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1.  Pharmacodynamics of Glucose Regulation by Methylprednisolone. II. Normal Rats 
A physiologic pharmacodynamic model was developed to jointly describe the effects of methylprednisolone (MPL) on adrenal suppression and glycemic control in normal rats. Six groups of animals were given MPL intravenously at 0, 10 and 50 mg/kg, or by subcutaneous 7 day infusion at rates of 0, 0.1 and 0.3 mg/kg/h. Plasma concentrations of MPL, corticosterone (CST), glucose and insulin were determined at various times up to 72 h after injection and 336 h after infusion. The pharmacokinetics of MPL was described by a two-compartment model. A circadian rhythm for CST was found in untreated rats with a stress-altered baseline caused by handling, which was captured by a circadian harmonic secretion rate with an increasing mesor. All drug treatments caused CST suppression. Injection of MPL caused temporary increases in glucose over 4 h. Insulin secretion was thereby stimulated yielding a later peak around 6 h. In turn, insulin can normalize glucose. However, long-term dosing caused continuous hyperglycemia during and after infusion. Hyperinsulinemia was achieved during infusion, but diminished immediately after dosing despite the high glucose concentration. The effects of CST and MPL on glucose production were described with a competitive stimulation function. A disease progression model incorporating reduced endogenous glucose uptake/utilization was used to describe glucose metabolism under different treatments. The results exemplify the roles of endogenous and exogenous hormones in mediating glucose dynamics. The pharmacokinetic/pharmacodynamic model is valuable for quantitating diabetogenic effects of corticosteroid treatments and provides mechanistic insights into the hormonal control of the metabolic system.
doi:10.1002/bdd.642
PMCID: PMC3712293  PMID: 19156669
corticosterone; methylprednisolone; pharmacodynamics; pharmacokinetics; glucose; insulin
2.  Pharmacodynamics of Glucose Regulation by Methylprednisolone. I. Adrenalectomized Rats 
Mechanisms related to the adverse effects of corticosteroids on glucose homeostasis were studied. Five groups of adrenalectomized (ADX) rats were given methylprednisolone (MPL) intravenously at 10 and 50 mg/kg, or a continuous 7 day infusion at rates of 0, 0.1, 0.3 mg/kg/h via subcutaneously implanted Alzet mini-pumps. Plasma concentrations of MPL, glucose and insulin were determined at various time points up to 72 h after injection or 336 h after infusion. The pharmacokinetics of MPL was captured with a two-compartment model. The Adapt II software was used in modeling. Injection of MPL caused a temporary glucose increase over 6 h by stimulating gluconeogenesis. The glucose changes stimulated pancreatic β-cell secretion yielding a later insulin peak at around 10 h. In turn, insulin can stimulate glucose disposition. However, long-term MPL treatment caused continuous hyperglycemia during and after infusion. Insulin was increased during infusion, and immediately returned to baseline after the infusion was terminated, despite the almost doubled glucose concentration. A disease progression model incorporating the reduced endogenous glucose disposition was included to capture glucose homeostasis under different treatments. The results exemplify the importance of the steroid dosing regimen in mediating pharmacological and adverse metabolic effects. This mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model quantitatively describes the induction of hyperglycemia and provides additional insights into metabolic disorders such as diabetes.
doi:10.1002/bdd.643
PMCID: PMC3712292  PMID: 19156931
corticosteroids; methylprednisolone; pharmacodynamics; pharmacokinetics; glucose; insulin
3.  Pharmacodynamic Modeling of Acute and Chronic Effects of Methylprednisolone on Hepatic Urea Cycle Genes in Rats* 
Corticosteroids (CS) regulate many enzymes at both mRNA and protein levels. This study used microarrays to broadly assess regulation of various genes related to the greater urea cycle and employs pharmacokinetic/pharmacodynamic (PK/PD) modeling to quantitatively analyze and compare the temporal profiles of these genes during acute and chronic exposure to methylprednisolone (MPL). One group of adrenalectomized male Wistar rats received an intravenous bolus dose (50 mg/kg) of MPL, whereas a second group received MPL by a subcutaneous infusion (Alzet osmotic pumps) at a rate of 0.3 mg/kg/hr for seven days. The rats were sacrificed at various time points over 72 hours (acute) or 168 hours (chronic) and livers were harvested. Total RNA was extracted and Affymetrix® gene chips (RG_U34A for acute and RAE 230A for chronic) were used to identify genes regulated by CS. Besides five primary urea cycle enzymes, many other genes related to the urea cycle showed substantial changes in mRNA expression. Some genes that were simply up- or down-regulated after acute MPL showed complex biphasic patterns upon chronic infusion indicating involvement of secondary regulation. For the simplest patterns, indirect response models were used to describe the nuclear steroid-bound receptor mediated increase or decrease in gene transcription (e.g. tyrosine aminotransferase, glucocorticoid receptor). For the biphasic profiles, involvement of a secondary biosignal was assumed (e.g. ornithine decarboxylase, CCAAT/enhancer binding protein) and more complex models were derived. Microarrays were used successfully to explore CS effects on various urea cycle enzyme genes. PD models presented in this report describe testable hypotheses regarding molecular mechanisms and quantitatively characterize the direct or indirect regulation of various genes by CS.
PMCID: PMC2733100  PMID: 19787073
urea cycle; corticosteroids; methylprednisolone; pharmacodynamics; genomics
4.  Meta-Modeling of Methylprednisolone Effects on Glucose Regulation in Rats 
PLoS ONE  2013;8(12):e81679.
A retrospective meta-modeling analysis was performed to integrate previously reported data of glucocorticoid (GC) effects on glucose regulation following a single intramuscular dose (50 mg/kg), single intravenous doses (10, 50 mg/kg), and intravenous infusions (0.1, 0.2, 0.3 and 0.4 mg/kg/h) of methylprednisolone (MPL) in normal and adrenalectomized (ADX) male Wistar rats. A mechanistic pharmacodynamic (PD) model was developed based on the receptor/gene/protein-mediated GC effects on glucose regulation. Three major target organs (liver, white adipose tissue and skeletal muscle) together with some selected intermediate controlling factors were designated as important regulators involved in the pathogenesis of GC-induced glucose dysregulation. Assessed were dynamic changes of food intake and systemic factors (plasma glucose, insulin, free fatty acids (FFA) and leptin) and tissue-specific biomarkers (cAMP, phosphoenolpyruvate carboxykinase (PEPCK) mRNA and enzyme activity, leptin mRNA, interleukin 6 receptor type 1 (IL6R1) mRNA and Insulin receptor substrate-1 (IRS-1) mRNA) after acute and chronic dosing with MPL along with the GC receptor (GR) dynamics in each target organ. Upon binding to GR in liver, MPL dosing caused increased glucose production by stimulating hepatic cAMP and PEPCK activity. In adipose tissue, the rise in leptin mRNA and plasma leptin caused reduction of food intake, the exogenous source of glucose input. Down-regulation of IRS-1 mRNA expression in skeletal muscle inhibited the stimulatory effect of insulin on glucose utilization further contributing to hyperglycemia. The nuclear drug-receptor complex served as the driving force for stimulation or inhibition of downstream target gene expression within different tissues. Incorporating information such as receptor dynamics, as well as the gene and protein induction, allowed us to describe the receptor-mediated effects of MPL on glucose regulation in each important tissue. This advanced mechanistic model provides unique insights into the contributions of major tissues and quantitative hypotheses for the multi-factor control of a complex metabolic system.
doi:10.1371/journal.pone.0081679
PMCID: PMC3847111  PMID: 24312573
5.  Effect of insulin and acute diabetes on plasma FFA and ketone bodies in the fasting rat 
Journal of Clinical Investigation  1970;49(9):1685-1693.
The metabolism of FFA and ketone bodies was studied in fasted rats by infusing at a constant rate tracer amounts of FFA-3H, β-hydroxybutyrate-14C or acetoacetate-14C for periods up to 2 hr. Blood that was removed for analyses was replaced by continuous transfusion. The rates of turnover of FFA, β-hydroxybutyrate, and acetoacetate in rats fasted for 2 days were, respectively, 3.2, 5.6, and 2.5 μmoles/100 g body weight per min.
Infusion of mannoheptulose with anti-insulin serum increased plasma glucose, FFA, and ketone body concentrations and decreased the specific activity of plasma FFA. Injection of insulin (20 mU i.v.) decreased almost simultaneously plasma glucose, FFA, and ketone body concentrations and increased the specific activity of FFA, but it did not affect the plasma concentration of FFA-3H. The findings indicate that insulin deprivation increased and insulin injection decreased the release of FFA from body tissues in fasting rats.
The plasma FFA concentration in fasting rats was increased by infusing chylomicrons and heparin, but this had very little effect on either plasma ketone body or glucose concentrations. Insulin injection (20 mU i.v.) lowered the plasma ketone body concentration in these animals. Studies using β-hydroxybutyrate-14C showed that insulin (50 mU i.v.) decreased ketogenesis in the presence of a sustained high plasma FFA concentration and had no effect on uptake of circulating ketone bodies.
The results indicate that plasma FFA concentration is not the sole determinant of plasma ketone body concentration and that insulin can suppress ketone body production through some means other than lowering plasma FFA concentration.
PMCID: PMC322652  PMID: 5452413
6.  Pharmacokinetic/Pharmacodynamic Modeling of Corticosterone Suppression and Lymphocytopenia by Methylprednisolone in Rats 
Journal of pharmaceutical sciences  2008;97(7):2820-2832.
Adrenal suppression and lymphocytopenia are commonly monitored pharmacological responses during systemic exposure to exogenously administered corticosteroids. The pharmacodynamics of plasma corticosterone (CS) and blood lymphocytes were investigated in 60 normal rats which received either 50 mg/kg methylprednisolone (MPL) or vehicle intramuscularly. Blood samples were collected between 0.5 and 96 h following treatment. Plasma CS displayed a transient suppression with re-establishment of a normal circadian rhythm 24 h following drug treatment. An indirect response model with suppression of production well captured plasma CS profiles. An early stress-induced rise in CS was also factored into the model. Blood lymphocyte numbers exhibited a sharp decline and then returned to a new circadian rhythm which was half of the original baseline level. An integrated pharmacodynamic (PD) model with inhibition of lymphocyte trafficking from tissue to blood by both MPL and CS and induction of cell apoptosis by MPL reasonably captured this lymphocytopenia. Rats and humans differ in lymphocyte responses with humans showing full recovery of baselines. Modeling provides a valuable tool in quantitative assessment of dual, complex drug responses.
doi:10.1002/jps.21167
PMCID: PMC3726057  PMID: 17828751
pharmacokinetics; pharmacodynamics; hormones; mathematical model; pharmacokinetic/pharmacodynamic models; corticosteroid; lymphocyte; cell trafficking; indirect response model; circadian rhythm
7.  Pharmacokinetic/Pharmacodynamic Modeling of Methylprednisolone Effects on iNOS mRNA Expression and Nitric Oxide During LPS-Induced Inflammation in Rats 
Pharmaceutical Research  2012;29(8):2060-2069.
Purpose
Increased expression of inducible nitric oxide synthase (iNOS) resulting in nitric oxide elevation represents an important component of inflammatory responses. We assess the effects of methylprednisolone (MPL) on these processes during endotoxin-induced acute inflammation and provide a mechanism-based model to quantitatively describe them.
Methods
Male Lewis rats were dosed with lipopolysaccharide (50 μg/kg LPS) alone or with methylprednisolone (10 and 50 mg/kg) and sacrificed at different time points. Plasma MPL, lung iNOS mRNA expression, plasma nitric oxide (NO) and other physiological factors were measured. Sodium nitrate (750 μmole/kg) was given to a separate cohort of rats to assess NO disposition kinetics. PK-PD modeling was performed with ADAPT 5.
Results
Disposition kinetics of plasma MPL and NO showed bi-exponential decline and were described by two-compartment models. LPS increased expression of iNOS mRNA in lung and increased plasma NO, while MPL dosing palliated this increase in a dose-dependent manner. These effects were well captured using tandem indirect response and precursor-pool models.
Conclusion
The model provides a quantitative assessment of the suppression of NO production by MPL and shows that the major effects are at the transcriptional level by reducing expression of iNOS mRNA.
doi:10.1007/s11095-012-0733-5
PMCID: PMC3400266  PMID: 22422321
corticosteroids; inflammation; iNOS; nitric oxide; PK-PD modeling
8.  Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat. 
Journal of Clinical Investigation  1996;97(12):2728-2735.
We asked whether the well known starvation-induced impairment of glucose-stimulated insulin secretion (GSIS) seen in isolated rat pancreas preparations also applies in vivo. Accordingly, fed and 18-24-h-fasted rats were subjected to an intravenous glucose challenge followed by a hyperglycemic clamp protocol, during which the plasma-insulin concentration was measured. Surprisingly, the acute (5 min) insulin response was equally robust in the two groups. However, after infusion of the antilipolytic agent, nicotinic acid, to ensure low levels of plasma FFA before the glucose load, GSIS was essentially ablated in fasted rats, but unaffected in fed animals. Maintenance of a high plasma FFA concentration by coadministration of Intralipid plus heparin to nicotinic acid-treated rats (fed or fasted), or further elevation of the endogenous FFA level in nonnicotinic acid-treated fasted animals by infusion of etomoxir (to block hepatic fatty acid oxidation), resulted in supranormal GSIS. The in vivo findings were reproduced in studies with the perfused pancreas from fed and fasted rats in which GSIS was examined in the absence and presence of palmitate. The results establish that in the rat, the high circulating concentration of FFA that accompanies food deprivation is a sine qua non for efficient GSIS when a fast is terminated. They also serve to underscore the powerful interaction between glucose and fatty acids in normal beta cell function and raise the possibility that imbalances between the two fuels in vivo could have pathological consequences.
PMCID: PMC507365  PMID: 8675683
9.  Mechanisms of Glucose Homeostasis After Roux-en-Y Gastric Bypass Surgery in the Obese, Insulin-Resistant Zucker Rat 
Annals of surgery  2009;249(2):277-285.
Objective
Obesity-related diabetes is caused by insulin resistance and β-cell dysfunction. The current study examines changes in food intake, weight loss, body fat depots, oxygen consumption, insulin sensitivity, and incretin levels as potential mechanisms for improved glucose tolerance after Roux-en-Y gastric bypass (RYGB).
Methods
Three groups of genetically obese Zucker rats were studied: RYGB, sham surgery pair-fed (PF), and sham surgery ad libitum (AL) fed rats. Changes in body weight, visceral and subcutaneous fat depots, oral glucose tolerance, insulin sensitivity, and the plasma concentrations of insulin, glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide, and peptide YY (PYY) were measured.
Results
Body weight and subcutaneous fat were decreased after RYGB, compared with the PF and AL groups. The reduction in visceral fat after RYGB appeared largely because of food restriction. Glucose tolerance and insulin sensitivity were significantly improved in only the RYGB group (P <0.05 vs. AL, PF). Euglycemic, hyperinsulinemic clamp studies indicated RYGB improved the ability of insulin to stimulate peripheral (eg, skeletal muscle) glucose uptake. Fasting total GLP-1, glucose-dependent insulinotropic peptide, and PYY levels were similar between the groups, whereas postprandial plasma levels of intact GLP-1 (7–36) amide, total GLP-1, and PYY were increased in the RYGB group compared with PF and AL controls.
Conclusions
Glucose homeostasis after RYGB is associated with decreased subcutaneous fat, increased postprandial PYY, GLP-1, and insulin, as well as improved insulin sensitivity/action. Changes in food intake and visceral fat do not seem to explain improvements in insulin action after RYGB in the Zucker rat model.
doi:10.1097/SLA.0b013e3181904af0
PMCID: PMC2748974  PMID: 19212182
gastric bypass; obesity; diabetes; incretin; GLP-1
10.  Fatty acids in the portal vein of the rat regulate hepatic insulin clearance. 
Journal of Clinical Investigation  1991;88(6):2054-2058.
The effects of FFA on hepatic insulin clearance were studied in the in situ perfused rat liver. Clearance decreased with increasing body weight (age) of the rats. When FFA were added to the perfusate a 40% reduction of hepatic removal of insulin was found over the normal, physiological range (less than 1,000 mumol/liter), less pronounced in heavier rats. When perfusion was started with high concentrations of FFA, inhibition was rapidly reversible, a phenomenon again blunted in heavier rats. In contrast to FFA, different glucose concentrations in the perfusate did not affect the hepatic insulin uptake in the presence of FFA within physiological concentrations. Thus, hepatic clearance of insulin is proportional to rat weight (age) and portal FFA concentrations. Other studies have recently shown that fatty acids inhibit insulin binding, degradation, and function in isolated rat hepatocytes, and that hepatic clearance is inversely dependent on hepatic triglyceride concentrations, both inhibitions reversible by prevention of fatty acid oxidation. It is suggested that the diminished hepatic clearance of insulin in heavier (older) rats is at least partly due to their relative obesity and increased hepatic triglyceride contents. This effect as well as that of portal FFA is probably mediated via fatty acid oxidation in the liver. This mechanism may have implications for the regulation of hepatic metabolism, and peripheral insulin concentrations.
PMCID: PMC295800  PMID: 1752963
11.  Tissue-Specific Gene Expression and Regulation in Liver and Muscle Following Chronic Corticosteroid Administration 
Although corticosteroids (CSs) affect gene expression in multiple tissues, the array of genes that are regulated by these catabolic steroids is diverse, highly tissue specific, and depends on their functions in the tissue. Liver has many important functions in performing and regulating diverse metabolic processes. Muscle, in addition to its mechanical role, is critical in maintaining systemic energy homeostasis and accounts for about 80% of insulin-directed glucose disposal. Consequently, a better understanding of CS pharmacogenomic effects in these tissues would provide valuable information regarding the tissue-specificity of transcriptional dynamics, and would provide insights into the underlying molecular mechanisms of action for both beneficial and detrimental effects.
We performed an integrated analysis of transcriptional data from liver and muscle in response to methylprednisolone (MPL) infusion, which included clustering and functional annotation of clustered gene groups, promoter extraction and putative transcription factor (TF) identification, and finally, regulatory closeness (RC) identification.
This analysis allowed the identification of critical transcriptional responses and CS-responsive functions in liver and muscle during chronic MPL administration, the prediction of putative transcriptional regulators relevant to transcriptional responses of CS-affected genes which are also potential secondary bio-signals altering expression levels of target-genes, and the exploration of the tissue-specificity and biological significance of gene expression patterns, CS-responsive functions, and transcriptional regulation.
The analysis provided an integrated description of the genomic and functional effects of chronic MPL infusion in liver and muscle.
doi:10.4137/GRSB.S13134
PMCID: PMC3956809  PMID: 24653645
liver; muscle; glucocorticoids; corticosteroids; gene expression; gene regulation; promoter analysis
12.  Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle After Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades 
Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
PMCID: PMC2733097  PMID: 19787081
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance
13.  Glucose and free fatty acid metabolism in non-insulin-dependent diabetes mellitus. Evidence for multiple sites of insulin resistance. 
Journal of Clinical Investigation  1989;84(1):205-213.
The effect of graded, physiologic hyperinsulinemia (+5, +15, +30, +70, +200 microU/ml) on oxidative and nonoxidative pathways of glucose and FFA metabolism was examined in nine lean non-insulin dependent diabetic patients (NIDDM) and in eight age- and weight-matched control subjects. Glucose and FFA metabolism were assessed using stepwise insulin clamp in combination with indirect calorimetry and infusion of [3H]3-glucose/[14C]palmitate. The basal rate of hepatic glucose production (HGP) was higher in NIDDM than in control subjects, and suppression of HGP by insulin was impaired at all but the highest insulin concentration. Glucose disposal was reduced in the NIDD patients at the three highest plasma insulin concentrations, and this was accounted for by defects in both glucose oxidation and nonoxidative glucose metabolism. In NIDDs, suppression of plasma FFA by insulin was impaired at all five insulin steps. This was associated with impaired suppression by insulin of plasma FFA turnover, FFA oxidation (measured by [14C]palmitate) and nonoxidative FFA disposal (an estimate of reesterification of FFA). FFA oxidation and net lipid oxidation (measured by indirect calorimetry) correlated positively with the rate of HGP in the basal state and during the insulin clamp. In conclusion, our findings demonstrate that insulin resistance is a general characteristic of glucose and FFA metabolism in NIDDM, and involves both oxidative and nonoxidative pathways. The data also demonstrate that FFA/lipid and glucose metabolism are interrelated in NIDDM, and suggest that an increased rate of FFA/lipid oxidation may contribute to the impaired suppression of HGP and diminished stimulation of glucose oxidation by insulin in these patients.
PMCID: PMC303971  PMID: 2661589
14.  Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats. 
Journal of Clinical Investigation  1997;99(12):2883-2889.
Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.
PMCID: PMC508139  PMID: 9185511
15.  Antihyperlipidemic and antidiabetic effects of umbelliferone in streptozotocin diabetic rats. 
The aim of the study was to evaluate blood glucose and lipid lowering effects of Umbelliferone (UMB) in streptozotocin (STZ) diabetic rats. Male albino Wistar rats (180 to 200 g) were induced diabetes by administration of STZ (40 mg/kg) intraperitonially. Normal and diabetic rats were treated with UMB in 10 percent dimethyl sulfoxide (DMSO) for 45 days. Diabetic rats had increased plasma glucose and decreased insulin, total proteins (TP), and albumin in addition to decreased food intake and body weight. Elevation in total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), triglycerides (TG), free fatty acids (FFA), and phospholipids (PL), and reduction in high density lipoprotein cholesterol (HDL-C) in the plasma were observed. Liver and kidney tissues of diabetic rats had elevation in the levels of TC, TG, FFA, and PL. Treatment with UMB decreased plasma glucose and increased insulin, TP, and albumin apart from food intake and body weight. In UMB-treated diabetic rats, plasma and tissue TC, TG, PL and FFA, and plasma LDL-C, VLDL-C, and HDL-C reversed to near normal. Thus, reduction of blood glucose and lipid profiles indicates that UMB has antidiabetic and antihyperlipidemic effects in diabetic rats.
PMCID: PMC2259150  PMID: 16720013
16.  Adenovirus-mediated chronic “hyper-resistinemia” leads to in vivo insulin resistance in normal rats 
Journal of Clinical Investigation  2004;114(2):224-231.
We investigated the chronic in vivo effect of resistin on insulin sensitivity and glucose metabolism by overexpressing resistin protein in male Wistar rats using intravenous administration of an adenovirus encoding mouse resistin. After 7 days of elevated resistin levels at a supraphysiological concentration, the animals displayed glucose intolerance and hyperinsulinemia during glucose tolerance tests, and insulin tolerance tests demonstrated an impaired glucose-lowering effect of insulin. The glucose clamp studies were performed at submaximal (4 mU/kg/min) and maximal (25 mU/kg/min) insulin infusion rates and demonstrated the presence of insulin resistance induced by elevated resistin levels. Indeed, the insulin-stimulated glucose infusion rate was decreased by 12–31%; suppression of hepatic glucose output was attenuated by 28–55%; and insulin suppression of circulating FFA levels was inhibited by 7%. Insulin receptor substrate–1 and –2 phosphorylation and Akt activation were impaired in muscle and adipose tissue. Interestingly, activation of AMP-activated protein kinase in skeletal muscle, liver, and adipose tissue was also significantly downregulated. Together, these results indicate that chronic “hyper-resistinemia” leads to whole-body insulin resistance involving impaired insulin signaling in skeletal muscle, liver, and adipose tissue, resulting in glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. Thus elevated resistin levels in normal rats fed a regular chow diet produce many of the features of human syndrome X.
doi:10.1172/JCI200420785
PMCID: PMC449745  PMID: 15254589
17.  No evidence of drug-induced pancreatitis in rats treated with exenatide for 13 weeks 
Diabetes, Obesity & Metabolism  2012;15(5):417-426.
Aims
The potential association of glucagon-like peptide receptor agonists (GLP-1RAs) with the development of pancreatitis or pancreatic malignancies in patients with diabetes has been suggested. This study evaluated the long-term effects of the GLP-1RA exenatide on pancreatic exocrine structure and function in the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes.
Methods
Rats received subcutaneous twice-daily injections of 0 (control), 6, 40 and 250 µg/kg/day exenatide for 3 months. Clinical signs, body and pancreas weight, food consumption, HbA1c, fasting serum amylase, lipase, glucose and insulin concentrations were evaluated during treatment and after a 28-day off-drug period to assess the reversibility of any observed effects. Morphometric analysis of pancreatic ductal cell proliferation and apoptosis were performed.
Results
Plasma exenatide concentrations were several-fold higher than therapeutic levels observed in humans. No exenatide-related effects were observed on clinical signs, lipase concentration, pancreatic weight, pancreatic histology, ductal cell proliferation or apoptosis. Exenatide improved animal survival, physical condition, glucose concentrations and HbA1c, decreased food intake, and increased serum insulin concentration. Total amylase concentrations, although within normal ranges, were slightly higher in exenatide-treated rats; following the off-drug period, total amylase concentrations were comparable in treated and untreated rats. Exenatide-related minimal-to-moderate islet hypertrophy was observed at doses ≥6 µg/kg/day, with dose-related increases in incidence and degree. These changes were still present after the off-drug period.
Conclusions
Chronic administration of exenatide in ZDF rats resulted in the expected metabolic benefits and improved animal survival, with no adverse effects noted on pancreatic exocrine structure and function.
doi:10.1111/dom.12040
PMCID: PMC3654567  PMID: 23163898
exenatide; exocrine pancreas; ZDF rat
18.  Pharmacodynamic characterization of insulin on MDMA-induced thermogenesis 
European journal of pharmacology  2009;615(1-3):257-261.
Sympathomimetic drugs (MDMA; Ecstasy) induce a potentially catastrophic hyperthermia that involves free fatty acid (FFA) activation of mitochondrial uncoupling proteins (UCP). Insulin is an important regulator of plasma FFA levels, although its role in thermogenesis is unclear. The aims of the present study were 1) to characterize the pharmacodynamic effects of MDMA on plasma insulin and glucose, 2) to examine the effects of insulin on MDMA-induced thermogenesis and 3) to examine MDMA-induced thermogenesis in an animal model of insulin resistance, the obese Zucker rat. Insulin levels peaked 15 min. after MDMA (40 mg/kg, sc), which preceded the peak temperature change at 60 min. Plasma glucose levels also peaked 15 min. after MDMA and remained elevated throughout the 90-min. monitoring period. Insulin pretreatment (10 units/kg, sc) 30 min. before a low dose of MDMA (5 mg/kg, sc) potentiated the thermogenic response. Insulin resistant, fa/fa (obese) Zucker rats demonstrated an attenuated thermogenic response to MDMA (40 mg/kg, sc). Consistent with the role for FFA in UCP3 expression, immunoblot analysis showed significantly increased levels of UCP3 protein in obese compared to lean Zucker skeletal muscle. In conclusion, the results of the present study suggest a potential role of insulin signaling in sympathomimetic-induced thermogenesis.
doi:10.1016/j.ejphar.2009.05.021
PMCID: PMC2740838  PMID: 19482019
3,4-methylenedioxymethamphetamine; thermogenesis; insulin; glucose; Zucker
19.  Atypical Antipsychotics Rapidly and Inappropriately Switch Peripheral Fuel Utilization to Lipids, Impairing Metabolic Flexibility in Rodents 
Schizophrenia Bulletin  2010;38(1):153-166.
Patients taking atypical antipsychotics are frequented by serious metabolic (eg, hyperglycemia, obesity, and diabetes) and cardiac effects. Surprisingly, chronic treatment also appears to lower free fatty acids (FFAs). This finding is paradoxical because insulin resistance is typically associated with elevated not lower FFAs. How atypical antipsychotics bring about these converse changes in plasma glucose and FFAs is unknown. Chronic treatment with olanzapine, a prototypical, side effect prone atypical antipsychotic, lowered FFA in Sprague–Dawley rats. Olanzapine also lowered plasma FFA acutely, concomitantly impairing in vivo lipolysis and robustly elevating whole-body lipid oxidation. Increased lipid oxidation was evident from accelerated losses of triglycerides after food deprivation or lipid challenge, elevated FFA uptake into most peripheral tissues (∼2-fold) except heart, rises in long-chain 3-hydroxylated acyl-carnitines observed in diabetes, and rapid suppression of the respiratory exchange ratio (RER) during the dark cycle. Normal rises in RER following refeeding, a sign of metabolic flexibility, were severely blunted by olanzapine. Increased lipid oxidation in muscle could be explained by ∼50% lower concentrations of the negative cytoplasmic regulator of carnitine palmitoyltransferase I, malonyl-CoA. This was associated with loss of anapleurotic metabolites and citric acid cycle precursors of malonyl-CoA synthesis rather than adenosine monophosphate-activated kinase activation or direct ACC1/2 inhibition. The ability of antipsychotics to lower dark cycle RER in mice corresponded to their propensities to cause metabolic side effects. Our studies indicate that lipocentric mechanisms or altered intermediary metabolism could underlie the FFA lowering and hyperglycemia (Randle cycle) as well as some of the other side effects of atypical antipsychotics, thereby suggesting strategies for alleviating them.
doi:10.1093/schbul/sbq053
PMCID: PMC3245588  PMID: 20494946
antipsychotics; fatty acid oxidation; metabolomics; side effects; malonyl-CoA; anaplerosis; obesity; diabetes; metabolic flexibility
20.  Rapid Pharmacokinetics of Intradermal Insulin Administered Using Microneedles in Type 1 Diabetes Subjects 
Abstract
Background
This study compared the pharmacokinetics, postprandial glycemic response, and pain associated with intradermal lispro insulin delivery using a microneedle with that of a conventional catheter.
Subjects and Methods
Five subjects with type 1 diabetes were administered a bolus infusion of lispro insulin using a 9-mm-long subcutaneous catheter (control treatment) and a 0.9-mm-long microneedle (study treatment), followed by consumption of a standardized meal. Blood samples were periodically assayed for plasma glucose and free insulin levels.
Results
Intradermal insulin infusion using microneedles reached peak insulin concentrations in approximately half the time and led to greater reduction in plasma glucose levels than subcutaneous catheters. Microneedles were also significantly less painful than the catheters.
Conclusion
The rapid pharmacokinetics and minimally invasive nature of intradermal insulin infusion using microneedles provide significant potential for improved diabetes management.
doi:10.1089/dia.2010.0204
PMCID: PMC3131988  PMID: 21355717
21.  Demonstration of a critical role for free fatty acids in mediating counterregulatory stimulation of gluconeogenesis and suppression of glucose utilization in humans. 
Journal of Clinical Investigation  1993;92(4):1617-1622.
In vitro studies indicate that FFA compete with glucose as an oxidative fuel in muscle and, in addition, stimulate gluconeogenesis in liver. During counterregulation of hypoglycemia, plasma FFA increase and this is associated with an increase in glucose production and a suppression of glucose utilization. To test the hypothesis that FFA mediate changes in glucose metabolism that occur during counterregulation, we examined the effects of acipimox, an inhibitor of lipolysis, on glucose production and utilization ([3-3H]glucose), and incorporation of [U-14C]-alanine into glucose during insulin-induced hypoglycemia. Eight normal volunteers were infused with insulin for 8 h to produce modest hypoglycemia (approximately 3 mM) on two occasions, first without acipimox (control) and then with acipimox administration (250 mg per os at 60 and 240 min). Despite identical plasma insulin concentrations, glucose had to be infused in the acipimox experiments (glucose-clamp technique) to maintain plasma glucose concentrations identical to those in control experiments. Acipimox completely prevented counterregulatory increases in lipolysis so that during the last 4 h plasma FFA were below baseline values and averaged 67 +/- 13 vs. 725 +/- 65 microM in control experiments, P < 0.001. Concomitantly, overall glucose production was reduced by 40% (5.5 +/- 11 vs. 9.3 +/- 0.7 mumol/kg per min, P < 0.001), and gluconeogenesis from alanine was reduced by nearly 70% (0.32 +/- 0.09 vs. 1.00 +/- 0.18 mumol/kg per min, P < 0.001), while glucose utilization increased by 15% (10.8 +/- 1.4 vs. 9.3 +/- 0.7 mumol/kg per min). We conclude that FFA play a critical role in mediating changes in glucose metabolism during counterregulation, and that under these conditions, FFA exert a much more profound effect on hepatic glucose production than on glucose utilization.
PMCID: PMC288319  PMID: 8408616
22.  Lipid infusion lowers sympathetic nervous activity and leads to increased β-cell responsiveness to glucose 
Journal of Clinical Investigation  1999;103(3):413-419.
We investigated the possible involvement of the autonomic nervous system in the effect of a long-term elevation of plasma free fatty acid (FFA) concentration on glucose-induced insulin secretion (GIIS) in rats. Rats were infused with an emulsion of triglycerides (Intralipid) for 48 hours (IL rats). This resulted in a twofold increase in plasma FFA concentration. At the end of infusion, GIIS as reflected in the insulinogenic index (ΔI/ΔG) was 2.5-fold greater in IL rats compared with control saline-infused rats. The ratio of sympathetic to parasympathetic nervous activities was sharply decreased in IL rats relative to controls. GIIS was studied in the presence of increasing amounts of α- and β-adrenoreceptor agonists and antagonists. The lowest concentrations of the α2A-adrenoreceptor agonist oxymetazoline, which were ineffective in control rats, reduced GIIS in IL rats. At the dose of 0.3 pmol/kg, GIIS became similar in IL and control rats. The use of β-adrenoreceptor agonist (isoproterenol) or antagonist (propranolol) did not result in a significant alteration in GIIS in both groups. GIIS remained as high in IL vagotomized rats as in intact IL rats, indicating that changes in parasympathetic tone were of minor importance. Altogether, the data show that lipid infusion provokes β-cell hyperresponsiveness in vivo, at least in part through changes in α2-adrenergic innervation.
PMCID: PMC407894  PMID: 9927503
23.  Carbohydrate metabolism in pregnancy 
Journal of Clinical Investigation  1970;49(7):1438-1446.
The effects of late pregnancy on adipose tissue metabolism have been examined in fed and fasted rats. Lumbar fat was excised from 19-day pregnant and age-matched virgin rats which had been given unrestricted access to food (“fed”) or fasted for 48 hr before sacrifice.
In the fed state, adipose tissue from pregnant rats displayed an increased content of free fatty acids (FFA). This coincided with augmented cleavage of preformed glycerides during incubation in vitro as evidenced by greater net production of FFA and glycerol, and altered disposition of labeled glucose. The enhanced lipolysis was independent of the availability of glucose and was not accompanied by impaired responsiveness to the antilipolytic or to the lipogenic actions of added insulin. In the presence of glucose and albumin, esterification as well as lipolysis was greater in adipose tissue from pregnant than nongravid animals. All the differences were exaggerated by prior fasting.
These properties of adipose tissue during late gestation have been ascribed to a primary activation of lipolysis rather than impaired esterification or resistance to insulin. It has been suggested that the hormones of pregnancy may be responsible. Although increased intake of food and heightened availability of insulin may offset the net lipolytic effects in the fed state, a heightened turnover of adipose stores is always present. Thus, the pregnant animal appears better poised to mobilize preformed fat whenever exogenous nutrients are withheld.
PMCID: PMC322617  PMID: 5432373
24.  Leptin Deficiency Causes Insulin Resistance Induced by Uncontrolled Diabetes 
Diabetes  2010;59(7):1626-1634.
OBJECTIVE
Depletion of body fat stores during uncontrolled, insulin-deficient diabetes (uDM) results in markedly reduced plasma leptin levels. This study investigated the role of leptin deficiency in the genesis of severe insulin resistance and related metabolic and neuroendocrine derangements induced by uDM.
RESEARCH DESIGN AND METHODS
Adult male Wistar rats remained nondiabetic or were injected with the β-cell toxin, streptozotocin (STZ) to induce uDM and subsequently underwent subcutaneous implantation of an osmotic minipump containing either vehicle or leptin at a dose (150 μg/kg/day) designed to replace leptin at nondiabetic plasma levels. To control for leptin effects on food intake, another group of STZ-injected animals were pair fed to the intake of those receiving leptin. Food intake, body weight, and blood glucose levels were measured daily, with body composition and indirect calorimetry performed on day 11, and an insulin tolerance test to measure insulin sensitivity performed on day 16. Plasma hormone and substrate levels, hepatic gluconeogenic gene expression, and measures of tissue insulin signal transduction were also measured.
RESULTS
Physiologic leptin replacement prevented insulin resistance in uDM via a mechanism unrelated to changes in food intake or body weight. This effect was associated with reduced total body fat and hepatic triglyceride content, preservation of lean mass, and improved insulin signal transduction via the insulin receptor substrate–phosphatidylinositol-3-hydroxy kinase pathway in the liver, but not in skeletal muscle or adipose tissue. Although physiologic leptin replacement lowered blood glucose levels only slightly, it fully normalized elevated plasma glucagon and corticosterone levels and reversed the increased hepatic expression of gluconeogenic enzymes characteristic of rats with uDM.
CONCLUSIONS
We conclude that leptin deficiency plays a key role in the pathogenesis of severe insulin resistance and related endocrine disorders in uDM. Treatment of diabetes in humans may benefit from correction of leptin deficiency as well as insulin deficiency.
doi:10.2337/db09-1918
PMCID: PMC2889761  PMID: 20424233
25.  Effects of phlorizin and acipimox on insulin resistance in STZ-diabetic rats. 
To evaluate the roles of hyperglycemia and increased plasma FFA level in the development of insulin resistance, we examined the effects of phlorizin and acipimox treatments on tissue sensitivity to insulin in streptozotocin(STZ)-diabetic rats. Insulin sensitivity was assessed with the glucose-insulin clamp technique. Blood glucose concentration was clamped at basal levels of control and diabetic states, and plasma insulin concentrations were clamped at the levels of basal, approximately 60 and approximately 1500 microU/ml. In diabetic rats, the basal blood glucose and plasma FFA levels in the fasting state were elevated, while the plasma insulin concentration was lower than in normal controls. Moreover, diabetic rats became glucose intolerant after intravenous injection of glucose. The metabolic clearance rate(MCR) of glucose showed a decrease of basal and insulin stimulated response in diabetic rats. As results of the glucose-insulin clamp study and intravenous glucose tolerance test, insulin resistance was developed in STZ-diabetic rats. Phlorizin treatment of diabetic rats recovered insulin sensitivity to nearly normal levels and improved glucose tolerance, but had no effect on insulin action in controls. Insulin sensitivity was also improved by acipimox treatment in diabetic rats, but did not reach normal levels. These results show that hyperglycemia is an obvious causative factor of insulin resistance, and increased FFA level may also act on the development of insulin resistance in STZ-diabetic rats.
PMCID: PMC3054119  PMID: 7598820

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