The original fld (fatty liver dystrophy) mutation arose spontaneously at The Jackson Laboratory in the Animal Resources BALB/cByJ colony in 1981, and was maintained by sibling mating for 47 generations, then backcrossed once in 2001 to a male BALB/cByJ via homozygous ovarian transplant, then sibling mating resumed. Homozygotes can be identified soon after birth by an enlarged, pale liver and smaller overall body size. Although the hepatic steatosis resolves to normal at wean age, a neurological phenotype manifests by day 14 as a tremor and an unsteady gait which is most pronounced in the rear legs (Sweet et al. 1988). Both phenotypes stem from improper cellular processing of lipid.
fld/fld mice are smaller than their normal littermates by three days of age and remain smaller throughout life. Hair growth is retarded and abnormal resulting in a ruffled, unkempt appearance in the adult. Homozygotes experience increased mortality between 19 and 35 days of age. Approximately half of the surviving females will breed, but they do not usually produce a first litter before 3 months of age. Homozygous males rarely breed and thus are generally considered sterile (Sweet et al. 1988; Langner et al., 1989).
Although no architectural changes or inflammation were detected in fatty livers from 12 day old fld/fld mice, the hepatocytes contain huge lipid droplets. Homozygous pups have hypertriglyceridemia with serum triglyceride levels up to 1000 mg/dl. There is a slight increase in diacylglyceride levels but no change in cholesterol, cholesterol ester, or phospholipid levels. Pre-wean pups have a liver-specific increase in mRNA levels for apolipoprotein A IV mRNA and apolipoprotein C-II mRNA that returns to normal in the adult. Hepatic lipase mRNA and serum hepatic lipase activity are decreased until the beginning of wean age. Between 13 and 15 days of age triglyceride levels abruptly drop to normal and at 30 days of age the hepatic lipid levels are normal. This return to normal is not delayed by extending suckling. (Langner et al., 1989).
Quantitative two-dimensional gel electrophoresis of liver extracts from 5 day old fld/fld pups revealed an increase in expression in twenty-two proteins (including keratin, B-actin, g-actin, and apolipoproteinA-IV) and a decrease in expression in two proteins relative to 5 day old wild-type controls. The majority of these proteins normally change their expression levels in response to peroxisome proliferating agents. These differences in protein expression resolve in the adult (Rehnmark et al., 1998) as does liver morphology. Hepatocytes taken from 12-day old fld/fld mice and cultured for 4days continue to show a decrease in hepatic lipase activity, an increase in apolipoproteinA IV mRNA, and an accumulation of lipid indicating that the fld defect is in cellular lipid metabolism. Extracts from these cultured hepatocytes have a lower rate of palmitate oxidation than do extracts from wild-type cultured hepatocytes. Extracts from adult fld/fld cultured hepatocytes have normal levels of palmitate oxidation activity (Rehnmark et al., 1998).
Both the brown and white fat pads of fld/fld mice are vastly reduced in mass, contain a paucity of lipid, and are morphologically distinct compared to wild-type sibling controls (Langner et al., 1989; Reue et al., 2000). This phenotype persists in the adult. The adipocytes are severely depleted of lipid. White adipose tissue has a decrease in lipoprotein lipase (LPL) activity and mRNA as well as a decrease in adipsin mRNA. Brown adipose tissue shows an increase in mRNA for PPARg and aP2 particularly in two-week old mice. Consistent with this decreased adipose fat, fld/fld mice have non-fasting hyperinsulinemia, impaired clearance of glucose, and reduced tissue response to insulin (Reue et al., 2000).
Representational difference analysis identified 22 mRNA species whose expression in liver differs between fld/fld and wild-type controls. In addition to novel sequences including a Ras homologue and a serine/threonine protein kinase homologue, a number of these differentially expressed cDNAs encode actin cytoskeleton-associated proteins such as actin, profilin, alpha-2 actinin, and myosin light chain. While fld/fld preadipocytes respond to serum treatment by normal elongation of actin stress fibers, they fail to develop membrane ruffles in response to insulin treatment (Klingenspor et al., 1999).
Feeding a high fat diet to adult fld/fld mice for 2 weeks did not re-induce hypertriglyceridemia or increases in liver apolipoprotein A-IV greater than in controls (Langner et al., 1989). A separate study looked at the development of aortic lesions in fld/fld adults maintained for 16 weeks on an atherogenic diet and found a 2-fold larger lesion area and more advanced lesions than in wild-type controls (Reue et al., 2000).
The fld/fld mice have a peripheral neuropathy which first presents shortly after 10 days of age as a tremor and unsteady gait which worsens gradually throughout life. When picked up by the tail, fld/fld mice clench their toes and clasp their hind feet together. The fld neuropathy is specific to the peripheral nervous system. Electron microscopy of sciatic nerves revealed thin, poorly compacted myelin sheaths, hypertrophic Schwann cells, myelin debris, degenerating axons, bands of Bugner, and regenerative clusters, but no evidence of inflammation (Langner et al, 1991). Western blot analysis of sciatic nerve from two-three month old fld/fld mice showed a 7-13-fold reduction in myelin P0, a vast increase in apoE, an increase in GAP-43, and no detectable myelin P2. It was also noted that an antibody to neurofilament 68K detected bands of lower molecular mass in fld/fld sciatic nerve extracts than in wild-type suggesting degradation of neurofilament 68K. Furthermore, TLC of fld/fld sciatic nerve lipids revealed decreased levels of phospholipids, glycosphingolipids, and some neutral lipids and increased levels of cholesterol esters. These combined findings support the postulate put forward by Langner et al. that fld/fld peripheral neuropathy involves "dysmyelination and concomitant demyelination" (Langner et al, 1991). In light of the role of insulin in Schwann cell and adipocyte differentiation and metabolism, it is noteworthy that fld/fld mice have been found to have altered insulin responsiveness (Klingenspor et al., 1999).
A spontaneous deletion of the Acads gene occurred on the BALB/cByJ background at approximately the same time the fld mutation arose. However, the Acads mutation is not present in the BALB/cByJ-fld mice (Reue and Cohen 1996. Mammalian Genome).
The fld critical region has been fine-mapped to a 0.42 cM interval on chromosome 12. RT-PCR of liver extracts from 6- and 21-day-old pups revealed an absence in fld/fld liver of transcript from one of the genes in this interval, formerly called Kiaa0188, now known to be the gene encoding lipin. (Peterfy et al. 1999; Peterfy et al. 2001)
