Novel proteomic signatures may indicate MRI-assessed intrahepatic fat state and changes: The DIRECT PLUS clinical trial.

BACKGROUND AND AIMS: We demonstrated in the randomized 18-month DIRECT PLUS trial (n = 294) that a Mediterranean (MED) diet, supplemented with polyphenol-rich Mankai duckweed, green tea, and walnuts and restricted in red/processed meat, caused substantial intrahepatic fat (IHF%) loss compared with 2 other healthy diets, reducing NAFLD by half, regardless of similar weight loss. Here, we investigated the baseline proteomic profile associated with IHF% and the changes in proteomics associated with IHF% changes induced by lifestyle intervention. APPROACH AND RESULTS: We calculated IHF% by proton magnetic resonance spectroscopy (normal IHF% <5% and abnormal IHF% ≥5%). We assayed baseline and 18-month samples for 95 proteomic biomarkers.Participants (age = 51.3 ± 10.8 y; 89% men; and body mass index = 31.3 ± 3.9 kg/m 2 ) had an 89.8% 18-month retention rate; 83% had eligible follow-up proteomics measurements, and 78% had follow-up proton magnetic resonance spectroscopy. At baseline, 39 candidate proteins were significantly associated with IHF% (false discovery rate <0.05), mostly related to immune function pathways (eg, hydroxyacid oxidase 1). An IHF% prediction based on the DIRECT PLUS by combined model ( R2 = 0.47, root mean square error = 1.05) successfully predicted IHF% ( R2 = 0.53) during testing and was stronger than separately inputting proteins/traditional markers ( R2 = 0.43/0.44). The 18-month lifestyle intervention induced changes in 18 of the 39 candidate proteins, which were significantly associated with IHF% change, with proteins related to metabolism, extracellular matrix remodeling, and immune function pathways. Thrombospondin-2 protein change was higher in the green-MED compared to the MED group, beyond weight and IHF% loss ( p = 0.01). Protein principal component analysis revealed differences in the third principal component time distinct interactions across abnormal/normal IHF% trajectory combinations; p < 0.05 for all). CONCLUSIONS: Our findings suggest novel proteomic signatures that may indicate MRI-assessed IHF state and changes during lifestyle intervention. Specifically, carbonic anhydrase 5A, hydroxyacid oxidase 1, and thrombospondin-2 protein changes are independently associated with IHF% change, and thrombospondin-2 protein change is greater in the green-MED/high polyphenols diet.

EMC10 modulates hepatic ER stress and steatosis in an isoform-specific manner.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) membrane protein complex subunit 10 (EMC10) has been implicated in obesity. Here we investigated the roles of the two isoforms of EMC10, including a secreted isoform (scEMC10) and an ER membrane-bound isoform (mEMC10), in metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: Manifold steatotic mouse models and HepG2 cells were employed to investigate the role of EMC10 in the regulation of hepatic PERK-eIF2α-ATF4 signaling and hepatosteatosis. The therapeutic effect of scEMC10-neutralizing antibody on mouse hepatosteatosis was explored. Associations of MASLD with serum scEMC10 and hepatic mEMC10 were determined in two cohorts of participants with MASLD. RESULTS: scEMC10 promoted, while mEMC10 suppressed, the activation of hepatic PERK-eIF2α-ATF4 signaling. Emc10 gene knockout exacerbated, while hepatic overexpression of mEMC10 ameliorated, hepatic ER stress and steatosis in mice challenged with either a methionine- and choline-deficient diet or tunicamycin, highlighting a direct, suppressive role of mEMC10 in MASLD via modulation of hepatic ER stress. Overexpression of scEMC10 promoted, whereas neutralization of circulating scEMC10 prevented, hepatosteatosis in mice with fatty liver, suggesting a role of scEMC10 in MASLD development. Clinically, serum scEMC10 was increased, while hepatic mEMC10 was decreased, in participants with MASLD. Correlative analysis indicated that serum scEMC10 positively, whereas hepatic mEMC10 negatively, correlated with liver fat content and serum ALT, AST, and GGT. CONCLUSIONS: These findings demonstrate a novel isoform-specific role for EMC10 in the pathogenesis of MASLD and identify the secreted isoform as a tractable therapeutic target for MASLD via antibody-based neutralization. IMPACT AND IMPLICATIONS: We have shown the role of EMC10 in the regulation of energy homeostasis and obesity. In this study, we determine the distinct roles of the two isoforms of EMC10 in the regulation of hepatic endoplasmic reticulum stress and steatosis in mice, and report on the associations of the different EMC10 isoforms with metabolic dysfunction-associated steatotic liver disease in humans. Our findings delineate a novel regulatory axis for hepatosteatosis and identify EMC10 as a modulator of the PERK-eIF2α-ATF4 signaling cascade that may be of broad physiological significance. Moreover, our pre-clinical and clinical studies provide evidence of the therapeutic potential of targeting scEMC10 in MASLD.

Consequences of COVID-19 on Adipose Tissue Signatures.

Since the emergence of coronavirus disease-19 (COVID-19) in 2019, it has been crucial to investigate the causes of severe cases, particularly the higher rates of hospitalization and mortality in individuals with obesity. Previous findings suggest that adipocytes may play a role in adverse COVID-19 outcomes in people with obesity. The impact of COVID-19 vaccination and infection on adipose tissue (AT) is currently unclear. We therefore analyzed 27 paired biopsies of visceral and subcutaneous AT from donors of the Leipzig Obesity BioBank that have been categorized into three groups (1: no infection/no vaccination; 2: no infection but vaccinated; 3: infected and vaccinated) based on COVID-19 antibodies to spike (indicating vaccination) and/or nucleocapsid proteins. We provide additional insights into the impact of COVID-19 on AT biology through a comprehensive histological transcriptome and serum proteome analysis. This study demonstrates that COVID-19 infection is associated with smaller average adipocyte size. The impact of infection on gene expression was significantly more pronounced in subcutaneous than in visceral AT and mainly due to immune system-related processes. Serum proteome analysis revealed the effects of the infection on circulating adiponectin, interleukin 6 (IL-6), and carbonic anhydrase 5A (CA5A), which are all related to obesity and blood glucose abnormalities.

Myeloid Cell-Specific IL-4 Receptor Knockout Partially Protects from Adipose Tissue Inflammation.

IL-4 receptor signaling is supposed to play a major role in anti-inflammatory polarization and proliferation of adipose tissue macrophages. In this study, we examined the metabolic and inflammatory phenotype of C57BL/6J mice (IIl4ra) with LysM-dependent knockout (IIl4ra Δmyel) of the IL-4 receptor α-chain (IL-4Rα), the mandatory signaling component of IL-4 and IL-13, on chow and high-fat diet. Lean IIl4ra Δmyel mice showed decreased insulin sensitivity, no divergent adipose tissue macrophage polarization, but an increased percentage of CD8+ T cells in visceral adipose tissue. After 20 wk of a high-fat diet, IIl4ra Δmyel mice exhibited higher glucose tolerance, no changes in the lymphocyte compartment and fewer M1 macrophages in visceral adipose tissue. In vivo adipose tissue macrophage proliferation measured by BrdU incorporation was unaffected by Il4ra knockout. Interestingly, we show that IL-4Rα signaling directly augmented Itgax (Cd11c) gene expression in bone marrow-derived macrophages and increased the amount of CD11c+ macrophages in adipose tissue explants. Myeloid cell-specific knockout of Il4ra deteriorated insulin sensitivity in lean mice but improved parameters of glucose homeostasis and partially protected from adipose tissue inflammation in obese mice. Hence, IL-4Rα signaling probably plays a minor role in maintaining the macrophage M2 population and proliferation rates in vivo. Moreover, our data indicate that IL-4 signaling plays a proinflammatory role in adipose tissue inflammation by directly upregulating CD11c on adipose tissue macrophages.

Antifibrotic Soluble Thy-1 Correlates with Renal Dysfunction in Chronic Kidney Disease.

Kidney fibrosis is a major culprit in the development and progression of chronic kidney disease (CKD), ultimately leading to the irreversible loss of organ function. Thymocyte differentiation antigen-1 (Thy-1) controls many core functions of fibroblasts relevant to fibrogenesis but is also found in a soluble form (sThy-1) in serum and urine. We investigated the association of sThy-1 with clinical parameters in patients with CKD receiving hemodialysis treatment compared to individuals with a preserved renal function. Furthermore, Thy-1 tissue expression was detected in a mouse model of diabetic CKD (eNOS-/-; db/db) and non-diabetic control mice (eNOS-/-). Serum and urinary sThy-1 concentrations significantly increased with deteriorating renal function, independent of the presence of diabetes. Serum creatinine is the major, independent, and inverse predictor of serum sThy-1 levels. Moreover, sThy-1 is not only predicted by markers of renal function but is also itself an independent and strong predictor of markers of renal function, i.e., serum creatinine. Mice with severe diabetic CKD show increased Thy-1 mRNA and protein expression in the kidney compared to control animals, as well as elevated urinary sThy-1 levels. Pro-fibrotic mediators, such as interleukin (IL)-4, IL-13, IL-6 and transforming growth factor ß, increase Thy-1 gene expression and release of sThy-1 from fibroblasts. Our data underline the role of Thy-1 in the control of kidney fibrosis in CKD and raise the opportunity that Thy-1 may function as a renal antifibrotic factor.

The effect of high-polyphenol Mediterranean diet on visceral adiposity: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Mediterranean (MED) diet is a rich source of polyphenols, which benefit adiposity by several mechanisms. We explored the effect of the green-MED diet, twice fortified in dietary polyphenols and lower in red/processed meat, on visceral adipose tissue (VAT). METHODS: In the 18-month Dietary Intervention Randomized Controlled Trial PoLyphenols UnproceSsed (DIRECT-PLUS) weight-loss trial, 294 participants were randomized to (A) healthy dietary guidelines (HDG), (B) MED, or (C) green-MED diets, all combined with physical activity. Both isocaloric MED groups consumed 28 g/day of walnuts (+ 440 mg/day polyphenols). The green-MED group further consumed green tea (3-4 cups/day) and Wolffia globosa (duckweed strain) plant green shake (100 g frozen cubes/day) (+ 800mg/day polyphenols) and reduced red meat intake. We used magnetic resonance imaging (MRI) to quantify the abdominal adipose tissues. RESULTS: Participants (age = 51 years; 88% men; body mass index = 31.2 kg/m2; 29% VAT) had an 89.8% retention rate and 79.3% completed eligible MRIs. While both MED diets reached similar moderate weight (MED: - 2.7%, green-MED: - 3.9%) and waist circumference (MED: - 4.7%, green-MED: - 5.7%) loss, the green-MED dieters doubled the VAT loss (HDG: - 4.2%, MED: - 6.0%, green-MED: - 14.1%; p < 0.05, independent of age, sex, waist circumference, or weight loss). Higher dietary consumption of green tea, walnuts, and Wolffia globosa; lower red meat intake; higher total plasma polyphenols (mainly hippuric acid), and elevated urine urolithin A polyphenol were significantly related to greater VAT loss (p < 0.05, multivariate models). CONCLUSIONS: A green-MED diet, enriched with plant-based polyphenols and lower in red/processed meat, may be a potent intervention to promote visceral adiposity regression. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03020186.

CHOP-ASO Ameliorates Glomerular and Tubular Damage on Top of ACE Inhibition in Diabetic Kidney Disease.

BACKGROUND: Maladaptive endoplasmic reticulum stress signaling in diabetic kidney disease (DKD) is linked to increased glomerular and tubular expression of the cell-death-promoting transcription factor C/EBP homologous protein (CHOP). Here, we determined whether locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) targeting CHOP ameliorate experimental DKD. METHODS: We determined the efficacy of CHOP-ASO in the early and late stages of experimental DKD (in 8- or 16-week-old db/db mice, respectively) alone or with an angiotensin-converting enzyme inhibitor (ACEi), after an in vivo dose-escalation study. We used renal functional parameters and morphologic analyses to assess the effect of CHOP-ASO and renal gene-expression profiling to identify differentially regulated genes and pathways. Several human CHOP-ASOs were tested in hyperglycemia-exposed human kidney cells. RESULTS: CHOP-ASOs efficiently reduced renal CHOP expression in diabetic mice and reduced markers of DKD at the early and late stages. Early combined intervention (CHOP-ASO and ACEi) efficiently prevented interstitial damage. At the later timepoint, the combined treatment reduced indices of both glomerular and tubular damage more efficiently than either intervention alone. CHOP-ASO affected a significantly larger number of genes and disease pathways, including reduced sodium-glucose transport protein 2 (Slc5a2) and PROM1 (CD133). Human CHOP-ASOs efficiently reduced glucose-induced CHOP and prevented death of human kidney cells in vitro . CONCLUSIONS: The ASO-based approach efficiently reduced renal CHOP expression in a diabetic mouse model, providing an additional benefit to an ACEi, particularly at later timepoints. These studies demonstrate that ASO-based therapies efficiently reduce maladaptive CHOP expression and ameliorate experimental DKD.

A novel compound heterozygous leptin receptor mutation causes more severe obesity than in Leprdb/db mice.

The leptin receptor (Lepr) pathway is important for food intake regulation, energy expenditure, and body weight. Mutations in leptin and the Lepr have been shown to cause early-onset severe obesity in mice and humans. In studies with C57BL/6NCrl mice, we found a mouse with extreme obesity. To identify a putative spontaneous new form of monogenic obesity, we performed backcross studies with this mouse followed by a quantitative trait locus (QTL) analysis and sequencing of the selected chromosomal QTL region. We thereby identified a novel Lepr mutation (C57BL/6N-LeprL536Hfs*6-1NKB), which is located at chromosome 4, exon 11 within the CRH2-leptin-binding site. Compared with C57BL/6N mice, LeprL536Hfs*6 develop early onset obesity and their body weight exceeds that of Leprdb/db mice at an age of 30 weeks. Similar to Leprdb/db mice, the LeprL536Hfs*6 model is characterized by hyperphagia, obesity, lower energy expenditure and activity, hyperglycemia, and hyperinsulinemia compared with C57BL/6N mice. Crossing Leprdb/wt with LeprL536Hfs*6/wt mice results in compound heterozygous LeprL536Hfs*6/db mice, which develop even higher body weight and fat mass than both homozygous Leprdb/db and LeprL536Hfs*6 mice. Compound heterozygous Lepr deficiency affecting functionally different regions of the Lepr causes more severe obesity than the parental homozygous mutations.

Circulating cell adhesion molecules in metabolically healthy obesity.

BACKGROUND/OBJECTIVES: People with metabolically healthy obesity (MHO) may still have an increased risk for cardiovascular mortality compared to metabolically healthy lean (MHL) individuals. However, the mechanisms linking obesity to cardiovascular diseases are not entirely understood. We therefore tested the hypothesis that circulating cell adhesion molecules (CAMs) are higher in MHO compared to MHL individuals. SUBJECTS/METHODS: Serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), E-selectin and P-selectin were measured in age- and sex-matched groups of MHL (n = 32), MHO categorized into BMI-matched insulin sensitive (IS, n = 32) or insulin resistant (IR) obesity (n = 32) and people with metabolically unhealthy obesity (MUO, n = 32). RESULTS: Indeed, individuals with MHO have significantly higher sICAM-1, E-selectin, and P-selectin serum concentrations compared to MHL people. However, these CAMs are still significantly lower in IS compared to IR MHO. There was no difference between the groups in sVCAM-1 serum concentrations. Compared to all other groups, circulating adhesion molecules were significantly higher in individuals with MUO. CONCLUSIONS: These findings suggest that obesity-related increased cardiovascular risk is reflected and may be mediated by significantly higher CAMs. The mechanisms causing elevated adhesion molecules even in the absence of overt cardio-metabolic risk factors and whether circulating CAMs could predict cardiovascular events need to be explored.

Leptin promotes adipocytes survival in non-vascularized fat grafting via perfusion increase.

BACKGROUND: Though autologous fat transplantation is regularly and successfully performed in plastic surgery, little is known about the factors that contribute to the rise of preadipocytes and how the viability of adipocytes is regulated. As sufficient blood supply is a key parameter for the transplant's survival, we opted to analyse the development of preadipocytes within the fat transplant via stimulation of tissue perfusion with the angiogenesis enhancing hormone leptin. METHODS: In a murine (C57BL/6N) model inguinal fat was autologously transplanted into a dorsal skinfold chamber. In the intervention group the fat transplant was treated with local administration of leptin (3 µg/ml) at days 3, 7 and 10 after transplantation. Saline solution was administered respectively in the control group. On the postoperative days 3, 7, 10, and 15 intra vital microscopy was done to assess the functional vessel density, vessel diameter, adipocyte survival and preadipocyte development. The study was completed by histological tissue analysis on days 15 after transplantation. RESULTS: Leptin administration leads to an increase of angiogenesis, which starts from day 7 after implantation and elevates perfusion as well as functional vessel density FVD at days 10 and 15 after transplantation. Perfusion develops first from the border zones of the transplant. Histological evaluation showed that the percentage of perilipin positive adipocytes increased markedly in the study group of mice. Moreover, fat transplants of mice of the leptin group disclosed significantly higher Pref-1 positive cells than fat transplants of the control group. The findings reported in this study indicate that the leptin can enhance the survival and the quality of grafted fat tissue, which may be due to induction of angiogenesis. CONCLUSION: Leptin administration to fat transplants induced an increase in angiogenesis in the transplanted tissue and may play a role in reducing the resorption rate of lipoaspirates.

Leptin Receptor Compound Heterozygosity in Humans and Animal Models.

Leptin and its receptor are essential for regulating food intake, energy expenditure, glucose homeostasis and fertility. Mutations within leptin or the leptin receptor cause early-onset obesity and hyperphagia, as described in human and animal models. The effect of both heterozygous and homozygous variants is much more investigated than compound heterozygous ones. Recently, we discovered a spontaneous compound heterozygous mutation within the leptin receptor, resulting in a considerably more obese phenotype than described for the homozygous leptin receptor deficient mice. Accordingly, we focus on compound heterozygous mutations of the leptin receptor and their effects on health, as well as possible therapy options in human and animal models in this review.

Effects of Whole-Body Adenylyl Cyclase 5 (Adcy5) Deficiency on Systemic Insulin Sensitivity and Adipose Tissue.

Genome-wide association studies have identified adenylyl cyclase type 5 (ADCY5) as candidate gene for diabetes-related quantitative traits and an increased risk of type 2 diabetes. Mice with a whole-body deletion of Adcy5 (Adcy5-/-) do not develop obesity, glucose intolerance and insulin resistance, have improved cardiac function and increased longevity. Here, we investigated Adcy5 knockout mice (Adcy5-/-) to test the hypothesis that changes in adipose tissue (AT) may contribute to the reported healthier phenotype. In contrast to previous reports, we found that deletion of Adcy5 did not confer any physiological or biochemical benefits. However, this unexpected finding allowed us to investigate the effects of Adcy5 depletion on AT independently of lower body weight and a metabolically healthier phenotype. Adcy5-/- mice exhibited an increased number of smaller adipocytes, lower mean adipocyte size and a distinct AT gene expression pattern with midline 1 (Mid1) as the most significantly downregulated gene compared to control mice. Our Adcy5-/- model challenges previously described beneficial effects of Adcy5 deficiency and suggests that targeting Adcy5 does not improve insulin sensitivity and may therefore limit the relevance of ADCY5 as potential drug target.

Treatment-Induced Neuropathy in Diabetes (TIND)-Developing a Disease Model in Type 1 Diabetic Rats.

Treatment-induced neuropathy in diabetes (TIND) is defined by the occurrence of an acute neuropathy within 8 weeks of an abrupt decrease in glycated hemoglobin-A1c (HbA1c). The underlying pathogenic mechanisms are still incompletely understood with only one mouse model being explored to date. The aim of this study was to further explore the hypothesis that an abrupt insulin-induced fall in HbA1c may be the prime causal factor of developing TIND. BB/OKL (bio breeding/OKL, Ottawa Karlsburg Leipzig) diabetic rats were randomized in three groups, receiving insulin treatment by implanted subcutaneous osmotic insulin pumps for 3 months, as follows: Group one received 2 units per day; group two 1 unit per day: and group three 1 unit per day in the first month, followed by 2 units per day in the last two months. We serially examined blood glucose and HbA1c levels, motor- and sensory/mixed afferent conduction velocities (mNCV and csNCV) and peripheral nerve morphology, including intraepidermal nerve fiber density and numbers of Iba-1 (ionized calcium binding adaptor molecule 1) positive macrophages in the sciatic nerve. Only in BB/OKL rats of group three, with a rapid decrease in HbA1c of more than 2%, did we find a significant decrease in mNCV in sciatic nerves (81% of initial values) after three months of treatment as compared to those group three rats with a less marked decrease in HbA1c <2% (mNCV 106% of initial values, p ≤ 0.01). A similar trend was observed for sensory/mixed afferent nerve conduction velocities: csNCV were reduced in BB/OKL rats with a rapid decrease in HbA1c >2% (csNCV 90% of initial values), compared to those rats with a mild decrease <2% (csNCV 112% of initial values, p ≤ 0.01). Moreover, BB/OKL rats of group three with a decrease in HbA1c >2% showed significantly greater infiltration of macrophages by about 50% (p ≤ 0.01) and a decreased amount of calcitonin gene related peptide (CGRP) positive nerve fibers as compared to the animals with a milder decrease in HbA1c. We conclude that a mild acute neuropathy with inflammatory components was induced in BB/OKL rats as a consequence of an abrupt decrease in HbA1c caused by high-dose insulin treatment. This experimentally induced neuropathy shares some features with TIND in humans and may be further explored in studies into the pathogenesis and treatment of TIND.

Tart Cherry Juice and Seeds Affect Pro-Inflammatory Markers in Visceral Adipose Tissue of High-Fat Diet Obese Rats.

BACKGROUND: Tart cherries (Prunus cerasus L.) are a rich source of anthocyanins. They are phytochemical flavonoids found in red and blue fruits, and vegetables that can reduce hyperlipidemia. Visceral Adipose Tissue (VAT) has emerged as a major player in driving obesity-related inflammatory response. METHODS: This study has investigated the potential positive effects of tart cherries on rats with Diet-Induced Obesity (DIO). In particular, the inflammatory status in retroperitoneal (RPW) and perigonadal (PGW) adipose tissue were studied. Rats were fed ad libitum for 17 weeks with a hypercaloric diet with the supplementation of tart cherries seeds powder (DS) and seeds powder plus tart cherries juice containing 1mg of anthocyanins (DJS). In RPW and PGW, expression of CRP, IL-1 ß, TNF-α, CCL2 and CD36, were measured by qRT-PCR, Western blot and immunohistochemistry techniques. RESULTS: No differences in the weight of RPW and PGW animals were found between DS and DJS groups compared to DIO rats. However, an increase of inflammatory markers was observed in DIO group in comparison with control lean rats. A modulation of these markers was evident upon tart cherry supplementation. CONCLUSION: Study results suggest that tart cherry enriched-diet did not modify the accumulation of visceral fat, but it decreased inflammatory markers in both tissues. Therefore, this supplementation could be useful, in combination with healthy lifestyles, to modify adipose tissue cell metabolism limiting-obesity related organ damage.

COMP-Ang-1 Improves Glucose Uptake in db/db Mice with Type 2 Diabetes.

Cartilage oligomeric matrix protein (COMP)-Angiopoietin-1 is a potent angiopoietin-1 (Ang-1) variant that possesses therapeutic potential in angiogenesis and vascular endothelial dysfunction. Noteworthy, we have shown that COMP-Ang-1 improves hyperglycemia and neuroregeneration in ob/ob mice. However, the mechanism of the antidiabetic effect of COMP-Ang-1 is completely unknown. Therefore, we elucidated the diabetes protective molecular mechanisms of COMP-Ang-1 in diabetic db/db mouse model. COMP-Ang-1 (0.5 ng/g body weight) or aqueous NaCl solution was injected intraperitoneally per day in 21 consecutive days into 3-month old, male db/db mice (n=10 per group). Blood glucose and HbA1c levels were determined at baseline and 21 days after COMP-Ang-1 or NaCl treatment. The effect of COMP-Ang-1 on glucose uptake was investigated by euglycemic-hyperinsulinemic clamp studies and key genes of glucose metabolism were studied by Western blot analysis. Our findings indicate that COMP-Ang-1 improves glucose metabolism in a tissue specific manner by regulating HIF-1α transcriptional genes of GLUT-1 expression.

Nicotinamide Nucleotide Transhydrogenase (Nnt) is Related to Obesity in Mice.

The C57BL/6J (B6J) mouse strain has been widely used as a control strain for the study of metabolic diseases and diet induced obesity (DIO). B6J mice carry a spontaneous deletion mutation in the nicotinamide nucleotide transhydrogenase (Nnt) gene eliminating exons 7-11, resulting in expression of a truncated form of Nnt, an enzyme that pumps protons across the inner mitochondrial membrane. It has been proposed that this mutation in B6J mice is associated with epigonadal fat mass and altered sensitivity to diet induced obesity. To define the role of Nnt in the development of diet induced obesity, we generated first backcross (BC1) hybrids of wild type Nnt C57BL/6NTac and mutated Nnt C57BL/6JRj [(C57BL/6NTac×C57BL/6JRj)F1×C57BL/6NTac]. Body weight gain and specific fat-pad depot mass were measured in BC1 hybrids under high fat diet conditions. Both sexes of BC1 hybrids indicate that mice with Nnt wild type allele are highly sensitive to DIO and exhibit higher relative fat mass. In summary, our data indicate that the Nnt mutation in mice is associated with sensitivity to DIO and fat mass.

Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis.

BACKGROUND & AIMS: The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm. METHODS: Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior. RESULTS: Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation. CONCLUSIONS: For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock. LAY SUMMARY: The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock.

microRNA-379 couples glucocorticoid hormones to dysfunctional lipid homeostasis.

In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)-379/410 genomic cluster as a key component of GC/GR-driven metabolic dysfunction. Particularly, miR-379 was up-regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR-dependent manner. Hepatocyte-specific silencing of miR-379 substantially reduced circulating very-low-density lipoprotein (VLDL)-associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR-379 effects on key receptors in hepatic TG re-uptake. As hepatic miR-379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR-controlled miRNA cluster not only defines a novel layer of hormone-dependent metabolic control but also paves the way to alternative miRNA-based therapeutic approaches in metabolic dysfunction.

Metabolic effects of genetic variation in the human REPIN1 gene.

BACKGROUND: Replication initiator 1 (Repin1) is a zinc finger protein highly expressed in liver and adipose tissue. The Repin1 resides within a quantitative trait locus (QTL) for body weight and triglyceride levels in the rat, and its hepatic deletion in mice results in improved insulin sensitivity and lower body weight. Here, we analyzed whether genetic variation within the Repin1 affects parameters of glucose and lipid metabolism. METHODS: We sequenced REPIN1 in 48 non-related Caucasian subjects. We discovered a 12 base pair deletion (12 bp del; rs3832490), which was subsequently genotyped in two well-characterized cohorts (N = 3013) to test for associations with metabolic traits. Functional consequences of the variant were investigated in HepG2 cells in vitro. RESULTS: In human cohorts, we show that the 12 bp del associates with improved glucose metabolism (lower fasting plasma glucose, fasting plasma insulin, and HOMA IR). Cells transfected with the plasmid carrying the 12 bp del variant are characterized by increased GLUT2 and fatty acid translocase CD36 expression and more lipid droplets. CONCLUSION: Our data suggest that genetic variation in human REPIN1 plays a role in glucose and lipid metabolism by differentially affecting the expression of REPIN1 target genes including glucose and fatty acid transporters.

Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high fat diet-induced obesity by counteracting adipose tissue inflammation in vivo.

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 -/-) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 -/- mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 -/-. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.