K. Kumar Changani

Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.

Rapid reversal of hepatic steatosis, and reduction of muscle triglyceride, by rosiglitazone: MRI/S studies in Zucker fatty rats.

Aim:  This study aimed to chart the time course and durability of the effects of rosiglitazone, a potent thiazolidinedione‐based peroxisome proliferator‐activated receptor γ agonist, on hepatic steatosis and intramyocellular lipid in an animal model of obesity, the Zucker Fatty (ZF) rat.

Evidence for altered hepatic gluconeogenesis in patients with cirrhosis using in vivo 31-phosphorus magnetic resonance spectroscopy

BACKGROUND AND AIMS Alterations in gluconeogenesis in the diseased liver can be assessed non-invasively using magnetic resonance spectroscopy by measuring changes in phosphomonoester resonance which contains information regarding several metabolites, including the phosphorylated intermediates of the gluconeogenic pathway. METHODS 31P magnetic resonance spectroscopy was used to determine changes in phosphomonoesters following bolus infusions of 2.8 mmol/kgl-alanine in five patients with functionally compensated cirrhosis and in five patients with functionally decompensated cirrhosis. RESULTS Compared with six healthy volunteers, baseline phosphomonoester values were elevated by 35% (p<0.05) in the compensated cirrhosis group and by 57% (p<0.01) in the decompensated cirrhosis group. Following alanine infusion, phosphomonoesters in healthy volunteers increased by 46% from baseline values (p<0.01), in patients with compensated cirrhosis by 27% (p<0.02) but those with decompensated cirrhosis showed no increase from baseline. There was a reduction in the percentage of inorganic phosphate signal in all subjects. CONCLUSIONS By analysing changes in phosphomonoester and inorganic phosphate resonances it is possible to discern clear metabolic differences between healthy volunteers and patients with cirrhosis of varying severity using magnetic resonance spectroscopy. Those patients with functionally decompensated cirrhosis have higher percentage baseline phosphomonoester values but the absence of phosphomonoester elevation following l-alanine infusion suggests that they are unable to mount a significant metabolic response with a progluconeogenic stimulus.

Non-invasive assessment of ATP regeneration potential of the preserved donor liver

We have developed a quick, non-invasive method for measuring the ability of an isolated preserved liver to regenerate high energy phosphate nucleotides without the need for biopsy. Using 31P MRS we have monitored the hepatic energetics of intact cold preserved pig liver using standard clinical harvesting and storage techniques. Following cold storage for 2 h the livers were hypothermically reperfused with oxygenated modified University of Wisconsin preservation fluid. Prior to reperfusion MRS detectable adenosine diphosphate plus adenosine triphosphate was negligible; however, the spectrum showed intense resonances from phosphomonoesters and inorganic phosphate, as a consequence of adenosine triphosphate hydrolysis during cold preservation. Following a 10-min period of hypothermic reperfusion, regeneration of adenosine triphosphate occurred with a concurrent decline in inorganic phosphate and phosphomonoester, both of which are associated with adenosine triphosphate synthesis. The capacity of the liver to regenerate adenosine triphosphate following a 24-h period of cold storage was reduced by approximately 40% (p < 0.01) of the total amount achieved following the shorter cold storage time. Adenosine triphosphate regeneration rates were biphasic and were decreased upon prolonged storage, with the initial rate being reduced from 40.6 x 10(-2).min-1 (standard deviation (sd) 2.70 x 10(-2).min-1) to 14.8 x 10(-2).min-1 (sd; 2.4 x 10(-2).min-1) and the secondary rate from 1.77 x 10(-2).min-1 (sd; 0.18 x 10(-2).min-1) to 0.84 x 10(-2).min-1 (sd; 0.45 x 10(-2).min-1). MR images of the liver during the period of hypothermic reperfusion were also performed providing an assessment for the degree of hepatic vascular perfusion. This non-invasive, 31P MRS assessment of hepatic energetics in a clinically relevant animal model has great potential for the understanding of graft preservation injury.

In vivo and in vitro hepatic phosphorus-31 magnetic resonance spectroscopy and electron microscopy in chronic ductopenic rejection of human liver allografts

Background—In vivo hepatic phosphorus-31 magnetic resonance spectroscopy (MRS) provides non-invasive information about phospholipid metabolism. Aims—To delineate MRS abnormalities in patients with chronic ductopenic rejection (CDR) and to characterise spectral changes by in vitro MRS and electron microscopy. Patients and methods—Sixteen liver transplant recipients (four with CDR; 12 with good graft function) and 29 controls (23 healthy volunteers; six patients with biliary duct strictures) were studied with in vivo 31P MRS. Peak area ratios of phosphomonoesters (PME) and phosphodiesters (PDE), relative to nucleotide triphosphates (NTP) were measured. In vitro MRS and electron microscopy were performed on biopsy specimens from five patients with CDR, freeze clamped at retransplantation. Phosphoethanolamine (PE), phosphocholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC) concentrations were measured. Results—The 12 patients with good graft function displayed no spectral abnormalities in vivo; the four patients with CDR showed significantly elevated PME:NTP (p<0.01) and PDE:NTP ratios (p<0.005). Patients with biliary strictures had significant differences in PME:NTP (p<0.01) from patients with CDR, but not in mean PDE:NTP. In vitro spectra from CDR patients showed elevated PE and PC, mirroring the in vivo changes in PME, but reduced GPE and GPC concentrations were observed, at variance with the in vivo PDE findings. On electron microscopy, there was no proliferation in hepatocyte endoplasmic reticulum. Conclusions—The increase in PME:NTP reflects altered phospholipid metabolism in patients with CDR, while the increase in PDE:NTP may represent a significant contribution from bile phospholipid.

Conflicting MRI signals from gliosis and neuronal vacuolation in prion diseases

Magnetic resonance imaging (MRI) has given inconsistent results when used as a non-invasive diagnostic tool for Creutzfeldt-Jakob disease (CJD). In order to understand this finding, we studied a hamster model of scrapie by in vivo MRI and histopathology. Vacuolation of neurones/neuropil and gliosis were found to correlate with hypo-intense and hyper-intense changes in the conventional T2-weighted MR images, respectively. These opposing effects were shown to give rise to normal images of a scrapie-affected brain undergoing severe neurodegeneration, and may underlie the variability of previous CJD MRI data.

Hepatic nucleotide triphosphate regeneration after hypothermic reperfusion in the pig model: an in vitro P-NMR study.

The aim of this study was to assess the possibility of regenerating nucleotide triphosphates (NTP) in the pig liver following its harvest and subsequent storage on ice. This study has used a pig model that allowed human donor liver retrieval techniques and methods of storage to be utilized. In vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy was used to evaluate the changes associated with phosphorus containing metabolites such as NTP, phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Po). During 4 hr storage NTP levels were reduced to undetectable levels but its regeneration was possible over a period of 2 hr of oxygenated hypothermic reperfusion. Resynthesized NTP reached values that were only 30% reduced from pre-harvest values. There was a corresponding reduction in Pi over the same period. Glycolytic intermediates, 3-phosphoglycerate and 2,3 diphosphoglycerate, both increased significantly during the period of storage and subsequently declined following hypothermic reperfusion. Cellular damage, indicated by the concentrations of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) was minimal during cold storage. However upon hypothermic reperfusion, concentrations of GPC and GPE reduced, indicating a degree of cellular damage caused by reperfusion. This study has shown for the first time that is possible to regenerate high energy phosphate nucleotides following a period of hypothermic reperfusion in a large, clinically related animal model. This technique warrants investigation clinically to improve the outcome of orthotopic liver transplantation. It also provides a method to study the effects of different preservation fluids and methods of storage and organ reperfusion.

Automatic Quantification of Changes in Bone in Serial MR Images of Joints

Recent innovations in drug therapies have made it highly desirable to obtain sensitive biomarkers of disease progression that can be used to quantify the performance of candidate disease modifying drugs. In order to measure potential image-based biomarkers of disease progression in an experimental model of rheumatoid arthritis (RA), we present two different methods to automatically quantify changes in a bone in in-vivo serial magnetic resonance (MR) images from the model. Both methods are based on rigid and nonrigid image registration to perform the analysis. The first method uses segmentation propagation to delineate a bone from the serial MR images giving a global measure of temporal changes in bone volume. The second method uses rigid body registration to determine intensity change within a bone, and then maps these into a reference coordinate system using nonrigid registration. This gives a local measure of temporal changes in bone lesion volume. We detected significant temporal changes in local bone lesion volume in five out of eight identified candidate bone lesion regions, and significant difference in local bone lesion volume between male and female subjects in three out of eight candidate bone lesion regions. But the global bone volume was found to be fluctuating over time. Finally, we compare our findings with histology of the subjects and the manual segmentation of bone lesions

A longitudinal magnetic resonance imaging (MRI) study of differences in abdominal fat distribution between normal mice, and lean overexpressors of mitochondrial uncoupling protein-3 (UCP-3)

Aim: To characterize evolution and distribution of abdominal adipose fat between 6 and 18 weeks of age in an animal model of energy consumption based on mice overexpressing the mitochondrial uncoupler protein 3 (UCP‐3).

Improved preservation solutions for organ storage: a dynamic study of hepatic metabolism.

BACKGROUND Organ cold storage times may be extended by modifications to organ preservation solutions. METHODS Three preservation solutions were investigated for their ability to maintain viable hepatic bioenergetics in stored pig livers: modified University of Wisconsin (mUW); mUW+adenosine (1.34 g/L), and mUW+ iloprost (10(-8)mol/L), a prostacyclin analogue. Using human liver retrieval and storage techniques, pig livers were stored on ice for either 2 or 16 hr, after which phosphorus-31 spectra were collected every 2 min during the period of cold ischemia and hypothermic reperfusion (HtR). During HtR, metabolite concentration changes associated with phosphomonoesters, inorganic phosphate, gamma-nucleotide triphosphate (NTP), and beta-NTP were measured for all solutions. RESULTS After a 2-hr storage, beta-NTP regeneration in mUW+iloprost produced +57.7% (P<0.01) more beta-NTP, at a faster initial rate of +66.3% (P<0.001), compared with mUW, and mUW+adenosine regenerated +35.6% (P<0.05) more beta-NTP, compared with mUW. Storage for 16 hr did not slow the rates of regeneration, and the total NTP produced during the course of the experiment remained unchanged for the respective preservation solutions. Cessation of HtR invoked a net accumulation of nucleotide diphosphate, indicating differential kinetics of adenine nucleotide hydrolysis. CONCLUSION This large animal model study suggests significant improvements to human organ preservation solutions using prostacyclin analogues and adenosine with respect to hepatic bioenergetics.