Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice

Abstract

In lipoatrophic diabetes, a lack of fat is associated with insulin resistance and hyperglycemia. This is in striking contrast to the usual association of diabetes with obesity. To understand the underlying mechanisms, we transplanted adipose tissue into A-ZIP/F-1 mice, which have a severe form of lipoatrophic diabetes. Transplantation of wild-type fat reversed the hyperglycemia, dramatically lowered insulin levels, and improved muscle insulin sensitivity, demonstrating that the diabetes in A-ZIP/F-1 mice is caused by the lack of adipose tissue. All aspects of the A-ZIP/F-1 phenotype including hyperphagia, hepatic steatosis, and somatomegaly were either partially or completely reversed. However, the improvement in triglyceride and FFA levels was modest. Donor fat taken from parametrial and subcutaneous sites was equally effective in reversing the phenotype. The beneficial effects of transplantation were dose dependent and required near-physiological amounts of transplanted fat. Transplantation of genetically modified fat into A-ZIP/F-1 mice is a new and powerful technique for studying adipose physiology and the metabolic and endocrine communication between adipose tissue and the rest of the body.

Figure 1

(a) Adipose tissue graft 3 weeks after transplantation. The graft (yellow, at center, originally 100 mg), attached to the skin (white), was dissected from the muscle (brown at bottom). Note the blood vessels supplying the graft. (b) Adipose tissue graft 13 weeks after transplantation. Hematoxylin and eosin stain. Original magnification, ×200. Note the blood vessel (V) and nerve (N). (c) A-ZIP/F-1 mice 13 weeks after transplantation. Skin was dissected from a sham-operated mouse (left) and from a mouse that received 900 mg of parametrial fat (right) in seven grafts (a ventral graft is not visible).

Figure 2

Fat transplantation improves plasma glucose and insulin levels and reduces postweaning growth in A-ZIP/F-1 mice. The sham-operated (filled circles [n = 5, except n = 3 at 13 weeks]) and transplanted animals (open circles [n = 6], 900 mg of parametrial fat) were significantly different beginning 5 weeks after transplantation for glucose, 2 weeks for insulin, and 3 weeks for body weight. The shaded region is the normal range for plasma/serum glucose for fed FVB/N mice (mean ± 2 SD; 150–306 mg/dL; n = 84).

Figure 3

Histology of the liver 13 weeks after transplantation of 900 mg of parametrial fat. Sections from sham operated (left) and transplanted (right) A-ZIP/F-1 mice were stained with hematoxylin and eosin. Original magnification, ×50. Note the large number of vacuolated hepatocytes (due to lipid deposition) located predominantly in the centrilobular zone of the liver in the sham-operated but not the transplanted mice.

Figure 4

Effect of fat transplantation on insulin sensitivity of A-ZIP/F-1 mice. (a) Female mice (n = 3–9/group; transplanted with 900 mg of adipose tissue 5 weeks earlier) were fasted for 8 hours, then glucose (2 mg/g, intraperitoneally) was injected and blood glucose was measured at the indicated times. *P < 0.001 WT versus sham; ⁺P < 0.02 transplanted versus sham. (b) Male mice (n = 3 to 9/group; transplanted with 900 mg of adipose tissue 8 weeks earlier) were fasted for 15–21 hours and then injected with insulin (0.75 mU/g), and blood glucose was measured at the indicated times. *P < 0.03 transplanted versus sham. (c) Tissue uptake of [¹⁴C]2-deoxyglucose in mice (n = 3/group, a subset of those in b) was measured 45 minutes after a single intraperitoneal injection. Muscle uptake was measured in gastrocnemius muscle and in either epididymal (WT) or transplanted adipose tissue. ⁺P = 0.07, sham A-ZIP/F-1 versus wild-type; *P = 0.004, transplanted versus sham A-ZIP/F-1. dpm, disintegrations per minute. (d) Female mice (n = 4/group; transplanted with 900 mg of adipose tissue 6 weeks earlier) were fasted for 13 hours, then [³H]2-deoxyglucose (2-DG) uptake into extensor digitorum longus muscle in the absence (basal) or presence of insulin was measured. *P < 0.001 versus basal WT; **P = 0.01 versus insulin-treated WT; ⁺P = 0.01 versus insulin-treated sham A-ZIP/F-1.

Figure 5

Inguinal and parametrial fat have similar effects on serum glucose, insulin, triglyceride, and FFA levels in the A-ZIP/F-1 mice. A-ZIP/F-1 were sham-operated (filled circles [n = 6]) or received 600 mg of parametrial (open circles [n = 7]) or inguinal (filled triangles [n = 6]) fat. Wild-type controls (open triangles [n = 5]) are age-matched mice from the experiment in Figure 8.

Figure 6

Effect of fat transplantation on RER. Resting RER was measured 10 weeks after transplantation at 24°C in mice either sham-operated (n = 5) or transplanted with 900 mg of parametrial fat (n = 6). The animals were fasted starting at 0900 hours, and data were collected for 2 hours starting 3 hours later. Fed data were obtained using the same animals at the same time of the day a day before the fast. The difference between sham and transplanted mice was significant in the fasted state (P = 0.02). The drop in RER with fasting was significant in both groups (P < 0.01).

Figure 7

Effect of a 24-hour fast on serum FFA and BUN levels in A-ZIP/F-1 mice 8 weeks after transplantation of 600 mg of fat. The A-ZIP/F-1 mice (n = 6–7/group) are from the series shown in Figure 5. The wild-type controls (n = 3) are age- and sex-matched mice. The fed (filled circles) sera were obtained a week before fasting (open circles) in the A-ZIP/F-1 mice and a day before fasting in the wild-type mice.

Figure 8

Fat transplantation increases serum leptin and improves insulin levels in a dose-dependent way. A-ZIP/F-1 mice (n = 5–7/group) were transplanted with 100, 300, or 900 mg of parametrial fat. (a) Serum leptin levels were measured 13 weeks after transplantation, with serum from ob/ob mice indicating the assay background. Leptin levels in the 900- and 300-mg groups were significantly different from the sham group. (b) Serum insulin, triglyceride, and FFA levels were measured at the indicated times in the sham-operated (filled circles), 100-mg (open circles), 300-mg (filled triangles), and 900-mg (open triangles) transplanted A-ZIP/F-1 mice and in wild-type controls (filled squares).