Preclinical Therapeutic Validation of Insparin

Type 2 diabetes mellitus (T2DM) is a major chronic health concern worldwide. While current treatments are helpful, not all patients benefit from them equally because diabetes is a heterogeneous disease, and often individualized treatment is needed. Most existing diabetes medications rely heavily on intact insulin signaling, which is often impaired in T2DM. Many of these treatments may over time lead to the development of hypoglycemia, sarcopenia, weight gain, and pancreatic beta cell exhaustion. Thus, there is a pressing need for new therapies that can function independent of insulin signaling. Insparin, a peptide derived from the E4orf1 gene of human adenovirus 36, is a promising novel agent with a unique mechanism of action. It activates distal insulin signaling through the Ras/PI3K/AKT pathway while bypassing the proximal insulin receptor pathway. Studies using virus vector mediated or transgenic delivery of Insparin improved glycemic control and reduced liver steatosis development without causing hypoglycemia. For clinically relevant delivery, Insparin was delivered via nano-liposomes to mice, reproducing the effects observed in proof of concept studies. The next steps included optimization of dose, frequency and duration of nano-liposome mediated Insparin delivery and determination of long-term efficacy and safety compared to established anti-diabetes drugs. To address these questions, male C57BL/6J mice were fed a 45% high-fat diet to induce hyperglycemia and insulin resistance. Insparin was encapsulated in liposome nanoparticles and administered subcutaneously at 145 µg per injection per mouse. Insparin was injected at varied time points to identify peak glucose-lowering effects (Aim 1). Tissue-specific glucose uptake was assessed both acutely and after chronic treatment using [³H]-2-deoxy-D-glucose (Aim 2). Long-term effects of Insparin administration were compared with that of anti-diabetes agent pioglitazone (Pio) over 8 weeks. During the study, glucose tolerance, HbA1c, insulin levels, food intake, body weight, and body composition were measured (Aim 3). The liver and pancreas were analyzed histologically, while molecular markers were assessed in the liver, white and brown adipose tissue, and muscle. Plasma was also analyzed for biomarkers and potential immunogenicity (Aim 4). Insparin showed a long-lasting glucose-lowering effect, with peaks between 12 to 18 hours post-injection (Aim 1). Acutely, did not alter tissue-specific glucose uptake. Chronic Insparin treatment changed heart metabolism. In heart, it reduced glucose uptake and genes of insulin signaling and lipid metabolism. Insparin increased CPT1b signaling and raised plasma triglyceride levels. Pio-treated mice reduced glucose uptake only in brown adipose tissue (Aim 2). After 8 weeks of treatment, Insparin improved glucose tolerance and HbA1c, reduced food intake and fat mass while preserving lean mass. Pio improved HbA1c but increased fat and body weight (Aim 3). Insparin lowered liver ALT and triglycerides. Both Insparin and Pio improved metabolic dysfunction-associated steatotic liver disease (MASLD) score, without significant differences in pancreatic histology. Immunogenicity was limited to weak response to the GST tag of Insparin, but not Insparin itself. Insparin was detected localized to the injection sites in inguinal adipose tissue (Aim 4). In conclusion, Insparin provides long-lasting glucose-lowering effects while protecting the liver from fat accumulation. It also outperforms Pio by reducing body weight mainly through fat loss. The results support Insparin as a safe and effective candidate for T2DM treatment and for related metabolic complications. This support continued development of Insparin as a new class of anti-diabetes drugs.