An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Macronutrients effects on satiety and food intake in older and younger adults: A randomised controlled trial.

Older adults are advised to increase their protein intake to maintain their muscle mass. However, protein is considered the most satiating macronutrient and this recommendation may cause a decrease in total energy intake. To date, satiety studies comparing all three macronutrients have been undertaken in young adults, and it is unclear if the same response is seen in older adults. The objective of this study was to compare the effect of preloads high in protein, fat, and carbohydrate but equal in energy (∼300 kcal) and volume (250 ml) on energy intake, perceived appetite, and gastric emptying in younger and older adults. Twenty older and 20 younger adults completed a single-blinded randomised crossover trial involving three study visits. Participants consumed a standard breakfast, followed by a preload milkshake high in either carbohydrate, fat, or protein. Three hours after the preload, participants were offered an ad libitum meal to assess food intake. Visual analogue scales were used to measure perceived appetite and gastric emptying was measured via the 13C-octanoic acid breath test. There was no significant effect of preload type or age on energy intake either at the ad libitum meal, self-recorded food intake for the rest of the test day or subjective appetite ratings. There was a significant effect of preload type on gastric emptying latency phase and ascension time, and an effect of age on gastric emptying latency and lag phase such that older adults had faster emptying. In conclusion, energy intake, and perceived appetite were not affected by macronutrient content of the preloads in both younger and older adults, but gastric emptying times differed.

Restoring polyamine levels by supplementation of spermidine modulates hepatic immune landscape in murine model of NASH.

AIMS: To determine if changes in polyamines metabolism occur during non-alcoholic steatohepatitis (NASH) in human patients and mice, as well as to assess systemic and liver-specific effects of spermidine administration into mice suffering from advanced NASH. MATERIALS AND METHODS: Human fecal samples were collected from 50 healthy and 50 NASH patients. For the preclinical studies C57Bl6/N male mice fed GAN or NIH-31 diet for 6 months were ordered from Taconic and liver biopsy was performed. Based on severity of liver fibrosis, body composition and body weight, the mice from both dietary groups were randomized into another two groups: half receiving 3 mM spermidine in drinking water, half normal water for subsequent 12 weeks. Body weight was measured weekly and glucose tolerance and body composition were assessed at the end. Blood and organs were collected during necropsy, and intrahepatic immune cells were isolated for flow cytometry analysis. RESULTS: Metabolomic analysis of human and murine feces confirmed that levels of polyamines decreased along NASH progression. Administration of exogenous spermidine to the mice from both dietary groups did not affect body weight, body composition or adiposity. Moreover, incidence of macroscopic hepatic lesions was higher in NASH mice receiving spermidine. On the other hand, spermidine normalized numbers of Kupffer cells in the livers of mice suffering from NASH, although these beneficial effects did not translate into improved liver steatosis or fibrosis severity. CONCLUSION: Levels of polyamines decrease during NASH in mice and human patients but spermidine administration does not improve advanced NASH.

FGF21 protects against hepatic lipotoxicity and macrophage activation to attenuate fibrogenesis in nonalcoholic steatohepatitis.

Analogues of the hepatokine fibroblast growth factor 21 (FGF21) are in clinical development for type 2 diabetes and nonalcoholic steatohepatitis (NASH) treatment. Although their glucose-lowering and insulin-sensitizing effects have been largely unraveled, the mechanisms by which they alleviate liver injury have only been scarcely addressed. Here, we aimed to unveil the mechanisms underlying the protective effects of FGF21 on NASH using APOE*3-Leiden.CETP mice, a well-established model for human-like metabolic diseases. Liver-specific FGF21 overexpression was achieved in mice, followed by administration of a high-fat high-cholesterol diet for 23 weeks. FGF21 prevented hepatic lipotoxicity, accompanied by activation of thermogenic tissues and attenuation of adipose tissue inflammation, improvement of hyperglycemia and hypertriglyceridemia, and upregulation of hepatic programs involved in fatty acid oxidation and cholesterol removal. Furthermore, FGF21 inhibited hepatic inflammation, as evidenced by reduced Kupffer cell (KC) activation, diminished monocyte infiltration, and lowered accumulation of monocyte-derived macrophages. Moreover, FGF21 decreased lipid- and scar-associated macrophages, which correlated with less hepatic fibrosis as demonstrated by reduced collagen accumulation. Collectively, hepatic FGF21 overexpression limits hepatic lipotoxicity, inflammation, and fibrogenesis. Mechanistically, FGF21 blocks hepatic lipid influx and accumulation through combined endocrine and autocrine signaling, respectively, which prevents KC activation and lowers the presence of lipid- and scar-associated macrophages to inhibit fibrogenesis.

High-calorie modern diets have contributed to growing rates of obesity-linked diseases. One such disease is non-alcoholic steatohepatitis or NASH for short, which affects about 5% of adults in the United States. The livers of people with this condition accumulate fat, become inflamed, and develop scar tissue. People with NASH are also at increased risk of developing liver cancer, type 2 diabetes, and heart disease. Currently, no drugs are available to treat the condition and prevent such severe complications. Previous research has shown the liver produces a stress hormone, called FGF21, in response to fat accumulation. This hormone boosts fat burning and so helps to reduce excess fat in the liver. Drugs that mimic FGF21 have already been developed for type 2 diabetes. But so far, it was unclear if such drugs could also help reduce liver inflammation and scarring in patients with NASH. Liu et al. show that increasing the production of FGF21 in mice with a NASH-like condition reduces fat accumulation, liver inflammation, and scarring. In the experiments, the researchers used gene therapy to ramp up FGF21 production in the livers of mice that develop obesity and a NASH-like condition when fed a high-fat diet for 23 weeks. Increasing FGF21 production prevented the mice from developing obesity while on the high fat diet by making the body burn more fat in the liver and brown fat tissue. The treatment also reduced inflammation and prevented scarring by reducing the number and activity of immune cells in the liver. Increasing the production of the stress hormone FGF21 prevents diet-induced obesity and NASH in mice fed a high-fat diet. More studies are necessary to determine if using gene therapy to increase FGF21 may also cause weight loss and could reverse liver damage in mice that already have NASH. If this approach is effective in mice, it may be tested in humans, a process that may take several years. If human studies are successful, FGF21-boosting therapy might provide a new treatment approach for obesity or NASH.

Peptide-YY3-36/glucagon-like peptide-1 combination treatment of obese diabetic mice improves insulin sensitivity associated with recovered pancreatic ß-cell function and synergistic activation of discrete hypothalamic and brainstem neuronal circuitries.

OBJECTIVE: Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. METHODS: In this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. RESULTS: Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous ß-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. CONCLUSIONS: These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous ß-cell function. These data also highlight the potential association between the gut-brain axis in control of metabolic homeostasis.

Resolution of NASH and hepatic fibrosis by the GLP-1R/GcgR dual-agonist Cotadutide via modulating mitochondrial function and lipogenesis.

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a GLP-1R/GcgR agonist, was shown to reduce blood glycemia, body weight and hepatic steatosis in patients with T2DM. Here, we demonstrate that the effects of Cotadutide to reduce body weight, food intake and improve glucose control are predominantly mediated through the GLP-1 signaling, while, its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signaling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with Cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, which are consistent with a unique therapeutic contribution of GcgR agonism by Cotadutide in vivo. Significantly, Cotadutide also alleviated fibrosis to a greater extent than Liraglutide or Obeticholic acid (OCA), despite adjusting dose to achieve similar weight loss in 2 preclinical mouse models of NASH. Thus Cotadutide, via direct hepatic (GcgR) and extra-hepatic (GLP-1R) effects, exerts multi-factorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

Combined loss of GLP-1R and Y2R does not alter progression of high-fat diet-induced obesity or response to RYGB surgery in mice.

OBJECTIVE: Understanding the mechanisms underlying the remarkable beneficial effects of gastric bypass surgery is important for the development of non-surgical therapies or less invasive surgeries in the fight against obesity and metabolic disease. Although the intestinal L-cell hormones glucagon-like peptide-1 (GLP-1) and peptide tyrosine-tyrosine (PYY) have attracted the most attention, direct tests in humans and rodents with pharmacological blockade or genetic deletion of either the GLP1-receptor (GLP1R) or the Y2-receptor (Y2R) were unable to confirm their critical roles in the beneficial effects gastric bypass surgery on body weight and glucose homeostasis. However, new awareness of the power of combinatorial therapies in the treatment of metabolic disease would suggest that combined blockade of more than one signaling pathway may be necessary to reverse the beneficial effects of bariatric surgery. METHODS: The metabolic effects of high-fat diet and the ability of Roux-en-Y gastric bypass surgery to lower food intake and body weight, as well as improve glucose handling, was tested in GLP1R and Y2R-double knockout (GLP1RKO/Y2RKO) and C57BL6J wildtype (WT) mice. RESULTS: GLP1RKO/Y2RKO and WT mice responded similarly for up to 20 weeks on high-fat diet and 16 weeks after RYGB. There were no significant differences in loss of body and liver weight, fat mass, reduced food intake, relative increase in energy expenditure, improved fasting insulin, glucose tolerance, and insulin tolerance between WT and GLP1RKO/Y2RKO mice after RYGB. CONCLUSIONS: Combined loss of GLP1R and Y2R-signaling was not able to negate or attenuate the beneficial effects of RYGB on body weight and glucose homeostasis in mice, suggesting that a larger number of signaling pathways is involved or that the critical pathway has not yet been identified.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution.

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Nonalcoholic steatohepatitis severity is defined by a failure in compensatory antioxidant capacity in the setting of mitochondrial dysfunction.

AIM: To comprehensively evaluate mitochondrial (dys) function in preclinical models of nonalcoholic steatohepatitis (NASH). METHODS: We utilized two readily available mouse models of nonalcoholic fatty liver disease (NAFLD) with or without progressive fibrosis: Lepob/Lepob (ob/ob) and FATZO mice on high trans-fat, high fructose and high cholesterol (AMLN) diet. Presence of NASH was assessed using immunohistochemical and pathological techniques, and gene expression profiling. Morphological features of mitochondria were assessed via transmission electron microscopy and immunofluorescence, and function was assessed by measuring oxidative capacity in primary hepatocytes, and respiratory control and proton leak in isolated mitochondria. Oxidative stress was measured by assessing activity and/or expression levels of Nrf1, Sod1, Sod2, catalase and 8-OHdG. RESULTS: When challenged with AMLN diet for 12 wk, ob/ob and FATZO mice developed steatohepatitis in the presence of obesity and hyperinsulinemia. NASH development was associated with hepatic mitochondrial abnormalities, similar to those previously observed in humans, including mitochondrial accumulation and increased proton leak. AMLN diet also resulted in increased numbers of fragmented mitochondria in both strains of mice. Despite similar mitochondrial phenotypes, we found that ob/ob mice developed more advanced hepatic fibrosis. Activity of superoxide dismutase (SOD) was increased in ob/ob AMLN mice, whereas FATZO mice displayed increased catalase activity, irrespective of diet. Furthermore, 8-OHdG, a marker of oxidative DNA damage, was significantly increased in ob/ob AMLN mice compared to FATZO AMLN mice. Therefore, antioxidant capacity reflected as the ratio of catalase:SOD activity was similar between FATZO and C57BL6J control mice, but significantly perturbed in ob/ob mice. CONCLUSION: Oxidative stress, and/or the capacity to compensate for increased oxidative stress, in the setting of mitochondrial dysfunction, is a key factor for development of hepatic injury and fibrosis in these mouse models.

A GLP-1:CCK fusion peptide harnesses the synergistic effects on metabolism of CCK-1 and GLP-1 receptor agonism in mice.

Combination approaches for the treatment of metabolic diseases such as obesity and diabetes are becoming increasingly relevant. Co-administration of a glucagon-like peptide-1 receptor (GLP-1R) agonist with a cholecystokinin receptor-1 (CCKR1) agonist exert synergistic effects on weight loss in obese rodents. Here, we report on the effects of a novel fusion peptide (C2816) comprised of a stabilized GLP-1R agonist, AC3174, and a CCKR1-selective agonist, AC170222. C2816 was constructed such that AC3174 was linked to the N-terminus of AC170222, thus preserving the C-terminal amide of the CCK moiety. In functional in vitro assays C2816 retained full agonism at GLP-1R and CCKR1 at lower potency compared to parent molecules, whereas a previously reported fusion peptide in the opposite orientation, (pGlu-Gln)-CCK-8/exendin-4, exhibited no activity at either receptor. Acutely, in vivo, C2816 increased cFos in key central nuclei relevant to feeding behavior, and reduced food intake in wildtype (WT), but less so in GLP-1R-deficient (GLP-1RKO), mice. In sub-chronic studies in diet-induced obese (DIO) mice, C2816 exerted superior reduction in body weight compared to co-administration of AC3174 and AC170222 albeit at a higher molar dose. These data suggest that the synergistic pharmacological effects of GLP-1 and CCK pathways can be harnessed in a single therapeutic peptide.

Superior reductions in hepatic steatosis and fibrosis with co-administration of a glucagon-like peptide-1 receptor agonist and obeticholic acid in mice.

OBJECTIVE: Nonalcoholic steatohepatitis (NASH) is an unmet need associated with metabolic syndrome. There are no approved therapies for NASH; however, glucagon-like peptide-1 receptor (GLP-1R) and farnesoid-X receptor (FXR) agonists are promising drug targets. We investigated the therapeutic effects of co-administration of a GLP-1R agonist, IP118, with FXR agonist obeticholic acid (OCA) in mice. METHODS: OCA and IP118 alone and in combination were sub-chronically administered to Lepob/Lepob mice with diet-induced NASH or diet-induced obese (DIO) mice. Metabolic (body weight and glucose) and liver (biochemical and histological) endpoints were assessed. NASH severity in Lepob/Lepob mice was graded using a customized integrated scoring system. RESULTS: OCA reduced liver weight and lipid in NASH mice (both by -17%) but had no effect on plasma ALT or AST levels. In contrast, IP118 significantly reduced liver weight (-21%), liver lipid (-15%), ALT (-29%), and AST (-27%). The combination of OCA + IP118 further reduced liver weight (-29%), liver lipid (-22%), ALT (-39%), and AST (-36%). Combination therapy was superior to monotherapies in reducing hepatic steatosis, inflammation, and fibrosis. Hepatic improvements with IP118 and OCA + IP118 were associated with reduced body weight (-4.3% and -3.5% respectively) and improved glycemic control in OCA + IP118-treated mice. In DIO mice, OCA + IP118 co-administration reduced body weight (-25.3%) to a greater degree than IP118 alone (-12.5%) and further improved glucose tolerance and reduced hepatic lipid. CONCLUSION: Our data suggest a complementary or synergistic therapeutic effect of GLP-1R and FXR agonism in mouse models of metabolic disease and NASH.

Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

Protective Role for B-1b B Cells and IgM in Obesity-Associated Inflammation, Glucose Intolerance, and Insulin Resistance.

OBJECTIVE: Little is known about the role(s) B cells play in obesity-induced metabolic dysfunction. This study used a mouse with B-cell-specific deletion of Id3 (Id3(Bcell KO)) to identify B-cell functions involved in the metabolic consequences of obesity. APPROACH AND RESULTS: Diet-induced obese Id3(Bcell KO) mice demonstrated attenuated inflammation and insulin resistance in visceral adipose tissue (VAT), and improved systemic glucose tolerance. VAT in Id3(Bcell KO) mice had increased B-1b B cells and elevated IgM natural antibodies to oxidation-specific epitopes. B-1b B cells reduced cytokine production in VAT M1 macrophages, and adoptively transferred B-1b B cells trafficked to VAT and produced natural antibodies for the duration of 13-week studies. B-1b B cells null for Id3 demonstrated increased proliferation, established larger populations in Rag1(-/-) VAT, and attenuated diet-induced glucose intolerance and VAT insulin resistance in Rag1(-/-) hosts. However, transfer of B-1b B cells unable to secrete IgM had no effect on glucose tolerance. In an obese human population, results provided the first evidence that B-1 cells are enriched in human VAT and IgM antibodies to oxidation-specific epitopes inversely correlated with inflammation and insulin resistance. CONCLUSIONS: NAb-producing B-1b B cells are increased in Id3(Bcell KO) mice and attenuate adipose tissue inflammation and glucose intolerance in diet-induced obese mice. Additional findings are the first to identify VAT as a reservoir for human B-1 cells and to link anti-inflammatory IgM antibodies with reduced inflammation and improved metabolic phenotype in obese humans.

Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue.

OBJECTIVE: Macrophages are important producers of obesity-induced MCP-1; however, initial obesity-induced increases in MCP-1 production precede M1 macrophage accumulation in visceral adipose tissue (VAT). The initial cellular source of obesity-induced MCP-1 in vivo is currently unknown. Preliminary reports based on in vitro studies of preadipocyte cell lines and adherent stroma-vascular fraction cells suggest that resident stromal cells express MCP-1. In the past several years, elegant methods of identifying adipocyte progenitor cells (AdPCs) have become available, making it possible to study these cells in vivo. We have previously published that global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet-induced obesity, but it is unclear if Id3 plays a role in diet-induced MCP-1 production. We sought to determine the initial cellular source of MCP-1 and identify molecular regulators mediating MCP-1 production. METHODS: Id3 (+/+) and Id3 (-/-) mice were fed either a standard chow or HFD for varying lengths of time. Flow cytometry, semi-quantitative real-time PCR, ELISAs and adoptive transfers were used to assess the importance of AdPCs during diet-induced obesity. Flow cytometry was also performed on a cohort of 14 patients undergoing bariatric surgery. RESULTS: Flow cytometry identified committed CD45(-)CD31 (-) Ter119(-)CD29(+)CD34(+)Sca-1(+)CD24(-) adipocyte progenitor cells as producers of high levels of MCP-1 in VAT. High-fat diet increased AdPC numbers, an effect dependent on Id3. Loss of Id3 increased p21(Cip1) levels and attenuated AdPC proliferation, resulting in reduced MCP-1 and M1 macrophage accumulation in VAT, compared to Id3 (+/+) littermate controls. AdPC rescue by adoptive transfer of 50,000 Id3 (+/+) AdPCs into Id3 (-/-) recipient mice increased MCP-1 levels and M1 macrophage number in VAT. Additionally, flow cytometry identified MCP-1-producing CD45(-)CD31(-)CD34(+)CD44(+)CD90(+) AdPCs in human omental and subcutaneous adipose tissue, with a higher percentage in omental adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in visceral adipose tissue. CONCLUSIONS: This study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21(Cip1) expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.

Helix-loop-helix factor inhibitor of differentiation 3 regulates interleukin-5 expression and B-1a B cell proliferation.

OBJECTIVE: Natural immunity is emerging as an important mediator of protection from atherogenesis. Natural IgM antibodies that recognize oxidation-specific epitopes on low-density lipoprotein or phospholipids and the B-1a B cells that produce them attenuate atherosclerosis. We previously demonstrated that Apoe(-/-) mice globally deficient in the helix-loop-helix protein inhibitor of differentiation 3 (Id3) develop early diet-induced atherosclerosis. Furthermore, B cell-mediated attenuation of atherosclerosis in B cell-deficient mice was dependent on Id3. Here, we sought to determine whether Id3 regulates B-1a B cells and the natural antibodies that they produce and identify mechanisms mediating these effects. APPROACH AND RESULTS: Mice lacking Id3 had significantly fewer B-1a B cells in the spleen and peritoneal cavity and reduced serum levels of the natural antibody E06. B cell-specific deletion of Id3 revealed that this effect was not because of the loss of Id3 in B cells. Interleukin (IL)-33 induced abundant, Id3-dependent IL-5 production in the recently identified innate lymphoid cell, the natural helper (NH) cell, but not Th2 or mast cells. In addition, delivery of IL-5 to Id3-deficient mice restored B-1a B cell proliferation. B-1a B cells were present in aortic samples also containing NH cells. Aortic NH cells produced IL-5, a B-1a B cell mitogen in response to IL-33 stimulation. CONCLUSIONS: These studies are the first to identify NH and B-1a B cells in the aorta and provide evidence that Id3 is a key regulator of NH cell IL-5 production and B-1a B cell homeostasis.

Loss of Id3 increases VCAM-1 expression, macrophage accumulation, and atherogenesis in Ldlr-/- mice.

OBJECTIVE: Inhibitor of differention-3 (Id3) promotes B cells homing to the aorta and atheroprotection in Apoe(-/-) mice. We sought to determine the impact of loss of Id3 in the Ldlr((-/-)) mouse model of diet-induced atherosclerosis and identify novel Id3 targets in the vessel wall. METHODS AND RESULTS: Ex vivo optical imaging confirmed that Id3((-/-)) Ldlr((-/-)) mice have significantly fewer aortic B cells than Id3((+/+)) Ldlr(-/-) mice. After 8 and 16 weeks of Western diet, Id3((-/-)) Ldlr((-/-)) mice developed significantly more atherosclerosis than Id3((+/+)) Ldlr((-/-)) mice, with Id3(+/-) Ldlr(-/-) mice demonstrating an intermediate phenotype. There were no differences in serum lipid levels between genotypes. Immunostaining demonstrated that aortas from Id3((-/-)) Ldlr((-/-)) mice had greater intimal macrophage density and C-C chemokine ligand 20 and vascular cell adhesion molecule 1 (VCAM-1) expression compared with Id3((+/+)) Ldlr(-/-) mice. Real-time polymerase chain reaction demonstrated increased VCAM-1 mRNA levels in the aortas of Id3(-/-) Ldlr(-/-) mice. Primary vascular smooth muscle cells from Id3((-/-)) mice expressed greater amounts of VCAM-1 protein compared with control. Gain and loss of function studies in primary vascular smooth muscle cells identified a role for Id3 in repressing VCAM-1 promoter activation. Chromatin immunoprecipitation demonstrated interaction of E12 with the VCAM-1 promoter, which is inhibited by Id3. CONCLUSIONS: Id3 is an atheroprotective transcription regulator with targets in both B cells and vessel wall cells leading to reduced macrophage accumulation and reduced atherosclerosis formation.

Inhibitor of differentiation-3 mediates high fat diet-induced visceral fat expansion.

OBJECTIVE: Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. METHODS AND RESULTS: Id3(-/-) mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3(-/-) mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. CONCLUSIONS: Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

B-cell aortic homing and atheroprotection depend on Id3.

RATIONALE: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. OBJECTIVE: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. METHODS AND RESULTS: Loss of Id3 in Apoe(-/-) mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3(-/-) Apoe(-/-) mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3(-/-) Apoe(-/-) mice into B-cell-deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3(+/+) Apoe(-/-) mice, but aortic reconstitution and B-cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. CONCLUSIONS: Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell-mediated protection from early atherosclerosis.

Deficiency of a transcriptional regulator, inhibitor of differentiation 3, induces glomerulonephritis in apolipoprotein E-deficient mice: a model linking hyperlipidemia and renal disease.

The clinical association between hyperlipidemia and renal disease is well established, yet hyperlipidemia as a cause for renal disease is rare. Apolipoprotein E-deficient (ApoE(-/-)) mice develop hyperlipidemia and are a model for atherosclerosis. Introducing deficiency of inhibitor of differentiation 3 (Id3) in ApoE(-/-) mice further exacerbates atherosclerosis. ID3 is a transcription regulator expressed in multiple cell types. Id3(-/-) mice develop antibodies to self-antigens and salivary gland autoimmunity. This study was undertaken to investigate a link between hyperlipidemia, autoimmunity, and renal disease. ApoE(-/-), Id3(-/-), and ApoE(-/-)Id3(-/-) double-knockout (DKO) mice were studied at different ages for renal pathological features and function. Serum samples were analyzed for the presence of autoantibodies. At 16 weeks, DKO mice developed mesangioproliferative glomerulonephritis (GN), leading to severe proteinuria. GN was associated with glomerular deposition of lipids and immune complexes and with macrophage infiltration. DKO mice had high levels of circulating autoantibodies. Although ApoE(-/-) mice had glomerular lipid deposits and Id3(-/-) mice had circulating autoantibodies, neither group of age-matched single-knockout mice developed GN. These data provide support for the hypothesis that induction of renal disease in hyperlipidemia is dictated by additional factors. Our study shows that some of these factors are regulated by ID3. Thus, ID3 is a novel risk factor linking cardiovascular and renal disease.

Increased 12/15-lipoxygenase enhances cell growth, fibronectin deposition, and neointimal formation in response to carotid injury.

OBJECTIVE: To determine whether increased 12/15-lipoxygenase (12/15LO) expression in vivo enhances neointimal formation in response to injury. METHODS AND RESULTS: 12/15LO expression in the vessel wall is increased in animal models of metabolic syndrome and diabetes mellitus. Increased expression of 12/15LO enhances cultured vascular smooth muscle cell (VSMC) proliferation, an effect mediated by the helix-loop-helix factor inhibitor of differentiation 3 (Id3). Carotid endothelial denudation was performed on apolipoprotein (Apo) E(-/-), ApoE(-/-)/12/15LO(-/-), C57BL/6, and 12/15LO-overexpressing transgenic mice. ApoE(-/-)/12/15LO(-/-) mice had attenuated and 12/15LO-overexpressing transgenic mice had enhanced neointimal formation compared with control mice. 12/15LO-overexpressing transgenic mice had greater postinjury carotid Id3 and Ki-67 expression, cell number, and fibronectin deposition compared with C57BL/6 mice. Loss of 12/15LO attenuated proliferation of cultured ApoE(-/-) VSMCs, whereas 12/15LO overexpression induced VSMC proliferation. Loss of Id3 enhanced immunoglobulin trascription factor (ITF)-2b binding to and activation of the p21(cip1) promoter and abrogated 12/15LO-induced VSMC proliferation. CONCLUSIONS: To our knowledge, these data are the first demonstration that increased expression of 12/15LO in the vessel wall enhances Id3-dependent cell proliferation, fibronectin deposition, and neointimal formation in response to injury. Results identify p21(cip1) as a potential target of the 12/15LO-Id3 pathway and suggest that modulation of this pathway may have therapeutic implications for targeting the increased risk of restenosis in patients with diabetes.