Publisher Correction: Targeting hepatic glutaminase activity to ameliorate hyperglycemia.

In the version of this article initially published, the "[13C2]α-ketoglutarate" label on Fig. 1g is incorrect. It should be "[13C5]α-ketoglutarate". Additionally, in Fig. 3b, the "AAV-GFP" group is missing a notation for significance, and in Fig. 3c, the "AAV-GLS2-sh" group is missing a notation for significance. There should be a double asterisk notating significance in both panels. Finally, in the Fig. 4g legend, "[13C6]UDP-glucose" should be "[13C3]UDP-glucose", and in the Fig. 4h legend, "[13C6]hexose" should be "[13C3]hexose". The errors have been corrected in the HTML and PDF versions of this article.

Targeting hepatic glutaminase activity to ameliorate hyperglycemia.

Glucagon levels increase under homeostatic, fasting conditions, promoting the release of glucose from the liver by accelerating the breakdown of glycogen (also known as glycogenolysis). Glucagon also enhances gluconeogenic flux, including from an increase in the hepatic consumption of amino acids. In type 2 diabetes, dysregulated glucagon signaling contributes to the elevated hepatic glucose output and fasting hyperglycemia that occur in this condition. Yet, the mechanism by which glucagon stimulates gluconeogenesis remains incompletely understood. Contrary to the prevailing belief that glucagon acts primarily on cytoplasmic and nuclear targets, we find glucagon-dependent stimulation of mitochondrial anaplerotic flux from glutamine that increases the contribution of this amino acid to the carbons of glucose generated during gluconeogenesis. This enhanced glucose production is dependent on protein kinase A (PKA) and is associated with glucagon-stimulated calcium release from the endoplasmic reticulum, activation of mitochondrial α-ketoglutarate dehydrogenase, and increased glutaminolysis. Mice with reduced levels of hepatic glutaminase 2 (GLS2), the enzyme that catalyzes the first step in glutamine metabolism, show lower glucagon-stimulated glutamine-to-glucose flux in vivo, and GLS2 knockout results in higher fasting plasma glucagon and glutamine levels with lower fasting blood glucose levels in insulin-resistant conditions. As found in genome-wide association studies (GWAS), human genetic variation in the region of GLS2 is associated with higher fasting plasma glucose; here we show in human cryopreserved primary hepatocytes in vitro that these natural gain-of-function missense mutations in GLS2 result in higher glutaminolysis and glucose production. These data emphasize the importance of gluconeogenesis from glutamine, particularly in pathological states of increased glucagon signaling, while suggesting a possible new therapeutic avenue to treat hyperglycemia.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Diets High in Fat or Fructose Differentially Modulate Bone Health and Lipid Metabolism.

Diets high in fat or carbohydrates can lead to obesity and diabetes, two interrelated conditions that have been associated with osteoporosis. Here, we contrasted the effects of a high fat (HF) versus fructose-enriched carbohydrate (CH) versus regular chow (SC) diet on bone morphology, fat content and metabolic balance in BALB/cByJ mice over a 15-week period. For 13 weeks, there were no differences in body mass between groups with small differences in the last 2 weeks. Even without the potentially confounding factor of altered body mass and levels of load bearing, HF consumption was detrimental to bone in the distal femur with lower trabecular bone volume fraction and thinner cortices than controls. These differences in bone were accompanied by twofold greater abdominal fat content and fourfold greater plasma leptin concentrations. High-fat feeding caused a decrease in de-novo lipid synthesis in the liver, kidney, white adipose and brown adipose tissue. In contrast to HF, the fructose diet did not significantly impact bone quantity or architecture. Fructose consumption also did not significantly alter leptin levels or de-novo lipid synthesis but reduced epididymal adipose tissue and increased brown adipose tissue. Cortical stiffness was lower in the CH than in HF mice. There were no differences in glucose or insulin levels between groups. Together, a diet high in fat had a negative influence on bone structure, adipose tissue deposition and lipid synthesis, changes that were largely avoided with a fructose-enriched diet.

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.