Factors Affecting Measurements of Glucose Metabolism and Lipolytic Rates in Porcine Adipose Tissue Slices in Vitro

Abstract

Porcine adipose tissue glucose metabolism and lipolytic rates have been measured for many years by numerous investigators. However, there is little or no documented indication of the effects of variation in tissue handling procedures or variations in incubation medium components on metabolic rates. We have systematically varied conditions to provide such documentation for these much used techniques. The temperature (18 to 38 C) of tissue during transport had little effect. The medium for tissue transport probably should be buffered. Use of Hepes buffer at greater than 10 or 25 mM in incubation media inhibited glucose metabolism and lipolysis. Calcium ion effects on glucose metabolism or lipolysis could not be demonstrated. Dimethyl sulfoxide should not be used routinely. Ascorbate at .56 mM did not inhibit glucose metabolism or lipolysis. Glucose metabolism was increased by glucose concentration to about 5 mM and not inhibited at higher concentrations; we recommend 10 or 20 mM glucose to ensure maximal rates. Insulin stimulated glucose metabolism but effects were slight, not related to insulin concentration and not consistently observed. Addition of some albumin preparations did not allow expression of insulin stimulation; we recommend albumin be omitted or, if included, carefully monitored. Lipolytic rates were dependent on albumin concentration, but rates were similar with all albumin preparations. Insulin markedly inhibited hormone-stimulated but not basal lipolysis. Adenosine, an inhibitor of lipolysis, did not affect glucose metabolism rates. An artificial oxygen carrier did not increase anabolic activity. Incubation in serum increased rates of glucose metabolism relative to lipolysis so that refinement of the incubation might lead to greater anabolic than catabolic rates in vitro to reflect the status of adipose tissue in growing pigs in vivo. Tissue handling and incubation conditions can markedly affect metabolic rates, and should be understood and controlled.

Overview of the effects of beta-adrenergic receptor agonists on animal growth including mechanisms of action.

“…Thus, although the physiology and pharmacology of an enzymatic step or of a pathway may be accurately described by the results, the quantitative use of the rates is not appropriate. Second, the observed results may stem from a particular addition or deletion to the incubation medium; for example, insulin usually will potently inhibit lipolytic rates (Mersmann and Hu, 1987) or the endogenous generation of adenosine will stimulate the adipocyte adenosine A 1 receptor to inhibit adenylyl cyclase (Carey, 1995). Third, the concentration of hormones or drugs (agonists and antagonists) may be pharmacological, and thus the effects may not represent the physiological response in vivo.…”

Effect of Full Feed and Early Feed Restriction on Broiler Performance, Abdominal Fat Level, Cellularity, and Fat Metabolism in Broiler Chickens

“…Unless otherwise indicated, lipolysis was measured in adipose tissue slices as described by Mersmann and Hu (1987) and Hu et al (1988 contained 5.56 mM glucose, .56 mM ascorbate, and 6% BSA. The basal lipolysis was measured in triplicate with no exogenous hormone added.…”

“…The tissue was sliced horizontally by passage across the moistened platform against the knife edge. Lipogenesis and glucose oxidation were measured by the modified radioactive method of Mersmann and Hu (1987). Krebs-Ringerbicarbonate (KRB) buffer medium containing 118 mM Nad, 4.71 mM KC1, .63 mM Ca 2 , 1.19 mM KH 2 P0 4/ 1.19 mM MgS0 4 7H20, and 25 mM NaHC0 3 was routinely gassed, using a gas dispersion tube, for 15 min with 5% CC»2 in oxygen.…”

GH and insulin affect fatty acid synthase activity in isolated porcine adipocytes in culture without any modifications of sterol regulatory element binding protein-1 expression

“…An optimum response for FAS activity was reached for a concentration as low as 1·7 nM in s.c. adipocytes, whereas higher concentrations of insulin are often used in isolated adipocytes from other species, such as rat (Briquet-Laugier et al 1994) or human (Moustaïd et al 1996). In pigs, adipose tissue and/or adipocyte response to insulin has been often reported to be inconsistent (Mills 1999), with reports of absence of any responsiveness (Rule et al 1987, Benmansour et al 1991, little response (Liu et al 1989, Harris et al 1993 but not related to insulin concentrations (Mersmann & Hu 1987), or clear stimulation of glucose incorporation into fatty acids and lipogenesis in vitro , Smith et al 1996, Mills 1999. One possible explanation for these discrepancies is that an exaggerated basal rate of fatty acid synthesis could mask insulin responses in porcine adipocytes (Mills 1999).…”

“…The more likely explanation is that the culture medium may interfere with the insulin response. In previous experiments, BSA has been shown to interfere with insulin response in pig adipose tissue slices , Mersmann & Hu 1987. For this reason, a BSA-free cell medium was used ).…”