Owen P. McGuinness

Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice

The Mouse Metabolic Phenotyping Center (MMPC) Consortium was established to address the need to characterize the growing number of mouse models of metabolic diseases, particularly diabetes and obesity. A goal of the MMPC Consortium is to propose standard methods for assessing metabolic phenotypes in mice. In this article, we discuss issues pertaining to the design and performance of various tests of glucose metabolism. We also propose guidelines for the description of methods, presentation of data and interpretation of results. The recommendations presented in this article are based on the experience of the MMPC Consortium and other investigators.

Insulin Signaling in α-cells Modulates Glucagon Secretion in vivo

Glucagon plays an important role in glucose homeostasis by regulating hepatic glucose output in both normo- and hypoglycemic conditions. In this study, we created and characterized alpha cell-specific insulin receptor knockout (alphaIRKO) mice to directly explore the role of insulin signaling in the regulation of glucagon secretion in vivo. Adult male alphaIRKO mice exhibited mild glucose intolerance, hyperglycemia, and hyperglucagonemia in the fed state and enhanced glucagon secretion in response to L-arginine stimulation. Hyperinsulinemic-hypoglycemic clamp studies revealed an enhanced glucagon secretory response and an abnormal norepinephrine response to hypoglycemia in alphaIRKO mice. The mutants also exhibited an age-dependent increase in beta cell mass. Furthermore, siRNA-mediated knockdown of insulin receptor in glucagon-secreting InR1G cells promoted enhanced glucagon secretion and complemented our in vivo findings. Together, these data indicate a significant role for intraislet insulin signaling in the regulation of alpha cell function in both normo- and hypoglycemic conditions.

Circadian disruption leads to insulin resistance and obesity

BACKGROUND Disruption of circadian (daily) timekeeping enhances the risk of metabolic syndrome, obesity, and type 2 diabetes. While clinical observations have suggested that insulin action is not constant throughout the 24 hr cycle, its magnitude and periodicity have not been assessed. Moreover, when circadian rhythmicity is absent or severely disrupted, it is not known whether insulin action will lock to the peak, nadir, or mean of the normal periodicity of insulin action. RESULTS We used hyperinsulinemic-euglycemic clamps to show a bona fide circadian rhythm of insulin action; mice are most resistant to insulin during their daily phase of relative inactivity. Moreover, clock-disrupted Bmal1-knockout mice are locked into the trough of insulin action and lack rhythmicity in insulin action and activity patterns. When rhythmicity is rescued in the Bmal1-knockout mice by expression of the paralogous gene Bmal2, insulin action and activity patterns are restored. When challenged with a high-fat diet, arhythmic mice (either Bmal1-knockout mice or wild-type mice made arhythmic by exposure to constant light) were obese prone. Adipose tissue explants obtained from high-fat-fed mice have their own periodicity that was longer than animals on a chow diet. CONCLUSIONS This study provides rigorous documentation for a circadian rhythm of insulin action and demonstrates that disturbing the natural rhythmicity of insulin action will disrupt the rhythmic internal environment of insulin sensitive tissue, thereby predisposing the animals to insulin resistance and obesity.

Deletion of the mouse Slc30a8 gene encoding zinc transporter-8 results in impaired insulin secretion.

The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid residue 325 of human ZnT-8 are associated with altered susceptibility to Type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in Type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analysed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioural changes or differences in body weight were observed between wild-type and ZnT-8-/- mice, and ZnT-8-/- mouse islets were indistinguishable from wild-type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8-/- mouse islets, as evaluated both by Timms histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16-week-old, 6 h fasted animals of either gender; however, plasma insulin concentrations were reduced in both female (approximately 31%) and male (approximately 47%) ZnT-8-/- mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8-/- mice, but glucose-stimulated insulin secretion from isolated islets was reduced approximately 33% relative to wild-type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKα2 subunit (α2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in α2-KD mice; expression of neuronal NOS (NOSμ) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 ± 8 and 50 ± 7%, respectively, in skeletal muscle of α2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSμ expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.

Estrogen Treatment After Ovariectomy Protects Against Fatty Liver and May Improve Pathway-Selective Insulin Resistance

Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a persons microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

NIH experiment in centralized mouse phenotyping: the Vanderbilt experience and recommendations for evaluating glucose homeostasis in the mouse

This article addresses two topics. We provide an overview of the National Institutes of Health Mouse Metabolic Phenotyping Center (MMPC) Program. We then discuss some observations we have made during the first eight years of the Vanderbilt MMPC regarding common phenotyping practices. We include specific recommendations to improve phenotyping practices for tests of glucose tolerance and insulin action. We recommend that methods for experiments in vivo be described in manuscripts. We make specific recommendations for data presentation, interpretation, and experimental design for each test. To facilitate and maximize the exchange of scientific information, we suggest that guidelines be developed for methods used to assess glucose tolerance and insulin action in vivo.

Ablation of ghrelin receptor reduces adiposity and improves insulin sensitivity during aging by regulating fat metabolism in white and brown adipose tissues

Aging is associated with increased adiposity in white adipose tissues and impaired thermogenesis in brown adipose tissues; both contribute to increased incidences of obesity and type 2 diabetes. Ghrelin is the only known circulating orexigenic hormone that promotes adiposity. In this study, we show that ablation of the ghrelin receptor (growth hormone secretagogue receptor, GHS‐R) improves insulin sensitivity during aging. Compared to wild‐type (WT) mice, old Ghsr−/− mice have reduced fat and preserve a healthier lipid profile. Old Ghsr−/− mice also exhibit elevated energy expenditure and resting metabolic rate, yet have similar food intake and locomotor activity. While GHS‐R expression in white and brown adipose tissues was below the detectable level in the young mice, GHS‐R expression was readily detectable in visceral white fat and interscapular brown fat of the old mice. Gene expression profiles reveal that Ghsr ablation reduced glucose/lipid uptake and lipogenesis in white adipose tissues but increased thermogenic capacity in brown adipose tissues. Ghsr ablation prevents age‐associated decline in thermogenic gene expression of uncoupling protein 1 (UCP1). Cell culture studies in brown adipocytes further demonstrate that ghrelin suppresses the expression of adipogenic and thermogenic genes, while GHS‐R antagonist abolishes ghrelin’s effects and increases UCP1 expression. Hence, GHS‐R plays an important role in thermogenic impairment during aging. Ghsr ablation improves aging‐associated obesity and insulin resistance by reducing adiposity and increasing thermogenesis. Growth hormone secretagogue receptor antagonists may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.

Circadian Clock Gene Bmal1 Is Not Essential; Functional Replacement with its Paralog, Bmal2

Most of the central circadian clock genes in the mouse exist as paralog pairs (Per1 and Per2, Cry1 and Cry2, Clock and Npas2) in which each gene of the pair must be knocked out to confer arrhythmicity. The only exception to this pattern is Bmal1 (also known as Mop3), the single knockout of which confers arrhythmicity, despite the presence of its paralog, Bmal2 (also known as Mop9). The knockout of Bmal1 also has significant effects on longevity, metabolism, etc. These results have led to the conclusion that Bmal1 is a singularly essential clock gene and that Bmal2 has a minimal role in the clock system. In contrast, we find that expression of Bmal2 from a constitutively expressed promoter can rescue the clock and metabolic phenotypes of Bmal1-knockout mice, including rhythmic locomotor activity, rhythmic metabolism, low body weight, and enhanced fat deposition. Combined with the data of Bunger and colleagues, who reported that knockout of Bmal1 downregulates Bmal2, we conclude that Bmal1 and Bmal2 form a circadian paralog pair that is functionally redundant and that, in the mouse, Bmal2 is regulated by Bmal1 such that knockout of Bmal1 alone results in a functionally double Bmal1 and Bmal2 knockout. Therefore, the role(s) of Bmal2 may be more important than has been appreciated heretofore.