Beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and a high-fat diet.

AIMS/HYPOTHESIS: We have previously demonstrated the therapeutic usefulness of leptin in lipoatrophic diabetes and insulin-deficient diabetes in mouse models and could also demonstrate its dramatic effects on lipoatrophic diabetes in humans. The aim of the present study was to explore the therapeutic usefulness of leptin in a mouse model of type 2 diabetes with increased adiposity. METHODS: To generate a mouse model mimicking human type 2 diabetes with increased adiposity, we used a combination of low-dose streptozotocin (STZ, 120 microg/g body weight) and high-fat diet (HFD, 45% of energy as fat). Recombinant mouse leptin was infused chronically (20 ng [g body weight](-1) h(-1)) for 14 days using a mini-osmotic pump. The effects of leptin on food intake, body weight, metabolic variables, tissue triacylglycerol content and AMP-activated protein kinase (AMPK) activity were examined. RESULTS: Low-dose STZ injection led to a substantial reduction of plasma insulin levels and hyperglycaemia. Subsequent HFD feeding increased adiposity and induced insulin resistance and further augmentation of hyperglycaemia. In this model mouse mimicking human type 2 diabetes (STZ/HFD), continuous leptin infusion reduced food intake and body weight and improved glucose and lipid metabolism with enhancement of insulin sensitivity. Leptin also decreased liver and skeletal muscle triacylglycerol content accompanied by an increase of alpha2 AMPK activity in skeletal muscle. Pair-feeding experiments demonstrated that leptin improved glucose and lipid metabolism independently of the food intake reduction. CONCLUSIONS/INTERPRETATION: This study demonstrates the beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity, indicating the possible clinical usefulness of leptin as a new glucose-lowering drug in humans.

Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway.

Ghrelin, an endogenous ligand for growth hormone secretagogue (GHS) receptor originally isolated from the stomach, occurs in the hypothalamic arcuate nucleus and may play a role in energy homeostasis. Synthetic GHSs have activated the hypothalamic arcuate neurons containing neuropeptide Y (NPY), suggesting the involvement of NPY in some of ghrelin actions. This study was designed to elucidate the role of ghrelin in the regulation of food intake. A single intracerebroventricular (ICV) injection of ghrelin (5-5,000 ng/rat) caused a significant and dose-related increase in cumulative food intake in rats. Ghrelin (500 ng/rat) was also effective in growth hormone-deficient spontaneous dwarf rats. Hypothalamic NPY mRNA expression was increased in rats that received a single ICV injection of ghrelin (500 ng/rat) (approximately 160% of that in vehicle-treated groups, P < 0.05). The ghrelin's orexigenic effect was abolished dose-dependently by ICV co-injection of NPY Y1 receptor antagonist (10-30 microg/rat). The leptin-induced inhibition of food intake was reversed by ICV co-injection of ghrelin in a dose-dependent manner (5-500 ng/rat). Leptin reduced hypothalamic NPY mRNA expression by 35% (P < 0.05), which was abolished by ICV co-injection of ghrelin (500 ng/rat). This study provides evidence that ghrelin is an orexigenic peptide that antagonizes leptin action through the activation of hypothalamic NPY/Y1 receptor pathway.

Transgenic overexpression of leptin rescues insulin resistance and diabetes in a mouse model of lipoatrophic diabetes.

Lipoatrophic diabetes is caused by a deficiency of adipose tissue and is characterized by severe insulin resistance, hypoleptinemia, and hyperphagia. The A-ZIP/F-1 mouse (A-ZIPTg/+) is a model of severe lipoatrophic diabetes and is insulin resistant, hypoleptinemic, hyperphagic, and shows severe hepatic steatosis. We have also produced transgenic "skinny" mice that have hepatic overexpression of leptin (LepTg/+) and no adipocyte triglyceride stores, and are hypophagic and show increased insulin sensitivity. To explore the pathophysiological and therapeutic roles of leptin in lipoatrophic diabetes, we crossed LepTg/+ and A-ZIPTg/+ mice, producing doubly transgenic mice (LepTg/+:A-ZIPTg/+) virtually lacking adipose tissue but having greatly elevated leptin levels. The LepTg/+:A-ZIPTg/+ mice were hypophagic and showed improved hepatic steatosis. Glucose and insulin tolerance tests revealed increased insulin sensitivity, comparable to LepTg/+ mice. These effects were stable over at least 6 months of age. Pair-feeding the A-ZIPTg/+ mice to the amount of food consumed by LepTg/+:A-ZIPTg/+ mice did not improve their insulin resistance, diabetes, or hepatic steatosis, demonstrating that the beneficial effects of leptin were not due to the decreased food intake. Continuous leptin administration that elevates plasma leptin concentrations to those of LepTg/+:A-ZIPTg/+ mice also effectively improved hepatic steatosis and the disorder of glucose and lipid metabolism in A-ZIP/F-1 mice. These data demonstrate that leptin can improve the insulin resistance and diabetes of a mouse model of severe lipoatrophic diabetes, suggesting that leptin may be therapeutically useful in the long-term treatment of lipoatrophic diabetes.

Change of the reactivity of sulfhydryl groups of the membrane proteins in response to amino acids in Chang liver cells.

Intensity of fluorescence induced by reaction of a SH-directed fluorogenic reagent with whole Chang liver cells or their plasma membrane preparations which were previously treated with 0.5 mM N-ethylmaleimide (NEM) plus 5 mM leucine was enhanced by 30% or more in the presence of 5 mM leucine or valine in assay medium consisting of Tris-Hepes buffer, pH 6.85, compared with the control. The dissociation constant for binding of leucine to the membrane protein(s) was estimated to be approximately 3 mM from the increments of fluorescence intensity with increasing leucine concentration. On the other hand, the amount of [14C]NEM bound to the membrane preparations pretreated with NEM plus leucine was significantly reduced in the presence of leucine in the reaction medium. A decrease of reactivity of SH groups near the binding sites of the membrane proteins and of mobility of the fluorescent adducts on binding leucine was suggested to explain all these results consistently.

Dietary conjugated linoleic acid reciprocally modifies ketogenesis and lipid secretion by the rat liver.

The effects of dietary conjugated linoleic acid (CLA) and linoleic acid (LA) on ketone body production and lipid secretion were compared in isolated perfused rat liver. After feeding the 1% CLA diet for 2 wk, the concentration of post-perfused liver cholesterol was significantly reduced by CLA feeding, whereas that of triacylglycerol remained unchanged. Livers from CLA-fed rats produced significantly more ketone bodies; and the ratio of beta-hydroxybutyrate to acetoacetate, an index of mitochondrial redox potential, tended to be consistently higher in the liver perfusate. Conversely, cumulative secretions of triacylglycerol and cholesterol were consistently lower in the livers of rats fed CLA, and the reduction in the latter was statistically significant. Thus dietary CLA appeared to exert its hypolipidemic effect at least in part through an enhanced beta-oxidation of fatty acids at the expense of esterification of fatty acid in the liver.

[Pathology and significance of leptin resistance in obesity].

Leptin, the protein product of the ob gene, is predominantly secreted from white adipose tissue, and acts on the brain to regulate food intake, energy expenditure, and neuroendocrine function. Obese rodent and humans are mostly associated with high circulating leptin levels. These findings have led to the conclusion that obese individuals are relatively insensitive to endogenous leptin termed 'leptin resistance'. The potential sites for leptin resistance include the blood-brain-barrier transport system and the leptin signaling mechanism in leptin-responsive neurons in the hypothalamus. In this review, we describe leptin, leptin receptor, and potential hypothesis of leptin resistance.

Statistical analysis of short-latency flash visual evoked cortical potentials with noncephalic reference.

Most of the reports on visual evoked cortical potentials have dealt with peak latency or amplitude. An early negative deflection at about 20 ms, however, was reported to coincide with the initial signal to the monkey cortex. We studied the initial signal to the human cortex in nine volunteers by taking into account prestimulus fluctuation. Visual evoked cortical potentials were recorded for an interval 10 ms before and 92.4 ms, after a flash stimulus, by means of a neck reference. The amplitudes of the response before the flash stimulus and at each succeeding 5-ms interval after the stimulus were tested with a t-test. The grand average of the visual evoked cortical potentials showed an initial negative peak at 22.3 ms over the frontal poles, a second negative peak at 52.4 ms over the central area, and a third negative peak at 74.7 ms over the occipital areas. The topographic maps corresponding to the peaks of the visual evoked cortical potentials were the same as those of the dynamic t-values. The visual evoked cortical potential and t-values with the bipolar and source derivations showed a marked response beginning around 40 ms over the posterior head. This statistical analysis is an easy method to standardize evoked potentials and to identify deflections.

Visual evoked potentials to a faint light: signal propagation analyzed with peak latency and topographic mapping.

Accurate localization for the process of recognition of a light stimulus is yet to be determined. We studied 19-channel VEP from nine healthy volunteers, evoked by physiologically faint light less than 200 Cd/m2, using a light emitting diode (5 mm phi, 0.3 degrees, energized for 5 msec). VEP was bandpass filtered from 0.16 to 120 Hz, and analyzed from 20 msec pre-stimulus to 184.2 msec post-stimulus. The grand average VEP suggested an initial positive peak at 115.8 msec at the frontal poles and at 136 msec over the occipital areas. An initial negative peak was noted at 156 msec at the frontal poles and at 179.2 msec over the occipital areas. This might suggest that the potentials evoked had propagated from the frontal poles to the occipital areas, taking about 20 msec. However, the topographic maps contained little evidence for such a propagation, but rather indicated the waxing and waning of positive or negative extremes.

Leptin as an adjunct of insulin therapy in insulin-deficient diabetes.

AIMS/HYPOTHESIS: The purpose of this study was to assess the therapeutic implication of leptin in insulin-deficient diabetes. METHODS: Insulin-deficient diabetes was induced by streptozotocin (STZ) in transgenic skinny mice overexpressing leptin. Plasma concentrations of glucose, insulin, and leptin were measured. The effects on body weight, food intake, and hypothalamic gene expressions were analyzed. After diabetes was induced, graded doses of insulin ranging from 0.4 to 51.2 mU.g(-1).day(-1) were injected. Co-administration of leptin and insulin was also carried out using osmotic pumps. RESULTS: After STZ injection, both transgenic and non-transgenic littermates developed marked hyperglycaemia as a result of severe hypoinsulinaemia [termed diabetic transgenic skinny mice overexpressing leptin (diabetic TGM) and diabetic non-transgenic littermates (diabetic WT) respectively], although diabetic TGM were more sensitive to exogenously administered insulin than diabetic WT. Diabetic WT were hypoleptinaemic and hyperphagic relative to non-diabetic WT, whereas diabetic TGM, which remained hyperleptinaemic, were less hyperphagic than diabetic WT. After STZ injection, hypothalamic expressions of orexigenic and anorexigenic peptide mRNAs were up-regulated and down-regulated, respectively, in diabetic WT, whereas they were unchanged in diabetic TGM. Diabetic TGM became normoglycaemic, when treated with insulin at such doses that did not improve hyperglycaemia in diabetic WT. We found that a sub-threshold dose of insulin that does not affect glucose homeostasis is effective in improving the diabetes in normal mice rendered diabetic by STZ injection, when combined with leptin. CONCLUSIONS/INTERPRETATION: This study suggests that leptin could be used as an adjunct of insulin therapy in insulin-deficient diabetes, thereby providing an insight into the therapeutic implication of leptin as an anti-diabetic agent.