An in situ perfusion protocol of rat epididymal adipose tissue useful in metabolic studies

Experimental approaches involving the perfusion of tissues and organs offer the advantage of improved physiological relevance over the use of isolated tissues or cells while at the same time being much more controlled and tissue-specific than studies in vivo. Nevertheless, there have been few metabolic studies performed in perfused white adipose tissue, largely because of the difficulty of the surgical technique involved. Although some methods have been described, they are difficult to use as perfusion protocols and their reproducibility is poor. We have modified a rat perfusion method, based on a modification of the Ho and Meng technique, for use with epididymal white adipose tissue (eWAT), and we present it here as a protocol to be reproduced. We also offer surgical solutions for the most common variants of vessel distributions in rats. Using the protocol described here, the perfused adipose tissue is viable and metabolically active, as indicated by the maintenance of tissue ATP levels and adiponectin secretion and by endogenous lipolysis regulation. Moreover, there is a high level of lipoprotein lipase activity in the endothelium of the tissue, which is heparin-releasable. Thus, this method is a useful and reproducible tool that allows the perfusion of eWAT for use in metabolic studies.     

Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin.

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.     

Regulation of lipoprotein lipase activity and mRNA content in rat epididymal adipose tissue in vitro by recombinant tumour necrosis factor.

Tumour necrosis factor (TNF) has previously been shown to decrease lipoprotein lipase (LPL) activity and mRNA levels in 3T3-L1 cells and in adipose tissue from rats and guinea pigs when injected in vivo, but not to alter LPL activity in human adipocytes incubated in vitro. The effect of recombinant human TNF on LPL activity and mRNA levels in rat epididymal adipose tissue incubated in vitro was examined. LPL activity and mRNA levels fell in adipose tissue taken from fed rats and incubated in Krebs-Henseleit bicarbonate medium with glucose. The addition of insulin and dexamethasone prevented these falls. TNF (400 ng/ml) produced a fall of approx. 50% in LPL activity after 2 h of incubation and of approx. 30% in LPL mRNA levels after 3 h. TNF did not decrease LPL activity in isolated adipocytes. These results demonstrate that rat adipose tissue incubated in vitro is responsive to TNF whereas isolated adipocytes are not.     

Preparation of adrenaline-sensitive lipid micelles from rat epididymal adipose tissue

Lipid micelles were prepared from rat epididymal adipose tissue. The micelles were mainly composed of triglyceride with small amounts of protein, sugar, phospholipid, choresterol and choresterol ester.Adrenaline stimulated lipolysis in intact micelles, but not when they were homogenized.     

Characterization of a colchicine receptor protein in rat epididymal adipose tissue

Colchicine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 × g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 ωM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37°C. Addition of guanosine triphosphate and Mg²⁺ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine.     

Ledakrin effect on free fatty acid and glycerol mobilization from epididymal adipose tissue of rat in vitro

It was demonstrated that Ledakrin, an antitumour drug, causes mobilization of free fatty acids and glycerol from the epididymal adipose tissue of rat in vitro. The disproportion observed in the release of glycerol and free fatty acids following Ledakrin administration suggested a biphasic effect of this drug, with initial stimulation and later inhibition of the processes of lipolysis and lipogenesis. Blockade of adrenergic membrane receptors (with propranolol or regitine) abolished the lipolytic effect of Ledakrin.     

Regulation of glycolysis and l-glycerol 3-phosphate concentration in rat epididymal adipose tissue in vitro. Role of phosphofructokinase

1. Attempts were made to define the role of phosphofructokinase in glycolytic control and the factors regulating the concentration of l-glycerol 3-phosphate in rat epididymal fat pads incubated in vitro. 2. Glycolysis rates were altered by anoxia or by additions of insulin, adrenaline or both to the incubation medium, and the changes in rate were related to changes in the steady-state concentrations of hexose phosphates, adenine nucleotides, l-glycerol 3-phosphate and citrate in the whole tissue. Measurements were also made of the lactate/pyruvate concentration ratio in the medium after incubation. 3. The mass-action ratios of phosphofructokinase, calculated from the whole-tissue concentrations of products and substrates, were less than 0.1% of the value of the ratio at pH7.4 at equilibrium. 4. Only in the presence of adrenaline could the observed stimulation of glycolytic flux be related to a possible activation of phosphofructokinase since, in this situation, the concentration of one substrate, fructose 6-phosphate, was not altered and the concentration of the other, ATP, was decreased. Increased glycolytic flux in the presence of insulin may be explained by an observed increase in the concentration of the substrate, fructose 6-phosphate. Under anaerobic conditions, glycolytic flux was decreased but this did not appear to be the result of inhibition of phosphofructokinase, since the concentrations of both substrates, fructose 6-phosphate and ATP, were decreased. The changes in glycolytic flux with insulin and anoxia may be secondary to changes in the rate of glucose uptake. 5. Changes in l-glycerol 3-phosphate concentration appear to be related both to changes in the concentration of dihydroxyacetone phosphate and to changes in the NADH/NAD(+) concentration ratio in the cytoplasm. They do not seem to be related directly to alterations in glycolytic rate.     

Characterization of a colchicine receptor protein in rat epididymal adipose tissue.

Colchincine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 x g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 muM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37 degree C. Addition of guanosine triphosphate and Mg-2+ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine.     

High sodium intake enhances insulin-stimulated glucose uptake in rat epididymal adipose tissue

Objective: This study investigated the effect of different sodium content diets on rat adipose tissue carbohydrate metabolism and insulin sensitivity. Methods and Procedures: Male Wistar rats were fed on normal‐ (0.5% Na⁺; NS), high‐ (3.12% Na⁺; HS), or low‐sodium (0.06% Na⁺; LS) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail‐cuff system. An intravenous insulin tolerance test (ivITT) was performed in fasted animals. At the end of each period, rats were killed and blood samples were collected for glucose and insulin determinations. The white adipose tissue (WAT) from abdominal and inguinal subcutaneous (SC) and periepididymal (PE) depots were weighed and processed for adipocyte isolation and measurement of in vitro rates of insulin‐stimulated 2‐deoxy‐d ‐[³H]‐glucose uptake (2DGU) and conversion of ‐[U‐¹⁴C]‐glucose into ¹⁴CO₂. Results: After 6 weeks, HS diet significantly increased the BP, SC and PE WAT masses, PE adipocyte size, and plasma insulin concentration. The sodium dietary content did not influence the whole‐body insulin sensitivity. A higher half‐maximal effective insulin concentration (EC₅₀) from the dose‐response curve of 2DGU and an increase in the insulin‐stimulated glucose oxidation rate were observed in the isolated PE adipocytes from HS rats. Discussion: The chronic salt overload enhanced the adipocyte insulin sensitivity for glucose uptake and the insulin‐induced glucose metabolization, contributing to promote adipocyte hypertrophy and increase the mass of several adipose depots, particularly the PE fat pad.