Improved adipose tissue metabolism after 5-year growth hormone replacement therapy in growth hormone deficient adults: The role of zinc-α2-glycoprotein

Abstract

Growth hormone (GH) supplementation therapy to adults with GH deficiency has beneficial effects on adipose tissue lipid metabolism, improving thus adipocyte functional morphology and insulin sensitivity. However, molecular nature of these effects remains unclear. We therefore tested the hypothesis that lipid-mobilizing adipokine zinc-α2-glycoprotein is causally linked to GH effects on adipose tissue lipid metabolism. Seventeen patients with severe GH deficiency examined before and after the 5-year GH replacement therapy were compared with age-, gender- and BMI-matched healthy controls. Euglycemic hyperinsulinemic clamp was used to assess whole-body and adipose tissue-specific insulin sensitivity. Glucose tolerance was determined by oGTT, visceral and subcutaneous abdominal adiposity by MRI, adipocyte size morphometrically after collagenase digestion, lipid accumulation and release was studied in differentiated human primary adipocytes in association with GH treatment and zinc-α2-glycoprotein gene silencing. Five-year GH replacement therapy improved glucose tolerance, adipose tissue insulin sensitivity and reduced adipocyte size without affecting adiposity and whole-body insulin sensitivity. Adipose tissue zinc-α2-glycoprotein expression was positively associated with whole-body and adipose tissue insulin sensitivity and negatively with adipocyte size. GH treatment to adipocytes in vitro increased zinc-α2-glycoprotein expression (>50%) and was paralleled by enhanced lipolysis and decreased triglyceride accumulation (>35%). Moreover, GH treatment improved antilipolytic action of insulin in cultured adipocytes. Most importantly, silencing zinc-α2-glycoprotein eliminated all of the GH effects on adipocyte lipid metabolism. Effects of 5-year GH supplementation therapy on adipose tissue lipid metabolism and insulin sensitivity are associated with zinc-α2-glycoprotein. Presence of this adipokine is required for the GH action on adipocyte lipid metabolism in vitro.

Keywords: ACC1, acetyl-CoA carboxylase 1; BSA, bovine serum albumin; DGAT, diacylglycerol acyltransferase; DMEM, Dulbecco's Modified Eagle Medium; EHC, euglycemic hyperinsulinemic clamp; FABP4, fatty acid binding protein 4; FAS, fatty acid synthase; FBS, fetal bovine serum; FFA, free fatty acids; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GH, growth hormone; GHD, growth hormone deficiency; GLUT4, glucose transporter 4; HSL, hormone sensitive lipase; IGF-1, insulin-like growth factor 1; IRS1, insulin receptor substrate 1; MRI, magnetic resonance imaging; PPARGC1A, peroxisome proliferator-activated receptor 1 gamma coactivator 1 α; RPL13A, ribosomal protein L13a; TG, triglycerides; ZAG, zinc-α2-glycoprotein.; adipocyte size; adipose tissue; glucose tolerance; growth hormone deficiency; growth hormone replacement therapy; insulin sensitivity; lipolysis; oGTT, oral glucose tolerance test; rhGH, recombinant human growth hormone.

Figure 1.

Positive effects of 5-year GH replacement therapy on the whole-body and adipose tissue metabolic phenotypes. The effect of 5-year GH replacement therapy on (A) glucose tolerance (2h glycemia in oral glucose tolerance test), (B) adipose tissue insulin sensitivity (ability of insulin to suppress FFA during EHC) and (C) adipocyte size (lines connect the data points of the “real follow-up” patients). Associations of adipose tissue ZAG gene expression with the (D) whole-body and (E) adipose tissue insulin sensitivity (ability of insulin to suppress FFA during EHC) and (F) adipocyte size (Δ controls; ○ baseline GH naive patients; GH treated GHD patients). GHD, patients with growth hormone deficiency; EHC, euglycemic hyperinsulinemic clamp; FFA, free fatty acids; ZAG, zinc-α2-glycoprotein; rhGH, recombinant human growth hormone; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Growth hormone regulates ZAG and inhibits lipid accumulation in 3T3-L1 adipocytes in vitro. (A) The effect of 6-day GH treatment on ZAG gene expression in differentiated 3T3-L1 and primary human adipocytes. (B) The effect of 6-day GH treatment on lipid accumulation and (C) expression of FAS, ACC1, DGAT mRNA in differentiated 3T3-L1 adipocytes. (D) Association of ZAG gene expression with DGAT mRNA. Effect of 6-day GH treatment on (E) basal and (F) isoproterenol-induced lipolysis in differentiated 3T3-L1 adipocytes. Presented data represent an average value of 3 (A, B) or 4 (C, D, E, F) independent experiments. ACC1, acetyl CoA carboxylase 1; DGAT, acyl CoA:diacylglycerol acyltransferase; FAS, fatty acid synthase; GH, growth hormone; ZAG, zinc-α2-glycoprotein; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Silencing ZAG eliminates GH effects on lipid metabolism in vitro. The effect of ZAG silencing on (A) ZAG gene and protein expression in differentiated human primary adipocytes. Representative western blot shows ZAG protein in siRNA transfected adipocytes when compared to control - scramble transfected cells. The effect of 6-day growth hormone treatment and transfection of ZAG/scramble siRNA on (B, C) lipid accumulation, (D) basal lipolysis (glycerol release in absence of insulin) and (E) insulin sensitivity (glycerol release in presence of 66 nM insulin) in differentiated human primary adipocytes. The results represent an average of 3 (A, B, C, D) or 4 (E) independent experiments. GH, growth hormone; ZAG, zinc-α2-glycoprotein; TG, triglycerides; picture presented in (C) was taken at magnification of 50x; *P < 0.05; **P < 0.01; ***P < 0.001.