Benjamin J. Renquist

Melanocortin-3 receptor regulates the normal fasting response

The melanocortin-3 receptor–deficient (MC3-R−/−) mouse exhibits mild obesity without hyperphagia or hypometabolism. MC3-R deletion is reported to increase adiposity, reduce lean mass and white adipose tissue inflammation, and increase sensitivity to salt-induced hypertension. We show here that the MC3-R−/− mouse exhibits defective fasting-induced white adipose tissue lipolysis, fasting-induced liver triglyceride accumulation, fasting-induced refeeding, and fasting-induced regulation of the adipostatic and hypothalamic-adrenal-pituitary axes. Close examination of the hypothalamic-pituitary-adrenal axis showed that MC3-R−/− mice exhibit elevated nadir corticosterone as well as a blunted fasting-induced activation of the axis. The previously described phenotypes of this animal and the reduced bone density reported here parallel those of Cushing syndrome. Thus, MC3-R is required for communicating nutritional status to both central and peripheral tissues involved in nutrient partitioning, and this defect explains much of the metabolic phenotype in the model.

Development of an assay for high-throughput energy expenditure monitoring in the zebrafish.

Energy homeostasis is maintained by balancing energy intake and expenditure. Many signals regulating energy intake are conserved between the human and teleost. However, before this work, there was no sensitive high-throughput system to monitor energy expenditure in the teleost. We exploit the nonfluorescent and fluorescent properties of resazurin and its reduced form resorufin (alamarBlue(®)) to monitor energy expenditure responses to drug application and genetic manipulation. We show that leptin, insulin, and alpha-melanocyte-stimulating hormone (α-MSH) increase energy expenditure dose dependently in the larval zebrafish. As previously established in the mouse, etomoxir, a carnitine palmitoyl transferase I inhibitor, blocks leptin-induced energy expenditure in the zebrafish. Metformin, the most commonly prescribed insulin sensitizer, increases the insulin-induced metabolic rate. Using genetic knockdown, we observed that α-MSH treatment increases the metabolic rate, as does knockdown of the melanocortin antagonist, agouti-related protein. The agouti-related protein and multiple melanocortin receptors are shown to be involved in these effects. These studies confirm that aspects of hormonal regulation of energy expenditure are conserved in the teleost, and suggest that this assay may provide a unique tool to perform in vivo screens for drugs or genes that affect the metabolic rate, including insulin or leptin sensitizers.

Hepatic lipid accumulation: Cause and consequence of dysregulated glucoregulatory hormones

Fatty liver can be diet, endocrine, drug, virus or genetically induced. Independent of cause, hepatic lipid accumulation promotes systemic metabolic dysfunction. By acting as peroxisome proliferator-activated receptor (PPAR) ligands, hepatic non-esterified fatty acids upregulate expression of gluconeogenic, beta-oxidative, lipogenic and ketogenic genes, promoting hyperglycemia, hyperlipidemia and ketosis. The typical hormonal environment in fatty liver disease consists of hyperinsulinemia, hyperglucagonemia, hypercortisolemia, growth hormone deficiency and elevated sympathetic tone. These endocrine and metabolic changes further encourage hepatic steatosis by regulating adipose tissue lipolysis, liver lipid uptake, de novo lipogenesis (DNL), beta-oxidation, ketogenesis and lipid export. Hepatic lipid accumulation may be induced by 4 separate mechanisms: (1) increased hepatic uptake of circulating fatty acids, (2) increased hepatic de novo fatty acid synthesis, (3) decreased hepatic beta-oxidation and (4) decreased hepatic lipid export. This review will discuss the hormonal regulation of each mechanism comparing multiple physiological models of hepatic lipid accumulation. Nonalcoholic fatty liver disease (NAFLD) is typified by increased hepatic lipid uptake, synthesis, oxidation and export. Chronic hepatic lipid signaling through PPARgamma results in gene expression changes that allow concurrent activity of DNL and beta-oxidation. The importance of hepatic steatosis in driving systemic metabolic dysfunction is highlighted by the common endocrine and metabolic disturbances across many conditions that result in fatty liver. Understanding the mechanisms underlying the metabolic dysfunction that develops as a consequence of hepatic lipid accumulation is critical to identifying points of intervention in this increasingly prevalent disease state.

Dietary restriction reduces the rate of estradiol clearance in sheep (Ovis aries)

Three experiments were designed to test the effect of dietary restriction on clearance of 17beta-estradiol (E(2)) in sheep. A preliminary experiment examined the effect of a 4-d fast on the rate of E(2) clearance in wethers. The second experiment tested the hypothesis that either long-term restriction (7 wk) or a 5-d fast would increase steroid-binding capacity of serum by increasing the concentration of sex hormone-binding globulin (SHBG) in the blood of ovariectomized ewes. In Exp. 3, we hypothesized that nutrition-dependent regulation of E(2) clearance by the liver would result in divergence in biliary extraction of E(2) in fed and fasted wethers receiving comparable levels of exogenous E(2). A marked difference in E(2) clearance between fed and fasted wethers was noted in the preliminary study. Relative to ad libitumfed wethers, a 4-d fast decreased E(2) clearance by 52%. Serum concentrations of SHBG were increased in long-term energy-restricted and fasted ewes, relative to the concentration in maintenancefed ewes (P = 0.015). Furthermore, a 5-d fast nearly doubled serum steroid-binding capacity in wethers. The E(2) concentration in bile was 2 times greater in fasted than in fed wethers. This fasting-dependent increase in biliary E(2) may be reflective of the increased serum E(2) in fasted animals, because each 1 pg/mL increase in serum E(2) increased bile E(2) by 0.86 +/- 0.12 pg/mL, independent of nutrition (P = 0.002). Our results demonstrate that the rate of clearance of E(2) is decreased during nutritional restriction. Additionally, these data indicate that altered SHBG expression, enterohepatic recirculation, or both are involved in the decreased E(2) clearance during dietary restriction.

Effects of supplementation and stocking rate on body condition and production parameters of multiparous beef cows

Fall-calving multiparous Angus × Hereford cows 3 to 10 years of age were stratified by age in a three by two factorial treatment arrangement to evaluate the efficacy of modifying stocking rate and supplementation strategy to manage cow body condition and production parameters over a 5-year study. Efficacy was evaluated quarterly in association with calving, breeding, weaning, and mid way between weaning and calving (i.e. in August). Three protein supplementation strategies (none, standard, strategic) were imposed across both a moderate (0·3 cows per ha) and a high (0·4 cows per ha) stocking rate. In the strategically supplemented group, protein supplement was provided to cows with a body condition score P P = 0·003 and P = 0·10, respectively). Standard, non-supplemented and strategically supplemented animals had estimated pregnancy rates of 0·83, 0·76, and 0·79, respectively ( P = 0·10). The effects of nutrition on both calving interval and birth weight were independent of the model employed. Animals that were not supplemented had extended calving intervals ( P = 0·06), but there was no effect of stocking rate ( P > 0·10). Birth weight was not affected by supplementation strategy or stocking rate ( P > 0·10). The lower 205-day weights of calves on a heavy compared with moderate stocking rate was independent of age ( P = 0·02). However, the increased 205-day weight of calves born to strategically supplemented cows compared with those born to unsupplemented cows was only evident when data were not corrected for differences in age among groups ( P = 0·03). Likewise, analyses of cow condition parameters using models without and with age resulted in different interpretations. These results suggest that strategic and standard supplementation result in similar animal performance and that the improvement in herd productivity associated with altering stocking rate and supplementation may partially be due to altered herd age dynamics.

High Throughput Danio Rerio Energy Expenditure Assay.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Altered Hepatic Transport by Fetal Arsenite Exposure in Diet-Induced Fatty Liver Disease

Non‐alcoholic fatty liver disease can result in changes to drug metabolism and disposition potentiating adverse drug reactions. Furthermore, arsenite exposure during development compounds the severity of diet‐induced fatty liver disease. This study examines the effects of arsenite potentiated diet‐induced fatty liver disease on hepatic transport in male mice. Changes were detected for Mrp2/3/4 hepatic transporter gene expression as well as for Oatp1a4/2b1/1b2. Plasma concentrations of Mrp and Oatp substrates were increased in arsenic exposure groups compared with diet‐only controls. In addition, murine embryonic hepatocytes and adult primary hepatocytes show significantly altered transporter expression after exposure to arsenite alone: a previously unreported phenomenon. These data indicate that developmental exposure to arsenite leads to changes in hepatic transport which could increase the risk for ADRs during fatty liver disease.

The hypophagic response to heat stress is not mediated by GPR109A or peripheral β-OH butyrate

Rising temperatures resulting from climate change will increase the incidence of heat stress, negatively impacting the labor force and food animal production. Heat stress elevates circulating β-OH butyrate, which induces vasodilation through GPR109a. Interestingly, both heat stress and intraperitoneal β-OH butyrate administration induce hypophagia. Thus, we aimed to investigate the role of β-OH butyrate in heat stress hypophagia in mice. We found that niacin, a β-OH butyrate mimetic that cannot be oxidized to generate ATP, also reduces food intake. Interestingly, the depression in food intake as a result of 8-h intraperitoneal niacin or 48-h heat exposure did not result from changes in hypothalamic expression of orexigenic or anorexigenic signals (AgRP, NPY, or POMC). Genetically eliminating GPR109a expression did not prevent the hypophagic response to heat exposure, intraperitoneal β-OH butyrate (5.7 mmol/kg), or niacin (0.8 mmol/kg). Hepatic vagotomy eliminated the hypophagic response to β-OH butyrate and niacin but did not affect the hypophagic response to heat exposure. We subsequently hypothesized that the hypophagic response to heat stress may depend on direct effects of β-OH butyrate at the central nervous system: β-OH butyrate induced hormonal changes (hyperinsulinemia, hypercorticosteronemia, and hyperleptinemia), or gene expression changes. To test these possibilities, we blocked expression of hepatic hydroxyl methyl glutaryl CoA synthase II (HMGCS2) to prevent hepatic β-OH butyrate synthesis. Mice that lack HMGCS2 maintain a hypophagic response to heat stress. Herein, we establish that the hypophagia of heat stress is independent of GPR109a, the hepatic vagus afferent nerve, and hepatic ketone body synthesis.

Transmembrane Protein 135 (TMEM135) is a Liver X Receptor Target Gene that Mediates an Auxiliary Peroxisome Matrix Protein Import Pathway

The liver x receptors (LXRs) are key regulators of systemic lipid metabolism. We identified transmembrane protein 135 (TMEM135), a peroxisomal protein with an unknown function, as a novel LXR target gene. The LXRs directly induce TMEM135 transcription in humans via an LXR response element in the 5’ untranslated region, but do not increase Tmem135 in murine cells. Functionally, knockdown of TMEM135 in vitro and in vivo results in many features typical of peroxisomal disorders such as steatosis and reduced peroxisomal β-oxidation. Mechanistically, proteomic and Western blot analyses indicated that TMEM135 mediates import of peroxisome matrix proteins necessary for β-oxidation and bile acid synthesis. These findings indicate that TMEM135 is an LXR-inducible regulator of peroxisome catabolic and anabolic processes by mediating an auxiliary matrix protein import pathway, and thus may represent a novel therapeutic target for disorders associated with peroxisome dysfunction.Abstract The liver x receptors (LXRs) are key regulators of systemic lipid metabolism. We determined whether transmembrane protein 135 (TMEM135) is an LXR target gene and its physiologic function. An LXR agonist increased TMEM135 mRNA and protein in human hepatocyte and macrophage cell lines, which was prevented by LXR knockdown. The human TMEM135 promoter contains an LXR response element that bound the LXRs via EMSA and ChIP, and mediated LXR-induced transcription in reporter assays. Knockdown of TMEM135 in HepG2 cells caused triglyceride accumulation despite reduced lipogenic gene expression, indicating a potential role in β-oxidation. To determine physiologic importance, TMEM135 was knocked-down via siRNA in livers of fed and fasted C57BL/6 mice. Fasting increased hepatic fatty acid and NADH concentrations in control mice, consistent with increased fatty acid uptake and β-oxidation. However, in fasted TMEM135 knockdown mice, there was a further significant increase in hepatic fatty acid concentrations and a significant decrease in NADH, indicating an impairment in β-oxidation by peroxisomes and/or mitochondria. Conversely, hepatic ketones tended to increase in fasted TMEM135 knockdown compared to control mice, and because ketogenesis is exclusively dependent on mitochondrial β-oxidation, this indicates peroxisomal β-oxidation was impaired in knockdown mice. Localization studies demonstrated that TMEM135 co-localized with peroxisomes but not mitochondria. Mechanistically, proteomic and Western blot analyses indicated that TMEM135 regulates concentrations of matrix enzymes within peroxisomes. In conclusion, TMEM135 is a novel LXR target gene in humans that mediates peroxisomal metabolism, and thus TMEM135 may be a therapeutic target for metabolic disorders associated with peroxisome dysfunction.

Environmental chamber heat stress responses and adaptations in crossbred Hereford steers

© The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. Transl. Anim. Sci. 2018.2:S185–S188 doi: 10.1093/tas/txy049