Adverse effects of dipeptidyl peptidases 8 and 9 inhibition in rodents revisited.

AIM: To evaluate the association between inhibition of dipeptidyl peptidase (DPP)-8 and/or DPP-9 organ toxicities and mortality in rodents. RESEARCH DESIGN AND METHODS: The relative selectivity of the DPP-4 inhibitor, vildagliptin, was determined by comparing its K(I) (concentration of compound yielding 50% inhibition of the enzyme) values for inhibition of recombinant human DPP-4, DPP-8 and DPP-9 assessed in vitro. In experiments performed in vivo, vildagliptin was administered by gavage for 13 weeks, at doses up to 1500 mg/kg/day in CD-1 mice and at doses up to 900 mg/kg/day in Wistar rats. Plasma concentrations of vildagliptin were assessed at week 12, and toxicities previously ascribed to inhibition of DPP-8 and/or DPP-9 were assessed at week 13. RESULTS: The K(I) values for vildagliptin-induced inhibition of DPP-4, DPP-8 and DPP-9 were 3, 810 and 95 nM respectively. The mean plasma concentration 24 h after dose after 12-week daily dosing with 1500 mg/kg/day in mice was 2279 nM. The mean plasma drug level 24 h after dose after 12-week daily dosing with 900 mg/kg/day in rats was 5729 nM. These high doses maintained plasma drug levels well above the K(I) values for DPP-8 and DPP-9 throughout a 24-h period. At these high doses, the toxicities of a selective DPP-8/DPP-9 inhibitor that were reported previously (100% mortality in mice, alopecia, thrombocytopenia, reticulocytopenia, enlarged lymph nodes, splenomegaly and 20% mortality in rats) were not observed. CONCLUSIONS: Inhibition of DPP-8 and DPP-9 per se does not lead to organ toxicities and mortality in rodents. Thus, a mechanism other than DPP-8/DPP-9 inhibition likely underlies the toxicity previously reported to be associated with a selective DPP-8/DPP-9 inhibitor.

Acute and chronic effects of the incretin enhancer vildagliptin in insulin-resistant rats.

The enzyme dipeptidyl peptidase-IV (DPP-4) inactivates the incretin hormone glucagon-like peptide-1 (GLP-1). Because GLP-1 has therapeutic effects in patients with type 2 diabetes, but its potential is limited by a short half-life, DPP-4 inhibition is a promising approach to diabetes treatment. This study examined acute (single dose) and chronic (once-a-day dosing for 21 days) effects of the DPP-4 inhibitor vildagliptin (0.03-10 mg/kg) on plasma DPP-4 activity, intact GLP-1, glucose, and insulin after an oral glucose load in insulin-resistant Zucker fatty rats and acute effects in mildly insulin-resistant high-fat-fed normal rats. A single oral dose of vildagliptin in Zucker rats produced a rapid and dose-related inhibition of DPP-4: the minimum effective dose (MED) was 0.3 mg/kg. Glucose-induced increases of intact GLP-1 were greatly but similarly enhanced by vildagliptin at doses > or =0.3 mg/kg. Postload glucose excursions decreased, and the insulinogenic index (Deltainsulin/Deltaglucose at 10 min) increased, with an MED of 0.3 mg/kg and a maximally effective dose of 3 mg/kg. The effects of vildagliptin after chronic treatment were nearly identical to those of acute administration, and vildagliptin had no effect on body weight. In fat-fed normal rats, vildagliptin (3 mg/kg) also decreased postload glucose excursions and increased the insulinogenic index, but these effects were smaller than those in Zucker rats. Thus, vildagliptin is an orally effective incretin enhancer with antihyperglycemic activity in insulin-resistant rats and exhibits no tachyphylaxis. GLP-1-mediated augmentation of glucose-induced insulin release seems to make the major contribution to the antidiabetic properties of vildagliptin.

The effect of 1,3-diaryl-[1H]-pyrazole-4-acetamides on glucose utilization in ob/ob mice.

This article provides evidence of a new class of compounds, 1,3-diaryl-[1H]-pyrazole-4-acetamides, initially identified from their ability to increase glucose transport in an adipocyte and muscle cell line and ultimately demonstrating dramatic glucose lowering in ob/ob mice, a diabetic animal model. The lead compound, 1, possessed some behavioral-like effects which were removed by structural variation during the course of this investigation. Specifically, 11g (R1 = meta-CF(3), Ar2 = 4'biphenyl, R3 = diethylamide) illustrated the potency of this series with ED(50) values for glucose lowering in ob/ob mice of 3.0 mg/kg/day. Concomitant with its effect on glucose lowering, 11g also caused a 50% reduction in insulin levels consistent with an agent that increases whole body insulin sensitivity. 11g showed favorable pharmacokinetic data with acceptable absorption, negligible metabolism, and good duration of action. 11g demonstrated no appreciable adipogenic effect through PPAR gamma agonism, a characteristic of the thiazolidinediones (TZD), and so represents a potentially new class of agents for the treatment of diabetes.

Beta 3-adrenoceptor agonists as anti-diabetic and anti-obesity drugs in humans.

In the early 1980s, an "atypical" beta-adrenergic receptor was discovered and subsequently called the beta (3)-adrenoceptor (beta(3)-AR). Agonists of the beta(3)-AR were observed to simultaneously increase lipolysis, fat oxidation, energy expenditure and insulin action leading to the belief that this receptor might serve as an attractive target for the treatment of diabetes and obesity. In vivo studies lent credence to this postulate with the finding that stimulation of this receptor by selective agonists lead to glycemic improvements and weight loss in rodent models of diabetes and obesity. This lead to intensive research efforts directed at developing beta(3)-AR selective agonists for the treatment of type 2 diabetes and obesity in humans. Unfortunately, endeavour been largely unsuccessful to date. Major obstacles have included the pharmacological differences between the rodent and human beta(3)-AR, the lack of selectivity of previous compounds for the beta(3)-AR over beta(1)-/beta(2)-ARs, and unsatisfactory oral bioavailability and pharmacokinetic properties. Cloning of the human beta(3)-AR has allowed for the development of novel compounds targeted specifically at the human receptor. Encouraging data has emerged from clinical studies wherein CL-316,243, a highly selective, albeit rodent specific beta(3)-AR agonist was observed to increase lipolysis, fat oxidation and insulin action in humans. More recently, beta(3)-AR agonists directed at the human receptor are showing promising results in their ability to increase energy expenditure in humans following a single dose. However, they do nor appear to be able to sustain their effects when administered chronically. Further clinical testing will be necessary, using compounds with improved oral bioavailability and potency, to help assess the physiology of the beta(3)-AR in humans and its attractiveness as a potential therapeutic for the treatment of type 2 diabetes and obesity.

Effects of pioglitazone on adipose tissue remodeling within the setting of obesity and insulin resistance.

Obesity and dysfunctional energy partitioning can lead to the development of insulin resistance and type 2 diabetes. The antidiabetic thiazolidinediones shift the energy balance toward storage, leading to an increase in whole-body adiposity. These studies examine the effects of pioglitazone (Pio) on adipose tissue physiology, accumulation, and distribution in female Zucker (fa/fa) rats. Pio treatment (up to 28 days) decreased the insulin-resistant and hyperlipidemic states and increased food consumption and whole-body adiposity. Magnetic resonance imaging (MRI) analysis and weights of fat pads demonstrated that the increase in adiposity was not only limited to the major fat depots but also to fat deposition throughout the body. Adipocyte sizing profiles, fat pad histology, and DNA content show that Pio treatment increased the number of small adipocytes because of both the appearance of new adipocytes and the shrinkage and/or disappearance of existing mature adipocytes. The remodeling was time dependent, with new small adipocytes appearing in clusters throughout the fat pad, and accompanied by a three- to fourfold increase in citrate synthase and fatty acid synthase activity. The appearance of new fat cells and the increase in fat mass were depot specific, with a rank order of responsiveness of ovarian > retroperitoneal > subcutaneous. This differential depot effect resulted in a redistribution of the fat mass in the abdominal region such that there was an increase in the visceral:subcutaneous ratio, as confirmed by MRI analysis. Although the increased adiposity is paradoxical to an improvement in insulin sensitivity, the quantitative increase of adipose mass should be viewed in context of the qualitative changes in adipose tissue, including the remodeling of adipocytes to a smaller size with higher lipid storage potential. This shift in energy balance is likely to result in lower circulating free fatty acid levels, ultimately improving insulin sensitivity and the metabolic state.

Effects of pioglitazone on promoting energy storage, not expenditure, in brown adipose tissue of obese fa/fa Zucker rats: comparison to CL 316,243.

Recent advances in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) include the use of thiazolidinediones (TZDs), agents that enhance insulin action, in part, through an activation of adipose tissue peroxisome proliferator-activated receptor gamma. Current evidence also indicates that these agents upregulate uncoupling protein 1 (UCP1) gene expression in brown adipocytes and increase interscapular brown adipose tissue (IBAT) mass in rodents, suggestive of a thermogenic component to their mechanism of action. In the present study, the TZD pioglitazone (PIO) and the beta3-adrenoceptor agonist CL 316,243 (CL), were used to determine whether the antidiabetic effects of PIO, like those of CL, may, in part, be mediated by an increase in either IBAT thermogenesis or whole-body energy expenditure. Treatment of obese, insulin resistant fa/fa Zucker rats with PIO for 10 days resulted in a 2- to 3-fold increase in IBAT mass, due largely to an increase in adipocyte size and number, and increased fatty acid biosynthesis. However, unlike the effects of CL, the PIO-induced IBAT changes were not associated with an increase in UCP1 expression or whole-body energy expenditure. In contrast to CL, PIO substantially increased body weight gains over the 10-day treatment period by increasing feeding efficiency. These data suggest that, unlike CL, the actions of PIO in the obese Zucker rat does not include increased energy expenditure, but rather strengthens its role as an adipogenic and lipogenic agent, which promotes energy storage.

Role of HDL phospholipid in efflux of cell cholesterol to whole serum: studies with human apoA-I transgenic rats.

Sera of transgenic rats expressing human apoA-I were tested for their ability to stimulate efflux of radiolabeled cholesterol from Fu5AH rat hepatoma cells. Expression of human apoA-I resulted in a dose-dependent increase in HDL, as measured by both HDL-cholesterol and HDL-phospholipid, and produced a decrease in rat apoA-I. In rats expressing high concentrations of human apoA-I (TgR[hAI]high, human apoA-I > 250 mg/dl), the increase in HDL-phospholipid was not proportional to the increase in human apoA-I, as illustrated by a HDL-PL/total apoA-I ratio of 0.84 +/- 0.19 compared to a ratio of 1.28 +/- 0.29 for control rats and of 1.28 +/- 0.39 for rats expressing low levels of human apoA-I (TgR[hAI]low, human apoA-I < 250 mg/dl). Compared to sera from control animals, efflux of cell cholesterol was increased by 26% in the sera from TgR[hAI]low, and by 76% in the TgR[hAI]high. An examination of the relationships between efflux and HDL-related parameters demonstrated a hyperbolic relationship between efflux and either HDL-cholesterol or HDL-apoA-I. In contrast, there was a strong linear association (r2 = 0.84) between cholesterol efflux and HDL-phospholipid, indicating that this parameter is the component of HDL that best reflects the serum's efflux efficiency. The importance of phospholipids in modulating cholesterol efflux was further explored by measuring the effect of supplementation of serum with dimyristoylphosphatidylcholine (DMPC) vesicles, apoA-I, or both DMPC vesicles and apoA-I. Whereas addition of human apoA-I had no effect on efflux, supplementation with DMPC vesicles produced a substantial increase in efflux that was further stimulated by the combination of DMPC vesicles and apoA-I. These results demonstrate that a major component of HDL that modulates cell cholesterol efflux is phospholipid.

Effect of experimental nephrosis on hepatic lipoprotein secretion and urinary lipoprotein excretion in rats expressing the human apolipoprotein A-I gene.

When human apolipoprotein A-I was expressed in transgenic rats, induction of the nephrotic syndrome resulted in plasma A-I levels exceeding 10 mg/ml. Plasma lipids were no higher than in non-transgenic nephrotic rats. To explain this, the livers from four groups of rats were perfused: wild-type controls (WC), high expressor human apoA-I transgenic controls (TrGC), wild-type nephrotics (WN), and high expressor transgenic nephrotics (TrGN). Compared to the WC group, TrGC rats secreted the same amount of d < 1.063 g/ml lipoproteins but 50% more high density lipoprotein (HDL), with a 5-fold increase in total apoA-I output due to human apoA-I. Compared to the WC group, nephrosis in the WN rats caused a 2-fold increase in both d < 1.063 g/ml lipoproteins and HDL secretion with a 4.6-fold increase in rat apoA-I output. Compared to the TrGC group, nephrosis in the TrGN rats did not increase d < 1.063 g/ml lipoprotein secretion, but caused a 50% increase in HDL secretion and a 6-fold increase in human apoA-I output. The hepatic levels of mRNA for apoB and for HMG-CoA reductase, as well as the degree of apoB mRNA editing, were unchanged. Examination of the perfusate HDL by electron microscopy revealed spherical particles averaging 30 nm in diameter in the WC and WN rats and 17 and 20 nm in the TrGC and TrGN rats. Urinary HDL particles from the TrGN rats did not contain rat apoA-I and averaged 8.2 nm versus 11 nm in the WN rats. We conclude that the size of the nascent HDL, and subsequently of the mature HDL, is determined by the primary structure of apoA-I. In the TrGN rats, the heterogeneous mature HDL has a population of smaller human HDL which is more readily lost in the urine, accounting for the failure of plasma HDL levels to rise above those in TrGC rats. The fact that plasma triglyceride levels in TrGN rats were also not increased may relate to the failure of hepatic apoB secretion to increase, which in turn may have been due to saturation of the protein synthetic capacity by human apoA-I production.

Overexpression of human apolipoprotein A-I in transgenic rats and the hyperlipoproteinemia associated with experimental nephrosis.

Hyperlipoproteinemia contributes both to kidney disease progression and the development of atherosclerosis. Elevated high density lipoprotein cholesterol and apolipoprotein A-I (apoA-I) serum levels are independent factors protective against the atherosclerotic process. We examined the effects in a transgenic rat model of human apoA-I expression on the hyperlipoproteinemia and edema after puromycin aminonucleoside-induced nephrosis in three groups of animals: low line (TgR[hAI]low, human plasma apoA-I = 16.0 mg/dl); high line (TgR[hAI]high, 284 mg/dl); and non-transgenic litter mates (TgR[hAI]non). Nephrosis increased total plasma apoA-I levels 2-fold in TgR[hAI]non rats (75 vs. 162 mg/dl) and 4-fold in the TgR[hAI]low (97 vs. 458 mg/dl) and TgR[hAI]high rats (356 vs. 1,346 mg/dl). In both transgenic lines, this increase was due mainly to elevations of serum human apoA-I. The hepatic steady-state levels of rat apoA-I mRNA increased 5- to 7-fold in all three groups, while human apoA-I mRNA levels increased 21- and 65-fold in the low and high expressing groups, respectively, indicating a different degree of responsiveness of the rat and human genes. While nephrotic TgR[hAI]non and TgR[hAI]low rats showed severe hyperlipoproteinemia and edema, much lower levels of edema and of serum triglycerides, phospholipids, and cholesterol were seen in the TgR[hAI]high group. Urinary excretion of apoA-I, phospholipids, and cholesterol was significantly increased in the TgR[hAI]high group, indicating this as one possible mechanism for the relatively lower serum levels of these lipids. We conclude that the human apoA-I gene is responsive to nephrosis and that human apoA-I-transgenic rats with this syndrome provide an animal model for the study of human high density lipoprotein and apoA-I metabolism.

Hepatic apolipoprotein J is secreted as a lipoprotein.

Apolipoprotein J (apoJ) is a unique glycoprotein thought to be involved in a variety of physiological processes, including lipid transport, regulation of complement function, sperm maturation, programmed cell death, and membrane recycling. In the plasma, apoJ is associated with apoA-I in high and very high density lipoproteins. In this report we demonstrate that HepG2 human hepatocellular carcinoma cells secrete apoJ in association with a significant amount of lipid, providing unequivocal evidence that apoJ can transport lipids. The HepG2 cell line has provided important clues about the structural organization of nascent lipoprotein particles. HepG2 cell apoJ-containing lipoproteins are dense and heterogenous in size, ranging from 100 to 910 kDa. Plasma and HepG2 cell apoJ-lipoproteins differ in size distribution. Both have alpha 2 electrophoretic mobility, although their average mobilities differ within the alpha 2 region. In contrast to plasma apoJ-HDL which contain little triglyceride and which can associate with apoA-I, HepG2 cell apoJ-lipoproteins are rich in triglyceride and lack apoA-I. By implication, nascent apoJ-lipoproteins undergo plasma remodeling that results in triglyceride depletion and apoA-I association. We propose that the metabolic consequences of this remodeling play an important role in lipid homeostasis in localized tissue environments, particularly where organs are isolated from the blood by cellular barriers such as in testis and brain. In such tissues, apoJ is expressed constitutively in high level compared to other lipid transport proteins.

The effects of vitamin K1 and warfarin on prothrombin expression in human hepatoblastoma (HepG2) cells.

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Intracellular processing of apolipoprotein J precursor to the mature heterodimer.

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.