso)lithocholic acid after METS-D FMT), inflammatory markers (decreased adipose tissue chemokine ligand 2 (CCL2) gene expression and plasma CCL2 after RYGB-D FMT) and changes in several intestinal microbiota taxa. CONCLUSION: Allogenic FMT using METS-D decreases insulin sensitivity in metabolic syndrome recipients when compared with using post-RYGB-D. Further research is needed to delineate the role of donor characteristics in FMT efficacy in human insulin-resistant subjects. TRIAL REGISTRATION NUMBER: NTR4327.

Fecal microbiota transplantation beyond Clostridioides difficile infections.

The importance of the commensal microbiota to human health and well-being has become increasingly evident over the past decades. From a therapeutic perspective, the popularity of fecal microbiota transplantation (FMT) to restore a disrupted microbiota and amend imbalances has increased. To date, most clinical experience with FMT originates from the treatment of recurrent or refractory Clostridioides difficile infections (rCDI), with resolution rates up to 90%. In addition to CDI, a role for the intestinal microbiome has been implicated in several disorders. FMT has been tested in several randomized controlled trials for the treatment of inflammatory bowel disease, irritable bowel disease and constipation with mixed results. FMT has also been explored for extra-gastrointestinal disorders such as metabolic syndrome, hepatic encephalopathy and graft-versus-host disease. With the exception of recurrent CDI, FMT is currently used in experimental settings only and should not yet be offered as standard care. In addition, it is critical to further standardize and optimize procedures for FMT preparation. This includes determination of active components of FMT to develop (personalized) approaches to treat disease.

Inflammation Improves Glucose Homeostasis through IKKß-XBP1s Interaction.

It is widely believed that inflammation associated with obesity has an important role in the development of type 2 diabetes. IκB kinase beta (IKKß) is a crucial kinase that responds to inflammatory stimuli such as tumor necrosis factor α (TNF-α) by initiating a variety of intracellular signaling cascades and is considered to be a key element in the inflammation-mediated development of insulin resistance. We show here, contrary to expectation, that IKKß-mediated inflammation is a positive regulator of hepatic glucose homeostasis. IKKß phosphorylates the spliced form of X-Box Binding Protein 1 (XBP1s) and increases the activity of XBP1s. We have used three experimental approaches to enhance the IKKß activity in the liver of obese mice and observed increased XBP1s activity, reduced ER stress, and a significant improvement in insulin sensitivity and consequently in glucose homeostasis. Our results reveal a beneficial role of IKKß-mediated hepatic inflammation in glucose homeostasis.

The role of BRD7 in embryo development and glucose metabolism.

Bromodomain-containing protein 7 (BRD7) is a member of bromodomain-containing protein family and its function has been implicated in several diseases. We have previously shown that BRD7 plays a role in metabolic processes. However, the effect of BRD7 deficiency in glucose metabolism and its role in in vivo have not been fully revealed. Here, we report the essential role of BRD7 during embryo development. Mice homozygous for BRD7 led to embryonic lethality at mid-gestation. Homozygous BRD7 knockout (KO) mice showed retardation in development, and eventually all BRD7 KO embryos died in utero prior to E16.5. Partial knockdown of Brd7 gene displayed mild changes in glucose metabolism.

BRD7 regulates XBP1s' activity and glucose homeostasis through its interaction with the regulatory subunits of PI3K.

Bromodomain-containing protein 7 (BRD7) is a member of the bromodomain-containing protein family that is known to play a role as tumor suppressors. Here, we show that BRD7 is a component of the unfolded protein response (UPR) signaling through its ability to regulate X-box binding protein 1 (XBP1) nuclear translocation. BRD7 interacts with the regulatory subunits of phosphatidylinositol 3-kinase (PI3K) and increases the nuclear translocation of both p85α and p85ß and the spliced form of XBP1 (XBP1s). Deficiency of BRD7 blocks the nuclear translocation of XBP1s. Furthermore, our in vivo studies have shown that BRD7 protein levels are reduced in the liver of obese mice, and reinstating BRD7 levels in the liver restores XBP1s nuclear translocation, improves glucose homeostasis, and ultimately reduces the blood glucose levels in the obese and diabetic mouse models.

IRS1Ser³°7 phosphorylation does not mediate mTORC1-induced insulin resistance.

Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1(Ser307) phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1(Ser307Ala) mice and controls. Our results demonstrate that blockade of IRS1(Ser307) phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance.

A role of the bile salt receptor FXR in atherosclerosis.

This study reviews current insights into the role of bile salts and bile salt receptors on the progression and regression of atherosclerosis. Bile salts have emerged as important modifiers of lipid and energy metabolism. At the molecular level, bile salts regulate lipid and energy homeostasis mainly via the bile salt receptors FXR and TGR5. Activation of FXR has been shown to improve plasma lipid profiles, whereas Fxr(-/-) mice have increased plasma triglyceride and very-low-density lipoprotein levels. Nevertheless, high-density lipoprotein cholesterol levels are increased in these mice, suggesting that FXR has both anti- and proatherosclerotic properties. Interestingly, there is increasing evidence for a role of FXR in "nonclassical" bile salt target tissues, eg, vasculature and macrophages. In these tissues, FXR has been shown to influence vascular tension and regulate the unloading of cholesterol from foam cells, respectively. Recent publications have provided insight into the antiinflammatory properties of FXR in atherosclerosis. Bile salt signaling via TGR5 might regulate energy homeostasis, which could serve as an attractive target to increase energy expenditure and weight loss. Interventions aiming to increase cholesterol turnover (eg, by bile salt sequestration) significantly improve plasma lipid profiles and diminish atherosclerosis in animal models. Bile salt metabolism and bile salt signaling pathways represent attractive therapeutic targets for the treatment of atherosclerosis.

Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells.

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.