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Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes.sup.1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation--all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment--reversing hepatic steatosis and glucose intolerance--were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes. A role and mechanism of action are identified for INSP3R1 in the stimulation of hepatic gluconeogenesis and mitochondrial oxidation by glucagon, suggesting that INSP3R1 may be a target for ameliorating dysregulation of hepatic glucose metabolism., Author(s): Rachel J. Perry [sup.1] [sup.2] , Dongyan Zhang [sup.1] , Mateus T. Guerra [sup.1] , Allison L. Brill [sup.2] , Leigh Goedeke [sup.1] , Ali R. Nasiri [sup.1] , [...]

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ζ (PKCζ) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCζinhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site [Thr.sup.1160] as a PKCζ substrate in the functionally critical INSR kinase activation loop. We hypothesized that [Thr.sup.1160] phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCζ in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue [Thr.sup.1150] ([Insr.sup.T1150A] mice) were protected from high fat diet-induced hepatic insulin resistance. InsrTm0A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR [Thr.sup.1160] phosphorylation and provide further mechanistic links between PKCζ and INSR in mediating NAFLD-induced hepatic insulin resistance., Introduction Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized nations (1). NAFLD is observed in nearly 100% of obese people with type 2 diabetes (T2D) [...]

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti-aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver-directed fashion could reduce oxidative damage and improve age-related metabolic disease and lifespan in diet-induced obese mice. Oral administration of CRMP (20 mg/[kg-day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74-week-old) high-fat diet (HFD)-fed C57BL/6J male mice, independently of changes in body weight, whole-body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long-term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94-104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex-specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof-of-concept data to support further studies investigating the use of liver-directed mitochondrial uncouplers to promote healthy aging in humans. [ABSTRACT FROM AUTHOR]

Summary Obesity is associated with colon cancer pathogenesis, but the underlying mechanism is actively debated. Here, we confirm that diet-induced obesity promotes tumor growth in two murine colon cancer models and show that this effect is reversed by an orally administered controlled-release mitochondrial protonophore (CRMP) that acts as a liver-specific uncoupler of oxidative phosphorylation. This agent lowered circulating insulin, and the reduction of tumor growth was abrogated by an insulin infusion raising plasma insulin to the level of high-fat-fed mice. We also demonstrate that hyperinsulinemia increases glucose uptake and oxidation in vivo in tumors and that CRMP reverses these effects. This study provides evidence that perturbations of whole-organism energy balance or hepatic energy metabolism can influence neoplastic growth. Furthermore, the data show that glucose uptake and utilization by cancers in vivo are not necessarily constitutively high but rather may vary according to the hormonal milieu. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Targeting regulators of adipose tissue lipoprotein lipase could enhance adipose lipid clearance, prevent ectopic lipid accumulation and consequently ameliorate insulin resistance and type 2 diabetes. Angiopoietin-like 8 (ANGPTL8) is an insulin-regulated lipoprotein lipase inhibitor strongly expressed in murine adipose tissue. However, Angptl8 knockout mice do not have improved insulin resistance. We hypothesised that pharmacological inhibition, using a second-generation antisense oligonucleotide (ASO) against Angptl8 in adult high-fat-fed rodents, would prevent ectopic lipid accumulation and insulin resistance by promoting adipose lipid uptake.Methods: ANGPTL8 expression was assessed by quantitative PCR in omental adipose tissue of bariatric surgery patients. High-fat-fed Sprague Dawley rats and C57BL/6 mice were treated with ASO against Angptl8 and insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamps in rats and glucose tolerance tests in mice. Factors mediating lipid-induced hepatic insulin resistance were assessed, including lipid content, protein kinase Cε (PKCε) activation and insulin-stimulated Akt phosphorylation. Rat adipose lipid uptake was assessed by mixed meal tolerance tests. Murine energy balance was assessed by indirect calorimetry.Results: Omental fat ANGPTL8 mRNA expression is higher in obese individuals with fatty liver and insulin resistance compared with BMI-matched insulin-sensitive individuals. Angptl8 ASO prevented hepatic steatosis, PKCε activation and hepatic insulin resistance in high-fat-fed rats. Postprandial triacylglycerol uptake in white adipose tissue was increased in Angptl8 ASO-treated rats. Angptl8 ASO protected high-fat-fed mice from glucose intolerance. Although there was no change in net energy balance, Angptl8 ASO increased fat mass in high-fat-fed mice.Conclusions/interpretation: Disinhibition of adipose tissue lipoprotein lipase is a novel therapeutic modality to enhance adipose lipid uptake and treat non-alcoholic fatty liver disease and insulin resistance. In line with this, adipose ANGPTL8 is a candidate therapeutic target for these conditions. [ABSTRACT FROM AUTHOR]

The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide ( PTHr P) expressed in the fibrous layer of the periosteum ( PO) drives the osteoclastic ( OC) resorption that models the metaphyseal-diaphyseal junction ( MDJ) in the proximal tibia and fibula during linear growth. PTHr P was conditionally deleted (c KO) in the PO via Scleraxis gene targeting ( Scx- Cre). In the lateral tibia, c KO of PTHr P led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand ( RANKL) and a 75% reduction in OC number ( P ≤ 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate ( P ≤ 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The c KO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3 D size of the entire bone, manifest as a 40-45% increase in the PO circumference at the MDJ ( P ≤ 0.001) as well as the mid-diaphysis ( P ≤ 0.001). These tibial and fibular phenotypes were reproduced in a Scx- Cre-driven RANKL c KO mouse. We conclude that PTHr P in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone. [ABSTRACT FROM AUTHOR]

The sites that receive ligament and tendon insertions (entheses) on the cortical surfaces of long bones are poorly understood, particularly regarding modeling and regulation. Entheses are classified as either fibrocartilaginous or fibrous based on their structures. Fibrous entheses typically insert into the metaphysis or diaphysis of a long bone, bear a periosteal component, and are modeled during long-bone growth. This modeling forms a root system by which the insertions attach to the cortical surface. In the case of the medial collateral ligament, modeling drives actual migration of the ligament along the cortical surface in order to accommodate linear growth, whereas in other sites modeling may excavate a deep cortical root system (eg, the teres major insertion) or a shallow root system with a large footprint (eg, the latissimus dorsi insertion). We report here that conditionally deleting parathyroid hormone-related protein (PTHrP) in fibrous entheses via Scleraxis-Cre targeting causes modeling to fail in these three iterations of osteoclast-driven enthesis excavation or migration. These iterations appear to represent formes frustes of a common modeling strategy, presumably differing from each other as a consequence of differences in biomechanical control. In sites in which PTHrP is not induced, either physiologically or because of conditional deletion, modeling does not take place and fibrocartilage is induced. These findings represent the initial genetic evidence that PTHrP regulates periosteal/intramembranous bone cell activity on cortical bone surfaces and indicate that PTHrP serves as a load-induced modeling tool in fibrous insertion sites during linear growth. © 2013 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Mechanisms coordinating pancreatic β cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of β cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phospho enol pyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and β cell health. • Enhanced mitochondrial GTP (mtGTP) production amplifies insulin secretion • mtGTP regulates PEPCK-M and cytosolic Ca2+, not OxPhos, to trigger insulin secretion • Exuberant secretion and enhanced insulin biosynthesis can coexist without toxicity • mtGTP turnover enhances insulin content, granule docking, and mitochondrial mass Jesinkey et al. report that mitochondrial GTP (mtGTP) is an integrative nutrient sentinel regulating β cell function. Signaling from mtGTP raises calcium independent of oxidative phosphorylation to promote insulin secretion. Without overworking the β cell, mtGTP cycling potentiates insulin secretion, nutrient sensing, and mitochondrial expansion alongside promoting health and increasing insulin reserves. [ABSTRACT FROM AUTHOR]