Fig 2: K414R mutation inhibits lipolysis. K414R lentivirus was used to infect 3T3‐L1 mature adipocytes. Following treatment with ISO 1 µM for 30 min and TNF‐α 50 ng/mL for 3 h, a decrease in the content of non‐esterified fatty acids (NEFA) and glycerol in the culture medium of the K414R group was observed (A‐B). K414R lentivirus was used to infect 3T3‐L1 mature adipocytes. Following treatment with ISO 1 µM for 30 min and TNF‐α 50 ng/mL for 3 h, ELISA showed a decrease in the content of leptin (C) and an increase in adiponectin content (D) in the culture medium of the K414R group. K414R lentivirus was used to infect 3T3‐L1 mature adipocytes. Western blot showed that the expression of fatty acid synthase (FAS) was increased, and the expression of the lipolytic enzymes HSL, p‐HSL and ATG was decreased, and the expression of perilipin was increased (E). Data are shown as the means ±standard deviations of three independent experiments performed in triplicate. *** P < .001

Fig 3: Analysis of the effects of LF on the phosphorylation of HSL and PLIN by PKA and determination of PKA activity.Phosphorylation of HSL and PLIN by PKA was detected in the presence or absence (0 min) of 1 mg/ml of LF. Phosphorylation levels normalized to protein expression levels of HSL and PLIN are shown. (A) Phosphorylation of HSL Ser660 and (B) PLIN Ser497 by PKA. (C) Analysis of PKA activity in adipocytes treated with LF. PKA activity in adipocytes was detected using an ELISA before (0 min) and after treatment with LF. Kinase activity normalized to the total protein determined by BCA is shown. The statistical significance of the data at each sampling time compared with the 0-min sample was evaluated using Dunnett’s multiple comparison test, and the data represent the mean ± SD values of triplicate determinations of one of three identical experiments. *p < 0.05, ***p < 0.001 HSL, hormone-sensitive lipase; LF, lactoferrin; PLIN, perilipin; PKA, protein kinase A; SD, standard deviation.

Fig 4: Hormone Sensitive Lipase.Total soleus HSL protein content in sedentary control (C, n = 6), sedentary diabetic (D, n = 7), and diabetes exercise (DX, n = 6). A representative blot run under the same experimental conditions (see methods) is shown. The representative blot has been cropped to show proteins of interest. There were no significant differences between groups (p = 0.556). Data are expressed as mean ± SE for each group.

Fig 5: UCP1, PGC1 alpha and HSL expression in histological sections of hRAN and hRAT. UCP1, PGC1 alpha and HSL expression was evaluated by immunohistochemistry in serial cuts of hRAN and hRAT. DAB staining quantification in the three tissue types was performed with Image J software (NIH). Histograms show mean ± SEM of five independent experiments. (a.u.: arbitrary units). *p < 0.01 hRAN versus hRAT. Representative photographs of hRAN- and hRAT-staining. Magnification: 10× and 40×. Arbitrary units (a.u) represent pixel quantification.