Leptin, lipodystrophy and insulin resistance
Simon Collins, HIV i-Base
The opening lecture at this year’s lipodystrophy workshop was given by Jeffrey Friedman, who is known for his work identifying genetic causes of obesity, and was titled leptin, lipodystrophy and insulin resistance. 
Earlier this year two papers looked at the role of leptin to treat diabetes cases of congenital lipodystrophy and to reverse insulin resistance and hepatic steatosis in patients with severe lipodystrophy who were HIV-negative, so this review in the context of an HIV-focused meeting was welcomed. [2, 3] In the second lecture of the meeting, Phillip Gorden from the research group behind these papers presented a summary of these results. 
Leptin and regulation of body weight
Leptin is a natural hormone produced by fat cells that works as an afferent signal in negative feedback loop linking nutrition to other physiologic systems.
This research was based on previous work with an obese mouse that continued to grow to three times normal weight with five times the normal amount of fat, and the discovery of the obi gene that altered the phenotype – making the mouse think it was in state of perceived starvation. As a result it ate more and burnt less in energy.
Friedman explained that circulating leptin is made by fat. If there are inadequate fat stores, leptin levels fall, signalling a response to starvation that includes increasing food intake and reducing energy levels and body temperature etc. When fat levels are high, leptin levels increase, signalling the hypothalamus to respond to obesity by reducing food intake and lipid content in tissue and increasing energy expenditure and insulin action. Leptin acts in the region of the hypothalamus known to regulate feeding and the ob/ob mouse has defective signalling resulting in similar reduced energy and increased food intake. When administered directly to the brain leptin produces higher doses than when given peripherally and a brain-specific knockout of the leptin receptor leads to obesity.
Leptin suppresses the firing rate of some neurons – NPOV and AGRB – and stimulates firing of POMC neurons. When leptin is absent one neuron fires continually and the other stops and vice versa, although the system is more complex than this and involves many other neurons that are less understood. For example, mutations in POMC reducing MC4R explains only 5% of severe obesity in humans and further research is expected to find others.
In addition to effects on metabolism via the hypothalamus, leptin also has a direct effect on T-cells, pancreatic B-cells and skeletal muscle. Immune defects in ob/ob mice include reduced CD4 and CD8 lymphocytes, reduced TH-1 responses, reduced thymus and spleen weight and reduced macrophage function. These same abnormalities are evident in starved mice and leptin corrects these abnormalities in both cases. Less research has been done in leptin deficiency.
Clinical utility of leptin
It was originally thought that leptin could be used to treat obesity in humans, but most overweight humans have normal levels of leptin and these are approximately 50 ng/ml higher than lean individuals – the assumption being that leptin resistance has developed. However, a subset of people have leptin levels less than 10 ng/ml
Some obese subjects have responded in trials, but with only one published study ongoing, research is still assessing whether individuals with lower levels of leptin will respond better and the threshold of response itself isn’t known. However, treating people who have congenital lipodystrophy and leptin levels under 3ng/ml has generated a response. Friedman observed that it is also easier to treat hormone deficiency than hormone resistance.
Congenital leptin deficiency associated with early onset obesity is a rare condition although around 20 cases have been described worldwide and this has been reported to have been reversed by the effect of recombinant leptin therapy in children (NEJM Sadif Farooqi et al 1999).
Leptin and lypodystrophy
Friedman then explained how leptin improves insulin sensitivity and lipid abnormalities indirectly via the CNS but that the exact mechanism for this effect is unknown. As well as improving insulin action it has been shown to improve glucose turnover in normal mice (Kamohara, Nature 1998) and correct insulin resistance and diabetes in lipodystrophic mice (Shimomura, Brown and Goldstein, Nature 1999). As food and diet (‘pair feeding’) had no effect in these studies this suggested leptin is doing something unique to homeostasis that cannot be achieved with diet alone.
The mechanisms of leptin’s effect on insulin sensitivity could be as hormone replacement but leptin deficiency per se is not associated with lipodystrophy and the effect of leptin is not immediate. Something else has to happen and intracellular leptin may reduce the content of peripheral tissues.
In lipodystrophic animals reduction of lipid levels in peripheral tissues were correlated with effects of six different doses of leptin (0-200ng/hr).
An effect on weight was seen at 100ng/ml and higher levels. However correction of hyperglycaemia and hyperinsulinaemia were seen at 50ng/hr without affecting liver triglycerides, which required doses of 200ng/hr. Studies of muscle lipid content and glucose clamps are currently ongoing. The effect of leptin deficiency on liver produces a much enlarged liver in ob/ob mice.
Affymetrix technology used to look for the genes responsible in eight experiments produced 12,000 genes and 96,000 data points ranked to increases in obi liver with stearoyl CoA desaturase (SCD), the rate limiting enzyme in the biosynthesis of monounsaturated fats, at the top of this list.
Leptin and stearoyl CoA desaturase-1 (SCD-1)
Friedman concluded that leptin reduces fat content in peripheral tissues by suppressing SCD-1 having studied the physiologic role of SCD-1 in SCD-1 knockout asebia mice. These mice have reduced weight, reduced adiposity and increased lean mass. They eat less and are resistant to high fat diet induced weight gain. Somehow deficiency of SCD-1 increases energy expenditure and food increases but not sufficiently and lean mass is reduced. Mice lacking SCD-1 also accumulate significantly less triglyceride in liver following EtOH feeding.
The mechanism for this is not known, but the hypothesis proposed is that in the absence of SCD-1 there are reduced triglycerides and reduced VLDL synthesis and that SCD-1 inhibits malonyl CoA (when malonyl reduced PST-1 is inhibited and oxidation is increased).
Alternative possibilities include altered levels of ligands for PPAR-a, PPAR-g or other nuclear hormone receptors, altered phospho-lipid composition changing signal transduction and/or membrane properties and direct or indirect effects of fatty acids on uncoupling proteins.
Limits to treatment based on this approach – and there are no SCD-1 inhibitors near the end of the pipeline – include the risk that increased free radicals could affect cardiovascular effects. Complete deficiency of this enzyme may not be a good thing and we would need to see if partial reduction might be useful.
The biochemical defects of leptin resistance are not known. The concept of insulin resistance for example was developed in the 1950s and yet the chemical molecular basis for this is only just being understood. The biochemical basis cannot be understood until we understand cellular mechanisms of resistance – and this is likely to occur in CNS as no cases of leptin resistance in peripherals has so far been found to lead to any change in phenotype.
The link to lipodystrophy in humans was then explored further in a plenary lecture by Phillip Gorden on the treatment of non-HIV congenital and acquired lipodystrophy with thiazolidinediones and leptin. 
Typical physical symptoms include lack of fat, hypertropic muscles and veins, acanthosis nigricans, hirsutism and virilization in women, enlarged liver and ovaries and increased body temperature and sweating.
Three types of clinical phenotypes were given as examples showing acquired lipodistrophy that was complete at age 14, two sisters with congenital lipodystophy and no body fat and an example of dunnigans ‘partial’ lipodystrophy that presented as normal or increased fat in upper body but complete loss of fat in arms and legs.
Genetics of these syndromes have shown the locus for genes for each case at 11q13, 9q34 and 1q21q22 affecting the Seipin, ASPAT2 and Lamin a/c proteins respectively.
Typical laboratory values include insulin resistance and hyperinsulinaemia, diabetes mellitus or impaired glucose tolerance, elevated triglyceride and FFA levels, decreased HDL-cholesterol levels, sometimes elevated LDL cholesterol and elevated androgen levels in women.
The rationale for treating with the PPAR-y agonist troglitazone includes promoting fat cell differentiation in vitro, insulin sensitising and triglyceride lowering in vivo. Arioglu E et al reported statistically significant reductions in HbA5, triglycerides, free fatty acids and liver volume and increases in percentages of fat oxidation and body fat in Ann Int Med, 2000 following six months treatment.
The rationale for leptin was largely outlined through the details in the previous lecture – as some patients with lipodystrophy have low levels of leptin and this was the group of patients studied (leptin levels under 4ng/ml).
Recombinant human methionyl-leptin (sc, BID) (Amgen corp).produces peak effect after 3-4 hours. Average concentrations increased as the dose increased over several months and showed a large variability between patients. Replacement dose was 0.02mg/kg/day for men aged 14 and over, 0.03mg/kg/day from women 14-18 years and 0.04mg/kg/day for women > 18 years. The protocol included a recommendation to adjust other anti-diabetic therapy down if needed, and the wide diversity in background of insulin resistance etc also led to very different results.
Symptoms for patient NIH-1 included an enlarged and distended stomach that was due to increased liver, and triglyceride levels in tens of thousands. In response to treatment and as dose increased to 200ng/ml, triglyceride levels fell, haemoglobin fell over time and liver size reduced by 40%. Benefit has so far continued out to 24 months (triglyceride values settled at 400-900; 200 = ULN) therefore this acquired form of genetic lipodystrophy has profound response.
However, no response to leptin was first found in a congenital case in a person with insulin resistance and diabetes type-1 – although some response may now be starting (after six months). Two sisters responded differently: one is OK. The other stopped treatment at four months – reverted, and then started again – and got a similar response. But the two different response rates in two sisters showed that genotype can produce different responses.
Grouped response showed 60-70% reductions and when contrasted to the troglitazone study showed an enormous difference in the quantitative response to different patients.
In conclusion, leptin was well tolerated, and led to significant decreases in glucose and haemoglobin HbA1c, triglycerides, free fatty acids, liver volume and liver fat as well as reducing resting metabolic rate and food intake. Six patients have been studied out to a year with a few to 18 months. Some neutralising antibodies have been detected but they have not appeared to have an effect on therapy. The dose has not been modified but from animal studies increasing the dose is expected to increase activity.
Future studies also need to determine whether there is an advantage for people who do not have such extreme leptin deficiency, and the protocol has since raised this level to 6ng/ml. No changes in intraabdomial fat have been recognised.
** Oral et al reported in the NEJM the benefits from use of recombinant methionyl human leptin (RL) administered sub-cutaneously every 12 hours for four months in nine women with lipodytrophy and serum leptin levels <4ng/ml. Eight women had diabetes mellitus and five had congenital generalised lipodystrophy. The physiologic replacement dose was estimated to be 0.03mg/kg body weight/day for girls under 18 years and 0.04 mg/kg/day for women over 18 years and this was approximately 10% of the doses used in previous obesity trials. Doses were escalated to achieve low, intermediate, and high physiologic replacement levels of leptin during the first, second and third/fourth months respectively.
Four months of therapy decreased average triglyceride levels by 60% (95% confidence interval, 43- 77% ; P<0.001) and liver volume by an average of 28% (95% confidence interval, 20-36%; P=0.002) in all nine patients and led to the discontinuation of or a large reduction in anti-diabetes therapy. Self-reported daily caloric intake and the measured resting metabolic rate also decreased significantly with therapy. Overall, recombinant leptin therapy was well tolerated. 
Petersen and colleagues from the same group reported in JCI the results from treating three patients with severe, generalised lipodystrophy associated with diabetes (fasting leptin concentration less than 4 ng/ml) with leptin replacement treatment for three months and six healthy nonsmoking women of similar age, weight, and body mass index as controls. 
Chronic leptin treatment improved insulin-stimulated hepatic and peripheral glucose metabolism in severely insulin-resistant lipodystrophic patients and was associated with a marked reduction in hepatic and muscle triglyceride content suggesting that leptin may represent an important new therapy to reverse the severe hepatic and muscle insulin resistance and associated hepatic steatosis in patients with lipodystrophy.
There was a marked reduction in the fasting plasma glucose concentration. The changes in hepatic and peripheral insulin sensitivity were associated with an 86% ± 8% reduction in hepatic triglyceride content (P = 0.008 compared with before leptin treatment) and a 33% ± 3% decrease in muscle triglyceride content (P = 0.006 compared with before leptin treatment). The leptin treatment–induced reduction in muscle triglyceride content was matched by an approximately 30% decrease in muscle total fatty acytl CoA concentrations. Leptin induced reduced food intake, rather than increased energy expenditure, was suggested as the mechanism for reduced triglycerides.
- Jeffrey Friedman – Leptin, lipodystrophy and insulin resistance. 4th Intl Workshop on Adverse Drug Reactions and Lipodystrophy in HIV. Sep 22-25 2002. San Diego. Keynote lecture.
- Oral EA – Leptin-Replacement Therapy for Lipodystrophy. New England Journal of Medicine February 21, 2002, Volume 346, Issue 8. 570-578.
- Petersen KF, Oral EA, Dufour S et al – Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. Jour Clin Invest May 2002. Vol109 No10 1345-1350.
- Phillip Gorden – Treatment of non-HIV lipodystrophy with thiazolidinediones and leptin. 4th Intl Workshop on Adverse Drug Reactions and Lipodystrophy in HIV. Sep 22-25 2002. San Diego. Plenary lecture.