Cellular program controlling the recovery of adipose tissue mass: An in vivo imaging approach

Abstract

The cellular program responsible for the restoration of adipose tissue mass after weight loss is largely uncharacterized. Leptin mRNA levels are highly correlated with adipose tissue mass, and leptin expression can thus be used as a surrogate for changes in the amount of adipose tissue. To further study the responses of adipocytes to changes in weight, we created a transgenic mouse expressing the luciferase reporter gene under the control of leptin regulatory sequences, which allows noninvasive imaging of the leptin expression of mice in vivo. We used these animals to show that weight loss induced by fasting or leptin treatment results in the retention of lipid-depleted adipocytes in adipose depots. To further study the cellular response to weight regain after leptin treatment, a leptin withdrawal protocol was used to induce a state of acute leptin deficiency in wild type mice. Acute leptin deficiency led to the transient deposition of large amounts of glycogen within pre-existing, lipid-depleted adipocytes. This was followed by rapid reaccumulation of lipid. Transcriptional profiling revealed that this cellular response was associated with induction of mRNAs for the entire pathway of enzymes necessary to convert glucose into acetyl-CoA and glycerol, key substrates for the synthesis of triglycerides.

Fig. 1.

Characterization of leptin-luciferase animals. (A) (Upper) Expression of the luciferase protein in tissues from leptin-luciferase mouse. Luciferase activity was normalized by the amount of protein of indicated tissue lysate. (mean ± SEM) The right panel displays in vivo imaging of a representative leptin-luciferase transgenic animal showing adipose-specific luciferase activity. (Lower) A schematic map of the leptin BAC RP24-69D4. Firefly luciferase is inserted in the translation start site in the second exon. (B) Luciferase activity recapitulates regulation of leptin. The relative expression of luciferase versus leptin was compared among fed, fasted, and ob/ob animals. (Lower) Endogenous leptin expression levels from fasted, fed, and ob/ob animals were measured by Taqman real time PCR normalized to cyclophilin levels. (Upper) Luciferase activity of adipose tissue lysates from fed, fasted, and ob/ob animals normalized to protein content (mean ± SEM). (C) In vivo imaging of fed, fasted, and ob/ob transgenic animals. The graph on the left shows quantitation of in vivo luciferase activity using Living Image 3.0 software (mean ± SEM).

Fig. 2.

Effects of fasting on adipose tissue and luciferase expression. (A) Time course luciferase activity in leptin-luciferase animals upon fasting and refeeding. The level of luciferase in animals was monitored during a 3-day fast and after refeeding. The data were collected for the same animal over time (n = 4). Luciferase activity fell gradually over the course of the fast. After refeeding, luciferase activity returned to baseline by 6 h. (Upper) Representative time-course images of fasted and re-fed animals are shown. (Lower) Quantitation of in vivo signals by Living Image 3.0 software (mean ± SEM). (B) Histological and immunohistochemical analysis of adipose tissue of fasted mouse. H&E staining of fed (Left) and fasted (Center) animals for comparison. Immunohistochemical staining of the adipocyte specific protein, aP2, in fasted animals (Right).

Fig. 3.

Adipose tissue response to leptin treatment and withdrawal. (A) In vivo imaging of leptin-luciferase animals upon leptin withdrawal. PBS (n = 6) or leptin at 2.5 μg/h (n = 5) was administered for 8 days by subcutaneously implanted osmotic pump. On day 8, leptin or PBS treatment was withdrawn by removing pumps under inhaled anesthesia. Animals were imaged before treatment, after 8 days of leptin treatment and daily for the 4 days following removal of leptin pumps. (Upper) Imaging results of representative animals form PBS and leptin-treated groups are shown. (Lower) Quantitation of in vivo signals by Living Image 3.0 software indicates a dramatic suppression of luciferase by exogenous leptin treatment (mean ± SEM). (B) Preservation of adipocyte identity during chronic leptin treatment. Leptin treatment results in near complete depletion of triglyceride droplets from white adipose tissue. White adipose tissue of mice treated for 8 days with leptin consists of dense islands of cells, as assessed by hematoxylin and eosin staining (Center). An identical magnification of control, PBS-treated white adipose tissue shows characteristic large, central lipid droplets and peripheral nuclei (Left). Note for comparison that nuclei are the same size. Immunohistochemical staining demonstrated high levels of the adipocyte-specific protein aP2 in the cytoplasm of high numbers of cells in delipidated white adipose tissue (Right, brown, peroxidase-positive material).

Fig. 4.

Accumulation of white adipose tissue glycogen during acute leptin deficiency. (A) White adipose tissue glycogen content rose 30- to 60-fold in leptin withdrawal animals (green and red bars) relative to PBS controls (blue bars) following withdrawal of exogenous leptin treatment, as measured by glucose liberated by amyloglucosidase (*, P < 0.05 vs. PBS; #, P < 0.065 vs. PBS). This increase occurred in both free-fed animals (Leptin-FF, green bars) and animals maintained at normocaloric intake levels (Leptin-NC, red bars), and increased glycogen content persisted for 3 and 4 days in these groups, respectively. (B) PAS staining of white adipose tissue on withdrawal day 1 and withdrawal day 3. PAS staining on withdrawal day 1 indicated large amounts of cytoplasmic glycogen content in leptin-FF and leptin-NC tissue sections (black arrows). This contrasted sharply with control PBS-treated white adipose tissue. On withdrawal day 3, glycogen accumulation decreased in parallel to the accumulation of lipid (red arrows). α-Amylase pretreatment of tissue sections eliminated PAS-positive granules in adipocyte cytoplasm, indicating specificity of staining for glycogen.

Fig. 5.

Pathway of acetyl-CoA and glycerol generating enzymes up-regulated by acute leptin deficiency. (A) Acute leptin deficiency induced the pathway of genes (blue ovals) necessary to synthesize cytoplasmic acetyl-CoA and glycerol from simple sugar precursors. Fold change for each gene on day 1 following leptin withdrawal is shown below each enzyme name for leptin-FF (Left) and leptin-NC (Right) mice with the associated P value immediately below each fold-change value. Potential sources of carbon for acetate or glycerol synthesis are in blue text. BP, bisphosphate; CoA, CoA; DH, dehydrognease; DHAP, dihydroxyacetone phosphate; G, glucose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, mannose; P, phosphate; PFK, phosphofructokinase; TPI, triose phosphate isomerase. (B) Average expression level for the genes in the cluster is shown in graphical format. Leptin-FF (green circles) and leptin-NC (red triangles) groups showed approximately equivalent induction relative to PBS controls (blue squares) following leptin withdrawal. Levels remained elevated for 4 days following cessation of exogenous leptin treatment.