Anne W. Thorburn

Estrogen deficiency causes central leptin insensitivity and increased hypothalamic neuropeptide Y.

OBJECTIVE: Altered fat distribution is a consequence of menopause, but the mechanisms responsible are unknown. Estrogen insufficiency in humans can be modeled using ovariectomized rats. We have shown that increased adiposity in these rats is due to reduced physical activity and transient hyperphagia, and can be reversed with 17β-estradiol treatment. The aims of this study were to examine whether this altered energy balance is associated with circulating leptin insufficiency, central leptin insensitivity, decreased hypothalamic leptin receptor (Ob-Rb) expression, and/or increased hypothalamic neuropeptide Y (NPY).METHODS: Plasma leptin levels, adipose tissue ob gene expression, energy balance responses to i.c.v. leptin, hypothalamic Ob-Rb expression and NPY concentration in five separate hypothalamic regions were measured in adult female rats after either ovariectomy or sham operations.RESULTS: Obesity was not associated with hypoleptinemia or decreased ob gene expression in ovariectomized rats; however, it was associated with insensitivity to central leptin administration. Food intake was less suppressed and spontaneous physical activity was less stimulated by leptin. This was not due to decreased hypothalamic Ob-Rb expression. NPY concentration in the paraventricular nucleus of the hypothalamus was elevated in the ovariectomized rats, consistent with leptin insensitivity; however this effect was transient and disappeared as body fat and leptin levels increased further and hyperphagia normalized.CONCLUSION: Impaired central leptin sensitivity and overproduction of NPY may contribute to excess fat accumulation caused by estrogen deficiency.

Intracellular glucose oxidation and glycogen synthase activity are reduced in non-insulin-dependent (type II) diabetes independent of impaired glucose uptake.

To examine whether reduced rates of oxidative (Gox) and non-oxidative (Nox) glucose metabolism in non-insulin-dependent diabetes mellitus (NIDDM) are due to reduced glucose uptake, intrinsic defects in intracellular glucose metabolism or increased fat oxidation (Fox), indirect calorimetry was performed at similar glucose uptake rates in eight nonobese NIDDM and eight comparable nondiabetic subjects. Three glucose clamp studies were performed: one in the nondiabetic and two in the NIDDM subjects. In the nondiabetic subjects, glucose uptake was increased to 7.62 +/- 0.62 mg/kg of fat-free mass (FFM) per min by increasing serum insulin to 309 pmol/liter at a glucose concentration of 5.1 mmol/liter. By raising the concentration of either serum glucose or insulin fourfold in the NIDDM subjects, glucose uptake was matched to nondiabetic subjects (8.62 +/- 0.49 and 8.59 +/- 0.51 mg/kg FFM per min, respectively, P = NS). Skeletal muscle glycogen synthase activity and plasma lactate levels were measured to characterize Nox. When glucose uptake was matched to nondiabetics by hyperglycemia or hyperinsulinemia, Gox was reduced by 26-28% in NIDDM (P less than 0.025) whereas Fox was similar. Nox was greater in NIDDM (P less than 0.01) and was accompanied by increases in circulating lactate levels. Glycogen synthase activity was reduced by 41% (P less than 0.025) when glucose uptake was matched by hyperglycemia. Glycogen synthase activity was normalized in NIDDM, however, when glucose uptake was matched by hyperinsulinemia. Therefore, a defect in Gox exists in nonobese NIDDM subjects which cannot be overcome by increasing glucose uptake or insulin. Since both glucose uptake and Fox were similar in the two subject groups these factors were not responsible for reduced Gox. Increased Nox in NIDDM is primarily into lactate. Reduced glycogen synthase activity in NIDDM is independent of glucose uptake but can be overcome by increasing the insulin concentration.

Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.

To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM.

Aromatase-deficient (ArKO) mice accumulate excess adipose tissue.

Aromatase is the enzyme which catalyses the conversion of C19 steroids into C18 estrogens. We have generated a mouse model wherein the Cyp19 gene, which encodes aromatase, has been disrupted, and hence, the aromatase knockout (ArKO) mouse cannot synthesise endogenous estrogens. We examined the consequences of estrogen deficiency on accumulation of adipose depots in male and female ArKO mice, observing that these animals progressively accrue significantly more intra-abdominal adipose tissue than their wildtype (WT) litter mates, reflected in increased adipocyte volume and number. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared to WT controls, as were elevated insulin levels, although blood glucose was unchanged. Associated with these changes, the livers of ArKO animals were characterised by a striking accumulation of lipid droplets. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.

Comparison of Insulin Secretory Function in Two Mouse Models with Different Susceptibility to β-Cell Failure

Type 2 diabetes is characterized by a susceptibility to beta-cell failure. However, subjects at risk of developing type 2 diabetes, such as those with obesity or a family history of diabetes, have been shown to display hyperinsulinemia. Although this hyperinsulinemia may be an adaptive response to insulin resistance, the possibility that insulin hypersecretion may be a primary defect has not been thoroughly investigated. The DBA/2 mouse is a model of pancreatic islet susceptibility. Unlike the resistant C57BL/6 mouse strain, the DBA/2 mouse islet fails when stressed with insulin resistance or when exposed to chronic high glucose concentrations. The aim of this study was to compare insulin secretory function in the DBA/2 and C57BL/6 strains in the absence of insulin resistance or high glucose. Insulin secretion was assessed in vivo using the iv glucose tolerance test and in vitro using isolated islets in static incubations. It was shown that DBA/2 mice hypersecreted insulin in vivo, compared with C57BL/6 mice, at 1 d and at 4 and 10 wk of age. This hypersecretion was not attributable to insulin resistance (as assessed by the insulin tolerance test) or increased parasympathetic nervous system outflow. Insulin hypersecretion was also demonstrated in vitro. This was associated with higher glycolysis and glucose oxidation, and elevated activity (but not protein levels) of islet glucokinase and hexokinase. Furthermore, GLUT2 protein levels were higher, which may explain an increase in glucokinase activity in DBA/2 mouse islets. In summary, the DBA/2 mouse, a model of islet failure, has increased glucose-mediated insulin secretion from a very early age, which is associated with an increase in glucose utilization. Further studies will determine whether there is a link between insulin hypersecretion and subsequent beta-cell failure.

Role of Impaired Intracellular Glucose Metabolism in the Insulin Resistance of Aging

The insulin resistance of aging is characterized by both reduced glucose uptake and impaired intracellular glucose metabolism. The aim of this study was to determine whether impaired intracellular glucose metabolism contributes to insulin resistance in the elderly independent of reduced glucose uptake. To address this question, glucose uptake in non-obese elderly males was matched to controls using the glucose clamp technique, and intracellular glucose metabolism was assessed in vivo by indirect calorimetry and in vitro by skeletal muscle biopsy for glycogen synthase activity. When elderly subjects were compared with controls at an equivalent basal glucose uptake of approximately 2.5 mg/kg fat-free mass (FFM)/min, muscle glycogen synthase activity was similar (fractional velocity of glycogen synthase at 0.1 mmol/L glucose-6-phosphate [FV0.1], 0.06 +/- 0.1 and 0.07 +/- 0.1), but whole-body rates of glucose oxidation were reduced (1.36 +/- 0.12 v 1.90 +/- 0.11 mg/kg FFM/min, P less than .05). During 40-mU/m2/min hyperinsulinemic clamps at matched rates of glucose uptake (approximately 10.7 mg/kg FFM/min in both groups), glycogen synthase activity was again similar (FV0.1, 0.15 +/- 0.02 and 0.14 +/- 0.02), and glucose oxidation remained reduced in the elderly (4.18 +/- 0.25 v 4.77 +/- 0.17 mg/kg FFM/min, P less than .05). Only during clamps in the maximal range of glucose uptake (approximately 29.5 mg/kg FFM/min) was glucose oxidation between the groups comparable (5.97 +/- 0.50 and 5.75 +/- 0.31 mg/kg FFM/min). Plasma free fatty acid (FFA) concentrations, fat oxidation, and protein oxidation were similar under all study conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel anti-obesity drugs

There is increasing evidence that body weight is homeostatically regulated and that in obesity this regulation maintains weight at a high level. Weight loss activates mechanisms that are designed to return individuals to their pre-existing weight. This explains the universally poor results of current strategies to maintain weight loss. On this basis, life-long drug therapy may be justified for those with significant obesity. Currently available drugs include selective serotonin re-uptake inhibitors (e.g., fluoxetine), noradrenergic re-uptake inhibitors (e.g., phentermine), a serotonin and noradrenergic re-uptake inhibitor (sibutramine) and an intestinal lipase inhibitor (orlistat). An active research program is underway to develop new agents based on the rapidly expanding knowledge of the complex mechanisms regulating body weight. Leptin, a hormone produced by adipocytes that inhibits food intake, has undergone clinical trials and analogues are currently being developed. Other agents include amylin, melanocortin-4 receptor agonists, neuropeptide Y antagonists, 3 adrenergic agonists and glucagon-like peptide-1 agonists. As some redundancy exists in the central regulatory system controlling body weight, some agents might need to be used in combination to be effective.

Impaired regulation of hepatic fructose-1,6-bisphosphatase in the New Zealand obese mouse model of NIDDM.

The New Zealand obese mouse, a model of NIDDM, is characterized by hyperglycemia, hyperinsulinemia, and hepatic and peripheral insulin resistance. The aim of this study was to investigate the biochemical basis of hepatic insulin resistance in NZO mice. Glycolytic and gluconeogenic enzyme activities were measured in fed and overnight fasted 19- to 20-wk-old NZO and control New Zealand chocolate mice. The NZO mice were twice as heavy as the NZC mice. The activity of the glycolytic enzymes glucokinase and pyruvate kinase was higher, whereas that of the gluconeogenic enzymes PEPCK and glucose-6-phosphatase was lower in fed and fasted NZO mice. These enzyme changes are consistent with a normal response to the hyperinsulinemia in NZO mice. In contrast, the activity of the third regulated gluconeogenic enzyme, fructose-1,6-bisphosphatase, was similar in fed and fasted NZO and NZC mice despite the higher insulin and glucose levels in the NZO mouse. This enzyme is primarily regulated by the powerful inhibitor fructose-2,6-bisphosphate. The levels of this metabolite were measured and found to be increased in both the fed and fasted states in the NZO mouse, suggesting that the activity of the bifunctional enzyme that regulates the level of inhibitor (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) is normally regulated in the NZO mouse. We conclude that most insulin-responsive gluconeogenic and glycolytic enzymes are normally regulated in the NZO mouse, but an abnormality in the regulation of fructose-1,6-bisphosphatase may contribute to the increase hepatic glucose production in these mice.

A circulatory model for calculating non-steady-state glucose fluxes. Validation and comparison with compartmental models

This study presents a circulatory model of glucose kinetics for application to non-steady-state conditions, examines its ability to predict glucose appearance rates from a simulated oral glucose load, and compares its performance with compartmental models. A glucose tracer bolus was injected intravenously in rats to determine parameters of the circulatory and two-compartment models. A simulated oral glucose tolerance test was performed in another group of rats by infusing intravenously labeled glucose at variable rates. A primed continuous intravenous infusion of a second tracer was given to determine glucose clearance. The circulatory model gave the best estimate of glucose appearance, closely followed by the two-compartment model and a modified Steele one-compartment model with a larger total glucose volume. The standard one-compartment model provided the worst estimate. The average relative errors on the rate of glucose appearance were: circulatory, 10%; two-compartment, 13%; modified one-compartment, 11%; standard one-compartment, 16%. Recovery of the infused glucose dose was 93+/-2, 94+/-2, 92+/-2 and 85+/-2%, respectively. These results show that the circulatory model is an appropriate model for assessing glucose turnover during an oral glucose load.

Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor.METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in vitro 125I-hGH receptor binding and cell proliferation.RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH.CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.