Melanocortin-4 Receptor Knockout Research Articles

Maid gene dysfunction promotes hyperobesity via the reduction of adipose tissue inflammation in Mc4r gene-deficient mice

The onset and progression mechanisms of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are being studied. We developed and analyzed a new mouse model of obesity by combining maternal Id-like molecule (Maid) and melanocortin-4 receptor (Mc4r) gene deletions. Four mice, each at 12 and 28 weeks of age, were analyzed for each genotype: Maid gene knockout, Mc4r gene knockout, combined Mc4r and Maid gene knockout, and Mc4r gene knockout with a high-fat diet. Mice with a combined deficiency of Mc4r and Maid gene showed significantly more severe obesity compared to all other genotypes, but no liver fibrosis or a decline in metabolic status were observed. In visceral white adipose tissue, Maid and Mc4r gene knockout mice had fewer CD11c-positive cells and lower mRNA expression of both inflammatory and anti-inflammatory cytokines. Furthermore, Maid and Mc4r gene knockout mice showed lower expression of adipocytokines in visceral white adipose tissue and uncoupling protein-1 in scapular brown adipose tissue. The expression of adipocytokines and uncoupling protein-1 is regulated by sympathetic nerve signaling that contribute severe obesity in Maid and Mc4r gene knockout mice. These mechanisms contribute hyperobesity in Maid and Mc4r gene knockout mice.

Direct and pleiotropic effects of antimicrobial peptide transgene integration on reproductive, growth regulating, and non-coding loci in channel catfish (Ictalurus punctatus)

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or Alligator sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P ​> ​0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P ​> ​0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

Pharmacological SERCA activation limits diet-induced steatohepatitis and restores liver metabolic function in mice

Metabolic dysfunction-associated steatotic liver disease is the most common form of liver disease and poses significant health risks to patients who progress to metabolic dysfunction-associated steatohepatitis. Fatty acid overload alters endoplasmic reticulum (ER) calcium stores and induces mitochondrial oxidative stress in hepatocytes, leading to hepatocellular inflammation and apoptosis. Obese mice have impaired liver sarco/ER Ca2+-ATPase (SERCA) function, which normally maintains intracellular calcium homeostasis by transporting Ca2+ ions from the cytoplasm to the ER. We hypothesized that restoration of SERCA activity would improve diet-induced steatohepatitis in mice by limiting ER stress and mitochondrial dysfunction. WT and melanocortin-4 receptor KO (Mc4r−/−) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were administered CDN1163 to activate SERCA, which reduced liver fibrosis and inflammation. SERCA activation also restored glucose tolerance and insulin sensitivity, improved histological markers of metabolic dysfunction-associated steatohepatitis, increased expression of antioxidant enzymes, and decreased expression of oxidative stress and ER stress genes. CDN1163 decreased hepatic citric acid cycle flux and liver pyruvate cycling, enhanced expression of mitochondrial respiratory genes, and shifted hepatocellular [NADH]/[NAD+] and [NADPH]/[NADP+] ratios to a less oxidized state, which was associated with elevated PUFA content of liver lipids. In sum, the data demonstrate that pharmacological SERCA activation limits metabolic dysfunction-associated steatotic liver disease progression and prevents metabolic dysfunction induced by WD feeding in mice.

Complementary role of peripheral and central autonomic nervous system on insulin-like growth factor-1 activation to prevent fatty liver disease.

Insulin-like growth factor-1 (IGF-1) is involved in the pathology of non-alcoholic fatty liver disease (NAFLD) and ameliorates fatty infiltration in the liver. It is activated by growth hormone (GH); however, the role of GH-IGF-1 axis in NAFLD developmental phase has not been well identified. Therefore, in this study, we focused on the effect of IGF-1 in NAFLD pathology and GH excretion activation from the pituitary gland by peripheral autonomic neural pathways relaying liver-brain-gut pathway and by central neuropeptides. GH and IGF-1 levels were assessed in wild-type and melanocortin-4 receptor knockout mice upon the development of diet-induced NAFLD. The contribution of the peripheral autonomic nervous system connecting the liver-brain-gut axis was assessed by its blockade using capsaicin and that of the central nervous system was assessed by the expression of hypothalamic brain-derived neurotrophic factor (BDNF) and corticotropin-releasing factor (CRH), which activates GH release from the pituitary gland. In the NAFLD mouse models, the levels of GH and IGF-1 increased (p < .05). Further, hepatic fatty infiltration was suppressed even under peripheral autonomic nervous system blockade (p < .001), which inhibited gastric ghrelin expression. In mice with peripheral autonomic nervous blockade, hypothalamic BDNF and CRH were inhibited (p < .05), resulting in GH and IGF-1 excretion, whereas other neuropeptides of somatostatin and cortistatin showed no changes. These complementary effects were canceled in melanocortin-4 receptor knockout mice, which diminished BDNF and CRH release control. Our study demonstrates that the release of IGF-1 by the nervous system is a key factor in maintaining the pathological homeostasis of NAFLD, suggesting its therapeutic potential.

CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

260-LB: The Effect of LXR Activation on Liver Metabolism and Diet-Induced Steatohepatitis in Mice

While nonalcoholic fatty liver disease (NAFLD) affects approx. 30% of the US population, there is currently no approved pharmaceutical treatment to inhibit the progression of NAFLD to its more severe stage of nonalcoholic steatohepatitis (NASH). The liver X receptors (LXRs) are key regulators of cholesterol and fatty acid homeostasis in liver. However, the effects of LXR activation on liver metabolism in NASH pathogenesis are not well understood. We hypothesize that LXR activation might reduce NAFLD progression by augmenting lipid composition and metabolism in hepatocytes. To test this hypothesis, 8-week-old male wild type (WT) or obese, melanocortin-4 receptor knockout (Mc4r-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were given WD supplemented with GW3965 (100mg/kg), a selective, orally active agonist for the LXRs. Animals fed GW3965-supplemented diet had increased gene expression of Abca1 and Lpcat3, both transcriptional targets of LXR action. LXR activation significantly decreased body mass of both WT and Mc4r-/- mice, which was correlated with reduction of subcutaneous fat. GW3965 supplementation greatly improved glucose homeostasis of Mc4r-/-. LXR activation significantly decreased hepatic triglycerides, cholesterol esters and ceramides content which was accompanied by decreased expression of lipogenic factors (Fasn, Dgat1 and 2, Fatp5, Elovl1 and 6) in Mc4r-/-. Contrary expression of genes involved in fatty acid β-oxidation together with pyruvate cycling fluxes (VPC, VPK+ME, VPEPCK) were elevated in Mc4r-/- fed diet supplemented with GW3965. These metabolic changes were related to decrease in hepatic fibrosis, expression of NF-κB pathway genes as well as inflammatory (Itgam, Cd274), oxidative (Sod2), and endoplasmic reticulum stress markers (Chop, Atf4 and 6). Taken together, these data suggest that LXR activation improves phenotype of genetically obese WD-fed animals and inhibits progression from NAFLD to NASH. Disclosure T. Bednarski: None. M. Rahim: None. N. Whittenbarger: None. A. J. Olichwier: None. J. Young: Consultant; Pfizer Inc. Stock/Shareholder; Metalytics, Inc. Consultant; Metalytics, Inc. Research Support; Merck Sharp &amp; Dohme Corp. Funding National Institutes of Health (R01DK106348); Vanderbilt University; University of Nebraska-Lincoln Nutrition and Health Sciences Fund

1608-P: Phenotype of Liver-Specific MC3R Knockout Mice

The melanocortin 3 receptor (MC3R) is a key regulator of energy homeostasis and feeding behavior. Global Mc3r knockout mice exhibit greater fat mass and reduced lean mass. Our previous research has found that Mc3r transcripts can be detected in liver and the MC3R deficiency phenotype is partially reversed in liver-specific MC3R recovery mice on a chow diet, in both male and female mice, and high fat diet, in females only. To further examine the role of hepatic MC3R, we produced a liver-specific Mc3r knockout model by crossing mice expressing albumin-Cre recombinase (Alb-Cre+/+) to mice with a floxed Mc3r gene (LoxP+/+), and placed them on a 45 kcal% energy from fat diet for 12 weeks. Female liver-specific Mc3r-deficient mice with diet-induced obesity displayed nonsignificant trends towards worsened glucose tolerance during a glucose tolerance test (glucose AUC Lox+/+, Alb-Cre+/+: n=10, 24036±1644; Alb-Cre+/+: n=10, 19745±1364; BL/6: n=11, 21072±1152 min*mg/dL, p=0.059) and insulin resistance during a glucose tolerance test (glucose AUC LoxP+/+, Alb-Cre+/+: n=10, 14938±812.2;Alb-Cre+/+: n=14, 13062±850; BL/6: n=11, 11725±1024 min*mg/dL, p=0.12), but no differences in morning serum C-peptide (LoxP+/+, Alb-Cre+/+: n=8, 0.74±0.07; Alb-Cre+/+: n=4, 0.55±0.15; BL/6: n=8, 0.29±0.05 ng/mL, p=0.32). Similar patterns were observed for total food consumption (LoxP+/+, Alb-Cre+/+: n=10, 37.2±1.2; Alb-Cre+/+: n=15, 34.5±0.7; BL/6: n=10, 35.7±1.4 g/15 days, p=0.082), with no differences in feeding efficiency (LoxP+/+, Alb-Cre+/+: n=10, 0.027±0.008; Alb-Cre+/+: n=15, 0.009±0.011; BL/6: n=10, 0.017±0.025 g/kcal, p=0.19). No notable trends were observed in male mice. These data suggest that hepatic Mc3r deficiency alone is at most a minor contributor to the obesity phenotypes associated with HFD-treated Mc3r-deficient mice. Additional analysis of tissue samples from these mice and future studies examining Mc3r function in the liver will further elucidate the role of hepatic MC3R. Disclosure J.Chen: None. J.K.Elmquist: Research Support; Regeneron Pharmaceuticals Inc. J.A.Yanovski: Research Support; Hikma Pharmaceuticals, Rhythm Pharmaceuticals, Inc., Soleno Therapeutics, Inc., Versanis Bio Inc. T.P.Patel: None. A.Jain: None. D.Elizondo: None. E.K.Altman: None. N.Tugarinov: None. M.Hollis: None. R.Roberson: None. A.Caron: None.

LBODP080 Mc3r Expression In Liver Tissue Is Partially Responsible For The Severity Of Murine MC3R Deficiency Associated Obesity

Abstract The Melanocortin 3 Receptor (MC3R) is highly expressed in the hypothalamus, where it regulates energy balance and pubertal development. Function-altering SNPs in MC3R have been found to be associated with greater adiposity in both children and adults. Global Mc3r knockout mice show a phenotype of increased fat mass and develop metabolic syndrome. However, in otherwise Mc3r null mice, recovery of Mc3r expression only in the CNS shows greater adiposity compared to controls. Thus, peripheral MC3R expression might have a role in regulating energy homeostasis. Using droplet digital PCR, we found evidence for Mc3r expression in murine liver tissue. A liver-specific MC3R recovery model was generated by crossbreeding mice with a floxed transcriptionally blocked Mc3r (Mc3rtb/tb) to mice expressing albumin-Cre recombinase (Alb-Cre+/+). MC3R reactivation in the liver caused a partial recovery from the obesity phenotype of Mc3rtb/tb mice exposed to 45 kcal% energy fat diet for 12 weeks, particularly in female mice. Recovery of hepatic Mc3r was sufficient to partially reverse the increased percentage fat mass of Mc3rtb/tb (Mc3rtb/tb: n=11, 55.3 ± 0.8% (SEM); Mc3rtb/tb, Alb-Cre+/+: n=13, 52.4 ± 0.8%; BL/6: n=14, 39.7 ± 2.1%, p=0. 017). Similar recovery was observed for total fat mass (p=0. 0012), % lean mass (p=0. 02), and body weight (p=0. 007), with no differences in total lean mass (p=0.1). Hepatic recovery mice also showed trends towards reduced total liver mass compared to the global knockout (p=0. 07). Glucose Area Under Curve (AUCg) analysis of ITT showed no improvement in insulin sensitivity with liver recovery (Mc3rtb/tb: n=11, 16223 ± 1223; Mc3rtb/tb, Alb-Cre+/+: n= 9, 14524 ± 1479; BL/6: n=9, 11725 ± 1024 min*mg/dL, p=0.3), and no evidence for recovery was observed in the GTT AUCg (Mc3rtb/tb: n=10, 31137 ± 2882; Mc3rtb/tb, Alb-Cre+/+: n=9, 27553 ± 1887; BL/6: n=9, 21355 ± 1205 min*mg/dL, p=0.32). In addition to liver-specific recovery, we generated a liver-specific Mc3r knockout mouse by crossing a floxed-Mc3r (LoxP­+/+) line with the Alb-Cre+/+ mouse line. In the liver-specific Mc3r knockout mice, no significant total body composition changes and a nonsignificant trend towards worsened ITT AUCg (Alb-Cre+/+: n=8, 12795 ± 943; LoxP+/+, Alb-Cre+/+: n=8, 14893 ± 855 min*mg/dL, p=0.12) were observed; however, GTT AUCg was higher (Alb-Cre+/+: n=8, 19691 ± 1351; LoxP+/+, Alb-Cre+/+: n=9, 24450 ± 1657 min*mg/dL, p=0. 042), and there was an increase in liver weight (Alb-Cre+/+: n=8, 1. 025 ± 0. 09 g; LoxP+/+, Alb-Cre+/+: n=9, 1.44 ± 0. 09 g, p=0. 0007). We conclude that hepatic Mc3r deficiency contributes to the obesity phenotype in Mc3r-deficient mice, and that MC3R in the liver is responsible for localized metabolic effects in peripheral tissues. Further analysis of tissue samples from these mice may guide a more specific understanding of the role of hepatic MC3R. Presentation: No date and time listed

Involvement of the liver-gut peripheral neural axis in nonalcoholic fatty liver disease pathologies via hepatic HTR2A.

ABSTRACTSerotonin (5-HT) is one of the key bioamines of nonalcoholic fatty liver disease (NAFLD). Its mechanism of action in autonomic neural signal pathways remains unexplained; hence, we evaluated the involvement of 5-HT and related signaling pathways via autonomic nerves in NAFLD. Diet-induced NAFLD animal models were developed using wild-type and melanocortin 4 receptor (MC4R) knockout (MC4RKO) mice, and the effects of the autonomic neural axis on NAFLD physiology, 5-HT and its receptors (HTRs), and lipid metabolism-related genes were assessed by applying hepatic nerve blockade. Hepatic neural blockade retarded the progression of NAFLD by reducing 5-HT in the small intestine, hepatic HTR2A and hepatic lipogenic gene expression, and treatment with an HTR2A antagonist reproduced these effects. The effects were milder in MC4RKO mice, and brain 5-HT and HTR2C expression did not correlate with peripheral neural blockade. Our study demonstrates that the autonomic liver-gut neural axis is involved in the etiology of diet-induced NAFLD and that 5-HT and HTR2A are key factors, implying that the modulation of the axis and use of HTR2A antagonists are potentially novel therapeutic strategies for NAFLD treatment. This article has an associated First Person interview with the first author of the paper.

Mutations in melanocortin-4 receptor: From fish to men.

Melanocortin-4 receptor (MC4R), expressed abundantly in the hypothalamus, is a critical regulator of energy homeostasis, including both food intake and energy expenditure. Shortly after the publication in 1997 of the Mc4r knockout phenotypes in mice, including increased food intake and severe obesity, the first mutations in MC4R were reported in humans in 1998. Studies in the subsequent two decades have established MC4R mutation as the most common monogenic form of obesity, especially in early-onset severe obesity. Studies in animals, from fish to mammals, have established the conserved physiological roles of MC4R in all vertebrates in regulating energy balance. Drug targeting MC4R has been recently approved for treating morbid genetic obesity. How the MC4R can be exploited for animal production is highly worthy of active investigation.

Cardiac Phenotype and Tissue Sodium Content in Adolescents With Defects in the Melanocortin System

Pro-opiomelanocortin (POMC) and the melanocortin-4 receptor (MC4R) play a pivotal role in the leptin-melanocortin pathway. Mutations in these genes lead to monogenic types of obesity due to severe hyperphagia. In addition to dietary-induced obesity, a cardiac phenotype without hypertrophy has been identified in MC4R knockout mice. We aimed to characterize cardiac morphology and function as well as tissue Na+ content in humans with mutations in POMC and MC4R genes. A cohort of 42 patients (5 patients with bi-allelic POMC mutations, 6 heterozygous MC4R mutation carriers, 19 obese controls without known monogenic cause, and 12 normal weight controls) underwent cardiac magnetic resonance (CMR) imaging and 23Na-MRI. Monogenic obese patients with POMC or MC4R mutation respectively had a significantly lower left ventricular mass/body surface area (BSA) than nonmonogenic obese patients. Left ventricular end-diastolic volume/BSA was significantly lower in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. Subcutaneous fat and skin Na+ content was significantly higher in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. In these compartments, the water content was significantly higher in patients with POMC and MC4R mutation than in control groups. Patients with POMC or MC4R mutations carriers had a lack of transition to hypertrophy, significantly lower cardiac muscle mass/BSA, and stored more Na+ within the subcutaneous fat tissue than nonmonogenic obese patients. The results point towards the role of the melanocortin pathway for cardiac function and tissue Na+ storage and the importance of including cardiologic assessments into the diagnostic work-up of these patients.

Time-Restricted Feeding Restored Insulin-Growth Hormone Balance and Improved Substrate and Energy Metabolism in MC4RKO Obese Mice

Background: Dysregulation of metabolic regulatory hormones often occurs during the progress of obesity. Key regulatory hormone insulin-growth hormone (GH) balance has recently been proposed to maintain metabolism profiles. Time-restricted feeding (TRF) is an effective strategy against obesity without detailed research on pulsatile GH releasing patterns. Methods: TRF was performed in an over-eating melanocortin 4 receptor-knockout (MC4RKO) obese mouse model using normal food. Body weight and food intake were measured. Series of blood samples were collected for 6-h pulsatile GH profile, glucose tolerance test, and insulin tolerance test at 5, 8, and 9 weeks of TRF, respectively. Indirect calorimetric recordings were performed by the Phenomaster system at 6 weeks for 1 week, and body composition was measured by nuclear magnetic resonance spectroscopy (NMR). Substrate- and energy metabolism-related gene expressions were measured in terminal liver and subcutaneous white adipose tissues. Results: TRF increased pulsatile GH secretion in dark phase and suppressed hyperinsulinemia in MC4RKO obese mice to reach a reduced insulin/GH ratio. This was accompanied by the improvement in insulin sensitivity, metabolic flexibility, glucose tolerance, and decreased glucose fluctuation, together with appropriate modification of gene expression involved in substrate metabolism and adipose tissue browning. NMR measurement showed that TRF decreased fat mass but increased lean mass. Indirect calorimeter recording indicated that TRF decreased the respiratory exchange ratio (RER) reflecting consumption of more fatty acid in energy production in light phase and increased the oxygen consumption during activities in dark phase. Conclusions: TRF effectively decreases hyperinsulinemia and restores pulsatile GH secretion in the overeating obese mice with significant improvement in substrate and energy metabolism and body composition without reducing total caloric intake.

Emerging roles of melanocortin receptor accessory proteins (MRAP and MRAP2) in physiology and pathophysiology.

Melanocortin-2 receptor accessory protein (MRAP) has an unusual dual topology and influences the expression, localisation, signalling and internalisation of the melanocortin receptor 2 (MC2); the adrenocorticotropic hormone (ACTH) receptor. Mutations in MRAP are associated with familial glucocorticoid deficiency type-2 and evidence is emerging of the importance of MRAP in adrenal development and ACTH signalling. Human MRAP has two functional splice variants: MRAP-α and MRAP-β, unlike MRAP-β, MRAP-α has little expression in brain but is highly expressed in ovary. MRAP2, identified through whole human genome sequence analysis, has approximately 40% sequence homology to MRAP. MRAP2 facilitates MC2 localisation to the cell surface but not ACTH signalling. MRAP and MRAP2 have been found to regulate the surface expression and signalling of all melanocortin receptors (MC1-5). Additionally, MRAP2 moderates the signalling of the G-protein coupled receptors (GCPRs): orexin, prokineticin and GHSR1a; the ghrelin receptor. Whilst MRAP appears to be mainly involved in glucocorticoid synthesis, an important role is emerging for MRAP2 in regulating appetite and energy homeostasis. Transgenic models indicate the importance of MRAP in adrenal gland formation. Like MC3R and MC4R knockout mice, MRAP2 knockout mice have an obese phenotype. In vitro studies indicate that MRAP2 enhances the MC3 and MC4 response to the agonist αMSH, which, like ACTH, is produced through precursor polypeptide proopiomelanocortin (POMC) cleavage. Analysis of cohorts of individuals with obesity have revealed several MRAP2 genetic variants with loss of function mutations which are causative of monogenic hyperphagic obesity with hyperglycaemia and hypertension. MRAP2 may also be associated with female infertility. This review summarises current knowledge of MRAP and MRAP2, their influence on GPCR signalling, and focusses on pathophysiology, particularly familial glucocorticoid deficiency type-2 and obesity.

74-OR: The Effect of SERCA2 Activation on Liver Metabolism and Diet-Induced Steatohepatitis in Mice

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease, but its progression to the severe form of nonalcoholic steatohepatitis (NASH) is unpredictable. Hepatocyte steatosis alters endoplasmic reticulum (ER) calcium stores and induces mitochondrial dysfunction leading to hepatocellular inflammation and apoptosis. Moreover, obese mice have impaired sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) function, responsible for transporting Ca2+ from the cytoplasm to ER. We thus hypothesized that restoration of SERCA2 activity may improve hepatocyte function during lipotoxicity. To test this hypothesis, 8-week-old male C57Bl/6J (wild type, WT) or melanocortin-4 receptor knockout (MC4R-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were also given injections of CDN1163 once every two weeks to activate SERCA2. Sodium [13C3]lactate was infused intravenously into conscious, unrestrained mice for 2 hours and 13C-enrichments of plasma glucose and liver metabolites were measured by GC-MS, then used to regress liver metabolic fluxes using the INCA modeling platform. CDN1163 treatment significantly decreased weight and fat mass of both WT and MC4R-/- WD-fed mice without affecting food intake or lean mass. Moreover, CDN1163 improved insulin sensitivity of WT and MC4R-/- mice. CDN1163 decreased NAFLD activity score in WT, but failed to reduce histological features of NASH in MC4R-/- mice. Liver free fatty acids, diglycerides and ceramides were not affected; however, CDN1163 decreased triglyceride content in WT mice. Liver metabolic fluxes analysis revealed that CDN1163 accelerated pyruvate cycling in WT and MC4R-/- mice, which may explain how CDN1163 helped hepatocytes adapt to an excess of energy substrates. Taken together, these data suggest that SERCA2 activation had a protective effect on liver when NAFLD was less severe (WT) but was insufficient to rescue lipotoxicity after progression to NASH had occurred (MC4R-/-). Disclosure T. Bednarski: None. M. Rahim: None. J. Young: Board Member; Self; Metalytics. Consultant; Self; Pfizer Inc. Funding National Institutes of Health (R01DK106348)

Optimized High-Yield Purification of Obesity-Associated Melanocortin 4 Receptor.

Obesity has emerged as a global public health challenge associated with increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically and in animal studies, which showed that severe obesity in MC4R knockout mice was caused by increased food intake and decreased energy consumption. Over 70 multiple different mis- -sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe early onset or hereditary obesity. The single amino acid variation (D90N) located in the second transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity. Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed normal cell surface expression and binding with agonist similar to the hMC4R wild-type TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation. It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their functional aspects. In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2 proteins under different chemical cleavage reaction times and purification procedures via SDS precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R- TM2 protein in the anisotropic phospholipid bicelles. The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium. In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography, dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h, 2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2. The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification. The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R- TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal. We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution- state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into the membrane.

SUN-672 SGLT2 Inhibitor Reduces Hyperinsulinemia and Restores Pulsatile Growth Hormone Secretion in Obese MC4RKO Mice

Insulin and growth hormone (GH) are crucial counter-regulatory hormones in regulating glucose and lipid metabolisms. Insulin promotes fat storage, while GH promotes lipolysis and fat oxidation. In obese individuals, reduced GH secretion (hyposomatotropism) and increased insulin secretion (hyperinsulinemia) co-exist. The imbalance of these two hormones exacerbates fat accumulation. Therapeutic approaches to correct such hormonal imbalance in obesity are limited. The sodium/glucose cotransporter 2 inhibitor (SGLT2i), which promotes urinary glucose excretion, is a novel drug for overt type 2 diabetes (T2D). However, little is known about its efficacy in obese individuals without T2D in the clinic, in particular with hormonal imbalance. By applying SGLT2i (dapagliflozin, 1 mg/kg/d for 10 weeks) to a hyperphagic obese melanocortin 4 receptor knockout (MC4RKO) mouse model, we observed a significant reduction of hyperinsulinemia (fasting: 1.36±0.19 vs. 4.93±1.04 ng/ml, p<0.01; fed: 9.50±3.37 vs. 31.11±5.85 ng/ml, p<0.05, n=8) and restored pulsatile GH secretion without changing secretion pattern (pulsatile GH: 185.3±18.37 vs. 56.28±13.22 ng/ml per 6h, p<0.001; GH mass per secretion pulse: 50.31±8.20 vs. 15.55±3.18 ng/ml, p<0.01; number of secretory pulse per 6h: 3.71±0.29 vs. 3.57±0.43, p=0.78, n=8) as early as 4 weeks after the initiation of the treatment. Lipolysis and lipid oxidation-related gene expression levels were increased by SGLT2i treatment, whereas lipogenesis and inflammation gene expression levels were reduced, leading to decreased whole-body fat mass. Following the treatment, glucose tolerance and insulin sensitivity were both improved. Although a null effect was observed in food intake and daily activity, the treatment significantly promoted lipid usage and shifted energy metabolism towards negative energy balance. In conclusion, 10-week SGLT2i treatment improved glucose and lipid metabolisms in the hyperphagic obese MC4RKO mice. Such improvement occurs alongside reduced hyperinsulinemia and restored pulsatile GH secretion. This work provides insights for the potential use of SGLT2i in obese individuals prior to overt T2D. The final version of this work is published (1).Acknowledgements: grant (NHMRC, University of Queensland) and scholarship (CSC and UQ International scholarship)Reference: (1) Huang, Zhengxiang, et al. “Dapagliflozin restores insulin and growth hormone secretion in obese mice.” Journal of Endocrinology 245.1 (2020): 1-12.Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

MON-639 Melanocortin 4 Receptor Contributes to Glucose Homeostasis by Regulating Kidney Glucose Reabsorption via the Glucose Transporter GLUT2

Melanocortin 4 receptor (MC4R) is essential for normal body weight and food intake. Deficiency of MC4R causes obesity in humans and mice. While the function of MC4R is well established in appetite regulation, its direct role in glucose homeostasis is unclear. Humans and mice with MC4R deficiency exhibit hyperinsulinemia and insulin resistance; however, they remain protected from fasting hyperglycemia/diabetes. To determine the role of MC4R in glucose homeostasis, we performed oral glucose and intra-peritoneal insulin tolerance tests (OGTT / ITT) in male and female Mc4r knockout (KO) and wild type (WT) mice. Remarkably, Mc4r KO mice exhibited improved glucose tolerance compared to WT mice (Area under the curve for OGTT, male: 29,125±2,028 vs. 38,493±1,161 mg/dL.min; female: 36,322±1,100 vs. 49,539±1,911 mg/dL.min, p<0.0001). The improvement in glucose tolerance was despite insulin resistance in Mc4r KO mice (Plasma insulin, male: 9.9±1.7 vs. 0.7±0.1 ng/mL, female: 6.2±2.0 vs. 1.1±0.3 ng/mL, p<0.05; Area under the curve for ITT, male: 13,174±1,073 vs. 8,132±255 mg/dL.min; female: 13,927±1,253 vs. 7,506±267 mg/dL.min, p<0.01). Based on our previous findings from POMC deficient mice, we hypothesized that the improved glucose tolerance in the Mc4r KO mice is due to their elevated glycosuria (excretion of glucose in urine). To test this hypothesis, we challenged Mc4r KO and WT mice with oral glucose (250 mg) and collected their 24h urine to evaluate glycosuria. Indeed, the KO mice demonstrated elevated glycosuria compared to their WT littermates (Urine glucose, male: 284±48 vs. 0.4±0.03 mg/24h, female: 63.4±14 vs. 1±0.6 mg/24h, p<0.002). To assess molecular mechanisms underlying elevated glycosuria in Mc4r KO mice, we measured the gene expression and levels of the kidney glucose transporters GLUT1, GLUT2, SGLT1 and SGLT2. Glut2 mRNA was reduced by ~ 40% and the protein level was decreased by ~ 20% in Mc4r KO mice compared to their WT littermates. The other glucose transporters remained unchanged. Altogether, our study demonstrates that MC4R contributes to glucose homeostasis by regulating kidney glucose reabsorption via GLUT2. These findings may explain why MC4R deficient mice or humans remain protected from diabetes despite their longstanding obesity and insulin resistance.

Acetyl-CoA carboxylase 1 and 2 inhibition ameliorates steatosis and hepatic fibrosis in a MC4R knockout murine model of nonalcoholic steatohepatitis.

Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in de novo lipogenesis, which is increased in the livers of patients with nonalcoholic steatohepatitis. GS-0976 (firsocostat), an inhibitor of isoforms ACC1 and ACC2, reduced hepatic steatosis and serum fibrosis biomarkers such as tissue inhibitor of metalloproteinase 1 in patients with nonalcoholic steatohepatitis in a randomized controlled trial, although the impact of this improvement on fibrosis has not fully been evaluated in preclinical models. Here, we used Western diet-fed melanocortin 4 receptor-deficient mice that have similar phenotypes to nonalcoholic steatohepatitis patients including progressively developed hepatic steatosis as well as fibrosis. We evaluated the effects of ACC1/2 inhibition on hepatic fibrosis. After the confirmation of significant hepatic fibrosis with a 13-week pre-feeding, GS-0976 (4 and 16 mg/kg/day) treatment for 9 weeks lowered malonyl-CoA and triglyceride content in the liver and improved steatosis, histologically. Furthermore, GS-0976 reduced the histological area of hepatic fibrosis, hydroxyproline content, mRNA expression level of type I collagen in the liver, and plasma tissue metalloproteinase inhibitor 1, suggesting an improvement of hepatic fibrosis. The treatment with GS-0976 was also accompanied by reductions of plasma ALT and AST levels. These data demonstrate that improvement of hepatic lipid metabolism by ACC1/2 inhibition could be a new option to suppress fibrosis progression as well as to improve hepatic steatosis in nonalcoholic steatohepatitis.

MC4R deficiency in pigs results in hyperphagia and ultimately hepatic steatosis without high-fat diet

Melanocortin 4 receptor (MC4R)-deficient mice had been used for several years to study human nonalcoholic steatohepatitis (NASH). However, although liver pathologic and biochemical indicators have been examined, mice models do not always faithfully display the phenotype of the human disease. In this study, we investigated the MC4R knockout phenotype in miniature pigs. We found that pigs lacking MC4R exhibited hyperorexia, insulin resistance, hyperinsulinemia, disordered lipid metabolism and their livers accumulated significant amounts of fat. We have shown that deletion of MC4R results in hyperphagia and increased body fat, ultimately leading to hepatic steatosis without atherogenic diet.

Generation of an MC3R knock-out pig by CRSPR/Cas9 combined with somatic cell nuclear transfer (SCNT) technology

BackgroundMelanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology.MethodFour CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis.ResultsFour vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R−/− and 10 MC3R+/− clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings.ConclusionA MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.