Abstract
beta 3-Adrenergic receptors (beta 3-ARs) are expressed predominantly in white and brown adipose tissue, and beta 3-selective agonists are potential anti-obesity drugs. However, the role of beta 3-ARs in normal physiology is unknown. To address this issue, homologous recombination was used to generate mice that lack beta 3-ARs. This was accomplished by direct injection of a DNA-targeting construct into mouse zygotes. Twenty-three transgenic mice were generated, of which two had targeted disruption of the beta 3-AR gene. Mice that were homozygous for the disrupted allele had undetectable levels of intact beta 3-AR mRNA, as assessed by RNase protection assay and Northern blotting, and lacked functional beta 3-ARs, as demonstrated by complete loss of beta 3-agonist (CL 316,243)-induced stimulation of adenylate cyclase activity and lipolysis. beta 3-AR-deficient mice had modestly increased fat stores (females more than males), indicating that beta 3-ARs play a role in regulating energy balance. Importantly, beta 1 but not beta 2-AR mRNA levels up-regulated in white and brown adipose tissue of beta 3-AR-deficient mice (brown more than white), strongly implying that beta 3-ARs mediate physiologically relevant signaling under normal conditions and that "cross-talk" exists between beta 3-ARs and beta 1-AR gene expression. Finally, acute treatment of normal mice with CL 316,243 increased serum levels of free fatty acids (FFAs) (3.2-fold) and insulin (140-fold), increased energy expenditure (2-fold), and reduced food intake (by 45%). These effects were completely absent in beta 3-AR-deficient mice, proving that the actions of CL are mediated exclusively by beta 3-ARs. beta 3-AR-deficient mice should be useful as a means to a better understanding of the physiology and pharmacology of beta 3-ARs.
Highlights
3-Adrenergic receptors (3-ARs) are expressed predominantly in white and brown adipose tissue, and 3-selective agonists are potential anti-obesity drugs
Mice that were homozygous for the disrupted allele had undetectable levels of intact 3-AR mRNA, as assessed by RNase protection assay and Northern blotting, and lacked functional 3-ARs, as demonstrated by complete loss of 3-agonist (CL 316,243)-induced stimulation of adenylate cyclase activity and lipolysis. 3-AR-deficient mice had modestly increased fat stores, indicating that 3-ARs play a role in regulating energy balance
This has led to the hypothesis that its primary function might be to maintain signaling during periods of sustained sympathetic stimulation, such as that which occurs in brown adipose tissue during periods of chronic cold exposure or dietinduced thermogenesis
Summary
Targeting Constructs—Using a mouse 129/SvJ genomic library (Stratagene) and published mouse 3-AR sequence [11], 3-AR genomic clones were obtained and mapped (Fig. 1). Injected zygotes were transferred to FVB/N pseudopregnant recipients as described previously [33], and genomic DNA was obtained from live born offspring for Southern blot analysis (Fig. 2). 3-AR and actin mRNA levels were determined in brown adipose tissue and liver samples using an RNase protection assay as described under “Experimental Procedures.”. Lipolysis and Adenylate Cyclase Assays—Adipocytes were isolated from epididymal fat pads of male mice using collagenase digestion as described previously [42, 43]. 1-, 2-, and 3-AR mRNA levels were determined by Northern blotting using 30 g of total RNA isolated from epididymal white adipose tissue and interscapular brown adipose tissue of 5 control (C) and 5 3-AR-deficient (K) 12-week-old male mice.
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