Male mice that lack the G-protein-coupled receptor GPR41 have low energy expenditure and increased body fat content.

SCFA are produced in the gut by bacterial fermentation of undigested carbohydrates. Activation of the Gαi-protein-coupled receptor GPR41 by SCFA in ß-cells and sympathetic ganglia inhibits insulin secretion and increases sympathetic outflow, respectively. A possible role in stimulating leptin secretion by adipocytes is disputed. In the present study, we investigated energy balance and glucose homoeostasis in GPR41 knockout mice fed on a standard low-fat or a high-fat diet. When fed on the low-fat diet, body fat mass was raised and glucose tolerance was impaired in male but not female knockout mice compared to wild-type mice. Soleus muscle and heart weights were reduced in the male mice, but total body lean mass was unchanged. When fed on the high-fat diet, body fat mass was raised in male but not female GPR41 knockout mice, but by no more in the males than when they were fed on the low-fat diet. Body lean mass and energy expenditure were reduced in male mice but not in female knockout mice. These results suggest that the absence of GPR41 increases body fat content in male mice. Gut-derived SCFA may raise energy expenditure and help to protect against obesity by activating GPR41.

Isolation and Purification of Rodent Pancreatic Islets of Langerhans.

This chapter describes the detailed protocol for the isolation and purification of islets of Langerhans from rodent pancreas using collagenase digestion. The first step of the process is to separate and isolate the insulin-producing islets of Langerhans from the rest of the pancreas. The pancreas is excised from the animal, trimmed of nonpancreatic tissues before being inflated and chopped into small pieces. The connective tissue is then broken down with a collagenase enzyme solution to selectively digest the bulk of the exocrine tissue while leaving the endocrine islets intact and separated from their surrounding non-islet tissue. Once this process is completed, the islets of Langerhans are separated from the remaining mixture by centrifugation and purified by the means of hand picking. Once isolated, the subsequent islets can be used for several varied experimental processes, including transplantation, the study of pathophysiological mechanisms in diabetic conditions, and in the screening of novel therapeutic approaches in pharmacological research.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Evidence from studies in rodents and in isolated adipocytes that agonists of the chemerin receptor CMKLR1 may be beneficial in the treatment of type 2 diabetes.

The literature is unclear on whether the adipokine chemerin has pro- or anti-inflammatory properties or plays any role in the aetiology of type 2 diabetes or obesity. To address these questions, and in particular the potential of agonists or antagonists of the chemerin receptor CMKLR1 in the treatment of type 2 diabetes and obesity, we studied the metabolic phenotypes of both male and female, CMKLR1 knockout and heterozygote mice. We also investigated changes in plasma chemerin levels and chemerin gene mRNA content in adipose tissue in models of obesity and diabetes, and in response to fasting or administration of the insulin sensitizing drug rosiglitazone, which also has anti-inflammatory properties. The effects of murine chemerin and specific C-terminal peptides on glucose uptake in wild-type and CMKLR1 knockout adipocytes were investigated as a possible mechanism by which chemerin affects the blood glucose concentration. Both male and female CMKLR1 knockout and heterozygote mice displayed a mild tendency to obesity and impaired glucose homeostasis, but only when they were fed on a high-fat died, rather than a standard low-fat diet. Obesity and impaired glucose homeostasis did not occur concurrently, suggesting that obesity was not the sole cause of impaired glucose homeostasis. Picomolar concentrations of chemerin and its C15- and C19-terminal peptides stimulated glucose uptake in the presence of insulin by rat and mouse wild-type epididymal adipocytes, but not by murine CMKLR1 knockout adipocytes. The insulin concentration-response curve was shifted to the left in the presence of 40 pM chemerin or its C-15 terminal peptide. The plasma chemerin level was raised in diet-induced obesity and ob/ob but not db/db mice, and was reduced by fasting and, in ob/ob mice, by treatment with rosiglitazone. These findings suggest that an agonist of CMKLR1 is more likely than an antagonist to be of value in the treatment of type 2 diabetes and to have associated anti-obesity and anti-inflammatory activities. One mechanism by which an agonist of CMKLR1 might improve glucose homeostasis is by increasing insulin-stimulated glucose uptake by adipocytes.

Endocrine pancreatic development: impact of obesity and diet.

During embryonic development, multipotent endodermal cells differentiate to form the pancreas. Islet cell clusters arising from the pancreatic bud form the acini tissue and exocrine ducts whilst pancreatic islets form around the edges of the clusters. The successive steps of islet differentiation are controlled by a complex network of transcription factors and signals that influence cell differentiation, growth and lineage. A Westernized lifestyle has led to an increased consumption of a high saturated fat diet, and an increase in maternal obesity. The developing fetus is highly sensitive to the intrauterine environment, therefore any alteration in maternal nutrition during gestation and lactation which affects the in-utero environment during the key developmental phases of the pancreas may change the factors controlling ß-cell development and ß-cell mass. Whilst the molecular mechanisms behind the adaptive programming of ß-cells are still poorly understood it is established that changes arising from maternal obesity and/or over-nutrition may affect the ability to maintain fetal ß-cell mass resulting in an increased risk of type 2 diabetes in adulthood.

ß2-adrenoceptor agonists can both stimulate and inhibit glucose uptake in mouse soleus muscle through ligand-directed signalling.

The ß-adrenoceptor agonists BRL37344 and clenbuterol have opposite effects on glucose uptake in mouse soleus muscle, even though the ß2-adrenoceptor mediates both effects. Different agonists may direct the soleus muscle ß2-adrenoceptor to different signalling mechanisms. Soleus muscles were incubated with 2-deoxy[1-(14)C]-glucose, ß-adrenoceptor agonists, other modulators of cyclic AMP, and inhibitors of intracellular signalling. The adenylyl cyclase activator forskolin (1 µM), the phosphodiesterase inhibitor rolipram (10 µM) and BRL37344 (10, but not 100 or 1,000, nM) increased, whereas clenbuterol (100 nM) decreased, glucose uptake. Forskolin increased, whereas clenbuterol decreased, muscle cyclic AMP content. BRL37344 (10 nM) did not increase cyclic AMP. Nevertheless, protein kinase A (PKA) inhibitors prevented the stimulatory effect of BRL37344. Nanomolar but not micromolar concentrations of adrenaline stimulated glucose uptake. After preincubation of muscles with pertussis toxin (100 ng/ml), 100 nM clenbuterol, 0.1-10 µM adrenaline and 100 nM BRL37344 stimulated glucose uptake. Clenbuterol increased the proportion of phosphorylated to total ß2-adrenoceptor. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and the stress-activated mitogen-activated protein kinase (MAPK), but not of the classical MAPK pathway, prevented stimulation of glucose uptake by BRL37344. Elevation of the cyclic AMP content of soleus muscle stimulates glucose uptake. Clenbuterol, and high concentrations of adrenaline and BRL37344 direct the ß2-adrenoceptor partly to Gαi, possibly mediated by ß2-adrenoceptor phosphorylation. The stimulatory effect of 10 nM BRL37344 requires the activity of PKA, PI3K and p38 MAPK, consistent with BRL37344 directing the ß2-adrenoceptor to Gαs. Ligand-directed signalling may explain why ß2-adrenoceptor agonists have differing effects on glucose uptake in soleus muscle.

Contrasts between the effects of zinc-α2-glycoprotein, a putative ß3/2-adrenoceptor agonist and the ß3/2-adrenoceptor agonist BRL35135 in C57Bl/6 (ob/ob) mice.

Previous studies by Tisdale et al. have reported that zinc-α(2)-glycoprotein (ZAG (AZGP1)) reduces body fat content and improves glucose homeostasis and the plasma lipid profile in Aston (ob/ob) mice. It has been suggested that this might be mediated via agonism of ß(3)- and possibly ß(2)-adrenoceptors. We compared the effects of dosing recombinant human ZAG (100 µg, i.v.) and BRL35135 (0.5 mg/kg, i.p.), which is in rodents a 20-fold selective ß(3)- relative to ß(2)-adrenoceptor agonist, given once daily for 10 days to male C57Bl/6 Lep(ob)/Lep(ob) mice. ZAG, but not BRL35135, reduced food intake. BRL35135, but not ZAG, increased energy expenditure acutely and after sub-chronic administration. Only BRL35135 increased plasma concentrations of glycerol and non-esterified fatty acid. Sub-chronic treatment with both ZAG and BRL35135 reduced fasting blood glucose and improved glucose tolerance, but the plasma insulin concentration 30 min after administration of glucose was lowered only by BRL35135. Both ZAG and BRL35135 reduced ß(1)-adrenoceptor mRNA levels in white adipose tissue, but only BRL35135 reduced ß(2)-adrenoceptor mRNA. Both ZAG and BRL35135 reduced ß(1)-adrenoceptor mRNA levels in brown adipose tissue, but neither influenced ß(2)-adrenoceptor mRNA, and only BRL35135 increased ß(3)-adrenoceptor and uncoupling protein-1 (UCP1) mRNA levels in brown adipose tissue. Thus, ZAG and BRL35135 had similar effects on glycaemic control and shared some effects on ß-adrenoceptor gene expression in adipose tissue, but ZAG did not display the thermogenic effects of the ß-adrenoceptor agonist, nor did it increase ß(3)-adrenoceptor or UCP1 gene expression in brown adipose tissue. ZAG does not behave as a typical ß(3/2)-adrenoceptor agonist.

A novel automated image analysis method for accurate adipocyte quantification.

Increased adipocyte size and number are associated with many of the adverse effects observed in metabolic disease states. While methods to quantify such changes in the adipocyte are of scientific and clinical interest, manual methods to determine adipocyte size are both laborious and intractable to large scale investigations. Moreover, existing computational methods are not fully automated. We, therefore, developed a novel automatic method to provide accurate measurements of the cross-sectional area of adipocytes in histological sections, allowing rapid high-throughput quantification of fat cell size and number. Photomicrographs of H&E-stained paraffin sections of murine gonadal adipose were transformed using standard image processing/analysis algorithms to reduce background and enhance edge-detection. This allowed the isolation of individual adipocytes from which their area could be calculated. Performance was compared with manual measurements made from the same images, in which adipocyte area was calculated from estimates of the major and minor axes of individual adipocytes. Both methods identified an increase in mean adipocyte size in a murine model of obesity, with good concordance, although the calculation used to identify cell area from manual measurements was found to consistently over-estimate cell size. Here we report an accurate method to determine adipocyte area in histological sections that provides a considerable time saving over manual methods.

Maternal Obesity and the Fetal Origins of the Metabolic Syndrome.

Over recent decades there has been a rapid rise in metabolic disorders throughout the world. Whilst lifestyle and societal habits have contributed to the obesity epidemic, there is now increasing evidence that the early developmental environment of an infant can play a pivotal role in the 'programming' of an adverse physiological phenotype in later life. Clinical evidence highlights that maternal over-nutrition and/or obesity during pregnancy presents not only adverse effects on maternal health, but also persistent and deleterious effects in the developing child. Animal models are providing essential information into the underlying cellular and molecular mechanisms that contribute to this adverse phenotype. The use of this information will aid our understanding of the programming signals related to maternal and paternal over-nutrition and the improved healthcare for both mother and infant.

The isolation and purification of rodent pancreatic islets of Langerhans.

This chapter describes the detailed protocol for the isolation and purification of islets of Langerhans from rodent pancreas using collagenase digestion. The first step of the process is to separate and isolate the insulin-producing islets of Langerhans from the rest of the pancreas. The pancreas is excised from the animal, trimmed of non-pancreatic tissues before being inflated and chopped into small pieces. The connective tissue is then broken down with a collagenase enzyme solution to selectively digest the bulk of the exocrine tissue while leaving the endocrine islets intact and separated from their surrounding non-islet tissue. Once this process is completed, the islets of Langerhans are separated from the remaining mixture by centrifugation and purified by the means of hand picking. Once isolated, the subsequent islets can be used for a number of experimental processes.

The measurement of insulin secretion from isolated rodent islets of Langerhans.

Insulin secretion plays an essential part in the modulation of glucose homeostasis. Pancreatic beta cells are extremely sensitive to small changes in the concentration of glucose, peptides, hormones and fatty acids and insulin secretion is stimulated in response to these factors. The measurement of insulin secretion from isolated islets with either initiators or potentiators is a useful tool for investigating their efficacy in vitro without using cell lines, which can be subject to modification. Static islet incubation is a fast and powerful tool for the investigation of dose-response curves in response to insulin secretagogues, while islet perifusion experiments are useful for the investigation of the kinetics of insulin secretion. These two methods for measuring insulin secretion from isolated rodent islets of Langerhans are described in detail in this chapter.