Amphiregulin from regulatory T cells promotes liver fibrosis and insulin resistance in non-alcoholic steatohepatitis.

Production of amphiregulin (Areg) by regulatory T (Treg) cells promotes repair after acute tissue injury. Here, we examined the function of Treg cells in non-alcoholic steatohepatitis (NASH), a setting of chronic liver injury. Areg-producing Treg cells were enriched in the livers of mice and humans with NASH. Deletion of Areg in Treg cells, but not in myeloid cells, reduced NASH-induced liver fibrosis. Chronic liver damage induced transcriptional changes associated with Treg cell activation. Mechanistically, Treg cell-derived Areg activated pro-fibrotic transcriptional programs in hepatic stellate cells via epidermal growth factor receptor (EGFR) signaling. Deletion of Areg in Treg cells protected mice from NASH-dependent glucose intolerance, which also was dependent on EGFR signaling on hepatic stellate cells. Areg from Treg cells promoted hepatocyte gluconeogenesis through hepatocyte detection of hepatic stellate cell-derived interleukin-6. Our findings reveal a maladaptive role for Treg cell-mediated tissue repair functions in chronic liver disease and link liver damage to NASH-dependent glucose intolerance.

Selective Inhibition of FOXO1 Activator/Repressor Balance Modulates Hepatic Glucose Handling.

Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.

Biochemical and cellular properties of insulin receptor signalling.

The mechanism of insulin action is a central theme in biology and medicine. In addition to the rather rare condition of insulin deficiency caused by autoimmune destruction of pancreatic ß-cells, genetic and acquired abnormalities of insulin action underlie the far more common conditions of type 2 diabetes, obesity and insulin resistance. The latter predisposes to diseases ranging from hypertension to Alzheimer disease and cancer. Hence, understanding the biochemical and cellular properties of insulin receptor signalling is arguably a priority in biomedical research. In the past decade, major progress has led to the delineation of mechanisms of glucose transport, lipid synthesis, storage and mobilization. In addition to direct effects of insulin on signalling kinases and metabolic enzymes, the discovery of mechanisms of insulin-regulated gene transcription has led to a reassessment of the general principles of insulin action. These advances will accelerate the discovery of new treatment modalities for diabetes.

An Updated Perspective on the Dual-Track Model of Enterocyte Fat Metabolism.

The small intestinal epithelium has classically been envisioned as a conduit for nutrient absorption, but appreciation is growing for a larger and more dynamic role for enterocytes in lipid metabolism. Considerable gaps remain in our knowledge of this physiology, but it appears that the enterocyte's structural polarization dictates its behavior in fat partitioning, treating fat differently based on its absorption across the apical versus the basolateral membrane. In this review, we synthesize existing data and thought on this dual-track model of enterocyte fat metabolism through the lens of human integrative physiology. The apical track includes the canonical pathway of dietary lipid absorption across the apical brush-border membrane, leading to packaging and secretion of those lipids as chylomicrons. However, this track also reserves a portion of dietary lipid within cytoplasmic lipid droplets for later uses, including the "second-meal effect," which remains poorly understood. At the same time, the enterocyte takes up circulating fats across the basolateral membrane by mechanisms that may include receptor-mediated import of triglyceride-rich lipoproteins or their remnants, local hydrolysis and internalization of free fatty acids, or enterocyte de novo lipogenesis using basolaterally absorbed substrates. The ultimate destinations of basolateral-track fat may include fatty acid oxidation, structural lipid synthesis, storage in cytoplasmic lipid droplets, or ultimate resecretion, although the regulation and purposes of this basolateral track remain mysterious. We propose that the enterocyte integrates lipid flux along both of these tracks in order to calibrate its overall program of lipid metabolism.

Sex-specific differences in metabolic outcomes after sleeve gastrectomy and intermittent fasting in obese middle-aged mice.

Despite the high prevalence of obesity among middle-aged subjects, it is unclear if sex differences in middle age affect the metabolic outcomes of obesity therapies. Accordingly, in this study, middle-aged obese female and male mice were randomized to one of three groups: sleeve gastrectomy (SG), sham surgery ad libitum (SH-AL), or sham surgery with weight matching to SG through intermittent fasting with calorie restriction (SH-IF). Comprehensive measures of energy and glucose homeostasis, including energy intake, body weight, energy expenditure, glucose and insulin tolerance, and interscapular brown adipose tissue (iBAT) sympathetic innervation density were obtained. At the end of 8 wk, SG and SH-IF females had better metabolic outcomes than their male counterparts. SG females had improved weight loss maintenance, preservation of fat-free mass (FFM), higher total energy expenditure (TEE), normal locomotor activity, and reduced plasma insulin and white adipose tissue (WAT) inflammatory markers. SH-IF females also had lower plasma insulin and WAT inflammatory markers, and higher TEE than SH-IF males, despite their lower FFM. In addition, SH-IF females had higher iBAT sympathetic nerve density than SG and SH-AL females, whereas there were no differences among males. Notably, SH-IF mice of both sexes had the most improved glucose tolerance, highlighting the benefits of fasting, irrespective of weight loss. Results from this study demonstrate that in middle-aged obese mice, female sex is associated with better metabolic outcomes after SG or IF with calorie restriction. Clinical studies are needed to determine if sex differences should guide the choice of obesity therapies.NEW & NOTEWORTHY SG or IF with calorie restriction produces better metabolic outcomes in females than in males. IF with calorie restriction prevents metabolic adaptation, even in the face of fat-free mass loss. IF with calorie restriction in females only, is associated with increased iBAT sympathetic innervation, which possibly mitigates reductions in energy expenditure secondary to fat-free mass loss. Lastly, IF leads to better glucose homeostasis than SG, irrespective of sex.

Differential effects of bariatric surgery on plasma levels of ANGPTL3 and ANGPTL4.

BACKGROUND AND AIM: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) are regulators of triglyceride storage and utilization. Bariatric surgery (BS) leads to profound changes in adipose tissue composition and energy metabolism. We evaluated the impact of BS on plasma levels of ANGPTL3 and ANGPTL4. METHODS AND RESULTS: Twenty-seven subjects affected by morbid obesity with or without type 2 diabetes (T2D) underwent Roux-en-Y gastric bypass (RYGB) and 18 patients with advanced T2D received Biliopancreatic Diversion (BPD). Fasting ANGPTL proteins levels, insulin sensitivity (evaluated by euglycemic hyperinsulinemic clamp), total bile acids (TBA) and free fatty acids (FFA) were measured at baseline and 1 year after surgery. Both surgical procedures resulted in the loss of fat mass, improved glucose control, and a ∼2-fold increase of insulin sensitivity. ANGPTL4 levels decreased significantly with both RYGB (26.6 ± 0.6 to 24.4 ± 0.3 ng/mL, p = 0.001) and BPD (27.9 ± 1.5 to 24.0 ± 0.5 ng/mL, p = 0.003). In contrast, ANGPTL3 concentrations did not change after RYGB but rose following BPD (225 ± 20 to 300 ± 15 ng/mL, p = 0.003). By multiple regression analysis, changes after BS in ANGPTL4 were independently associated with changes in blood glucose, (p = 0.0169) whereas changes in ANGPTL3 were associated with variations in FFA (p = 0.008) and insulin sensitivity (p = 0.043). CONCLUSION: Circulating ANGPTL4 is reduced by BS, probably due to the loss of fat mass and improved insulin sensitivity. Conversely, ANGPTL3 levels increased after BPD, but not after RYGB, presumably because of the metabolic changes induced by the malabsorptive effect of BPD.

Bile acid composition regulates the manganese transporter Slc30a10 in intestine.

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis.

Bile acid composition regulates GPR119-dependent intestinal lipid sensing and food intake regulation in mice.

OBJECTIVES: Lipid mediators in the GI tract regulate satiation and satiety. Bile acids (BAs) regulate the absorption and metabolism of dietary lipid in the intestine, but their effects on lipid-regulated satiation and satiety are completely unknown. Investigating this is challenging because introducing excessive BAs or eliminating BAs strongly impacts GI functions. We used a mouse model (Cyp8b1-/- mice) with normal total BA levels, but alterations in the composition of the BA pool that impact multiple aspects of intestinal lipid metabolism. We tested two hypotheses: BAs affect food intake by (1) regulating production of the bioactive lipid oleoylethanolamide (OEA), which enhances satiety; or (2) regulating the quantity and localisation of hydrolysed fat in small intestine, which controls gastric emptying and satiation. DESIGN: We evaluated OEA levels, gastric emptying and food intake in wild-type and Cyp8b1-/- mice. We assessed the role of the fat receptor GPR119 in these effects using Gpr119-/- mice. RESULTS: Cyp8b1-/- mice on a chow diet showed mild hypophagia. Jejunal OEA production was blunted in Cyp8b1-/- mice, thus these data do not support a role for this pathway in the hypophagia of Cyp8b1-/- mice. On the other hand, Cyp8b1 deficiency decreased gastric emptying, and this was dependent on dietary fat. GPR119 deficiency normalised the gastric emptying, gut hormone levels, food intake and body weight of Cyp8b1-/- mice. CONCLUSION: BAs regulate gastric emptying and satiation by determining fat-dependent GPR119 activity in distal intestine.

Inhibition of PU.1 ameliorates metabolic dysfunction and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Obesity is a well-established risk factor for type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH), but the underlying mechanisms remain incompletely understood. Herein, we aimed to identify novel pathogenic factors (and possible therapeutic targets) underlying metabolic dysfunction in the liver. METHODS: We applied a tandem quantitative proteomics strategy to enrich and identify transcription factors (TFs) induced in the obese liver. We used flow cytometry of liver cells to analyze the source of the induced TFs. We employed conditional knockout mice, shRNA, and small-molecule inhibitors to test the metabolic consequences of the induction of identified TFs. Finally, we validated mouse data in patient liver biopsies. RESULTS: We identified PU.1/SPI1, the master hematopoietic regulator, as one of the most upregulated TFs in livers from diet-induced obese (DIO) and genetically obese (db/db) mice. Targeting PU.1 in the whole liver, but not hepatocytes alone, significantly improved glucose homeostasis and suppressed liver inflammation. Consistently, treatment with the PU.1 inhibitor DB1976 markedly reduced inflammation and improved glucose homeostasis and dyslipidemia in DIO mice, and strongly suppressed glucose intolerance, liver steatosis, inflammation, and fibrosis in a dietary NASH mouse model. Furthermore, hepatic PU.1 expression was positively correlated with insulin resistance and inflammation in liver biopsies from patients. CONCLUSIONS: These data suggest that the elevated hematopoietic factor PU.1 promotes liver metabolic dysfunction, and may be a useful therapeutic target for obesity, insulin resistance/T2D, and NASH. LAY SUMMARY: Expression of the immune regulator PU.1 is increased in livers of obese mice and people. Blocking PU.1 improved glucose homeostasis, and reduced liver steatosis, inflammation and fibrosis in mouse models of non-alcoholic steatohepatitis. Inhibition of PU.1 is thus a potential therapeutic strategy for treating obesity-associated liver dysfunction and metabolic diseases.

Glucose-6-Phosphate Regulates Hepatic Bile Acid Synthesis in Mice.

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient-sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose-6-phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver-specific glucose-6-phosphatase knockout (L-G6pc-/- ) mice, and mice treated with a pharmacological inhibitor of the G6P transporter. Hepatic G6P accumulation induces sterol 12α-hydroxylase (Cyp8b1) expression, which is mediated by the major glucose-sensitive transcription factor, carbohydrate response element-binding protein (ChREBP). Activation of the G6P-ChREBP-CYP8B1 axis increases the relative abundance of cholic-acid-derived bile acids and induces physiologically relevant shifts in bile composition. The G6P-ChREBP-dependent change in bile acid hydrophobicity associates with elevated plasma campesterol/cholesterol ratio and reduced fecal neutral sterol loss, compatible with enhanced intestinal cholesterol absorption. Conclusion: We report that G6P, the primary intracellular metabolite of glucose, controls hepatic bile acid synthesis. Our work identifies hepatic G6P-ChREBP-CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.

Metformin and AMP Kinase Activation Increase Expression of the Sterol Transporters ABCG5/8 (ATP-Binding Cassette Transporter G5/G8) With Potential Antiatherogenic Consequences.

OBJECTIVE: The mechanisms underlying the cardiovascular benefit of the anti-diabetic drug metformin are poorly understood. Recent studies have suggested metformin may upregulate macrophage reverse cholesterol transport. The final steps of reverse cholesterol transport are mediated by the sterol transporters, ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8), which facilitate hepato-biliary transport of cholesterol. This study was undertaken to assess the possibility that metformin induces Abcg5 and Abcg8 expression in liver and to elucidate the underlying mechanisms. APPROACH AND RESULTS: Metformin-treated mouse or human primary hepatocytes showed increased expression of Abcg5/8 and the bile salt export pump, Bsep. Administration of metformin to Western-type diet-fed mice showed significant upregulation of Abcg5/8 and Bsep. This resulted in increased initial clearance of 3H-cholesteryl ester HDL (high-density lipoprotein) from plasma. However, fecal 3H-cholesterol output was only marginally increased, possibly reflecting increased hepatic Ldlr (low-density lipoprotein receptor) expression, which would increase nonradiolabeled cholesterol uptake. Abcg5/8 undergo strong circadian variation. Available chromatin immunoprecipitation-Seq data suggested multiple binding sites for Period 2, a transcriptional repressor, within the Abcg5/8 locus. Addition of AMPK (5' adenosine monophosphate-activated protein kinase) agonists decreased Period 2 occupancy, suggesting derepression of Abcg5/8. Inhibition of ATP citrate lyase, which generates acetyl-CoA from citrate, also decreased Period 2 occupancy, with concomitant upregulation of Abcg5/8. This suggests a mechanistic link between feeding-induced acetyl-CoA production and decreased cholesterol excretion via Period 2, resulting in inhibition of Abcg5/8 expression. CONCLUSIONS: Our findings provide partial support for the concept that metformin may provide cardiovascular benefit via increased reverse cholesterol transport but also indicate increased Ldlr expression as a potential additional mechanism. AMPK activation or ATP citrate lyase inhibition may mediate antiatherogenic effects through increased ABCG5/8 expression.

Unexplained reciprocal regulation of diabetes and lipoproteins.

PURPOSE OF REVIEW: Type 2 diabetes is associated with a characteristic dyslipidemia that may exacerbate cardiovascular risk. The causes of, and the effects of new antihyperglycemia medications on, this dyslipidemia, are under investigation. In an unexpected reciprocal manner, lowering LDL-cholesterol with statins slightly increases the risk of diabetes. Here we review the latest findings. RECENT FINDINGS: The inverse relationship between LDL-cholesterol and diabetes has now been confirmed by multiple lines of evidence. This includes clinical trials, genetic instruments using aggregate single nucleotide polymorphisms, as well as at least eight individual genes - HMGCR, NPC1L1, HNF4A, GCKR, APOE, PCKS9, TM6SF2, and PNPLA3 - support this inverse association. Genetic and pharmacologic evidence suggest that HDL-cholesterol may also be inversely associated with diabetes risk. Regarding the effects of diabetes on lipoproteins, new evidence suggests that insulin resistance but not diabetes per se may explain impaired secretion and clearance of VLDL-triglycerides. Weight loss, bariatric surgery, and incretin-based therapies all lower triglycerides, whereas SGLT2 inhibitors may slightly increase HDL-cholesterol and LDL-cholesterol. SUMMARY: Diabetes and lipoproteins are highly interregulated. Further research is expected to uncover new mechanisms governing the metabolism of glucose, fat, and cholesterol. This topic has important implications for treating type 2 diabetes and cardiovascular disease.

Cyp8b1 ablation prevents Western diet-induced weight gain and hepatic steatosis because of impaired fat absorption.

Bile acids (BAs) are cholesterol derivatives that regulate lipid metabolism, through their dual abilities to promote lipid absorption and activate BA receptors. However, different BA species have varying abilities to perform these functions. Eliminating 12α-hydroxy BAs in mice via Cyp8b1 knockout causes low body weight and improved glucose tolerance. The goal of this study was to determine mechanisms of low body weight in Cyp8b1-/- mice. We challenged Cyp8b1-/- mice with a Western-type diet and assessed body weight and composition. We measured energy expenditure, fecal calories, and lipid absorption and performed lipidomic studies on feces and intestine. We investigated the requirement for dietary fat in the phenotype using a fat-free diet. Cyp8b1-/- mice were resistant to Western diet-induced body weight gain, hepatic steatosis, and insulin resistance. These changes were associated with increased fecal calories, due to malabsorption of hydrolyzed dietary triglycerides. This was reversed by treating the mice with taurocholic acid, the major 12α-hydroxylated BA species. The improvements in body weight and steatosis were normalized by feeding mice a fat-free diet. The effects of BA composition on intestinal lipid handling are important for whole body energy homeostasis. Thus modulating BA composition is a potential tool for obesity or diabetes therapy.

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing.

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.

Alteration of serum bile acids in amyotrophic lateral sclerosis.

Hydrophilic endogenous bile acids ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), and glucourosodeoxycholic acid (GUDCA) have suggested neuroprotective effects. We performed a case-control study to examine the association between ALS diagnosis and serum levels of bile acids. Sporadic and familial ALS patients, age- and sex-matched healthy controls, and presymptomatic gene carriers who donated blood samples were included. Non-fasted serum samples stored at -80°C were used for the analysis. Serum bile acid levels were measured by liquid chromatography-mass spectrometry (LC-MS). Concentrations of 15 bile acids were obtained, 5 non-conjugated and 10 conjugated, and compared between ALS versus control groups (presymptomatic gene carriers + healthy controls) using the Wilcoxon-Rank-Sum test. In total, 80 participants were included: 31 ALS (17 sporadic and 14 familial ALS); 49 controls (22 gene carriers, 27 healthy controls). The mean age was 50 years old and 50% were male. In the ALS group, 45% had familial disease with a pathogenic variant in C9orf72 (29%), TARDBP (10%), FUS (3%), and CHCHD10 (3%) genes. In the control group, 43% carried pathogenic variants: C9orf72 (27%), SOD1 (10%), and FUS (6%). The serum levels of UDCA, TUDCA, and GUDCA trended higher in the ALS group compared to controls (median 27 vs. 7 nM, 4 vs. 3 nM, 110 vs. 47 nM, p-values 0.04, 0.06, 0.04, respectively). No significant group differences were found in other bile acids serum levels. In conclusion, the serum level of UDCA, TUDCA, GUDCA trended higher in ALS patients compared to controls, and no evidence of deficiencies was found.

Insulin sensitization by hepatic FoxO deletion is insufficient to lower atherosclerosis in mice.

Background­: Type 2 diabetes is associated with an increased risk of atherosclerotic cardiovascular disease. It has been suggested that insulin resistance underlies this link, possibly by altering the functions of cells in the artery wall. We aimed to test whether improving systemic insulin sensitivity reduces atherosclerosis. Methods­: We used mice that are established to have improved systemic insulin sensitivity: those lacking FoxO transcription factors in hepatocytes. Three hepatic FoxO isoforms (FoxO1, FoxO3, and FoxO4) function together to promote hepatic glucose output, and ablating them lowers glucose production, lowers circulating glucose and insulin, and improves systemic insulin sensitivity. We made these mice susceptible to atherosclerosis in two different ways, by injecting them with gain-of-function AAV8.mPcsk9D377Y and by crossing with Ldlr-/- mice. Results­: We verified that hepatic FoxO ablation improves systemic insulin sensitivity in these atherosclerotic settings. We observed that FoxO deficiency caused no reductions in atherosclerosis, and in some cases increased atherosclerosis. These phenotypes coincided with large increases in circulating triglycerides in FoxO-ablated mice. Conclusions­: These findings suggest that systemic insulin sensitization is insufficient to reduce atherosclerosis.

The 16α-hydroxylated Bile Acid, Pythocholic Acid Decreases Food Intake and Increases Oleoylethanolamide in Male Mice.

Modulation of bile acid (BA) structure is a potential strategy for obesity and metabolic disease treatment. BAs act not only as signaling molecules involved in energy expenditure and glucose homeostasis, but also as regulators of food intake. The structure of BAs, particularly the position of the hydroxyl groups of BAs, impacts food intake partly by intestinal effects: (1) modulating the activity of N-acyl phosphatidylethanolamine phospholipase D, which produces the anorexigenic bioactive lipid oleoylethanolamide (OEA) or (2) regulating lipid absorption and the gastric emptying-satiation pathway. We hypothesized that 16α-hydroxylated BAs uniquely regulate food intake because of the long intermeal intervals in snake species in which these BAs are abundant. However, the effects of 16α-hydroxylated BAs in mammals are completely unknown because they are not naturally found in mammals. To test the effect of 16α-hydroxylated BAs on food intake, we isolated the 16α-hydroxylated BA pythocholic acid from ball pythons (Python regius). Pythocholic acid or deoxycholic acid (DCA) was given by oral gavage in mice. DCA is known to increase N-acyl phosphatidylethanolamine phospholipase D activity better than other mammalian BAs. We evaluated food intake, OEA levels, and gastric emptying in mice. We successfully isolated pythocholic acid from ball pythons for experimental use. Pythocholic acid treatment significantly decreased food intake in comparison to DCA treatment, and this was associated with increased jejunal OEA, but resulted in no change in gastric emptying or lipid absorption. The exogenous BA pythocholic acid is a novel regulator of food intake and the satiety signal for OEA in the mouse intestine.

MAD2-Dependent Insulin Receptor Endocytosis Regulates Metabolic Homeostasis.

Insulin activates insulin receptor (IR) signaling and subsequently triggers IR endocytosis to attenuate signaling. Cell division regulators MAD2, BUBR1, and p31comet promote IR endocytosis on insulin stimulation. Here, we show that genetic ablation of the IR-MAD2 interaction in mice delays IR endocytosis, increases IR levels, and prolongs insulin action at the cell surface. This in turn causes a defect in insulin clearance and increases circulating insulin levels, unexpectedly increasing glucagon levels, which alters glucose metabolism modestly. Disruption of the IR-MAD2 interaction increases serum fatty acid concentrations and hepatic fat accumulation in fasted male mice. Furthermore, disruption of the IR-MAD2 interaction distinctly changes metabolic and transcriptomic profiles in the liver and adipose tissues. Our findings establish the function of cell division regulators in insulin signaling and provide insights into the metabolic functions of IR endocytosis. ARTICLE HIGHLIGHTS: The physiological role of IR endocytosis in insulin sensitivity remains unclear. Disruption of the IR-MAD2 interaction delays IR endocytosis and prolongs insulin signaling. IR-MAD2 controls insulin clearance and glucose metabolism. IR-MAD2 maintains energy homeostasis.

FOXO1 Is Present in Stomach Epithelium and Determines Gastric Cell Distribution.

BACKGROUND AND AIMS: Stomach cells can be converted to insulin-producing cells by Neurog3, MafA, and Pdxl over-expression. Enteroendocrine cells can be similarly made to produce insulin by the deletion of FOXO1. Characteristics and functional properties of FOXO1-expressing stomach cells are not known. METHODS: Using mice bearing a FOXO1-GFP knock-in allele and primary cell cultures, we examined the identity of FOXO1-expressing stomach cells and analyzed their features through loss-of-function studies with red-to-green fluorescent reporters. RESULTS: FOXO1 localizes to a subset of Neurog3 and parietal cells. FOXO1 deletion ex vivo or in vivo using Neurog3-cre or Atp4b-cre increased numbers of parietal cells, generated insulin- and C-peptide-immunoreactive cells, and raised Neurog3 messenger RNA. Gene expression and ChIP- seq experiments identified the cell cycle regulator cyclin E1 (CCNE1) as a FOXO1 target. CONCLUSION: FOXO1 is expressed in a subset of stomach cells. Its ablation increases parietal cells and yields insulin-immunoreactive cells, consistent with a role in lineage determination.

Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway.

Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway.