The impact of glucagon to support postabsorptive glucose flux and glycemia in healthy rats and its attenuation in male Zucker diabetic fatty rats.

Hyperglucagonemia is a hallmark of type 2 diabetes (T2DM), yet the role of elevated plasma glucagon (P-GCG) to promote excessive postabsorptive glucose production and contribute to hyperglycemia in patients with this disease remains debatable. We investigated the acute action of P-GCG to safeguard/support postabsorptive endogenous glucose production (EGP) and euglycemia in healthy Zucker control lean (ZCL) rats. Using male Zucker diabetic fatty (ZDF) rats that exhibit the typical metabolic disorders of human T2DM, such as excessive EGP, hyperglycemia, hyperinsulinemia, and hyperglucagonemia, we examined the ability of hyperglucagonemia to promote greater rates of postabsorptive EGP and hyperglycemia. Euglycemic or hyperglycemic basal insulin (INS-BC) and glucagon (GCG-BC) clamps were performed in the absence or during an acute setting of glucagon deficiency (GCG-DF, ∼10% of basal), either alone or in combination with insulin deficiency (INS-DF, ∼10% of basal). Glucose appearance, disappearance, and cycling rates were measured using [2-3H] and [3-3H]-glucose. In ZCL rats, GCG-DF reduced the levels of hepatic cyclic AMP, EGP, and plasma glucose (PG) by 50%, 32%, and 50%, respectively. EGP fell in the presence GCG-DF and INS-BC, but under GCG-DF and INS-DF, EGP and PG increased two- and threefold, respectively. GCG-DF revealed the hyperglucagonemia present in ZDF rats lacked the ability to regulate hepatic intracellular cyclic AMP levels and glucose flux, since EGP and PG levels fell by only 10%. We conclude that the liver in T2DM suffers from resistance to all three major regulatory factors, glucagon, insulin, and glucose, thus leading to a loss of metabolic flexibility.NEW & NOTEWORTHY In postabsorptive state, basal plasma insulin (P-INS) and plasma glucose (PG) act dominantly to increase hepatic glucose cycling and reduce endogenous glucose production (EGP) and PG in healthy rats, which is only counteracted by the acute action of basal plasma glucagon (P-GCG) to support EGP and euglycemia. Hyperglucagonemia, a hallmark of type 2 diabetes (T2DM) present in Zucker diabetic fatty (ZDF) rats, is not the primary mediator of hyperglycemia and high EGP as commonly thought; instead, the liver is resistant to glucagon as well as insulin and glucose.

Concordance between Thioacetamide-Induced Liver Injury in Rat and Human In Vitro Gene Expression Data.

The immense resources required and the ethical concerns for animal-based toxicological studies have driven the development of in vitro and in silico approaches. Recently, we validated our approach in which the expression of a set of genes is uniquely associated with an organ-injury phenotype (injury module), by using thioacetamide, a known liver toxicant. Here, we sought to explore whether RNA-seq data obtained from human cells (in vitro) treated with thioacetamide-S-oxide (a toxic intermediate metabolite) would correlate across species with the injury responses found in rat cells (in vitro) after exposure to this metabolite as well as in rats exposed to thioacetamide (in vivo). We treated two human cell types with thioacetamide-S-oxide (primary hepatocytes with 0 (vehicle), 0.125 (low dose), or 0.25 (high dose) mM, and renal tubular epithelial cells with 0 (vehicle), 0.25 (low dose), or 1.00 (high dose) mM) and collected RNA-seq data 9 or 24 h after treatment. We found that the liver-injury modules significantly altered in human hepatocytes 24 h after high-dose treatment involved cellular infiltration and bile duct proliferation, which are linked to fibrosis. For high-dose treatments, our modular approach predicted the rat in vivo and in vitro results from human in vitro RNA-seq data with Pearson correlation coefficients of 0.60 and 0.63, respectively, which was not observed for individual genes or KEGG pathways.

Toxicant-Induced Metabolic Alterations in Lipid and Amino Acid Pathways Are Predictive of Acute Liver Toxicity in Rats.

Liver disease and disorders associated with aberrant hepatocyte metabolism can be initiated via drug and environmental toxicant exposures. In this study, we tested the hypothesis that gene and metabolic profiling can reveal commonalities in liver response to different toxicants and provide the capability to identify early signatures of acute liver toxicity. We used Sprague Dawley rats and three classical hepatotoxicants: acetaminophen (2 g/kg), bromobenzene (0.4 g/kg), and carbon tetrachloride (0.3 g/kg), to identify early perturbations in liver metabolism after a single acute exposure dose. We measured changes in liver genes and plasma metabolites at two time points (5 and 10 h) and used genome-scale metabolic models to identify commonalities in liver responses across the three toxicants. We found strong correlations for gene and metabolic profiles between the toxicants, indicative of similarities in the liver response to toxicity. We identified several injury-specific pathways in lipid and amino acid metabolism that changed similarly across the three toxicants. Our findings suggest that several plasma metabolites in lipid and amino acid metabolism are strongly associated with the progression of liver toxicity, and as such, could be targeted and clinically assessed for their potential as early predictors of acute liver toxicity.

Mechanistic identification of biofluid metabolite changes as markers of acetaminophen-induced liver toxicity in rats.

Acetaminophen (APAP) is the most commonly used analgesic and antipyretic drug in the world. Yet, it poses a major risk of liver injury when taken in excess of the therapeutic dose. Current clinical markers do not detect the early onset of liver injury associated with excess APAP-information that is vital to reverse injury progression through available therapeutic interventions. Hence, several studies have used transcriptomics, proteomics, and metabolomics technologies, both independently and in combination, in an attempt to discover potential early markers of liver injury. However, the casual relationship between these observations and their relation to the APAP mechanism of liver toxicity are not clearly understood. Here, we used Sprague-Dawley rats orally gavaged with a single dose of 2 g/kg of APAP to collect tissue samples from the liver and kidney for transcriptomic analysis and plasma and urine samples for metabolomic analysis. We developed and used a multi-tissue, metabolism-based modeling approach to integrate these data, characterize the effect of excess APAP levels on liver metabolism, and identify a panel of plasma and urine metabolites that are associated with APAP-induced liver toxicity. Our analyses, which indicated that pathways involved in nucleotide-, lipid-, and amino acid-related metabolism in the liver were most strongly affected within 10 h following APAP treatment, identified a list of potential metabolites in these pathways that could serve as plausible markers of APAP-induced liver injury. Our approach identifies toxicant-induced changes in endogenous metabolism, is applicable to other toxicants based on transcriptomic data, and provides a mechanistic framework for interpreting metabolite alterations.

Comparison of the physiological relevance of systemic vs. portal insulin delivery to evaluate whole body glucose flux during an insulin clamp.

To understand the underlying pathology of metabolic diseases, such as diabetes, an accurate determination of whole body glucose flux needs to be made by a method that maintains key physiological features. One such feature is a positive differential in insulin concentration between the portal venous and systemic arterial circulation (P/S-IG). P/S-IG during the determination of the relative contribution of liver and extra-liver tissues/organs to whole body glucose flux during an insulin clamp with either systemic (SID) or portal (PID) insulin delivery was examined with insulin infusion rates of 1, 2, and 5 mU·kg(-1)·min(-1) under either euglycemic or hyperglycemic conditions in 6-h-fasted conscious normal rats. A P/S-IG was initially determined with endogenous insulin secretion to exist with a value of 2.07. During an insulin clamp, while inhibiting endogenous insulin secretion by somatostatin, P/S-IG remained at 2.2 with PID, whereas, P/S-IG disappeared completely with SID, which exhibited higher arterial and lower portal insulin levels compared with PID. Consequently, glucose disappearance rates and muscle glycogen synthetic rates were higher, but suppression of endogenous glucose production and liver glycogen synthetic rates were lower with SID compared with PID. When the insulin clamp was performed with SID at 2 and 5 mU·kg(-1)·min(-1) without managing endogenous insulin secretion under euglycemic but not hyperglycemic conditions, endogenous insulin secretion was completely suppressed with SID, and the P/S-IG disappeared. Thus, compared with PID, an insulin clamp with SID underestimates the contribution of liver in response to insulin to whole body glucose flux.

Glucotoxicity targets hepatic glucokinase in Zucker diabetic fatty rats, a model of type 2 diabetes associated with obesity.

A loss of glucose effectiveness to suppress hepatic glucose production as well as increase hepatic glucose uptake and storage as glycogen is associated with a defective increase in glucose phosphorylation catalyzed by glucokinase (GK) in Zucker diabetic fatty (ZDF) rats. We extended these observations by investigating the role of persistent hyperglycemia (glucotoxicity) in the development of impaired hepatic GK activity in ZDF rats. We measured expression and localization of GK and GK regulatory protein (GKRP), translocation of GK, and hepatic glucose flux in response to a gastric mixed meal load (MMT) and hyperglycemic hyperinsulinemic clamp after 1 or 6 wk of treatment with the sodium-glucose transporter 2 inhibitor (canaglifrozin) that was used to correct the persistent hyperglycemia of ZDF rats. Defective augmentation of glucose phosphorylation in response to a rise in plasma glucose in ZDF rats was associated with the coresidency of GKRP with GK in the cytoplasm in the midstage of diabetes, which was followed by a decrease in GK protein levels due to impaired posttranscriptional processing in the late stage of diabetes. Correcting hyperglycemia from the middle diabetic stage normalized the rate of glucose phosphorylation by maintaining GK protein levels, restoring normal nuclear residency of GK and GKRP under basal conditions and normalizing translocation of GK from the nucleus to the cytoplasm, with GKRP remaining in the nucleus in response to a rise in plasma glucose. This improved the liver's metabolic ability to respond to hyperglycemic hyperinsulinemia. Glucotoxicity is responsible for loss of glucose effectiveness and is associated with altered GK regulation in the ZDF rat.

Mechanism-based identification of plasma metabolites associated with liver toxicity.

Early diagnosis of liver injuries caused by drugs or occupational exposures is necessary to enable effective treatments and prevent liver failure. Whereas histopathology remains the gold standard for assessing hepatotoxicity in animals, plasma aminotransferase levels are the primary measures for monitoring liver dysfunction in humans. In this study, using Sprague Dawley rats, we investigated whether integrated analyses of transcriptomic and metabolomic data with genome-scale metabolic models (GSMs) could identify early indicators of injury and provide new insights into the mechanisms of hepatotoxicity. We obtained concurrent measurements of gene-expression changes in the liver and kidneys, and expression changes along with metabolic profiles in the plasma and urine, from rats 5 or 10 h after exposing them to one of two classical hepatotoxicants, acetaminophen (2 g/kg) or bromobenzene (0.4 g/kg). Global multivariate analyses revealed that gene-expression changes in the liver and metabolic profiles in the plasma and urine of toxicant-treated animals differed from those of controls, even at time points much earlier than changes detected by conventional markers of liver injury. Furthermore, clustering analysis revealed that both the gene-expression changes in the liver and the metabolic profiles in the plasma induced by the two hepatotoxicants were highly correlated, indicating commonalities in the liver toxicity response. Systematic GSM-based analyses yielded metabolites associated with the mechanisms of toxicity and identified several lipid and amino acid metabolism pathways that were activated by both toxicants and those uniquely activated by each. Our findings suggest that several metabolite alterations, which are strongly associated with the mechanisms of toxicity and occur within injury-specific pathways (e.g., of bile acid and fatty acid metabolism), could be targeted and clinically assessed for their potential as early indicators of liver damage.

Assessing Chemical-Induced Liver Injury In Vivo From In Vitro Gene Expression Data in the Rat: The Case of Thioacetamide Toxicity.

Consumers are exposed to thousands of chemicals with potentially adverse health effects. However, these chemicals will never be tested for toxicity because of the immense resources needed for animal-based (in vivo) toxicological studies. Today, there are no viable in vitro alternatives to these types of animal studies. To develop an in vitro approach, we investigated whether we could predict in vivo organ injuries in rats with the use of RNA-seq data acquired from tissues early in the development of toxicant-induced injury, by comparing gene expression data from RNA isolated from these rat tissues with those obtained from in vitro exposure of primary liver and kidney cells. We collected RNA-seq data from the liver and kidney tissues of Sprague-Dawley rats 8 or 24 h after exposing them to vehicle (control), low (25 mg/kg), or high (100 mg/kg) doses of thioacetamide, a known liver toxicant that promotes fibrosis; we used these doses and exposure times to cause only mild toxicant-induced injury. For the in vitro study, we treated two cell types from Sprague-Dawley rats, primary hepatocytes (vehicle; low, 0.025 mM; or high, 0.125 mM dose), and renal tube epithelial cells (vehicle; low, 0.125 mM; or high, 0.500 mM) dose) with the thioacetamide metabolite, thioacetamide-S-oxide, selecting in vitro doses and exposure times to recreate the early-stage toxicant-induced injury model that we achieved in vivo. RNA-seq data were collected 9 or 24 h after application of vehicle or thioacetamide-S-oxide. We found that our modular approach for the analysis of gene expression data derived from in vivo RNA-seq strongly correlated (R2 > 0.6) with the in vitro results at two different dose levels of thioacetamide/thioacetamide-S-oxide after 24 h of exposure. The top-ranked liver injury modules in vitro correctly identified the ensuing development of liver fibrosis.

Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat.

The liver-a central metabolic organ that integrates whole-body metabolism to maintain glucose and fatty-acid regulation, and detoxify ammonia-is susceptible to injuries induced by drugs and toxic substances. Although plasma metabolite profiles are increasingly investigated for their potential to detect liver injury earlier than current clinical markers, their utility may be compromised because such profiles are affected by the nutritional state and the physiological state of the animal, and by contributions from extrahepatic sources. To tease apart the contributions of liver and non-liver sources to alterations in plasma metabolite profiles, here we sought to computationally isolate the plasma metabolite changes originating in the liver during short-term fasting. We used a constraint-based metabolic modeling approach to integrate central carbon fluxes measured in our study, and physiological flux boundary conditions gathered from the literature, into a genome-scale model of rat liver metabolism. We then measured plasma metabolite profiles in rats fasted for 5-7 or 10-13 h to test our model predictions. Our computational model accounted for two-thirds of the observed directions of change (an increase or decrease) in plasma metabolites, indicating their origin in the liver. Specifically, our work suggests that changes in plasma lipid metabolites, which are reliably predicted by our liver metabolism model, are key features of short-term fasting. Our approach provides a mechanistic model for identifying plasma metabolite changes originating in the liver.

Genome-wide gene expression changes associated with exposure of rat liver, heart, and kidney cells to endosulfan.

Endosulfan was once the most commonly used pesticide in agriculture and horticulture. It is an environmentally persistent organochlorine compound with the potential to bioaccumulate as it progresses through the food chain. Its acute and chronic toxicity to mammals, including humans, is well known, but the molecular mechanisms of its toxicity are not fully understood. To gain insight to these mechanisms, we examined genome-wide gene expression changes of rat liver, heart, and kidney cells induced by endosulfan exposure. We found that among the cell types examined, kidney and liver cells were the most sensitive and most resilient, respectively, to endosulfan insult. We acquired RNA sequencing information from cells exposed to endosulfan to identify differentially expressed genes, which we further examined to determine the cellular pathways that were affected. In kidney cells, exposure to endosulfan was uniquely associated with altered expression levels of genes constituting the hypoxia-inducible factor-1 (HIF-1) signaling pathway. In heart and liver cells, exposure to endosulfan altered the expression levels of genes for many members of the extracellular matrix (ECM)-receptor interaction pathway. Because both HIF-1 signaling and ECM-receptor interaction pathways directly or indirectly control cell growth, differentiation, proliferation, and apoptosis, our findings suggest that dysregulation of these pathways is responsible for endosulfan-induced cell death.

Metabolic network-based predictions of toxicant-induced metabolite changes in the laboratory rat.

In order to provide timely treatment for organ damage initiated by therapeutic drugs or exposure to environmental toxicants, we first need to identify markers that provide an early diagnosis of potential adverse effects before permanent damage occurs. Specifically, the liver, as a primary organ prone to toxicants-induced injuries, lacks diagnostic markers that are specific and sensitive to the early onset of injury. Here, to identify plasma metabolites as markers of early toxicant-induced injury, we used a constraint-based modeling approach with a genome-scale network reconstruction of rat liver metabolism to incorporate perturbations of gene expression induced by acetaminophen, a known hepatotoxicant. A comparison of the model results against the global metabolic profiling data revealed that our approach satisfactorily predicted altered plasma metabolite levels as early as 5 h after exposure to 2 g/kg of acetaminophen, and that 10 h after treatment the predictions significantly improved when we integrated measured central carbon fluxes. Our approach is solely driven by gene expression and physiological boundary conditions, and does not rely on any toxicant-specific model component. As such, it provides a mechanistic model that serves as a first step in identifying a list of putative plasma metabolites that could change due to toxicant-induced perturbations.

Correcting Postprandial Hyperglycemia in Zucker Diabetic Fatty Rats With an SGLT2 Inhibitor Restores Glucose Effectiveness in the Liver and Reduces Insulin Resistance in Skeletal Muscle.

Ten-week-old Zucker diabetic fatty (ZDF) rats at an early stage of diabetes embody metabolic characteristics of obese human patients with type 2 diabetes, such as severe insulin and glucose intolerance in muscle and the liver, excessive postprandial excursion of plasma glucose and insulin, and a loss of metabolic flexibility with decreased lipid oxidation. Metabolic flexibility and glucose flux were examined in ZDF rats during fasting and near-normal postprandial insulinemia and glycemia after correcting excessive postprandial hyperglycemia using treatment with a sodium-glucose cotransporter 2 inhibitor (SGLT2-I) for 7 days. Preprandial lipid oxidation was normalized, and with fasting, endogenous glucose production (EGP) increased by 30% and endogenous glucose disposal (E-Rd) decreased by 40%. During a postprandial hyperglycemic-hyperinsulinemic clamp after SGLT2-I treatment, E-Rd increased by normalizing glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake; activation of glucokinase was restored and insulin action was improved, stimulating muscle glucose uptake in association with decreased intracellular triglyceride content. In conclusion, SGLT2-I treatment improves impaired glucose effectiveness in the liver and insulin sensitivity in muscle by eliminating glucotoxicity, which reinstates metabolic flexibility with restored preprandial lipid oxidation and postprandial glucose flux in ZDF rats.

G6PC2: a negative regulator of basal glucose-stimulated insulin secretion.

Elevated fasting blood glucose (FBG) is associated with increased risk for the development of type 2 diabetes and cardiovascular-associated mortality. Genome-wide association studies (GWAS) have linked polymorphisms in G6PC2 with variations in FBG and body fat, although not insulin sensitivity or glucose tolerance. G6PC2 encodes an islet-specific, endoplasmic reticulum-resident glucose-6-phosphatase catalytic subunit. A combination of in situ perfused pancreas, in vitro isolated islet, and in vivo analyses were used to explore the function of G6pc2 in mice. G6pc2 deletion had little effect on insulin sensitivity and glucose tolerance, whereas body fat was reduced in female G6pc2 knockout (KO) mice on both a chow and high-fat diet, observations that are all consistent with human GWAS data. G6pc2 deletion resulted in a leftward shift in the dose-response curve for glucose-stimulated insulin secretion (GSIS). As a consequence, under fasting conditions in which plasma insulin levels were identical, blood glucose levels were reduced in G6pc2 KO mice, again consistent with human GWAS data. Glucose-6-phosphatase activity was reduced, whereas basal cytoplasmic calcium levels were elevated in islets isolated from G6pc2 KO mice. These data suggest that G6pc2 represents a novel, negative regulator of basal GSIS that acts by hydrolyzing glucose-6-phosphate, thereby reducing glycolytic flux.