Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • Lactate induced GPR activation was confirmed with a

    2021-09-27

    Lactate-induced GPR81 activation was confirmed with a fluorimetric imaging plate reader (FLIPR) assay, in which lactate induced a [Ca2+] flux in CHO cells stably expressing Gqi5 and HA-GPR81 but not vector control (Fig. 1C). The EC50 of lactate in the FLIPR assay was 4.3±0.73mM, a value well within the physiological range observed in man following exercise [18], [19], [20]. To determine if GPR81, like GPR109A and GPR109B, is Gi-coupled we employed a 35S-GTPγS exchange assay with membranes prepared from cells pretreated with or without pertussis toxin (PTX), an inhibitor of Gi signaling. Similar to nicotinic Pazopanib Hydrochloride sale stimulation of GPR109A [9], [11], [12], lactate-mediated activation of HA-GPR81 was completely inhibited by the pretreatment of PTX (Fig. 2A) and is thus Gi-coupled. Consistent with this conclusion, we found lactate inhibited forskolin-stimulated cAMP production in CHO cells transfected with HA-GPR81 (IC50=2.15±0.26mM (Fig. 2B) but not with GPR109A (data not shown). Lactate has previously been shown to reduce lipolysis in human and rat adipose tissues [7], [8]. We extended this observation by showing that lactate can also suppress lipolysis in mouse fat pads in an ex vivo assay (Fig. 3A). Inhibition of both basal and, to a greater extent, isoproterenol-stimulated lipolysis was evident. Similar to an effect of nicotinic acid, lactate-mediated suppression of lipolysis were blocked by the pretreatment of mice with pertussis toxin (Fig. 3B), indicating a role of a Gi-coupled receptor in the process. The ability of lactate to suppress lipolysis in mouse fat pads allowed us to genetically test whether GPR81 was required for this process. To this end we employed fat pads from GPR81-deficient mice and wild-type littermate controls. As expected, lactate suppressed lipolysis in the fat pads from wild-type control mice but failed to do so in the GPR81-deficient fat pads (Fig. 3C), indicating a critical role of GPR81 in lactate-mediated suppression of adipose lipolysis.
    Discussion Our results clearly show that lactate can activate the adipose-localized GPCR GPR81 at concentrations well within the physiological range observed at states of relative oxygen deficit such as exercise (may reach up to 30mM, 18–19). Specifically, we show using a cell based cAMP assay or a [Ca2+] flux assay that the EC50 for lactate-mediated activation of GPR81 can be as low as 2.1mM. Our demonstration that GPR81 is Gi coupled is consistent with the fact that infusion of lactate into humans can reduce plasma FFA. This is proposed to occur in a manner similar to the mechanism by which niacin lowers plasma FFA—specifically, by agonizing the highly related Gi-coupled GPCR GPR109A, reduction of intracellular cAMP and thus protein kinase A activity, and in turn the reduced phosphorylation of the lipid droplet coat protein perilipin and hormone sensitive lipase, which are key components of the TG hydrolytic system that generates FFA [11], [13]. Glucose and FFA are considered the major metabolic fuels for energy needs. Under conditions of oxygen deficit, the working muscles can not utilize FFA as a substrate. This shift in metabolism during oxygen deficit often results in a drastic elevation of plasma levels of lactate [6]. Results from the current study suggest the following hypothesis. Under the state of oxygen deficit, while many factors, such as decreased circulating insulin levels, increased sympathetic tone and circulating norepinephrine levels [22], are signaling for an increase of FFA, an elevation of plasma lactate serves as a signal to the adipose tissues to reduce lipolysis. GPR81 on adipocytes may function as such sensor. In addition to the production of lactate by muscle contraction during exercise [18], [19], [20], recent studies show that adipose tissues may also produce significant amounts of lactate [23]. The amount of lactate produced in adipocytes appears to correlate with cell size [24]. Lactate levels rise with obesity and decline with weight loss [23], [25]. The results from the work presented here suggest that lactate produced in adipocytes could potentially function as an autocrine signal to regulate energy storage in adipose via activation of GPR81.